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CA 02209659 1997-07-04
WO 97/17432 PCT/US96/18003
INSECTICIDAL PROTEIN TOXINS FROM PHOTORHABDUS
,
15
Field of the Invention
The present invention relates to toxins isolated from
bacteria and the use of said toxins as insecticides.
Background of the Invention
Many insects are widely regarded as pests to homeowners, to
picnickers, to gardeners, and to farmers and others whose
investments in agricultural products are often destroyed or
diminished as a result of insect damage to field crops.
Particularly in areas where the growing season is short,
significant insect damage can mean the loss of all profits to
growers and a dramatic decrease in crop yield. Scarce supply of
particular agricultural products invariably results in higher
costs to food processors and, then, to the ultimate consumers of
food plants and products derived from those plants.
Preventing insect damage to crops and flowers and
eliminating the nuisance of insect pests have typically relied on
strong organic pesticides and insecticides with broad toxicities.
These synthetic products have come under attack by the general
population as being too harsh on the environment and on those
exposed to such agents. Similarly in non-agricultural settings,
homeowners would be satisfied to have insects avoid their homes
or outdoor meals without needing to kill the insects.
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The extensive use of chemical insecticides has raised
environmental and health concerns for farmers, companies that
produce the insecticides, government agencies, public interest
groups, and the public in general. The development of less
intrusive pest management strategies has been spurred along both
by societal concern for the environment and by the development of
biological tools which exploit mechanisms of insect management.
Biological control agents present a promising alternative to
chemical insecticides.
Organisms at every evolutionary development level have
devised means to enhance their own success and survival.= The use
of biological molecules as tools of defense and aggression is
known throughout the animal and plant kingdoms. In addition, the
relatively new tools of the genetic engineer allow modifications
to biological insecticides to accomplish particular solutions to
particular problems.
One such agent, Bacillus thuringiensis (Bt), is an effective
insecticidal agent, and is widely commercially used as such. In
fact, the insecticidal agent of the Bt bacterium is a protein
which has such limited toxicity, it can be used on human food
crops on the day of harvest. To non-targeted organisms, the Bt
toxin'is a digestible non-toxic protein.
Another known class of biological insect control agents are
certain genera of nematodes known to be vectors of transmission
for insect-killing bacterial symbionts. Nematodes containing
insecticidal bacteria invade insect larvae. The bacteria then
kill the larvae. The nematodes reproduce in the larval cadaver.
The nematode progeny then eat the cadaver from within. The
bacteria-containing nematode progeny thus produced can then
invade additional larvae.
In the past, insecticidal nematodes in the Steinernema and
Heterorhabditis genera were used as insect control agents.
Apparently, each genus of nematode hosts a particular species of
bacterium. In nematodes of the Heterorhabditis genus, the
symbiotic bacterium is Photorhabdus luminescens.
Although these nematodes are effective insect control
agents, it is presently difficult, expensive, and inefficient to
produce, maintain, and distribute nematodes for insect control.
It has been known in the art that one may isolate an
insecticidal toxin from Photorhabdus luminescens that has
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activity only when injected into Lepidopteran and Coleopteran
insect larvae. This has made it impossible to effectively
exploit the insecticidal properties of the nematode or its
bacterial symbiont. What would be useful would be a more
practical, less labor-intensive wide-area delivery method of an
insecticidal toxin which would retain its biological properties
after delivery. It would be quite desirous to discover toxins
with oral activity produced by the genus Photorhabdus. The
isolation and use of these toxins are desirous due to
efficacious reasons. Until applicants' discoveries, these
toxins had not been isolated or characterized.
Summary of the Invention
The native toxins are protein complexes that are
produced and secreted by growing bacteria cells of the genus
Photorhabdus, of interest are the proteins produced by the
species Photorhabdus luminescens. The protein complexes, with
a molecular size of approximately 1,000 kDa, can be separated
by SDS-PAGE gel analysis into numerous component proteins. The
toxins contain no hemolysin, lipase, type C phospholipase, or
nuclease activities. The toxins exhibit significant toxicity
upon exposure administration to a number of insects.
The present invention provides an easily administered
insecticidal protein as well as the expression of toxin in a
heterologous system.
The present invention also provides a method for
delivering insecticidal toxins that are functional active and
effective against many orders of insects.
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CA 02209659 2006-09-06
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In one aspect, the invention provides a
polynucleotide that is operably associated with a heterologous
promoter, wherein said polynucleotide encodes a protein that
has toxin activity against an insect pest wherein a nucleotide
molecule that codes for said protein maintains hybridization
with the complement of a nucleic acid sequence after
hybridization and wash wherein said hybridization is conducted
at 60 C in solution containing 10% w/v PEG (polyethylene
glycol, M.W. approximately 8000), 0.6X SSC, 7% w/v SDS, 10 mM
sodium phosphate buffer, 5 mM EDTA, and 100 mg/ml denatured
salmon sperm DNA, said wash is conducted in 0.25X SSC and
0.2% SDS at 60 C, and said nucleic acid sequence is selected
from the group consisting of SEQ ID N0:11 and SEQ ID NO:46.
In another aspect, the invention provides a
polynucleotide that encodes a protein having toxin activity
against an insect pest wherein said protein comprises the amino
acid sequence of SEQ ID NO:12, and SEQ ID NO:47, wherein said
polynucleotide is operably associated with a heterologous
promoter.
In another aspect, the invention provides a
polynucleotide comprising a nucleotide sequence that encodes
the protein of SEQ ID NO: 12 operably associated with a
heterologous promoter.
In another aspect, the invention provides a
polynucleotide comprising a nucleotide sequence that encodes
the protein of SEQ ID NO:47 operably associated with a
heterologous promoter.
In another aspect, the invention provides a
transgenic plant cell comprising a polynucleotide as described
above.
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In another aspect, the invention provides a
recombinant protein having toxin activity against an insect
pest wherein a nucleotide sequence that codes for said protein
maintains hybridization with a nucleic acid sequence under
hybridization and wash wherein said hybridization is conducted
at 60 C in solution containing 10% w/v PEG (polyethylene
glycol, M.W. approximately 8000), 0.6X SSC, 7% w/v SDS, 10 mM
sodium phosphate buffer, 5 mM EDTA, and 100 mg/ml denatured
salmon sperm DNA, wherein said wash is conducted in 0.25X SSC
and 0.2% SDS at 60 C and wherein said nucleic acid sequence is
selected from the group consisting of SEQ ID NO:11 and SEQ ID
NO:46.
In another aspect, the invention provides a
recombinant protein having toxin activity against an insect
pest wherein said protein comprises an amino acid sequence
selected from the group consisting of SEQ ID NO:12 and SEQ ID
NO:47.
In another aspect, the invention provides a method
of controlling an insect pest wherein said method comprises
feeding a protein as described above to said pest.
In another aspect, the invention provides a
substantially pure culture of a Photorhabdus strain of ATCC
553597.
Objects, advantages, and features of the present
invention will become apparent from the following
specification.
Brief Description of the Drawings
Fig. 1 is an illustration of a match of cloned DNA
isolates used as a part of sequence genes for the toxin of the
present invention.
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Fig. 2 is a map of three plasmids used in the
sequencing process.
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2 9 3 5 5-1 CA 02209659 2001-01-19
Fig. 3 is a map 11ustrating che inter-relationship of
s-everal partial DNA fragments.
Fig. 4 is an illustration of a hcmology analysis becweeii the
protein sequences of TcbAii and TcaBii proteins.
Fig. 5 is a phenogram of Photorhabdus strains. Relaticnship
of Phctorhabdus Strains was defined by rep-PCR.
The upper axis of Fig. 5 measures the percentage similaricy of
strains based on scoring of rep-PCR products (i.e., 0.0 (no
similarity] to 1.0 (100% similarity)). At the right axis, ttie
numbers and letters indicate the various strains tested; 14=W-14,
Hm=Hm, H9=H9, 7=vdX-7, 1=WX-1, 2=WX-2, 88=HP88, NC-1=NC-1, 4=-WX-4,
9=WX-9, B=Wx-8, 10=Wx-10, WIR=WIR, 3=wX-3, 11=Wx-11, 5=W"/,-5,
6=WX-6, 12=WX-12, x14=14X-14, 15=WX-15, Hb=Hb, B2=B2, 48 through
52=ATCC 43948 through ATCC 43952. Vertical lines separating
horizontal lines indicate the degree of relatedness (as read from
the extrapolated intersection of the vertical line with the upper
axis) between strains or groups of strains at the base of the
horizontal lines (e.g., strain W-14 is approximately 60% si:nilar
to strains H9 and Hm).
Fig. 6 is an illustration of the genomic maps of the W-14
Strain.
Detailed DescriDtion of the Invention
The present inventions are directed to the discovery cF- a
unique class of insecticidal protein toxins from the genus
Phocorhabdus that have oral toxicity against insects. A unique
feature of Photorhabdus is its bioluminescence. Photorhabdus may
be isolated from a variety of sources. One such source is
nematodes, more particularly nematodes of the genus
Heterorhabdicis. Another such source is from human clinical
samples from wounds, see Farmer et al. 1989 J. Clin. Microbicl.
27 pp. 1594-1600. These saprohytic strains are deposited in the
American Type Culture Collection (Rockville, MD) ATCC #s 43948,
43949, 43950, 43951, and 43952.
It is possible that other sources could harbor.
Photorhabdus bacteria that produce insecticidal toxins. Suciz
sources in the environment could be either terrestrial or aQuatlc
based.
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The genus =hecor' abdus is taxonomically def ined as a mecrl :~L
of che Family .'ncereoaccar=aceae, although it has cercain tra=c=
acypical of this family. For example, scrains of this genus are
nitrate reduction negacive, yellow and red pigmenc producing anc
bioluminescenc. This latter trait is otherwise unknown within
the =ncerooaccer:aceae. Photorhabdus has only recently been
described as a genus separate from the Xenorhabdus (Boemare
et al., 1993 Int. J. Syst. Bacteriol. 43, 249-255). This
differentiation is based on DPtA-DNA hybridization studies,
phenotypic differences (e.g., presence (Photorhabdus; or absence
(Xenorhabdus) of cacalase and bioluminescence) and the Family of
the nematode host (Xenorhabdus; Steinernematidae, Phocorhabdus;
Heterorhabd=cidae). Comparative, cellular fatty-acid analyses
(Janse et al. 1990, Lett. Appl. Microbiol 10, 131-135; Suzuki
et al. 1990, J. Gen. Appl. Microbiol., 36, 393-401) support the
separation of Phocorhabdus from Xenorhabdus.
In order to establish that the strain collection disclosed
herein was comprised of Photorhabdus straiiis, the strains were
characterized based on reccgnized traits which define
Photorhabdus and differentiate it from other cnterobacceriaceae
and Xenorhabdus species. (Farmer, 1984 Bergey's Manual of
Systemic Bacteriology Vol. 1 pp.510-511; Akhurst and Boemare 1988,
J. Gen. Microbiol. 134 pp.1835-1845; Boemare et al. 1993 Int. J.
Syst. Bacteriol. 43 pp.249-255.
The traits studied were the following: gram stain
negative rods, organism size, colony pigmentation, inclusion
bodies, presence of catalase, ability to reduce nitrace,
bioluminescence, dye uptake, gelatin hydrolysis, growth on
selective media, growth temperature, survival under anerobic
conditions and motility. Fatty acid analysis was used to confirm
that the strains herein all belong to the single genus
Photorhabdus.
Currently, the bacterial genus Photorhabdus is comprised of
a single defined species, Photorhabdus luminescens (ATCC T'ype
strain #29999, Poinar et al., 1977, Nematologica 23, 97-102). A
variety of related strains have been described in the literacure
(e.g. Akhurst et al. 1988 J. Gen. Microbiol., 134, 1835-1845;
Boemare et al. 1993 Izt. J. Syst. Bacteriol. 43 pp. 249-255; Pucz
ec al. 1990, appl. Environ. Microbiol. , 56, 181-186) Numerous
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WO 97/17432 PCT/US96/18003
Photorhabdus strains have been characterized herein. Such
strains are listed in Table 18 in the Examples. Because there is
currently only one species (luminescens) defined within the genus
Photorhabdus, the luminescens species traits were used to
characterize the strains herein. As can be seen in Fig. 5, these ~
strains are quite diverse. It is not unforeseen that in the
future there may be other Photorhabdus species that will have
some of the attributes of the luminescens species as well as some
different characteristics that are presently not defined as a
trait of Photorhabdus luminescens. However, the scope of the
invention herein is to any Photorhabdus species or strains which
produce proteins that have functional activity as insect control
agents, regardless of other traits and characteristics.
Furthermore, as is demonstrated herein, the bacteria of the
genus Photorhabdus produce proteins that have functional activity
as defined herein. Of particular interest are proteins produced
by the species Photorhabdus luminescens. The inventions herein
should in no way be limited to the strains which are disclosed
herein. These strains illustrate for the first time that
proteins produced by diverse isolates of Photorhabdus are toxic
upon exposure to insects. Thus, included within the inventions
described herein are the strains specified herein and any mutants
thereof, as well as any strains or species of the genus
Photorhabdus that have the functional activity described herein.
There are several terms that are used herein that have a
particular meaning and are as follows:
By "functional activity" it is meant herein that the protein
toxins function as insect control agents in that the proteins are
orally active, or have a toxic effect, or are able to disrupt or
deter feeding, which may or may not cause death of the insect.
when an insect comes into contact with an effective amount of
toxin delivered via transgenic plant expression, formulated
protein compositions(s), sprayable protein composition(s), a bait
matrix or other delivery system, the results are typically death
of the insect, or the insects do not feed upon the source which
makes the toxins available to the insects.
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The protein toxins discussed herein are typically
referred to as "insecticides". By insecticides it is meant
herein that the protein toxins have a "functional activity" as
further defined herein and are used as insect control agents.
By the use of the term "oligonucleotides" it is meant
a macromolecule consisting of a short chain of nucleotides of
either RNA or DNA. Such length could be at least one
nucleotide, but typically are in the range of about 10 to about
12 nucleotides. The determination of the length of the
oligonucleotide is well within the skill of an artisan and
should not be a limitation herein. Therefore, oligonucleotides
may be less than 10 or greater than 12.
By the use of the term "toxic" or "toxicity" as used
herein it is meant that the toxins produced by Photorhabdus
have "functional activity" as defined herein.
By the use of the term "genetic material" herein, it
is meant to include all genes, nucleic acid, DNA and RNA.
By use of the term "Photorhabdus toxin" it is meant
any protein produced by a Photorhabdus microorganism strain
which has functional activity against insects, where the
Photorhabdus toxin could be formulated as a sprayable
composition, expressed by a transgenic plant, formulated as a
bait matrix, delivered via a Baculovirus, or delivered by any
other applicable host or delivery system.
Fermentation broths from selected strains reported in
Table 18 were used to determine the following: breadth of
insecticidal toxin production by the Photorhabdus genus, the
insecticidal spectrum of these toxins, and to provide source
material to purify the toxin complexes. The strains
characterized herein have been shown to have oral toxicity
against a variety of insect orders. Such insect orders include
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but are not limited to Coleoptera, Homopotera, Lepidoptera,
Diptera, Acarina, Hymenoptera and Dictyoptera.
As with other bacterial toxins, the rate of mutation
of the bacteria in a population causes many related toxins
slightly different in sequence to exist. Toxins of interest
here are those which produce protein complexes toxic to a
variety of insects upon exposure, as described herein.
Preferably, the toxins are active against Lepidoptera,
Coleoptera, Homopotera, Diptera, Hymenoptera, Dictyoptera and
Acarina. The inventions herein are intended to capture the
protein toxins homologous to protein toxins produced by the
strains herein and any derivative
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strains chereof, as we?1 as any protein toxins produced by
Phocorhabdus. These homologous oroceins may differ in sequence,
but do not differ in function from those toxins described her=in.
Homologous toxins are meant to include protein complexes oE
between 300 kDa to 2,000 kDa and are comprised of ac least t,=.
(2) subunits, where a subunit is a peptide which may or may n~t
be the same as the ocher subunit. Various protein subunits have
been identified and are taught in the Examples herein.
Typically, the protein subunits are between about 18 kDa to about
230 kDa; between about 160 kDa to about 230 kDa; 100 kDa to 160
kDa; about 80 kDa to about 100 kDa; and about 50 kDa to about 80
kDa.
As discussed above, some Photorhabdus strains can be
isolated from nematodes. Some nematodes, elongated cylindrical
parasitic worms of the phylum Nemacoda, have evolved an ability
to exploit insect larvae as a favored growth environment. The
insect larvae provide a source of food for growing nematodes and
an environment in which to reproduce. One dramatic effect that
follows invasion of larvae by certain nematodes is larval death.
Larval death results from the presence of, in certain nematodes,
bacteria that produce an insecticidal toxin which arrests lar-,a1
growth and inhibits feeding activity.
Interestingly, it appears that each genus of insect
parasitic nematode hosts a particular species of bacterium,
uniquely adapted for symbiotic growth with that nematode. In the
interim since this research was initiated, the name of the
bacterial genus Xenorhabdus was reclassified into the Xenorhabdus
and the Photorhabdus. Bacteria of the genus Photorhabdus are
characterized as being symbionts of Keterorhabditus nematodes
while Xenorhabdus species are symbionts of the Steinernema
species. This change in nomenclature is reflected in this
specification, but in no way should a change in nomenclature
alter the scope of the inventions described herein.
The peptides and genes that are disclosed herein are named
according to the guidelines recently published in the Journai of
Bacteriology "Instructions to Authors" p. i-xii (Jan. 1996).
The following peptides and genes were isolated from Photorhabdus
strain W-14.
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Peptide / Gene Nomenclature
Toxin complex (Tc)
Peptide Gene Patent
Name Name Sequence ID#
tca genomic region
TcaA tcaA 12
TcaAiii tcaA 4
TcaBi tcaB 3 (19, 20)
TcaBii tcaB 5
TcaC tcaC 2
tcb genomic region
TcbA tcbA 16
TcbAi tcbA (pro-peptide)
TcbAii tcbA 1 (21, 22, 23, 24)
TcbAiii tcbA 40
tcc genomic region
TccA tccA 8
TccB tccB 7
tcd genomic region
TcdAi tcdA (pro-peptide)
TcdAii tcdA 13, (38, 39
17, 18)
TcdAiii tcdA 41, (42, 43)
TcdB tcdB 14
(bracket sequence indicates internal amino acid sequence obtained
by tryptic digests)
The sequences listed above are grouped by genomic region.
The tcbA gene was expressed in E. coli as two protein fragments
TcbA and TcbAiii as illustrated in the Examples. It may be
beneficial to have proteolytic clippage of some sequences to
obtain the higher activity of the toxins for commercial
transgenic applications.
The toxins described herein are quite unique in that the
toxins have functional activity, which is key to developing an
insect management strategy. In developing an insect management
strategy, it is possible to delay or circumvent the protein
degradation process by injecting a protein directly into an
organism, avoiding its digestive tract. In such cases, the protein
administered to the organism will retain its function
until it is denatured, non-specifically degraded, or eliminated
by the immune system in higher organisms. Injection into insects
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of an insecticidal toxin has potential application only in the
laboratory, and then only on large insects which are easily
injected. The observation that the insecticidal protein toxins
herein described exhibits their toxic activity after oral
ingestion or contact with the toxins permits the development of
an insect management plan based solely on the ability to
incorporate the protein toxins into the insect diet. Such a plan
could result in the production of insect baits.
The Photorhabdus toxins may be administered to insects in a
purified form. The toxins may also be delivered in amounts from
about 1 to about 100 mg / liter of broth. This may vary upon
formulation condition, conditions of the inoculum source,
techniques for isolation of the toxin, and the like. The toxins
may be administered as an exudate secretion or cellular protein
originally expressed in a heterologous prokaryotic or eukaryotic
host. Bacteria are typically the hosts in which proteins are
expressed. Eukaryotic hosts could include but are not limited to
plants, insects and yeast. Alternatively, the toxins may be
produced in bacteria or transgenic plants in the field or in the
insect by a baculovirus vector. Typically the toxins will be
introduced to the insect by incorporating one or more of the
toxins into the insects' feed.
Complete lethality to feeding insects is useful but is not
required to achieve useful toxicity. If the insects avoid the
toxin or cease feeding, that avoidance will be useful in some
applications, even if the effects are sublethal. For example, if
insect resistant transgenic crop plants are desired, a reluctance
of insects to feed on the plants is as useful as lethal toxicity
to the insects since the ultimate objective is protection of the
plants rather than killing the insect.
There are many other ways in which toxins can be
incorporated into an insect's diet. As an example, it is
possible to adulterate the larval food source with the toxic
protein by spraying the food with a protein solution, as
disclosed herein. Alternatively, the purified protein could be
genetically engineered into an otherwise harmless bacterium,
which could then be grown in culture, and either applied to the
food source or allowed to reside in the soil in an area in which
insect eradication was desirable. Also, the protein could be
genetically engineered directly into an insect food source. For
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instance, the major food source of many insect larvae is plant
material.
By incorporacing genetic material that encodes the
insecticidal propercies of the Photorhabdus toxins into che
genome of a plant eaten by a particular insect pest, the adult or
larvae would die after consuming the food plant. Numerous
members of the mcnocotyledonous and dictyledenous genera have
been transforrned. Transgenic agronmonic crops as well as fruics
and vegetables are of commercial interest. Such crops include
buc are not limited to maize, rice, soybeans, canola, sunflower,
alfalfa, sorghum, wheat, cotton, peanuts, tomatoes, potatoes, and
the like. Several techniques exist for introducing foreign
genetic material into plant cells, and for obtaining plants that
stably maintain and express the introduced gene. Such techniques
include acceleracion of genetic material coated onto
mic:oparticles directly into cells(U.S. Patents 4,945,050 to
Cornell and 5,141,131 to DowElanco). Plants may be transformed
using Agrobacceriuun technology, see U.S. Patent 5,177;010 to
University of Toledo, 5,104,310 to Texas A&M, European Yatent
Application 0131624B1, European Patent Applications 120516,
159418B1 and 176,112 to Schilperoot, U.S. Patents 5,149,645,
5,469',976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot,
7-uropean Patent Applications 116718, 290799, 320500 all to
MaxPlanck, European Patent Applications 604662 and 627752 to
Japan Tobacco, European Patent Applications 0267159, and 0292435
and U.S. Patent 5,231,019 all to Ciba Geigy, U.S. Patents
5,463,174 and 4,762,785 both to Calgene, and U.S. Patents
5,004,863 and 5,159,135 both to Agracetus. Other transformation
technology includes whiskers technology, see U.S. Patents
5,302,523 and 5,464,765 both to Zeneca. Electroporation
technology has also been used to transform plants, see WO
87/06614 to Boyce Thompson Institute, 5,472,869 and 5,384,253
both to Dekalb, W09209696 and W09321335 both to PGS.
In addition to numerous technologies for transforming
plants, the type of tissue which is contacted with the foreign
genes may vary as well. Such tissue would include but would not
be limited to embryogenic tissue, callus tissue type I and II,
hypocotyl, meristem, and the like. Almost all plant tissues may
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be tr3nsfor:rle=.i dur:na jed1LL -rent_atlon using 3ppropr_-a_a
c-'?,=h~.iques wichin che skill of an artisan.
Anoche= variable is che choice of a seleccable marker. The
preference for a particular marker is at the discretion ot the
artisan, buc any of che following seleccable markers may be used
along wich any ocher gene noc listed herein which could flrnction
as a selectable marker. Such seleccable markers include but are
noc limited co aminoglycoside phosphotransferase gene of
transposon Tn5 (Aph II) which encodes resistance to the
antibiocics kanamycin, neomycin and G418, as well as those genes
which code for resistance or tolerance to glyphosace; hygromycin;
methotrexate; phosphinothricin (bialophos); imidazolincnes,
sulfonylureas and triazolopyrimidine herbicides, such as
chlorosulfuron; bromoxynil, dalapon and the like.
In addition to a seleccable marker, it may be desirous co
use a reporter gene. In some instances a reporcer gene may be
used without a selectable marker. Reporter genes are genes which
are cypically noc presenc or expressed in the recipient organism
or tissue. The reporter gene typically encodes for a protein
which provides fcr some phenotypic change or enzymacic property.
Examples c'_ such genes are provided in K. Weising et a!. Ann.
Rev. Genetics, 22, 421 (1988),
A preferred reporter gene is the glucuronidase (GUS) gene.
Regardless of transformation technique, the gene is
preferably incorporated inco a gene transfer vector adapted to
express the Phocorhabdus toxins in the planc cell by including in
the vector a plant promoter. in addition to plant promoters,
promoters from a variety of sources can be used efficiencly in
plant cells to express foreign genes. For example. promocers of
bacterial origin, such as the octopine synthase promoter, the
nopaline synthase promoter, the mannopine synthase promocer;
promoters of viral origin, such as the cauliflower mosaic =:irus
(35S and 19S)and the like may be used. Plant promocers incl+lde,
but are not limiced to ribulose-l,6-bisphosphate (RUEP)
carboxylase small subunit (ssu), beta-conglycinin promoter,
phaseolin promoter, ADH promoter, heat-shock promoters and tissue
specific promoters. Prcmoters may also contain certain enhancer
se-uence elemencs that may improve the cranscription efficiency.
?ypical enhancers include but are noc limiced to Adh-intron I and
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Adh-intron 6. Constitutive promoters may be used. Constitutive
promoters direct continuous gene expression in all cells types
and at all times (e.g., actin, ubiquitin, CaMV 35S). Tissue
specific promoters are responsible for gene expression in
specific cell or tissue types, such as the leaves or seeds (e.g.,
zein, oleosin, napin, ACP) and these promoters may also be used.
Promoters may also be are active during a certain stage of the
plants' development as well as active in plant tissues and
organs. Examples of such promoters include but are not limited
to pollen-specific, embryo specific, corn silk specific, cotton
fiber specific, root specific, seed endosperm specific promoters
and the like.
Under certain circumstances it may be desirable to use an
inducible promoter. An inducible promoter is responsible for
expression of genes in response to a specific signal, such as:
physical stimulus (heat shock genes); light (RUBP carboxylase);
hormone (Em); metabolites; and stress. Other desirable
transcription and translation elements that function in plants
may be used. Numerous plant-specific gene transfer vectors are
known to the art.
In addition, it is known that to obtain high expression of
bacterial genes in plants it is preferred to reengineer the
bacterial genes so that they are more efficiently expressed in
the cytoplasm of plants. Maize is one such plant where it is
preferred to reengineer the bacterial gene(s) prior to
transformation to increase the expression level of the toxin in
the plant. One reason for the reengineering is the very low G+C
content of the native bacterial gene(s) (and consequent skewing
towards high A+T content). This results in the generation of
sequences mimicking or duplicating plant gene control sequences
that are known to be highly A+T rich. The presence of some A+T-
rich sequences within the DNA of the gene(s) introduced into
plants (e.g., TATA box regions normally found in gene promoters)
may result in aberrant transcription of the gene(s). On the
other hand, the presence of other regulatory sequences residing
in the transcribed mRNA (e.g., polyadenylation signal sequences
(AAUAAA), or sequences complementary to small nuclear RNAs
involved in pre-mRNA splicing) may lead to RNA instability.
Therefore, one goal in the design of reengineered bacterial
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gene(s), more oreferably referred to as plant optimized gene!s),
is to generate a DNA sequence having a higher G+C content, and
preferably one close to that of plant genes coding for metabolic
enzymes. Another goal in the design of the plant optimized
gene(s) is to generate a DNA sequence that not only has a higher
G+C content, but by modifying the sequence changes, should be
made so as to not hinder translation.
An example of a plant that has a high G+C content is maize.
The table below illustrates how high the G+C content is in maize.
As in maize, it is thought that G+C content in other plants is
also high.
Table I
Compilation of G+C contents of protein coding regions
of maize genes
Protein Classa Range %G+C Mean %G+C
Metabolic Enzymes (40) 44.4-75.3 59.0 (8.0)
Storage Proteins
Group I (23) 46.0-51.9 48.1 (1.3)
Group II (13) 60.4-74.3 67.5 (3.2)
Group I + II (36) 46.0-74.3 55.1 (9.6) c
Structural Proteins (18) 48.6-70.5 63.6 (6.7)
Regulatory Proteins (5) 57.2-68.9 62.0 (4.9)
Uncharacterized Proteins (9) 41.5-70.3 64.3 (7.2)
All Proteins (108) 44.4-75.3 60.8 (5.2)
a Number of genes in class given in parentheses.
b Standard deviations given in parentheses.
~ Combined groups mean ignored in calculation of
overall mean.
For the data in Table 1, coding regions of the genes were
extracted from GenBank (Release 71) entries, and base
compositions were calculated using the MacVectorT"" program (IBI,
New Haven, CT). Intron sequences were ignored in the
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-alculations. Group I and II storage protein gene sequences were
distinguished by their marked difference in base composition.
Due to the plasticity afforded by the redundancy of the
genetic code (i.e., some amino acids are specified by more than
one codon), evolution of the genomes of different organisms or
classes or organisms has resulted in differential usage of
redundant codons. This "codon bias" is reflected in the mean base
composition of protein coding regions. For example, organisms
with relatively low G+C contents utilize codons having A or T in
the third position of redundant codons, whereas those having
higher G+C contents utilize codons having G or C in the third
position. It is thought that the presence of "minor" codons
within a gene's mRNA may reduce the absolute translation rate of
that mRNA, especially when the relative abundance of the charged
tRNA corresponding to the minor codon is low. An extension of
this is that the diminution of translation rate by individual
minor codons would be at least additive for multiple minor
codons. Therefore, mR1VAs having high relative contents of minor
codons would have correspondingly low translation rates. This
rate would be reflected by the synthesis of low levels of the
encoded protein.
In order to reengineer the bacterial gene(s), the codon bias
of the plant is determined. The codon bias is the statistical
codon distribution that the plant uses for coding its proteins.
After determining the bias, the percent frequency of the codons
in the gene(s) of interest is determined. The primary codons
preferred by the plant should be determined as well as the second
and third choice of preferred codons. The amino acid sequence of
the protein of interest is reverse translated so that the
resulting nucleic acid sequence codes for the same protein as the
native bacterial gene, but the resulting nucleic acid sequence
corresponds to the first preferred codons of the desired plant.
The new sequence is analyzed for restriction enzyme sites that
might have been created by the modification. The identified
sites are further modified by replacing the codons with second or
third choice preferred codons. other sites in the sequence which
could affect the transcription or translation of the gene of
interest are the exon:intron 5' or 3' junctions, poly A addition
signals, or RNA polymerase termination signals. The sequence is -15-
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f,lrt!ier anal~ zed snd modified to reduce the Ereq'1P-ncy oE T. ,r
doublets. Ln addit:.on co che doublets, G or C sequence b!ocks
thaC have more than about four residues thac are the same can
affect transcripCion of the sequence. Therefore, these blocks
are 3lso modified by replacing the codons of Eirst or second
choice, etc. with the next oreferred codon of choice. It is
preferred that the plant ootimized gene(s) contains abouc 630 oE
firsC choice codons, between about 22t co about 37% second choice
codons, and between 15o and 01 third choice codons, wherein the
lU Cocal percentage is 1001. Most preferred the plant optimized
gene(s) contain about 63% of first choice codons, at le3st about
221 second choice codons, about 7.5e third choice codons, and
about 7.5% fourth choice codons, wherein the total percentage i.3
100:. The method described above enables one skilled in the 3rt
to modify gene(s) ttlat are foreign to a particular plant so that
the genes are optimally expressed in plants. The mec'-cd is
Eurcher illustraCed in International PublicaLion W097/13402.
Thus, in order to design plant optimized gene(s) the amino
acid sequence of the toxins are reverse translated into a DNA
sequence, utilizing a nonredundant genetic code established E=om
a codon bias table compiled for the gene DNA sequence Ear the
particular plant being transformed. The resulting DNR sequence,
2:5 which is complecely homogeneous in codon usage, is further
modified to establish a DNA sequence that, besides having a
higher degree of codon diversity, also contains strategically
placed restriction enzyme recognition sites, desirable base
composition, and a lack of sequences that might interfere with
transcription of the gene, or translation of the product mPSIA.
It is theorized that bacterial genes may be more easily
expressed in plants if the bacterial genes are expressed in the
plastids. Thus, it may be possible to express bacterial genes in
plants, without=optimizing the genes Eor plant expression, and
obtain high express of the protein. See U.S. Patent Clos.
4,762,785; 5,451,513 and 5,545,817.
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~ne of the issues regarding commercial exploiting transgenic
plants is resistance management. This is of particular concern
with Bacillus thuringiensis toxins. There are numerous companies
commerically exploiting Bacillus thuringiensis and there has been
much concern about Bt toxins becoming resistant. One strataegy =
for insect resistant management would be to combine the toxins
produced by Photorhabdus with toxins such as Bt, vegetative
insect proteins (Ciba Geigy) or other toxins. The combinations
could be formulated for a sprayable application or could be
molecular combinations. Plants could be transformed with
Photorhabdus genes that produce insect toxins and other insect
toxin genes such as Bt as with other insect toxin genes such as
Bt.
European Patent Application 0400246A1 describes
transformation of 2 Bt in a plant, which could be any 2 genes.
Another way to produce a transgenic plant that contains more than
one insect resistant gene would be to produce two plants, with
each plant containing an insect resistant gene. These plants
would be backcrossed using traditional plant breeding techniques
to produce a plant containing more than one insect resistant
gene.
In addition to producing a transformed plant containing
plant optimized gene(s), there are other delivery systems where
it may be desirable to reengineer the bacterial gene(s). Along
the same lines, a genetically engineered, easily isolated protein
toxin fusing together both a molecule attractive to insects as a
food source and the insecticidal activity of the toxin may be
engineered and expressed in bacteria or in eukaryotic cells using
standard, well-known techniques. After purification in the
laboratory such a toxic agent with "built-in" bait could be
packaged inside standard insect trap housings.
Another delivery scheme is the incorporation of the genetic
material of toxins into a baculovirus vector. Baculoviruses
infect particular insect hosts, including those desirably
targeted with the Photorhabdus toxins. Infectious baculovirus
harboring an expression construct for the Photorhabdus toxins
could be introduced into areas of insect infestation to thereby
intoxicate or poison infected insects.
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Transfer of the insecticidal properties requires nucleic
acid sequences encoding the coding the amino acid sequences for
the Photorhabdus toxins integrated into a protein expression
vector appropriate to the host in which the vector will reside.
One way to obtain a nucleic acid sequence encoding a protein with
insecticidal properties is to isolate the native genetic material
which produces the toxins from Photorhabdus, using information
deduced from the toxin's amino acid sequence, large portions of
which are set forth below. As described below, methods of
purifying the proteins responsible for toxin activity are also
disclosed.
Using N-terminal amino acid sequence data, such as set forth
below, one can construct oligonucleotides complementary to all,
or a section of, the DNA bases that encode the first amino acids
of the toxin. These oligonucleotides can be radiolabeled and
used as molecular probes to isolate the genetic material from a
genomic genetic library built from genetic material isolated from
strains of Photorhabdus. The genetic library can be cloned in
plasmid, cosmid, phage or phagemid vectors. The library could be
transformed into Escherichia coli and screened for toxin
production by the transformed cells using antibodies raised
against the toxin or direct assays for insect toxicity.
This approach requires the production of a battery of
oligonucleotides, since the degenerate genetic code allows an
amino acid to be encoded in the DNA by any of several three-
nucleotide combinations. For example, the amino acid arginine
can be encoded by nucleic acid triplets CGA, CGC, CGG, CGT, AGA,
and AGG. Since one cannot predict which triplet is used at those
positions in the toxin gene, one must prepare oligonucleotides
with each potential triplet represented. More than one DNA
molecule corresponding to a protein subunit may be necessary to
construct a sufficient number of oligonucleotide probes to
recover all of the protein subunits necessary to achieve oral
toxicity.
From the amino acid sequence of the purified protein,
genetic materials responsible for the production of toxins can
readily be isolated and cloned, in whole or in part, into an
expression vector using any of several techniques well-known to
one skilled in the art of molecular biology. A typical
expression vector is a DNA plasmid, though other transfer means
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including, but not limited to, cosmids, phagemids and phage are
also envisioned. In addition to features required or desired for
plasmid replication, such as an origin of replication and
antibiotic resistance or other form of a selectable marker such
as the bar gene of Streptomyces hygroscopicus or
viridochromogenes, protein expression vectors normally
additionally require an expression cassette which incorporates
the cis-acting sequences necessary for transcription and
translation of the gene of interest. The cis-acting sequences
required for expression in prokaryotes differ from those required
in eukaryotes and plants.
A eukaryotic expression cassette requires a transcriptional
promoter upstream (5') to the gene of interest, a transcriptional
termination region such as a poly-A addition site, and a ribosome
binding site upstream of the gene of interest's first codon. In
bacterial cells, a useful transcriptional promoter that could be
included in the vector is the T7 RNA Polymerase-binding promoter.
Promoters, as previously described herein, are known to
efficiently promote transcription of mRNA. Also upstream from
the gene of interest the vector may include a nucleotide sequence
encoding a signal sequence known to direct a covalently linked
protein to a particular compartment of the host cells such as the
cell surface.
Insect viruses, or baculoviruses, are known to infect and
adversely affect certain insects. The affect of the viruses on
insects is slow, and viruses do not stop the feeding of insects.
Thus viruses are not viewed as being useful as insect pest
control agents. Combining the Photorhabdus toxins genes into a
baculovirus vector could provide an efficient way of transmitting
the toxins while increasing the lethality of the virus. In
addition, since different baculoviruses are specific to different
insects, it may be possible to use a particular toxin to
selectively target particularly damaging insect pests. A
particularly useful vector for the toxins genes is the nuclear
polyhedrosis virus. Transfer vectors using this virus have been
described and are now the vectors of choice for transferring
foreign genes into insects. The virus-toxin gene recombinant may
be constructed in an orally transmissible form. Baculoviruses
normally infect insect victims through the mid-gut intestinal
mucosa. The toxin gene inserted behind a strong viral coat
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procein promocer would be expressed and should rapidly kill the
infected insect.
In addition to an insect virus or baculovirus or transgenic
plant delivery syscem for che protein toxins of the presenc
invention, the proteins may be encapsulated using Baci:lus
_huringiensis encapsulacion technology such as but not limited to
U.S. Patent rJos. 4,695,455; 4,695,462; 4,861,595,
Another delivery system for
the protein toxins of the present invention is formulation of the
protein into a bait matrix, which could then be used in above and
below ground insect bait stations. Examples of such technology
include but are not limited to PCT Patent Application WO
93/23998.
As is described above, it might become necessary to modify
the sequence encoding the protein when expressing it in a non-
native hosc, since the codon preferences of other hoscs may
differ from that of Phocorhabdus. In such a case, translation
may be quite inefficient in a new host unless compensating
modifications to the coding sequence are made. Additionally,
modifications to the amino acid sequence might be desirable to
avoid inhibitory cross-reactivity with proteins of the new host,
or to refine the insecticidal properties of the protein in the
new host. A genetically modified toxin gene might encode a toxin
exhibiting, for example, enhanced or reduced toxicity, altered
insect resistance develcpment, altered stability, or modified
target species specificity.
In addition to the Photorhabdus genes encoding the toxins,
the scope of the present invention is intended to include relaced
nucleic acid sequences which encode amino acid biopolymers
homologous to the toxin proteins and which retain the toxic
effect of the Photorhabdus proteins in insect species after oral
ingestion.
For instance, the toxins used in the present invention seem
to first inhibit larval feeding before death ensues. By
manipulating the nucleic acid sequence of Photorhabdus toxins or
its controlling sequences, genetic engineers placing the toxin
gene into plants could modulace ics potency or its mode of action
to, for example, keep the eating-inhibito ry activity while
eliminating the absolute toxicity to the larvae. This change
could permit the transformed planc to survive until harvest
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without having the unnecessarily dramatic effect on the
ecosystem of wiping out all target insects. All such
modifications of the gene encoding the toxin, or of the protein
encoded by the gene, are envisioned to fall within the scope of
the present invention.
Other envisioned modifications of the nucleic acid
include the addition of targeting sequences to direct the toxin
to particular parts of the insect larvae for improving its
efficiency.
Strains ATCC 55397, 43948, 43949, 43950, 43951, 43952
have been deposited in the American Type Culture Collection,
12301 Parklawn Drive, Rockville, MD 20852 USA. Amino acid and
nucleotide sequence data for the W-14 native toxin
(ATCC 55397)* is presented below. Isolation of the genomic DNA
for the toxins from the bacterial hosts is also exemplified
herein.
Standard and molecular biology techniques were
followed and taught in the specification herein. Additional
information may be found in Sambrook, J., Fritsch, E.F, and
Maniatis, T. (1989), Molecular Cloning, A Laboratory Manual,
Cold Spring Harbor Press.
*Xenorhabdus luminescens, W-14 was deposited on March 5, 1993
in the American Type Culture Collection under accession number
55397.
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The following abbreviations are used throughout the
Examples: Tris = tris (hydroxymethyl) amino methane; SDS =
sodium dodecyl sulfate; EDTA = ethylenediaminetetraacetic acid,
IPTG = isopropylthio-B-galactoside, X-gal = 5-bromo-4-chloro-3-
indoyl-B-D-galactoside, CTAB = cetyltrimethylammonium bromide;
kbp = kilobase pairs; dATP, dCTP, dGTP, dTTP, I = 2'-deoxy-
nucleoside 5'-triphosphates of adenine, cytosine, guanine,
thymine, and inosine, respectively; ATP = adenosine 5'
triphosphate.
Example 1
Purification of toxin from P. luminescens and Demonstration of
toxicity after oral delivery of purified toxin
The insecticidal protein toxin of the present
invention was purified from P. luminescens strain W-14, ATCC
Accession Number 55397. Stock cultures of P. luminescens were
maintained on petri dishes containing 2% Proteose Peptone No. 3
(i.e., PP3, Difco Laboratories, Detroit MI) in 1.5% agar,
incubated at 25 C and transferred weekly. Colonies of the
primary form of the bacteria were inoculated into 200 ml of PP3
broth supplemented with 0.5%
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polyoxyethylene sorbitan mono-stearate (Tween 60, Sigma Chemical
Company=, St. Louis t4O) in a one liter flask. The broth cultures
were grown for 72 hours at 30 C on a rotary shaker. The toxin
proteins can be recovered from cuitures grown in the presence or
absence of Tween; however, the absence of Tween can affect the
form of the bacteria grow-n and the profile of proteins produced
by the bacteria. In the absence of Tween;' a variant shift occurs
insofar as the molecular weight of at least one identified toxin
subunit shifts from about 200 kDa to about 185 kDa.
The 72 hour cultures were centri'uged at 10,000 x g for 30
minutes to remove cells and debris. The supernatant fraction
that contained the insecticidal activ4-=y was decanted and brought
to 50 mM K2HPO4 by adding an appropriate volume of 1.0 M KtHPO,.
The pH was adjusted to 8.6 by adding potassium hydroxide. This
supernatant fraction was then mixed with DEAE-Sephacei (Pharmacia
LKB Biotechnology) which had been equilibrated with 50 mM K2HPO..
The toxic activity was adsorbed to the DEAE resin. This mixture
was then poured into a 2.6 x 40 cm column and washed with 50 mM
K,HPO4 at room temperature at a flow rate of 30 ml/hr until the
effluent reached a steady baseline W absorbance at 280 nm. The
column was then washed with 150 mM KC1 until the effluent again
reached a steady 280 nm baseline. Finally the column was washed
with 300 mM KC1 and fractions were collected.
Fractions containing the toxin were pooled and filter
sterilized using a 0.2 micron pore membrane filter. The toxin
was then concentrated and equilibrated to 100 mM KPO., pH 6.9,
using an ultrafiltration membrane with a molecular weight cutoff
of 100 kDa at 4 C (Centriprep 100, Amicon Division-W.R. Grace and
Company). A 3 ml sample of the toxin concentrate was applied to
the top of a 2.6 x 95 cm Sephacryl S-400 HR gel filtration column
(Pharmacia LKB Biotechnology) . The eluent buffer was 100 mM KPO4,
pH 6.9, which was run at a flow rate of 17 ml/hr, at 4 C. The
effluent was monitored at 280 nm.
Fractions were collected and tested for toxic activity.
Toxicity of chromatographic fractions was examined in a
biological assay using Manduca sexta larvae. Fractions were
either applied directly onto the insect diet (Gypsy moth wheat
germ diet, ICN Biochemicals Division - ICN Biomedicals, Inc.) or
administered by intrahemocelic injection of a 5 ul sample through
the first proleg of 4th or 5th instar larva using a 30 gauge
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needle. The weighc of each larva wichin a creatment group was
recorded ac 24 hour intervals. Toxicicy was presumed if the
insect ceased feeding and died within several days of consuming
treated insect diet or if death occurred within 24 hours after
injection of a fraction.
The toxic fractions were pooled and concentrated using the
Cencriprep-100 and were then analyzed by HPLC using a 7.5 mm x 60
cm TSK-GEL * G-4000 SW gel permeacion column with 100 mM potassium
phosphate, pH 6.9 eluent buffer running at 0.4 ml/min. This
analysis revealed the toxin protein to be contained within a
single sharp peak that eluted from the column with a retention
time of approximately 33.6 minutes. This retention time
corresponded to an estimated molecular weight of 1,000 kDa. Peak
fractions were collected for further purification while fractions
not containing this protein were discarded. The peak eluted from
the HPLC absorbs W lighc at 218 and 280 nm but did not absorb at
405 nm. Absorbance at 405 nm was shown to be an attribute of
xenorhabdin antibiotic compounds.
Electrophoresis of the pooled peak fractions in a non-
denaturing agarose gel (Metaphor*Agarose, b'MC BioProducts) showed
that two protein complexes are present in the peak. The peak
material, buffered in 50 mM Tris-HC1, pH 7.0, was separated on a
1.5% agarose stacking gel buffered with 100 mM Tris-HC1 at pH 7.0
and 1.9% agarose resolving gel buffered with 200 mM Tris-borate
ac pH 8.3 under standard buffer conditions (anode buffer 1M Tris-
HCI, pH 8.3; cathode buffer 0.025 M Tris, 0.192 M glycine). The
gels were run at 13 mA constant current at 15 C until the phenol
red tracking dye reached the end of the gel. Two protein bands
were visualized in the agarose gels using Coomassie brilliant
blue staining.
The slower migrating band was referred to as 'protein band
1" and faster migrating band was referred to as protein band 2."
The two protein bands were present in approximately equal
amounts. The Coomassie stained agarose gels were used as a guide
to precisely e:ccise the two protein bands from unstained portions
of the gels. The excised pieces containing the protein bands
were macerated and a small amounc of sterile water was added. As
a control, a portion of the gel that contained no procein was
also excised and treated in the same manner as the gel pieces
concaining the protein. Protein was recovered from the gel
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pieces by electroelution into 100 mM Tris-borate pH 3.3, ar_ iOn
volts (constant voltage) for two hours. Alternatively, protein
was passively eluted frorn the gel pieces by adding an equal
volume of 50 mM Tris-HC1, pH 7.0, to the gel pieces, then
incubating at 30 C for 16 hours. This allowed the protein to
diffuse from the gel into the buffer, which was then collected.
Results of insect toxicity tests using HPLC-purified toxin
(33.6 min. peak) and agarose gel purified toxin demonstrated
toxicity of the extracts. Injection of 1.5 ug of the HPLC
purified protein kills within 24 hours. Both protein bands 1 and
2, recovered from agarose gels by passive elution or
electroelution, were lethal upon injection. The protein
concentration estimated for these samples was less than 50
ng/larva. A comparison of the weight gain and the mortality
between the groups of larvae injected with protein bands 1 cr 2
indicate that protein band 1 was more toxic by injection
delivery.
When HPLC-purified toxin was applied to larval diet at a
concentration of 7.5 jig/larva, it caused a halt in larval weight
gain (24 larvae tested). The larvae begin to feed, but after
consuming only a very small portion of the toxin treated diet
they began to show pathological symptoms induced by the toxii: and
the larvae cease feeding. The insect frass became discolorei and
most larva showed signs of diarrhea. Significant insect
mortality resulted when several 5 tig toxin doses were applied to
the diet over a 7-10 day period.
Agarose-separated protein band 1 significantly inhibited
larval weight gain at a dose of 200 ng/larva. Larvae fed similar
concentrations of protein band 2 were not inhibited and gained
weight at the same rate as the control larvae. Twelve larva~
were fed eluted protein and 45 larvae were fed protein-containing
agarose pieces. These two sets of data indicate that proteiii
band 1 was orally toxic to Manduca sexta. In this experimenT: it
appeared that protein band 2 was not toxic to Manduca sexta.
Further analysis of protein bands 1 and 2 by SDS-PAGE urider
denaturing conditions showed that each band was composed of
several smaller protein subunits. Proteins were visualized k;y
Coomassie brilliant blue staining followed by silver staining to
achieve maximum sensitivity.
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The protein subunits in the two bands were very similar.
Protein band i contains 8 protein subunits of 25.1, 56.2, 60.8,
65.6, 166, 171, 184 and 208 kDa. Protein band 2 had an identical
profile except that the 25.1, 60.8, and 65.6 kDa proteins were
not present. The 56.2, 60.8, 65.6, and 184 kDa proteins were
present in the complex of protein band 1 at approximately equal
concentrations and represent 80% or more of the total protein
content of that complex.
The native HPLC-purified toxin was further characterized as
follows. The toxin was heat labile in that after being heated to
60 C for 15 minutes it lost its ability to kill or to inhibit
weight gain when injected or fed to M. sexta larvae. Assays were
designed to detect lipase, type C phospholipase, nuclease or red
blood cell hemolysis activities and were performed with purified
toxin. None of these activities were present. Antibiotic zone
inhibition assays were also done and the purified toxin failed to
inhibit growth of Gram-negative or -positive bacteria, yeast or
filamentous fungi, indicating that the toxic is not a xenorhabdin
antibiotic.
The native HPLC-purified toxin was tested for ability to
kill insects other than Manduca sexta. Table 2 lists insects
killed by the HPLC-purified P. luminescens toxin in this study.
Table 2
Insects Killed by P: luminescens Toxin
Genus and Route of
Common Name Order species Delivery
Tobacco Lepidoptera Manduca sexta Oral and
horn worm injected
Mealworm Coleoptera Tenebrio molitor Oral
Pharaoh ant Hymenoptera Monomorium pharoanis Oral
German Dictyoptera BZattel.la gerrnanica Oral and
cockroach injected
Mosquito Diptera Aedes aegypti Oral
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Examcle 2
Insecticide Utilitv
The Phccorhabdus lurninescens utility and toxicity were
further characterized. Phocorhabdus Iuminescens (strain W-1,+;
culture broch was produced as follows. The production medium was
295 Bacto Proteose Peptone Number 3 (PP3, Difco Laboratories,
Detroit, Michigan) in Milli-Q deionized water. Seed culture
flasks consisted of 175 ml medium placed in a 500 ml tribaffied
flask with a Delong neck, covered with a Kaput and autoclaved
for 20 minutes, T=250 F. Producticn flasks consisted of 500 inls
in a 2.8 liter 500 mi tribaffled flask with a Delong neck,
covered by a Shin-etsu silicon foam closure. These were
autoclaved for 45 minutes, T=250'F. The seed culture was
incubated at 28"C at 150 rpm in a gyrotory shaking incubator .:ith
a 2 inch throw. After 16 hours of growth, 1% of the seed cul.ture
was placed in the production flask which was allowed to grow for
24 hours before harvest. Production of the toxin appears to be
during log phase growth. The microbial broth was transferred to
a IL centrifuge bortle and the cellular biomass was pelleted i30
minutes at 2500 RPM at 4'C, (R.C.F. =-16001 HG-4L Rotor RC3
Sorval centrifuge, Dupont, Wilmington, Delaware). The primary
broth was chilled at 4*C for 8 - 15 hours and recentrifuged at
least 2 hours (conditions above) to further clarify the brotti by
removal of a putative mucopolysaccharide which precipitated upon
standing. (An alternative processing method combined both :;teps
and involved the use of a 16 hour clarification centrifugatiori,
same conditions as above.) This broth was then stored at 4C
prior to bioassay or filtration.
Photorhabdus culture broth and protein toxin(s) purified
from this broth showed activity (mortality and/or growth
inhibition, reduced adult emergence) against a number of insects.
More specifically, the activity is seen against corn rootworni
(larvae and adult), Colorado potato beetle, and turf grubs, whicb.
are members of the insect order Coleoptera. Other members of the
Coleoptera include wireworms, pollen beetles, flea beetles, seed
beetles and weevils. Activity has also been observed against
aster leafhopper, which is a member of the order, Homopcera.
Other members of the Homopcera include planthoppers, pear pysila,
apple sucker, scale insects, whiteflies, and spittle bugs, as
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well as numerous hosc specific aphid species. The broth and
purified fractions are also accive againsc beet armyworm, cabbage
looper, black cutworm, tobacco budworm, European corn borer, corn
earworm, and codling moch, which are members of the order
I.epidoptera. Other typical members of this order are clothes
moch, Indian mealmoth, leaf rollers, cabbage worm, cotton
bollworn, bagworm, Eastern tent caterpillar, sod webworm, and
fall armyworm. Activity is also seen against fruitfly and
mosquito larvae, which are members of the order Diptera. Other
members of the order Dipcera are pea midge, carrot fly, cabbage
root fly, turnip root fly, onion fly, crane fly, house fly, and
various mosquito species. Activity is seen against carpenter ant
and Argentine ant, which are members of the order that also
includes fire ants, oderous house ants, and little black ants.
The broth/fraction is useful for reducing populations of
insects and were used in a mechod of inhibiting an insect
pepulation. The method may comprise applying to a locus of the
insect an effective insecc inactivating amount of the active
described. Results are reported in Table 3.
Activity against corn rootworm larvae was tested as follows.
Photorhabdus culture broth (filter sterilized, cell-free) or
purified HPLC fractions were applied directly to the surface
(-1.5 cm2) of 0.25 ml of artificial diet in 30 l aliquots
following dilucion in control medium or 10 mM sodium phosphate
buffer, pH 7.0, respectively. The diet plates were allowed to
air-dry in a sterile flow-hood and the wells were infested with
single, neonate Diabrotica undecimpunctata howardi (Southern corn
rootworin, SCR) hatched from sterilized eggs, with second instar
SCR grown on artificial diet or with second instar Diabrotica
virgifera virgifera (Western corn rootworm, WCR) reared on corn
seedlings gros,m in Metromix . Second instar larvae were weighed
prior to addition to the diet. The plates were sealed, placed in
a humidified growth chamber and maintained at 27 C for the
appropriate period (4 days for neonate and adult SCR, 2-5 days
for WCR larvae, 7-14 days for second instar SCR). Mortality and
weight determinations were scored as indicated. Generally, 16
inseccs per treatment were used in all scudies. Control
mortalities were as follows: neonate larvae, <5%, adult beetles,
5s. .
*Trade-mark
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Activity against Colorado potato beetle was tested as
follows. Photorhabdus culture broth or control medium was applied
to the surface (-2.0 cm2) of 1.5 ml of standard artificial diet
held in the wells of a 24-well tissue culture plate. Each well
received 50 l of treatment and was allowed to air dry.
Individual second instar Colorado potato beetle (Leptinotarsa
decemlineata, CPB) larvae were then placed onto the diet and
mortality was scored after 4 days. Ten larvae per treatment were
used in all studies. Control mortality was 3.3%.
Activity against Japanese beetle grubs and beetles was
tested as follows. Turf grubs (Popillia japonica, 2-3rd instar)
were collected from infested lawns and maintained in the
laboratory in soil/peat mixture with carrot slices added as
additional diet. Turf beetles were pheromone-trapped locally and
maintained in the laboratory in plastic containers with maple
leaves as food. Following application of undiluted Photorhabdus
culture broth or control medium to corn rootworm artificial diet
(30 .l/1.54 cm2, beetles) or carrot slices (larvae), both stages
were placed singly in a diet well and observed for any mortality
and feeding. In both cases there was a clear reduction in the
amount of feeding (and feces production) observed.
Activity against mosquito larvae was tested as follows. The
assay was conducted in a 96-well microtiter plate. Each well
contained 200 l of aqueous solution (Photorhabdus culture broth,
control medium or H20) and approximately 20, 1-day old larvae
(Aedes aegypti). There were 6 wells per treatment. The results
were read at 2 hours after infestation and did not change over
the three day observation period. No control mortality was seen.
Activity against fruitflies was tested as follows.
Purchased Drosophila melanogaster medium was prepared using 50%
dry medium and a 50% liquid of either water, control medium or
Photorhabdus culture broth. This was accomplished by placing
8.0 ml of dry medium in each of 3 rearing vials per treatment and
adding 8.0 ml of the appropriate liquid. Ten late instar
Drosophila melanogaster maggots were then added to each vial.
The vials were held on a laboratory bench, at room temperature,
under fluorescent ceiling lights. Pupal or adult counts were
made after 3, 7 and 10 days of exposure. Incorporation of
Photorhabdus culture broth into the diet media for fruitfly
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maggots caused a slight (17%) buc significant reduction in day-10
adult emergence as compared to wacer and control medium !30
reduction).
Activity against aster leafhopper was tested as follows.
The ingestion assay for aster leafhopper (Macrosceles severini)
is designed to allow ingestion of the active without other
excernal contac--. The reservoir for the active/'food solution
is made by making 2 holes in the center of the bottom portion of
a 35 x 10 mm Petri dish. A 2 inch ParafileM square is placed
across the top of the dish and secured with an "O" ring. A I oz.
plastic cup is then infested with approximately 7 leafhoppers and
the reservoir is placed on top of the cup, Parafilm down. The
test solution is then added to the reservoir through the holes.
in tescs using undiluted Photorhabdus culture broth, the broth
and concrol medium were dialyzed against water to reduce control
mortality. Morcalicy is reported at day 2 where 26.5% concrol
mortality was seen. In the tests using purified fractions (200
mg protein/ml ) a final concentration of 5% sucrose was used in
all treatments to improve surrivability of the aster leafhoppers.
The assay was held in an incubator at 28 C, 70% RH with a 16/8
photoperiod. The assay was graded for mortality at 72 hours.
Control mortality was 5.5%.
Activity against Argentine ants was tested as follows. A
1.5 ml aliquot of 100% Photorhabdus culture broth, control medium
or water was pipetted into 2.0 ml clear glass vials. The vials
were plugged with a piece of cotton dental wick that was
moistened with the appropriate treatment. Each vial was placed
into a separate 60x16mm Petri dish with 8 to 12 adult Argentine
ants (Linepithema humile). There were three replicates per
treatment. Bioassay plates were held on a laboratory bench, at
room temperature under fluorescent ceiling lights. Mortality
readings were made after 5 days of exposure. Control mortality
was 24%.
Activity against carpenter ant was tested as follows. Black
carpenter ant workers (Camponocus pennsylvanicus) were collected
from trees on DowElanco property in Indianapolis, IN. Tests with
Photorhabdus culture broth were perzormed as follows. Each
plastic bioassay container (7 1/8' x 3") held fifteen workers, a
paper harborage and 10 mi cf broth or concrol media in a piascic
shot glass. A cotton wick delivered the treatment to the ants
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through a hole in the shot glass lid. All treatments contained
5% sucrose. Bioassays were held in the dark at room temperature
and graded at 19 days. Control mortality was 9%. Assays
delivering purified fractions utilized artificial ant diet mixed
with the treatment (purified fraction or control solution) at a
rate of 0.2 ml treatment/2.0 g diet in a plastic test tube. The
final protein concentration of the purified fraction was less
than 10 g/g diet. Ten ants per treatment, a water source,
harborage and the treated diet were placed in sealed plastic
containers and maintained in the dark at 27 C in a humidified
incubator. Mortality was scored at day 10. No control mortality
was seen.
Activity against various lepidopteran larvae was tested as
follows. Photorhabdus culture broth or purified fractions were
applied directly to the surface (-1.5 cm2) of 0.25 ml of standard
artificial diet in 30 l aliquots following dilution in control
medium or 10 mM sodium phosphate buffer, pH 7.0, respectively.
The diet plates were allowed to air-dry in a sterile flow-hood
and the wells were infested with single, neonate larva. European
corn borer (Ostrinia nubilalis) and corn earworm (Helicoverpa
zea) eggs were supplied from commercial sources and hatched in-
house, whereas beet armyworm (Spodoptera exigua), cabbage looper
(Trichaplusia ni), tobacco budworm (Heliothis virescens), codling
moth (Laspeyresia pomonella) and black cutworm (Agrotis ipsilon)
larvae were supplied internally. Following infestation with
larvae, the diet plates were sealed, placed in a humidified
growth chamber and maintained in the dark at 27 C for the
appropriate period. Mortality and weight determinations were
scored at days 5-7 for Photorhabdus culture broth and days 4-7
for the purified fraction. Generally, 16 insects per treatment
were used in all studies. Control mortality ranged from 4-12.5%
for control medium and was less than 10% for phosphate buffer.
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Table 3
Effect of Photorhabdus luminescens (strain W-l4)
Culture Broth and Purified Toxin Fraction on Mortality and Growth
Inhibition of Different Insect Orders/Species
Insect Order/Species Broth Purified Fraction
o Mort. % G.I. % Mort. % G.I.
COLEOPTERP, =
Corn Rootworm
Southern/neonate larva 100 na 100 na
Southern/2"d instar na 38.5 nt nt
Southern/adult 45 nt nt nt
Western/2"instar na 35 nt nt
Colorado Potato
Beetle 93 nt nt nt
2d instar
Turf Grub na a.f. nt nt
3'0 instar na a.f. nt nt
adult
DIPTERA
Fruit Fly (adult 17 nt nt nt
emergence) 100 'na nt nt
Mosquito larvae
HOMOPTERA
Aster Leafhopper 96.5 na 100 na
HYMENOPTERA
Argentine Ant 75 na nt na
Carpenter Ant 71 na 100 na
L$PIDOPT$RA
Beet Armyworm 12.5 36 18.75 41.4
Black Cutworm nt nt 0 71.2
Cabbage Looper nt nt 21.9 66.8
Codling Moth nt nt 6.25 45.9
Corn Earworm 56.3 94.2 97.9 na
European Corn Borer 96.7 98.4 100 na
Tobacco Budworm 13.5 52.5 19.4 85.6
Mort. = mortality, G.I. = growth inhibition,
na = not applicable, nt = not tested, a.f. = anti-feedant
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Example 3
insectir-ide Utility Upon Soil Application
Photorhabdus luminescens (strain W-14) culture broth was
shown to be active against corn rootworm when applied directly to
soil or a soil-mix (Metromix"). Activity against neonate SCR and
WCR in Metromix was tested as follows (Table 4). The test was
run using corn seedlings (United Agriseeds brand CL614) that were
germinated in the light on moist filter paper for 6 days. After
roots were approximately 3-6 cm long, a single kernel/seedling
was planted in a 591 ml clear plastic cup with 50 gm of dry
Metromix . Twenty neonate SCR or WCR were then placed directly on
the roots of the seedling and covered with Metromix' . Upon
infestation, the seedlings were then drenched with 50 ml total
volume of a diluted broth solution. After drenching, the cups
were sealed and left at room temperature in the light for 7 days.
Afterwards, the seedlings were washed to remove all Metromix"' and
the roots were excised and weighed. Activity was rated as the
percentage of corn root remaining relative to the control plants
and as leaf damage induced by feeding. Leaf damage was scored
visually and rated as either -, +, ++, or +++, with -
representing no damage and +++ representing severe damage.
Activity against neonate SCR in soil was tested as follows
(Table 5). The test was run using corn seedlings (United
Agriseeds brand CL614) that were germinated in the light on moist
filter paper for 6 days. After the roots were approximately 3-6
cm long, a single kernel/seedling was planted in a 591 ml clear
plastic cup with 150 gm of soil from a field in Lebanon, IN
planted the previous year with corn. This soil had not been
previously treated with insecticides. Twenty neonate SCR were
then placed directly on the roots of the seedling and covered
with soil. After infestation, the seedlings were drenched with
50 ml total volume of a diluted broth solution. After drenching,
the unsealed cups were incubated in a high relative humidity
chamber (80%) at 78 F. Afterwards, the seedlings were washed to
remove all soil and the roots were excised and weighed. Activity
was rated as the percentage of corn root remaining relative to
the control plants and as leaf damage induced by feeding. Leaf
damage was scored visually and rated as either -, +, ++, or +++,
with - representing no damage and +++ representing severe damage.
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Table 4
Effect of Photorhabdus luminescens (strain W-14) Culture
Broth on Rootworm Larvae After Post-Infestation Drenching
(Metromix )
Treatment Larvae Leaf Damage Root Weight (g) 'o
Southern Corn Rootworm
Water - - 0.4916 0.023 100 Medium (2.0% v/v) - - 0.4416 0.029 100
Broth (6.25%v/v) - - 0.4641 0.081 100
Water + +++ 0.1410 0.006 28.7
Media (2.0% v/v) + +++ 0.1345 0.028 30.4
Broth (1.56% v/v) + - 0.4830 0.031 104
Western Corn Rootworm
Water - - 0.4446 0.019 100
Broth (2.0% v/v) - - 0.4069 0.026 100
Water + - 0.2202 0.015 49
Broth (2.0% v/v) + - 0.3879 0.013 95
Table 5
Effect of Photorhabdus luminescens (strain W-14) Culture Broth on
Southern Corn Rootworm Larvae After Post-Infestation Drenching
(Soil)
Treatment Larvae Le- af Damage Root Weight(g) %
Water - - 0.2148 0.014 100
Broth (509. v/v) - - 0.2260 0.016 103
Water + +++ 0.0916 0.009 43
Broth (50% v/v) + - 0.2428 0.032 113
Activity of Photorhabdus luminescens (strain W-14) culture
broth against second instar turf grubs in Metromix' was observed
in tests conducted as follows (Table 6). Approximately 50 gm of
dry Metromix" was added to a 591 ml clear plastic cup. The
Metromix* was then drenched with 50 ml total volume of a 50% (v/v)
diluted Photorhabdus broth solution. The dilution of crude broth
was made with water, with 50% broth being prepared by adding 25
ml of crude broth to 25 ml of water for 50 ml total volume. A 1%
(w/v) solution of proteose peptone #3 (PP3), which is a 50%
dilution of the normal media concentration, was used as a broth
control. After drenching, five second instar turf grubs were
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placed on the top of the moistened Metromix . Healthy turf grub
larvae burrowed rapidly into the Metromix9. Those larvae that did
not burrow within lh were removed and replaced with fresh larvae.
The cups were sealed and placed in a 28 C incubator, in the dark.
After seven days, larvae were removed from the Metromix and
scored for mortality. Activity was rated the percentage of
mortality relative to control.
Table 6
Effect of Photorhabdus luminescens (strain W-14) Culture Broth on
Turf Grub After Pre-Infestation Drenching (Metromix')
Treatment Mortality* Mortality ~
Water 7/15 47
Control medium
(1.0% w/v) 12/19 63
Broth
(50% v/v) 17/20 85
*expressed as a ratio of dead/living larvae
Example 4
Insecticide Utility Upon Leaf Application
Activity of Photorhabdus broth against European corn borer
was seen when the broth was applied directly to the surface of
maize leaves (Table 7). In these assays Photorhabdus broth was
diluted 100-fold with culture medium and applied manually to the
~
surface of excised maize leaves at a rate of -6.0 l/cm' of leaf
surface. The leaves were air dried and cut into equal sized
strips approximately 2 x 2 inches. The leaves were rolled,
secured with paper clips and placed in 1 oz plastic shot glasses
with 0.25 inch of 2% agar on the bottom surface to provide
moisture. Twelve neonate European corn borers were then placed
onto the rolled leaf and the cup was sealed. After incubation
for 5 days at 27 C in the dark, the samples were scored for
feeding damage and recovered larvae.
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Table 7
Effect of Phctorhabdus luminescens (strain W-14) Culture Brot'r_ on
European Corn Borer Larvae Following Pre-Infestation Application
to Excised Maize Leaves
Treatment Leaf Damage Larvae Recovered Weight(mg)
Water Extensive 55/120 0.42 mg
Control Medium Extensive 40/120 0.50 mg
Broth (1.0% v/v) Trace 3/120 0.15 mg
Activity of the culture broth against neonate tobacco
budworm (Heliothis virescens) was demonstrated using a leaf dip
methodology. Fresh cotton leaves were excised from the plant and
leaf disks were cut with an 18.5 mm cork-borer. The disks were
individually emersed in control medium (PP3) or Photorhabdus
luminescens (strain W-14) culture broth which had been
concentrated approximately 10-fold using an Amicon (Beverly, MA),
Proflux M12 tangential filtration system with a 10 kDa filter.
Excess liquid was removed and a straightened paper clip was
placed through the center of the disk. The paper clip was then
wedged into a plastic, 1.0 oz shot glass containing approximately
2.0 ml of 1% Agar. This served to suspend the leaf disk above
the agar. Following drying of the leaf disk, a single neonate
tobacco budworm larva was placed on the disk and the cup was
capped. The cups were then sealed in a plastic bag and placed in
a darkened, 270C incubator for 5 days. At this time the
remaining larvae and leaf material were weighed to establish a
measure of leaf damage (Table 8).
Table 8
Effect of Photorhabdus luminescens (Strain W-14) Culture Broth on
Tobacco Budworm Neonates in a Cotton-Leaf Dip Assay
Final Weights (mg)
Treatment Leaf Disk Larvae
Control leaves 55.7 1.3 na*
Control Medium 34.0 2.9 4.3 0.91
Photorhabdus broth 54.3 1.4 0.0**
*- not applicable, ** - no live larvae found
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Example 5, Part A
Characterization of Toxin Peptide Components
In a subsequent analysis, the toxin protein subunits of the
bands isolated as in Example 1 were resolved on a 7% SDS
polyacrylamide electrophoresis gel with a ratio of 30:0.8
(acrylamide:BIS-acrylamide). This gel matrix facilitates better
resolution of the larger proteins. The gel system used to
estimate the Band 1 and Band 2 subunit molecular weights in
Example 1 was an 18% gel with a ratio of 38:0.18 (acrylamide:BIS-
acrylamide), which allowed for a broader range of size
separation, but less resolution of higher molecular weight
components.
In this analysis, 10, rather than 8, protein bands were
resolved. Table 9 reports the calculated molecular weights of
the 10 resolved bands, and directly compares the molecular
weights estimated under these conditions to those of the prior
example. It is not surprising that additional bands were
detected under the different separation conditions used in this
example. Variations between the prior and new estimates of
molecular weight are also to be expected given the differences in
analytical conditions. In the analysis of this example, it is
thought that the higher molecular weight estimates are more
accurate than in Example 1, as a result of improved resolution.
However, these are estimates based'on SDS PAGE analysis, which
are typically not analytically precise and result in estimates of
peptides and which may have been further altered due to post- and
co-translational modifications.
Amino acid sequences were determined for the N-terminal
portions of five of the 10 resolved peptides. Table 9 correlates
the molecular weight of the proteins and the identified
sequences. In SEQ ID NO:2, certain analyses suggest that the
proline at residue 5 may be an asparagine (asn). In SEQ ID NO:3,
certain analyses suggest that the amino acid residues at
positions 13 and 14 are both arginine (arg). In SEQ ID NO:4,
certain analyses suggest that the amino acid residue at position
6 may be either alanine (ala) or serine (ser). In SEQ ID NO:5,
certain analyses suggest that the amino acid residue at position
3 may be aspartic acid (asp).
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Table 9
EXAMPLE 1
ESTI14ATE NEW ESTIMATE* SEQ. LISTING
208 200.2 kDa SEQ ID NO:1
184 175.0 kDa SEQ ID NO:2
65.6 68.1 kDa SEQ ID NO:3
60.8 65.1 kDa SEQ ID NO:4
56.2 58.3 kDa SEQ ID NO:5
25.1 23.2 kDa SEQ ID NO:15
*New estimates are based on SDS PAGE and are not based on
gene sequences. SDS PAGE is not analytically precise.
Example 5, Part B
Characterization of Toxin Peptide Components
New N-terminal sequence, SEQ ID NO:15, Ala Gln Asp Gly Asn
Gln Asp Thr Phe Phe Ser Gly Asn Thr, was obtained by further N-
terminal sequencing of peptides isolated from Native HPLC-
purified toxin as described in Example 5, Part A, above. This
peptide comes from the tcaA gene. The peptide labeled TcaAii,
starts at position 254 and goes to position 491, where the
TcaAiii peptide starts, SEQ ID NO:4. The estimated size of the
peptide based on the gene sequence is 25,240 Da.
Example 6
Characterization of Toxin Peptide Components
In yet another analysis, the toxin protein complex was re-
isolated from the Photorhabdus luminescens growth medium (after
culture without Tween) by performing a 10% - 80% ammonium sulfate
precipitation followed by an ion exchange chromatography step
(Mono Q) and two molecular sizing chromatography steps. These
conditions were like those used in Example 1. During the first
molecular sizing step, a second biologically active peak was
found at about 100 10 kDa. Based upon protein measurements,
this fraction was 20 - 50 fold less active than the larger, or
primary, active peak of about 860 100 kDa (native). During
this isolation experiment, a smaller active peak of about 325
50 kDa that retained a considerable portion of the starting
biological activity was also resolved. It is thought that the
325 kDa peak is related to or derived from the 860 kDa peak.
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A 56 kDa protein was resolved in this anaiysis. The N-
terminal sequence of this protein is presented in SEQ ID N0:6.
It is noteworthy that this protein shares significant identity
and conservation with SEQ ID NO:5 at the N-terminus, suggestin.:r
that the two may be encoded by separate members of a gene family
and that the proteins produced by each gene are sufficiently
similar to both be operable in the insecticidal toxin complex.
A second, prominent 185 kDa protein was consistently present
in amounts comparable to that of protein 3 from Table 9, and m,ay
be the same protein or protein fragment. The N-terminal sequence
of this 185 kDa protein is shown at SEQ ID NO:7.
Additional N-terminal amino acid sequence data were also
obtained from isolated proteins. None of the determined N-
terminal sequences appear identical to a protein identified in
Table 9. Other proteins were present in isolated preparation.
One such protein has an estimated molecular weight of 108 kDa and
an N-terminal sequence as shown in SEQ ID NO:8. A second such
protein has an estimated molecular weight of 80 kDa and an N-
terminal sequence as shown in SEQ ID NO:9.
When the protein material in the approximately 325 kDa
active peak was analyzed by size, bands of approximately 51, 31,
28, and 22 kDa were observed. As in all cases in which a
molecular weight was determined by analysis of electrophoretic
mobility, these molecular weights were subject to error effect=
introduced by buffer ionic strength differences, electrophoresis
power differences, and the like. One of ordinary skill would
understand that definitive molecular weight values cannot be
determined using these standard methods and that each was subject
to variation. It was hypothesized that proteins of these sizes
are degradation products of the larger protein species (of
approximately 200 kDa size) that were observed in the larger
primary toxin complex.
Finally, several preparations included a protein having ttie
N-terminal sequence shown in SEQ ID NO:10. This sequence was
strongly homologous to known chaperonin proteins, accessory
proteins known to function in the assembly of large protein
complexes. Although the applicants could not ascribe such an
assembly function to the protein identified in SEQ ID NO:10, it
was consistent with the existence of the described toxin protein
complex that such a chaperonin protein could be involved in its
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3ss?mbli . moreover, alzhough such proteins have not direcclf
been suggested co have toxic accivity, this protein may be
impor*_ant ro determining the overall structural nature of the
protein toxin, and thus, may contribute to the toxic activity or
~urability of the complex in viro after oral delivery.
Subseq=uent analysis of the stability of the protein coxin
complex co proteinase K was undertaken. It was determined that
after 24 hour incubation of the complex in the presence of a 10-
fold molar excess of proteinase K, activity was virtually
1() eliminated (mortality on oral application dropped to about 5e).
These data confirm the proteinaceous nature of the toxin.
The toxic activity was also retained by a dialysis membrane,
again confirming the large size of the native toxin complex.
Examule 7
Isolat'_on, Characterization and Partial Amino Acid
Sequencing of Phocor:iabdus Toxins
Isolatien and N-Terminal Amino Acid Sequencing: In a set of
experiments conducted in parallel to Examples 5 and 6, ammenium
sulfate precipitation of Phocorhabdus proteins was performed by
adjusting Photorhabdus broth, typically 2-3 liters, to a final
concentration of either 10% or 20% 'oy the slow addition of
ammonium sulfate crystals. After stirring for 1 hour at 4 C, the
material was centrifuged at 12,000 x g for 30 minutes. The
supernatant was adjusted to 80t ammonium sulfate, stirred ac 4 C
for 1 hour, and centrifuged at 12,000 x g for 60 minutes. The
pellet was resuspended in one-tenth the volume of 10 mM Na:=POs,
pH 7.0 and dialyzed against the same phosphate buffer overnight
at 4 C. The dialyzed material was centrifuged at 12,000 x g for
1 hour prior to ion exchange chromatography.
A. HR 16/50 Q Sepharose"(Pharmacia) anion exchange column was
equilibrated with 10 mM Nai=PO4, pH 7Ø Centrifuged, dialyzed
ammonium sulfate pellet was applied to the Q Sepharose column at
a rate of 1.5 ml/min and washed extensively at 3.0 ml/min with
equilibration buffer until the optical density (O.D. 280) reached
less than 0.100. Next, either a 60 minute NaCl gradient ranging
from 0 to 0.5 M at 3 ml/min, or a series of step elutions using
0.1 M, 0.4 M and finally 1.0 NaCl for 60 minutes each was acplied
to the column. Fractions were pooled and concentrated using a
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C~encriprep 100. Alternacively, proceins could be eluted by a
single 0.4 M NaC1 wash without prior elution with 0.1 M NaCl.
Two milliliter aliquocs of concer.trated Q Sepharose samples
were loaded ac 0.5 ml/min onto a HR 16/50 Superose 12 (Pharmacia)
gel filtratien column equilibraced wich 10 mM Nai=PO4, pH 7Ø
The column .ras washed wich the same buffer for 240 min at 0.5
mi/min and 2 min samples were collected. The void volume
macerial was collected and concentrated using a Centriprep 100.
Two millilicer aliquots of concentrated Superose* 12 samples -4ere
loaded at 0.5 ml/min onto a HR 16/50 Sepharose 4B-CL (Pharmacia)
gel filtration column equilibrated with 10 mM Na)=PO4, pH 7Ø
The column was washed with the same buffer for 240 min at 0.5
ml/min and 2 min samples were collected.
The excluded protein peak was subjected to a second
=ractionacion by application to a gel filtration column that used
a Sepharose CL-4B resin, which separates proteins ranging from
-30 kDa to 1000 kDa. This fraction was resolved into two peaks;
a minor peak at the void volume (>1000 kDa) and a major peak
which eluted ac,an apparenc molecular weight of abouc 860 kDa.
Over a one week period subsequent samples subjected to gel
filtration showed the gradual appearance of a third peak
(approximately 325 kDa) that seemed to arise from the major peak,
perhaps by limited proteolysis. Bioassays performed on the ch:-ee
peaks showed that the void peak had no activity, while the 860
kDa toxin complex fraction was highly active, and the 325 kD3
peak was less active, although quite potent. SDS PAGE analysis
of Sepharose CL-4B toxin complex peaks from different
fermentation productions revealed two distinct peptide patterns,
denoted =P" and S. The two patterns had marked differences in
che molecular weights and concentrations of peptide componencs in
their fractions. The "S" pattern, produced most frequently, had
4 high molecular weight peptides (> 150 kDa) while the "P"
pattern had 3 high molecular weight peptides. In addition, che
"S" peptide fraction was found to have 2-3 fold more activitr
against European Corn Borer. This shift may be related to
variations in protein expression due to age of inoculum and/or
other factors based on growth parameters of aged cultures.
Milligram quantities of peak toxin complex fractions
determined to be "P" or "S" peptide patterns were subjected to
preparative SDS PAGE, and transblotted with TRIS-glycine
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(Seprabuff'T" to PVDF membranes (ProBlott'''", Applied Biosystems)
for 3-4 hours. Blots were sent for amino acid analysis and N-
terminal amino acid sequencing at Harvard MicroChem and Cambridge
ProChem, respectively. Three peptides in the "S" pattern had
unique N-terminal amino acid sequences compared to the sequences
identified in the previous example. A 201 kDa (TcdAii) peptide
set forth as SEQ ID NO:.13 below shared between 33% amino acid
identity and 50% similarity with SEQ ID NO:l (TcbAii)(Table 10,
in Table 10 vertical lines denote amino acid identities and
colons indicate conservative amino acid substitutions). A second
peptide of 197 kDa, SEQ ID NO:14 (TcdB), had 42% identity and 58%
homology with SEQ ID NO:2 (TcaC). Yet a third peptide of 205 kDa
was denoted TcdAii. In addition, a limited N-terminal amino acid
sequence, SEQ ID NO:16 (TcbA), of a peptide of at least 235 kDa
was identical in homology with the amino acid sequence, SEQ ID
NO:12, deduced from a cloned gene (tcbA), SEQ ID NO:11,
containing a deduced amino acid sequence corresponding to SEQ ID
NO:1 (TcbAii). This indicates that the larger 235+ kDa peptide
was proteolytically processed to the 201 kDa peptide, (TcbAii),
(SEQ ID NO:1) during fermentation, possibly resulting in
activation of the molecule. In yet another sequence, the
sequence originally reported as SEQ ID NO:5 (TcaBii) reported in
Example 5 above, was found to contain an aspartic acid residue
(Asp) at the third position rather than glycine (Gly) and two
additional amino acids Gly and Asp at the eighth and ninth
positions, respectively. In yet two other sequences, SEQ ID NO:2
(TcaC) and SEQ ID NO:3 (TcaBi), additional amino acid sequence was
obtained. Densitometric quantitation was performed using a
sample that was identical to the "S" preparation sent for N-
terminal analysis. This analysis showed that the 201 kDa and 197
kDa peptides represent 7.0% and 7.2%, respectively, of the total
Coomassie brillant blue stained protein in the "S" pattern and
are present in amounts similar to the other abundant peptides.
It is speculated that these peptides may represent protein
homologs, analogous to the situation found with other bacterial
toxins, such as various CryI Et toxins. These proteins vary from
40-90% homology at their N-terminal amino acid sequence, which
encompasses the toxic fragment.
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Internal Amino Acid Sequencing: To facilitate cloning of
toxin peptide genes, internal amino acid sequences of selected
peptides were obtained as followed. Milligram quantities of peak
2A fractions determined to be "P" or "S" peptide patterns were
subjected to preparative SDS PAGE, and transblotted with TRIS-
glycine (SeprabuffT" to PVDF membranes (ProBlottT'4, Applied
Biosystems) for 3-4 hours. Blots were sent for amino acid
analysis and N-terminal amino acid sequencing at Harvard
MicroChem and Cambridge ProChem, respectively. Three peptides,
referred to as TcbAii (containing SEQ ID NO:1), TcdAii, and TcaBi
(containing SEQ ID NO:3) were subjected to trypsin digestion by
Harvard MicroChem followed by HPLC chromatography to separate
individual peptides. N-terminal amino acid analysis was
performed on selected tryptic peptide fragments. Two internal
peptides were sequenced for the peptide TcaBi (205 kDa peptide)
referred to as TcaBi-PT111 (SEQ ID NO:17) and TcaBz-PT79 (SEQ ID
NO:18). Two internal peptides were sequenced for the peptide
TcaB; (68 kDa peptide) referred to as TcaB!-PT158 (SEQ ID NO:19)
and TcaBi-PT108 (SEQ ID NO:20). Four internal peptides were
sequenced for the peptide TcbAii (201 kDa peptide) referred to as
TCBAII-PT103 (SEQ ID NO:21), TcbAii-PT56 (SEQ ID NO:22), TcbAii-
PT81(a) (SEQ ID NO:23), and TcbAii-PT81(b) (SEQ ID NO:24).
Table 10
N-Terminal Amino Acid Sequences -
201 kDa (33% identity & 50% similarity to SEQ ID NO.1)
L I G Y N N Q F S LIGYNNQFSGA A SEQ ID NO:13
. i I I . I
F I Q G Y S D L F G N - A SEQ ID NO:1
197 kDa (42% identity & 58% similarity SEQ ID NO.2)
M Q N S Q T F S V G E L SEQ ID NO.14
I I. I i.. I
M Q D S P E V S I T T L SEQ ID NO.2
Example 8
Construction of a cosmid library of Photorhabdus lurninescens W-14
genomic DNA and its screening to isolate genes encoding peptides
comprising the toxic protein preparation
As a prerequisite for the production of Photorhabdus insect
toxic proteins in heterologous hosts, and for other uses, it is
- 45 necessary to isolate and characterize the genes that encode those
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peptides. 'rni.s oD7ective was pursueci in parallel. One approach,
described later, was based on the use of monoclonal and
polyclonal antibodies raised against the purified toxin which
were then used to isolate clones from an expression library. The
other approach, described in this example, is based on the use of
the N-terminal and internal amino acid sequence data to design
degenerate oligonucleotides for use in PCR amplication. Either
method can be used to identify DNA clones that contain the
peptide-encoding genes so as to permit the isolation of the
respective genes, and the determination of their DNA base
sequence.
GENOMIC DNA ISOLATION: Photorhabdus luminescens strain W-14
(ATCC accession number 55397) was grown on 2% proteose peptone #3
agar (Difco Laboratories, Detroit, MI) and insecticidal toxin
competence was maintained by repeated bioassay after passage,
using the method described in Example 1 above. A 50 ml shake
culture was produced in a 175 ml baffled flask in 2% proteose
peptone #3 medium, grown at 28 C and 150 rpm for approximately 24
hours. 15 ml of this culture was pelleted and frozen in its
medium at -20 C until it was thawed for DNA isolation. The
thawed culture was centrifuged, (700 x g, 30 min) and the
floating orange mucopolysaccharide material was removed. The
remaining cell material was centrifuged (25,000 x g, 15 min) to
pellet the bacterial cells, and the medium was removed and
discarded.
Genomic DNA was isolated by an adaptation of the CTAB method
described in section 2.4.1 of Current Protocols in Molecular
Biology (Ausubel et al. eds, John Wiley & Sons, 1994) (modified
to include a salt shock and with all volumes increased 10-fold].
The pelleted bacterial cells were resuspended in TE buffer (10 mM
Tris-HC1, 1 mM EDTA, pH 8.0) to a final volume of 10 ml, then 12
ml of 5 M NaCl was added; this mixture was centrifuged 20 min at
15,000 x g. The pellet was resuspended in 5.7 ml TE and 300 ml
of 10% SDS and 60 ml of 20 mg/ml proteinase K (Gibco BRL
Products, Grand Island, NY; in sterile distilled water) were
added to the suspension. This mixture was incubated at 37 C for
I hr; then approximately 10 mg lysozyme (Worthington Biochemical =
Corp., Freehold, NJ) was added. After an additional 45 min, I ml
of 5 M NaCl and 800 ml of CTAB/NaCl solution (10% w/v CTAB, 0.7 M
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NaCl) were aaded. 'rnis preparation was incuoaced 10 min at 63c'_'.
then gently agitated and zurther incubated and agitaced for
approximately 20 min to assist clearing of the cellular material.
An equal volume of chloroform/isoamyl alcohol solution (24:1,
v/v) was added, mixed gently and centrifuged. After two
extractions with an equal volume of PCI
(phenol/chloro=o r=n/isoamyl alcohol; 50:49:1, v/v/v; equilibrated
with I M Tris-HC1, pH 8.0; Intermountain Scientific Corporation,
Kaysville, UT), the DNA was precipitated with 0.6 volume of
isopropanol. The DNA precipitate was gently removed with a glass
rod, washed twice with 70% ethanol, dried, and dissolved in 2 cnl
STE (10 mM Tris-HC1 pH 8.0, 10 mM NaCl, 1 mM EDTA). This
preparation contained 2.5 mg/ml DNA, as determined by optical
density at 260 nm (i.e., OD26a) .
The molecular size range of the isolated genomic DNA was
evaluated for suitability for library construction. CHEF gel
analysis was performed in 1.5% agarose (Seakeni LE, FMC
BioProducts, Rockland, ME) gels with 0.5 X TBE buffer (44.5 mti
Tris-HCI pH 8.0, 44.5 mM H)BOI, 1 mM EDTA) on a BioRad CHEF-DR IZ
apparatus with a Pulsewave 760 Switcher (Bio-Rad Laborator;.es.
Inc., Richmond, CA). The running parameters were: initial A
time,"3 sec: final A time, 12 sec; 200 volts; running
temperature, 4-18 C; r.in time, 16.5 hr. Ethidium bromide
staining and examination of the gel under ultraviolet light
indicated the DNA ranged from 30-250 kbp in size.
CONSTRUCTiON OF LIBRARY: A partial Sau3A I digest was i::.;d=
of this Phoccrhabdus genomic DNA preparation. The method was
based on section 3.1.3 of Ausubel (supra.). Adaptions included
running smaller scale reactions under various conditions until
nearly optimal results were achieved. Several scaled-up large
reactions with varied conditions were run, the results analyzed
on CHEF gels, and only the best large scale preparation was
carried forward. In the optimal case, 200 g of Phocorhabdus
genomic DNA was incubated with 1.5 units of Sau3A 1(New England
Biolabs, "NEB", Beverly, MA) for 15 min at 37 C in 2 ml total
volume of 1X NEB 4 buffer (supplied as lOX by the manufacturer).
The reaction was stopped by adding 2 ml of PCI and centrifuging
at 8000 x g for 10 min. To the supernatant were added 200 l ci
5 M NaCl plus 6 ml of ice-cold ethanol. This preparation was
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chilled for 30 min at -20 C, then centrifuged at 12,000 :-: g for
15 min. The supernatant was removed and the precipitate was
dried in a vacuum oven at 40 C, then resuspended in 400 l STE.
Spectrophotometric assay indicated about 40% recovery of the
input DNA. The digested DNA was size fractionated on a sucrose
gradient according to section 5.3.2 of CPMB (op. cit.). A 10%
to 40% (w/v) linear sucrose gradient was prepared with a gradient
maker in Ultra-C1earT"' tubes (Beckman Instruments, Inc., Palo
Alto, CA) and the DNA sample was layered on top. After
centrifugation, (26,000 rpm, 17 hr, Beckman SW41 rotor, 20 C!,
fractions (about 750 l) were drawn from the top of the gradient
and analyzed by CHEF gel electrophoresis (as described earlier).
Fractions containing Sau3A 1 fragments in the size range 20-40
kbp were selected and DNA was precipitated by a modification
(amounts of all solutions increased approximately 6.3-fold) -,f
the method in section 5.3.3 of Ausubel (supra.). After overnight
precipitation, the DNA was collected by centrifugation (17,O(j0 x
g, 15 min), dried, redissolved in TE, pooled into a final volume
of 80 .l, and reprecipitated with the addition of 8 l 3 M s~.-dium
acetate and 220 l ethanol. The pellet collected by
centrifugation as above was resuspended in 12 l TE.
Concentration of the DNA was determined by Hoechst 33258 dye
(Polysciences, Inc., Warrington, PA) fluorometry in a Hoefer
TKO100 fluorimeter (Hoefer Scientific Instruments, San Francisco,
CA). Approximately 2.5 g of the size-fractionated DNA was
recovered.
Thirty g of cosmid pWE15 DNA (Stratagene, La Jolla, CA) was
digested to completion with 100 units of restriction enzyme b3mH
1(NEB) in the manufacturer's buffer (final volume of 200 l,
37 C, 1 hr). The reaction was extracted with 100 l of PCI and
DNA was precipitated from the aqueous phase by addition of 20 l
3M sodium acetate and 550 l -20 C absolute ethanol. After :0
min at -70 C, the DNA was collected by centrifugation (17,000 x
g, 15 min), dried under vacuum, and dissolved in 180 l of 10 mM
Tris-HC1, pH B.O. To this were added 20 N.1 of lOX CIP buffe!_
(100 mM Tris-HC1, pH 8.3; 10 mM ZnC12; 10 mM MgC12), and 1 l
(0.25 units) of 1:4 diluted calf intestinal alkaline phosphatase
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(9oehringer Mannheim Corporation. Indianapolis, IN). After 30
min at 37 C, the following additions were made: 2 l 0.5 M EDTA,
pH 8.0; 10 l 10% SDS; 0.5 l of 20 mgiml proteinase K (as
above), followed by incubation at 55 C for 30 min. Following
sequential extractions with 100 l of PCI and 100 l phenol
(Incermountain Scientific Corporation, equilibrated with I M
Tris-HCI, pH 8.0), the dephosphorylated DNA was precipitated by
addition of 72 l of 7.5 M ammonium acetate and 550 l -20 C
ethanol, incubation on ice for 30 min, and centrifugation as
lU above. The pelleted DNA was washed once with 500 l -20 C 70e
ethanol, dried under vacuum, and dissolved in 20 l of TE buffer.
Ligation of the size-fractionated Sau3A 1 fragments to the
BamH 1-digesced and phosphatased pWE15 vector was accomplished
using T4 ligase (NEB) by a modification (i.e., use of premixed
lOX ligation buffer supplied by the manufacturer) of the protoci1
in section 3.33 of Ausubel. Ligation was carried ouc overnight
in a total volume of 20 l at 15 C, followed by storage at -
C.
Four l of the cosmid DNA ligation reaction, containing
20 about 1 g of DNA, was packaged into bacteriophage lambda usinq a
commercial packaging extract (Gigapac)~ III Gold Packaging
Extract, Stratagene), following the manufacturer's directions.
The packaged preparation was stored at 4 C until use. The
packaged cosmid preparation was used to infect Escherichia coli
XL1 Blue MR cells (Stratagene) according to the Gigapack' III ~.=Ad
protocols ("Titering the Cosmid Library"), as follows. :CL1 Hlue
MR cells were grown in LB medium (g/L: Bacto-tryptone, 10; Bacto-
yeast extract, 5; Bacto-agar, 15; NaCl, 5; (Difco Laboratories,
Detroit, MIl) containing 0.2% (w/v) maltose plus 10 mM MgSO4, at
37 C. After 5 hr growth, cells were pelleted at 700 x g (15 min)
and resuspended in 6 ml of 10 mM MgSO.. The culture density was
adjusted with 10 mM MgSO. to OD,soo= 0.5. The packaged cosmid
library was diluted 1:10 or 1:20 with sterile SM medium (0.1 M
NaCl, 10 mM MgS04,50 mM Tris-HCI pH 7.5, 0.01% w/v gelatin), an-3
25 l of the diluted preparation was mixed with 25 l of the
diluted XLI Blue MR cells. The mixture was incubated at 25 i_ E.-)r
30 min (without shaking), then 200 l of LB broth was added, aild
incubation was continued for approximately 1 hr with occasional
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gefltle shaking. ?liquots (20-40 l) of this culture were spre_=i
on LB agar plates containing 100 mg/1 ampicillin (i.e. , LB-An:n;..,)
and incubated overnight at 37 C. To store the library withouc
amplification, single colonies were picked and inoculated into
individual wells of sterile 96-well microwell plates; each weil
containing 75 l of Terrific Broth (TB media: 12 g/1 Bacto-
tryptone, 24 g/1 Bacto-yeast extract, 0.4% v/v glycerol. 17 mM
KH2PO4, 72 mM K2HP04) plus 100 mg/1 ampicillin (i.e., TB-Amp;) and
incubated (without shaking) overnight ac 37 C. After replicaci.ig
lU the 96-well plate into a copy plate, 75 l/well of filter-
sterilized TB:glycerol (1:1, v/v; with, or without, 100 mg/i
ampicillin) was added to the plate, it was shaken briefly at 100
rpm, 37 C, and then closed with Parafilm' (American National I:aii,
Greenwich, CT) and placed in a-70 C freezer for storage. Cupy
plates were grown and processed identically to the master plac=s.
A total of 40 such master plates (and their copies) were
prepared.
SCREENING OF THE LI3RARY WITH rZADIOL?.BELED DNA PROBES : T::
prepare colony filters for probing with radioactively labeled
probes, ten 96-well plates of the library were thawed at 25 C
(bench top at room temperature). A replica plating tool wit!i 2i
prongs was used to inoculate a fresh 96-well copy plate
containing 75 )tl/well of TB-Amploo. The copy plate was grown
overnight (stationary) at 37 C, then shaken about 30 min at 100
rpm at 37 C. A total of 800 colonies was represented in these
copy plates, due to nongrowth of some isolates. The replica tool
was used to inoculate duplicate impressions of the 96-well aLrays
onto Magna tIT (MSI, Westboro, MA) nylon membranes (0.45 micr=n.
220 x 250 mm) which had been placed on solid LB-Amptoo (100
mi/dish) in Bio-assay plastic dishes (Nunc. 243 x 243 x 18 mm;
Curtin Mathison Scientific, Inc., Wood Dale, IL). The colonies
were grown on the membranes at 37 C for about 3 hr.
A positive control colony (a bacterial clone containing a
GZ4 sequence insert, see below) was grown on a separate Magn:t t1T
membrane (Nunc, 0.45 micron, 82 mm circle) on LB medium
supplemented with 35 mg/1 chloramphenicol (i.e., LB-Cam,;), az:d
processed alongside the library colony membranes. Bacterial
colonies on the membranes were lysed, and the DNA was denatured
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and neutralized according to a protocol taken from the Genius'M
System User's Guide version 2.0 (Boehringer Mannheim,
Indianapolis, IN). Membranes were placed colony side up on
filter paper soaked with 0.5 N NaOH plus 1.5 M NaCl for 15 min to
denature, and neutralized on filter paper soaked with I M Tris-
HC1 pH 8.0, 1.5 M NaCl for 15 min. After UV-crosslinking using a
Stratagene W Stratalinker set on auto crosslink, the membraries
were stored dry at 25 C until use. Membranes were trimmed irito
strips containing the duplicate impressions of a single 96-well
plate, then washed extensively by the method of section 6.4.1 in
CPMB (op. cit.): 3 hr at 25 C in 3X SSC, 0.1% (w/v) SDS, followed
by 1 hr at 65 C in the same solution, then rinsed in 2X SSC in
preparation for the hybridization step (20X SSC = 3 M NaCl, 0.3 M
sodium citrate, pH 7.0).
Amplification of a specific genomic fragment of a tcaC -rene.
Based on the N-terminal amino acid sequence determined for ttie
purified TcaC peptide fraction [disclosed herein as SEQ ID NO:2],
a pool of degenerate oligonucleotides (pool S4Psh) was
synthesized by standard 0-cyanoethyl chemistry on an Applied
BioSystem AB1394 DNA/RNA Synthesizer (Perkin Elmer, Foster City,
CA). The oligonucleotides were deprotected 8 hours at 55 C,
dissolved in water, quantitated by spectrophotometric
measurement, and diluted for use. This pool corresponds to the
determined N-terminal amino acid sequence of the TcaC peptide.
The determined amino acid sequence and the corresponding
degenerate DNA sequence are given below, where A, C, G, and T are
the standard DNA bases, and I represents inosine:
Amino Met Gln Asp Ser Pro Glu Val
Acid
S4Psh 5' ATG CA(A/G) GA(T/C) (T/A)(C/G)(T/A) CCI GA(A/G) GT 3'
Another set of degenerate oligonucleotides was synthesi-ed
(pool P2.3.5R), representing the complement of the coding strand
for the determined amino acid sequence of the SEQ ID NO:17:
Amino
Acid Ala Phe Asn Ile Asp Asp Val
Codons 5' GCN TT(T/C) AA(T/C) AT(A/T/C) GA(T/C) GA(T/C) GT 3'
P2.3.5R 3'CG(A/C/G/T) AA(A/G) TT(A/G) TA(T/A/G) CT(A/G) CT(A/G) CA 5'
These oligonucleotides were used as primers in Polymerase
Chain Reactions (PCe, Roche Molecular Systems, Branchburg, NJ) to
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amplify a specitic UNA tragment from genomic DNA prepared from
Photorhabdus strain W-14 (see above). A typical reaction (50 1)
contained 125 pmol of each primer pool P2Psh and P2.3.5R, 253 ng
of genomic template DNA, 10 nmol each of dATP, dCTP, dGTP, and
dTTP, 1X GeneAmp") PCR buffer, and 2.5 units of AmpliTaq'9 DNA
polymerase (both from Roche Molecular Systems; lOX GeneAmpo buffer
is 100 mM Tris-HC1 pH 8.3, 500 mM KC1, 0.01% w/v gelatin).
Amplifications were performed in a Perkin Elmer Cetus DNA Thermal
Cycler (Perkin Elmer, Foster City, CA) using 35 cycles of 94 C
(1.0 min), 55 C (2.0 min), 72 C (3.0 min), followed by an
extension period of 7.0 min at 72 C. Amplification products were
analyzed by electrophoresis through 2% w/v NuSieve 3:1 agarose
(FMC BioProducts) in TEA buffer (40 mM Tris-acetate, 2 mM EDTA,
pH 8.0). A specific product of estimated size 250 bp was
observed amongst numerous other amplification products by
ethidium bromide (0.5 g/ml) staining of the gel and examination
under ultraviolet light.
The region of the gel containing an approximately 250 bp
product was excised, and a small plug (0.5 mm dia.) was removed
and used to supply template for PCR amplification (40 cycles).
The reaction (50 l) contained the same components as above,
minus genomic template DNA. Following amplification, the ends of
the fragments were made blunt and were phosphorylated by
incubation at 25 C for 20 min with 1 unit of T4 DNA polymerase
(NEB), 1 nmol ATP, and 2.15 units of T4 kinase (Pharmacia Biotech
Inc., Piscataway, NJ).
DNA fragments were separated from residual primers by
electrophoresis through 1% w/v GTe agarose (FMC) in TEA. A gel
slice containing fragments of apparent size 250 bp was excisecl,
and the DNA was extracted using a Qiaex kit (Qiagen Inc.,
Chatsworth, CA).
The extracted DNA fragments were ligated to plasmid vector
pBC KS(+) (Stratagene) that had been digested to completion with
restriction enzyme Sma 1 and extracted in a manner similar to
that described for pWE15 DNA above. A typical ligation reaction
(16.3 l) contained 100 ng of digested pBC KS(+) DNA, 70 ng oF
250 bp fragment DNA, 1 nmol 1Co(NH3)61C1,, and 3.9 Weiss units of
T4 DNA ligase (Collaborative Biomedical Products, Bedford, MA),
in IX ligation buffer (50 mM Tris-HC1, pH 7.4; 10 mM MgClz; 10 mM
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dithiothreitol; 1 mM spermidine, 1 mM ATP, 100 mg/ml bovine serum
albumin). Following overnight incubation at 14 C, the ligate%.l
= products were transformed into frozen, competent Escherichia .=oii
DH5a cells (Gibco BRL) according to the suppliers'
recommendations, and plated on LB-Cam3splates, containing IPT13
(119 g/ml) and X-gal (50 g/ml). Independent white colonies
were picked, and plasmid DNA was prepared by a modified alkalinF-
lysis/PEG precipitation method (PRISMT'" Ready Reaction DyeDeor.YTM
Terminator Cycle Sequencing Kit Protocols; ABI/Perkin Elmer).
The nucleotide sequence of both strands of the insert DNA was
determined, using T7 primers [pBC KS(+) bases 601-623:
TAAAACGACGGCCAGTGAGCGCG) and LacZ primers [pBC KS(+) bases 792-
816: ATGACCATGATTACGCCAAGCGCGC) and protocols supplied with the
PRISMTM sequencing kit (ABI/Perkin Elmer). Nonincorporated dye-
terminator dideoxyribonucleotides were removed by passage through
Centri-Sep 100 columns (Princeton Separations, Inc., Adelphia,
NJ) according to the manufacturer's instructions. The DNA
sequence was obtained by analysis of the samples on an ABI Model
373A DNA,Sequencer (ABI/Perkin Elmer). The DNA sequences of two
isolates, GZ4 and HB14, were found to be as illustrated in Figure
1.
This sequence illustrates the following features: 1) bases
1-20 represent one of the 64 possible sequences of the S4Psh
degenerate oligonucleotides, ii) the sequence of amino acids 1-3
and 6-12 correspond exactly to that determined for the N-terminus
of TcaC (disclosed as SEQ ID NO:2), iii) the fourth amino acid
encoded is a cysteine residue rather than serine. This difference
is encoded within the degeneracy for the serine codons (see
above), iv) the fifth amino acid encoded is proline,
corresponding to the TcaC N-terminal sequence given as SEQ ID
NO:2, v) bases 257-276 encode one of the 192 possible sequences
designed into the degenerate pool, vi) the TGA termination codon
introduced at bases 268-270 is the result of complementarity to
the degeneracy built into the oligonucleotide pool at the
corresponding position, and does not indicate a shortened reading
frame for the corresponding gene.
Labeling of a TcaC peptide gene-specific probe. DNA
fragments corresponding to the above 276 bases were amplified (35
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=.:cies) by PCR' in a 100 l reaction volume, using 100 pmcl each
of'P2Psh and P2.3.5R primers, 10 ng of piasmids GZ4 or HB14 as
cemplaces, 20 nmol each of dAT?, dCT?, dGTP, and dTTP, 5 units of
AmpliTAq' DNA polymerase, and IX concencracion of GeneAmp" buf'er,
under the same temperature regimes as described above. The
amplification pr-~ducts were extracted from a 190- GTG' agarose gel
by Qiae *kit and quantitated by fluoromecry.
The extracted amplification products from plasmid HB14
template (approximately 400 ng) were split into five aliquots and
labeled with ''P-dCTP using the High Prime Labeling Mix
(Boehringer Mannheim) according to the manufacturer's
instructions. Nonincorporated radioisotope was removed by
passage through NucTrap' Probe Purification Columns (Stracagene),
according to the supplier's instructions. The specific accivity
of the labeled DNA product was determined by scintillation
counting to be 3.11 x l0jdpm/ g. This labeled DNA was used to
probe membranes prepared frcm 800 members of the gencmic Library.
Screening with a TcaC-neptide gene specific Drobe. The
radiolabeled HB14 probe was boiled approximately 10 min, then
added to "minimal hyb" solution. (Note: The "minimal hyb" method
is taken from a CERES protocol; "Restriction Fragment Length
Polymorphism Laboratory Manual version 4.0", sections 4-40 and 4-
4'; CERES/NPI, Salt Lake City, UT. NPI is now defunct, with its
successors operating as Linkage Genetics) . "Minimal hyb"
solution contains 10% w/v PEG (polyethylene glycol, M.W. approx.
8000), 7% w/v SDS; 0.6X SSC, 10 mM sodium phosphate buffer (from
a 1M stock containing 95 g/l NaH2PO4-1H2O and 84.5 g/l
Na,HPO4=7H2O), 5 mM EDTA, and 100 mg/ml denatured salmon sperm
DNA. Membranes were blotted dry briefly then, without
prehybridization, 5 strips of membrane were placed in each of 2
plastic boxes containing 75 ml of =minimal hyb" and 2.6 ng/ml ol.
radiolabeled HB14 probe. These were incubated overnight with
slow shaking (50 rpm) at 60 C. The filters were washed three
times for approximately 10 min each at 25 C in "minimal hyb wash
solution" (0.25X SSC, 0.2% SDS), followed by two 30-min washes
with slow shaking at 60 C in the same solution. The filters were
placed on paper covered with Saran wrap' (Dow Brands,
Indianapolis, IN) in a light-tight autoradiographic cassette and
exposed to X-Omat*X-ray film (Kodak, Rochescer, NY) with two
*Trade-mark -51-
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DuPont CrCn :: L13hCZ=nQ-?Lus~
-1 enhancers (Sigma C iemlcal Cc.,
Sc, Louis, MO), for 4 hr 3C -70 C._ Upon developmenc (scandarci
phocographic procedures) , significant signals were evident in
boch replicates amongst a high background of weaker, more
irregular signals. The filters were again washed for about 4 hr
ac 69 C in "minimal hyb wash solucicn" and then placed again in
che casseccas and film was exposed overnight at -70 C. Tuelve
possible posi:ives were identified due to strong signals cn boch
of che duplicate 96-well colony impressions. No signal was seen
wich negaci=re concrol membranes (colonies of XL1 Blue MP, cells
concaining pWE15), and a very strong signal was seen wizh
positive concrol membranes (DH5a cells containing the GZ4 isolace
of the PCR producc) that had been processed concurrently with the
experimental samples.
The twelve pucative hybridization-positive colonies were
retrieved from the frozen 96-well library plates and grc,rn
overnighc at 37 C on solid LB-Amp,oo medium. They were then
patched (3/plate, plus three negative controls: XL1 31ue MR cells
containing the pWE15 vector) onto solid LB-Amptoo= Two sets of
membranes (Magna NT nylon, 0.45 micron) were prepared for
hybridization. The first sec was prepared by placing a filter
direccly onto the colonies on a patch plate. then removing it
with adherent bacterial cells, and processing as below. ?il--ers
of che secor.d set -were placed on plates containing L3-,ampt0q
medium, then inoculated by transferring cells from the patch
plates onto the filters. Afcer overnight growth at 37 C, che
filters were removed from the plates and processed.
3acterial cells on the filters were lysed and DNA denatured
by placing each filter colony-side-up on a pool (1.0 ml) o: 0.5 tt
ttaOH in a plastic plate for 3 min. The filters were blocced dr-I
on a paper towel, then the process was repeaced with fresh 0.5 ~1
NaOH. After blotting dry, the filters were neutralized by
placing each on a 1.0 ml pool of 1 M Tris-HC1, pH 7.5 for 3 min,
blotted dry, and reneutralised with fresh buffer. Th'-s was
followed by two similar soakings (5 min each) on pools of 0.5 t4
Tris-HC1 pH 7.5 plus 1.5 M NaC1. After blotting dry, the DNA was
UV crosslinked to the filter (as above), and the filzers -.:ere
washed (25 C, 100 rpm) in about 100 ml of 3X SSC plus 0.1a;=~~v)
SDS (4 times, 30 min each with fresh solution for each wash).
They were then placed in a minimal volume of prehybridlz3tion
*Trade-mark
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soi':ticn (6X SSC plus 1l w.'v each of Fico11* 400 (Pharmacia;
polyvinylpyrroiidone (av. 4I.W. 360,000; Sigma ) and bovine ser,lm
albumin Fraction V; (Sigma)J for 2 hr ac 65 C, 50 rpm. The
prehybridization solution was removed, and replaced with the HB14
''P-labeled probe that had been saved frcm the previous
hybridization of the library membranes and which had been
denatured at 95 C for 5 min. Hybridization was performed at 60 C
for 16 hr with shaking at 50 rpm.
Following removal of the labeled probe solution, the
membranes were washed 3 times at 25 C (50 rpm, 15 min) in 3X SSC
(about 150 ml each wash). They were then washed for 3 hr ac 63 C
(50 rpm) in 0.25X SSC plus 0.2% SDS (minimal hyb wash solution),
and exposed to X-ray film as described above for 1.5 hr at 25 C
(no enhancer screens). This exposure revealed very strong
hybridization signals to cosmid isolates 22G12, 25A10, 26A5, and
26310, and a very weak signal with cosmid isolate 8310. No
signal was seen wi:h the negative control (pWE15) colonies, and a
very strong signal was seen with positive control membranes (DH5(X
cells containing the GZ4 isolate of the PCR product) that had
been processed concurrently with the experimental samples.
AmDlification of a sDecific gencmic fragment of a c=a? gene.
Based on the N-terminal amino acid sequence determined for the
purified TcaB, peptide fraction (disclosed here as SEQ ID 170:3) a
pool of degenerate oligonucleotides (pool P8F) was synthesized as
described for peptide TcaC. The determined amino acid sequence
and the corresponding degenerate DNA sequence are given below,
where A, C, G, and T are the standard DNA bases, and I represents
inosine:
>mino
Acid Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg
P8F 5' TTT ACI CA(A/G) ACI (C/T)TI AAA GAA CCI (A/C)C 3'
(C/T)TI
Another set of degenerate oligonucleotides was synthesized
(pool P8.108.3R), representing the complement of the coding
strand for the determined amino acid seqsence of the TcaBi-PT108
internal peptide (disclosed herein as SEQ ID NO:20):
Amino
Acid Met 'I'yr T'yr Ile Gln Ala GLn Gin
*Trade-mark -53-
CA 02209659 1997-07-04
WO 97/17432 PCT/US96/18003
Codons ATG TA(T/C) TA(T/C) AT(T/C/A) CA(?./G) GC(A/C/G!T) CA(A/G Ca(;;iG)
c'8.1 3F. 3' AT(A/G) AT(A/G) TA(A/G/T) GT(T/C) CGI GT(T,'C) GT 5'
TAC
These oligonucleotides were used as primers for PCR using
HotStart 50 TubesTM' (Molecular Bio-Products, Inc., San Diego, CA)
to amplify a specific DNA fragment from genomic DNA prepared from
Photorhabdus strain W-14 (see above). A typical reaction (50 l)
contained (bottom layer) 25 pmol of each primer pool PBF and
P8.108.3R, with 2 nmol each of dATP, dCTP, dGTP, and dTTP, in 1X
GeneAmp'9 PCR buffer, and (top layer) 230 ng of genomic template
DNA, 8 nmol each of dATP, dCTP, dGTP, and dTTP, and 2.5 units of
AmpliTaq'0 DNA polymerase, in 1X GeneAmpo PCR buffer.
Amplifications were performed by 35 cycles as described for the
TcaC peptide. Amplification products were analyzed by
electrophoresis through 0.7% w/v SeaKemA LE agarose (FMC) in TEA
buffer. A specific product of estimated size 1600 bp was
observed.
Four such reactions were pooled, and the amplified DNA was
extracted from a 1.0% SeaKee LE gel by Qiaex kit as described for
the TcaC peptide. The extracted DNA was used directly as the
template for sequence determination (PRISMTM Sequencing Kit) using,
the P8F and P8.108.3R primer pools. Each reaction contained
about 100 ng template DNA and 25 pmol of one primer pool, and was
processed according to standard protocols as described for the
TcaC peptide. An analysis of the sequence derived from extension
of the P8F primers revealed the short DNA sequence (and encoded
amino acid sequence):
GAT GCA TTG NTT GCT
Asp Ala Leu (Val) Ala
which corresponds to a portion of the N-terminal peptide sequence
disclosed as SEQ ID NO:3 (TcaBi).
Labeling of a TcaBi-peptide gene-specific probe.
Approximately 50 ng of gel-purified TcaBi DNA fragment was
labeled with "-P-dCTP as described above, and nonincorporated
radioisotopes were removed by passage through a NICK Column
(Pharmacia). The specific activity of the labelled DNA was
determined to be 6 x 10" dpm/ g. This labeled DNA was used to
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probe colony membranes prepared from members of the genomic
library that had hybridized to the TcaC-peptide specific probe.
The membranes containing the 12 colonies identified in the
TcaC-probe library screen (see above) were stripped of
radioactive TcaC-specific label by boiling twice for
approximately 30 min each time in 1 liter of 0.1X SSC plus 0.1 %
SDS. Removal of radiolabel was checked with a 6 hr film
exposure. The stripped membranes were then incubated with the
TcaBi peptide-specific probe prepared above. The labeled DNA was
denatured by boiling for 10 min, and then added to the filters
that had been incubated for 1 hr in 100 ml of "minimal hyb"
solution at 60 C. After overnight hybridization at this
temperature, the probe solution was removed, and the filters were
washed as follows (all in 0.3X SSC plus 0.1% SDS): once for 5 min
at 25 C, once for 1 hr at 60 C in fresh solution, and once for 1
hr at 63 C in fresh solution. After 1.5 hr exposure to X-ray
film by standard procedures, 4 strongly-hybridizing colonies were
observed. These were, as with the TcaC-specific probe, isolates
22G12, 25A10, 26A5, and 26B10.
The same TcaBiprobe solution was diluted with an equal
volume (about 100 ml) of "minimal hyb" solution, and then used to
screen the membranes containing the 800 members of the genomic
library. After hybridization, washing, and exposure to X-ray
film as described above, only the four cosmid clones 22G12,
25A10, 26A5, and 26B10, were found to hybridize strongly to this
probe.
ISOLATION OF SUBCLONES CONTAINING GENES ENCODING TcaC PlID
TcaBi PEPTIDES, AND DETERMINATION OF DNA BASE SEQUENCE THEP.EOF:
Three hybridization-positive cosmids in strain XL1 Blue MR were
grown with shaking overnight (200 rpm) at 30 C in 100 ml TB-
Ampt,,. After harvesting the cells by centrifugation, cosmid DNA
was prepared using a commercially available kit (BIGprepTM', 5
Prime 3 Prime, Inc., Boulder, CO), following the manufacturer's
protocols. Only one cosmid, 26A5, was successfully isolated by
this procedure. When digested with restriction enzyme EcoR 1
(NEB) and analyzed by gel electrophoresis, fragments of
approximate sizes 14, 10, 8 (vector), 5, 3.3, 2.9, and 1.5 kbp
were detected. A second attempt to isolate cosmid DNA from the 40 same three
strains (8 ml cultures; TB-Ampluu, 30 C) utilized a -55-
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boiling miniprep method (Evans G. and G. Wahl., 1987, "Cosmid
vectors for genomic walking and rapid restriction mapping." in
Guide to Molecular Cloning Techniques. Meth. Enzymology, vol.
152, S. Berger and A. Kimmel, eds., pgs. 604-610). Only one
cosmid, 25A10, was successfully isolated by this method. When
digested with restriction enzyme EcoR 1(NEB) and analyzed by gel
electrophoresis, this cosmid showed a fragmentation pattern
identical to that previously seen with cosmid 26A5.
A 0.15 .g sample of 26A5 cosmid DNA was used to transform 50
ml of E. co1.i DH5a cells (Gibco BRL), by the supplier's
protocols. A single colony isolate of that strain was inoculated
into 4 ml of TB-Ampl:.,u, and grown for 8 hr at 37 C.
Chloramphenicol was added to a final concentration of 225 g/ml,
incubation was continued for another 24 hr, then cells were
harvested by centrifugation and frozen at -200C. Isolation of
the 26A5 cosmid DNA was by a standard alkaline lysis miniprep
(Maniatis et al., op. cit., p. 382), modified by increasing all
volumes by 50% and with stirring or gentle mixing, rather than
vortexing, at every step. After washing the DNA pellet in 70%
ethanol, it was dissolved in TE containing 25 g/ml ribonuclease
A (Boehringer Mannheim).
Identification of EcoR 1 fragments hybridizing to GZ4-
derived and TcaBi- probes. Approximately 0.4 g of cosmid 25A10
(from XL1 Blue MR cells) and about 0.5 g of cosmid 26A5 (from
chloramphenicol-amplified DH5(x cells) were each digested with
about 15 units of EcoR 1 (NEB) for 85 min, frozen overnight, then
heated at 65 C for five min, and electrophoresed in a 0.7%
agarose gel (Seakem" LE, 1X TEA, 80 volts, 90 min). The DNA was
stained with ethidium bromide as described above, and
photographed under ultraviolet light. The EcoR 1 digest of
cosmid 25A10 was a complete digestion, but the sample of cosmid
26A5 was only partially digested under these conditions. The
agarose gel containing the DNA fragments was subjected to
depurination, denaturation and neutralization, followed by
Southern blotting onto a Magna NT nylon membrane, using a high
salt (20X SSC) protocol, all as described in section 2.9 of
Ausubel et al. (CPMB, op. cit.). The transferred DNA was then
UV-crosslinked to the nylon membrane as before.
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An TcaC-peptide specific DtIA fragment corresponding to the
insert of plasmid isolate GZ4 was amplified by PCR in a 100 ml
reaction volume as described previously above. The amplification
products from three such reactions were pooled and were extracted
from a 1% GTG' agarose gel by Qiaex kit, as described above, and
quantitated by fluorometry. The gel-purified DNA (100 ng) was
labeled with "P-dCTP using the High Prime Labeling Mix
(Boehringer Mannheim) as described above, to a specific activity
of 6.34 x 108 dpm/ g.
The 32P-labeled GZ4 probe was boiled 10 min, then added to
"minimal hyb" buffer (at 1 ng/ml), and the Southern blot membranA
containing the digested cosmid DNA fragments was added, and
incubated for 4 hr at 60 C with gentle shaking at 50 rpm. The
membrane was then washed 3 times at 25 C for about 5 miri each
(minimal hyb wash solution), followed by two washes for :0 min
each at 60 C. The blot was exposed to film !with enhancer
screens) for about 30 min at -70 C. The GZ4 probe hybridized
strongly to the 5.0 kbp (apparent size) EcoR 1 fragmeiit ~.>f both
these two cosmids, 26A5 and 25A10.
The membrane was stripped of radioactivity by boiling for
about 30 min in 0.1X SSC plus 0.1 % SDS, and absence of
radiolabel was checked by exposure to film. It was then
hybridized at 60 C for 3.5 hours with the (denatured) TcaBi probe
in "minimal hyb" buffer previously used for screening the colony
membranes (above), washed as described previously, and exposed to
film for 40 min at -70 C with two enhancer screens. With both
cosmids, the TcaBi probe hybridized lightly with the about 5.0
kbp EcoR 1 fragment, and strongly with a fragment of
approximately 2.9 kbp.
The sample of cosmid 26A5 DNA previously described, (from
DH5a cells) was used as the source of D14A from which to subclone
the bands of interest. This DNA (2.5 ug) was digested with about
3 units of EcoR 1 (NEB) in a total volume of 30 l for 1.5 hr, to
give a partial digest, as confirmed by gel electrophoresis. Ten
g of pBC KS (+) DNA (Stratagene) were digested for 1.5 hr with
20 units of EcoR 1 in a total volume of 20 l, leading to total
digestion as confirmed by electrophoresis. Both EcoR 1-cut DNA
preparations were diluted to 50 l with water, to each an equal
volume of PCI was added, the suspension was gently mixed, spun in
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a microcentrifuge and the aqueous supernatant was collected. Ltin
was precipitated by 150 41 ethanol, and the mixture was placed ac
-20 C overnight. Following centrifugation and drying, the EcoR
1-digested pBC KS (+) was dissolved in 100 41 TE; the partially
digested 26A5 was dissolved in 20 l TE. DNA recovery was
checked by fluorometry.
In separate reactions, approximately 60 ng of EcoR 1-
digested pBC KS(+) DNA was ligated with approximately 180 ng or
270 ng of partially digested cosmid 26A5 DNA. Ligations were
carried out in a volume of 20 l at 15 C for 5 hr, using T4
ligase and buffer from New England BioLabs. The ligation
mixture, diluted to 100 l with sterile TE, was used to transform
frozen, competent DH5a cells (Gibco BRL) according to the
supplier's instructions. Varying amounts (25-200 l) of the
transformed cells were plated on freshly prepared solid LB-Cam;S
medium with 1 mM IPTG and 50 mg/l X-gal. Plates were incubated
at 37 C about 20 hr, then chilled in the dark for approximately 3
hr to intensify color for insert selection. White colonies were
picked onto patch plates of the same composition and incubated
overnight at 37 C.
Two colony lifts of each of the selected patch plates were
prepared as follows. After picking white colonies to fresh
plates, round Magna NT nylon membranes were pressed onto the
patch plates, the membrane was lifted off, and subjected to
denaturation, neutralization and W crosslinking as described
above for the library colony membranes. The crosslinked colony
lifts were vigorously washed, including gently wiping off the
excess cell debris with a tissue. One set was hybridized with
the GZ4(TcaC) probe solution described earlier, and the other set
was hybridized with the TcaBi probe solution described earlier,
according to the 'minimal hyb' protocol, followed by washing and
film exposure as described for the library colony membranes.
Colonies showing hybridization signals either only with the
GZ4 probe, with both GZ4 and TcaBi probes, or only with the TcaBi
probe, were selected for further work and cells were streaked for
single colony isolation onto LB-Cam35 media with IPTG and X-gal as
before. Approximately 35 single colonies, from 16 different
isolates, were picked into liquid LB-Cam35 media and grown
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overnight at 37 C; the cells were collected by centrifugation and
plasmid DNA was isolated by a standard alkaline lysis miniprep
according to Maniatis et al. (op. cit. p. 368). DNA pellets were
dissolved in TE + 25 g/ml ribonuclease A and DNA concentration
was determined by fluorometry. The EcoR 1 digestion pattern was
analyzed by gel electrophoresis. The following isolates were
picked as useful. Isolate A17.2 contains religated pBC F:S(+)
only and was used for a (negative) control. Isolates D38.3 and
C44.1 each contain only the 2.9 kbp, TcaBi -hybridizing EcoR 1
fragment inserted into pBC KS(+). These plasmids, named pDAB2000
and pDAB2001, respectively, are illustrated in Fig. 2.
Isolate A35.3 contains only the approximately 5 kbp, GZ4)-
hybridizing EcoR 1 fragment, inserted into pBC KS(+). This
plasmid was named pDAB2002 (also Fig. 2). These isolates
provided templates for DNA sequencing.
Plasmids pDAB2000 and pDAB2001 were prepared using the
BIGprepr" kit as before. Cultures (30 ml) were grown overnight in
TB-Cam35 to an ODsoo of 2, then plasmid was isolated according to
the manufacturer's directions. DNA pellets were redissolved in
100 ul TE each, and sample integrity was checked by EcoR 1
digestion and gel electrophoretic analysis.
Sequencing reactions were run in duplicate, with one
replicate using as template pDAB2000 DNA, and the other replicate
using as template pDAB2001 DNA. The reactions were carried out
using the dideoxy dye terminator cycle sequencing method, as
described above for the sequencing of the GZ4/HB14 DNAs. Initial
sequencing runs utilized as primers the LacZ and T7 primers
described above, plus primers based on the determined sequence of
the TcaB1 PCR amplification product (TH1 =
aTTGCAGACTGCCAATCGCTTCGG, TH12 = GAGAGTATCCAGACCGCGGATGATCTG).
After alignment and editing of each sequencing output, each
was truncated to between 250 to 350 bases, depending on the
integrity of the chromatographic data as interpreted by the
Perkin Elmer Applied Biosystems Division SeqEd 675 software.
Subsequent sequencing "steps" were made by selecting appropriate
sequence for new primers. With a few exceptions, primers
(synthesized as described above) were 24 bases in length with a 50% G+C
composition. Sequencing by this method was carried out
on both strands of the approximately 2.9 kbp EcoR 1 fragment.
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To further sert:e as template for DNA sequencing, plasmid DtrA
from isolate pDAB2002 was prepared by BIGprep"kit. Sequencing
reactions were performed and analyzed as described above.
Initially, a T3 primer (pBS SK (+) bases 774-796:
CGCGCAATTAACCCTCACTAAAG) and a T7 primer (pBS KS (+) bases 621-
643: GCGCGTAATACGACTCACTATAG) were used to prime the sequencing
reactions from the flanking vector sequences, reading into the
insert DNA. Another set of primers, (GZ4F:
GTATCGATTACAACGCTGTCACTTCCC; TH13: GGGAAGTGACAGCGTTGTAATCGATAC;
TH14: ATGTTGGGT(3CGTCGGCTAATGGACATAAC; and LW1-204:
GGGAAGTGACAGCGTTGTAATCGATAC) was made to prime from internal
sequences, which were determined previously by degenerate
oligonucleotide-mediated sequencing of subcloned TcaC-peptide PCR
products. From the data generated during the initial rounds of
sequencing, new sets of primers were designed and used to walk
the entire length of the -5 kbp fragment. A total of 55 oligo
primers was used, enabling the identification of 4832 total bp of
contiguous sequence.
When the DNA sequence of the EcoR 1 fragment insert of
pDAB2002 is combined with part of the determined sequence of the
pDaB2000/pDAB2001 isolates, a total contiguous sequence of 6005
bp was generated (disclosed herein as SEQ ID NO:25). When long
open reading frames were translated into the corresponding amino
acids, the sequence clearly shows the TcaBi N-terminal peptide
(disclosed as SEQ ID NO:3), encoded by bases 19-75, immediately
following a methionine residue (start of translation). Upstream
lies a potential ribosome binding site (bases 1-9), and
downstream, at bases 166-228 is encoded the TcaBi-PT158 internal
peptide (disclosed herein as SEQ ID NO:19). Further downstream,
in the same reading frame, at bases 1738-1773, exists a secr.ience
encoding the TcaBi-PT108 internal peptide (disclosed herein as
SEQ ID NO:20). Also in the same reading frame, at bases 1897-
1923, is encoded the TcaBii N-terminal peptide (disclosed herein
as SEQ ID NO:5), and the reading frame continues uninterrupted t~:)
a translation termination codon at nucleotides 3586-3588.
The lack of an in-frame stop codon between the end of the
sequence encoding TcaBi-PT108 and the start of the TcaBii encoding
region, and the lack of a discernible ribosome binding site
immediately upstream of the TcaBii coding region, indicate that
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peptides TcaBii and TcaBi are encoded by a single open readir.-a
frame of 3567 bp beginning at base pair 16 in SEQ ID NO:25), and
are most likely derived from a single primary gene product of
1189 amino acids (131,586 Daltons; disclosed herein as SEQ ID
NO:26) by post-translational cleavage. If the amino acid
immediately preceding the TcaBii N-terminal peptide represents
the C-terminal amino acid of peptide TcaBi, then the predicted
mass of TcaBii (627 amino acids) is 70,814 Daltons (disclosed
herein as SEQ ID NO:28), somewhat higher than the size observed
by SDS-PAGE (68 kDa). This peptide would be encoded by a
contiguous stretch of 1881 base pairs (disclosed herein as SEQ ID
N0:27). It is thought that the native C-terminus of TcaBi lies
somewhat closer to the C-terminus of TcaBi-PT108. The molecular
mass of PT108 [3.438 kDa; determined during N-terminal amino acid
sequence analysis of this peptidej predicts a size of 30 amino
acids. Using the size of this peptide to designate the C-
terminus of the TcaBi coding region [Glu at position 604 of SEQ
ID NO:281, the derived size of TcaBi is determined to be 604
amino acids or 68,463 Daltons, more in agreement with
experimental observations.
Translation of the TcaBii peptide coding region of 1686 base
pairs (disclosed herein as SEQ ID NO:29) yields a protein of 562
amino acids (disclosed herein as SEQ ID NO:30) with predicted
mass of 60,789 Daltons, which corresponds well with the cbserved
61 kDa.
A potential ribosome binding site (bases 3633-3638) is found
48 bp downstream of the stop codon for the tcaB open reading
frame. At bases 3645-3677 is found a sequence encoding the N-
terminus of peptide TcaC, (disclosed as SEQ ID NO.2). The open
reading frame initiated by this N-terminal peptide continues
uninterrupted to base 6005 (2361 base pairs, disclosed herein as
the first 2361 base pairs of SEQ ID NO.31). A gene (tcaC)
encoding the entire TcaC peptide, (apparent size -165 kDa; -1500
amino acids), would comprise about 4500 bp.
Another isolate containing cloned EcoR 1 fragments of cosmid
26A5, E20.6, was also identified by its homology to the
previously mentioned GZ4 and TcaBiprobes. Agarose gel analysis
of EcoR 1 digests of the DNA of the plasmid harbored by this
strain (pDAB2004, Fig. 2), revealed insert fragments of estimated
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sizes 2.9, 5, and 3.3 kbp. DNA sequence analysis initiated from
primers designed from the sequence of plasmid pDAB2002 revealed
that the 3.3 kbp EcoR 1 fragment of pDAB2004 lies adjacent to the
kbp EcoR 1 fragment represented in pDAB2002. The 2361 base
5 pair open reading frame discovered in pDAB2002 continues
uninterrupted for another 2094 bases in pDAB2004 [disclosed
herPin as base pairs 2362 to 4458 of SEQ ID NO:31]. DNA sequence
analysis using the parent cosmid 26A5 DNA as template confirmed
the continuity of the open reading frame. Altogether, the open
reading frame (TcaC SEQ ID NO:31) comprises 4455 base pairs, and
encodes a protein (TcaC) of 1485 amino acids [disclosed herein as
SEQ ID NO:32]. The calculated molecular size of 166,214 Daltons
is consistent with the estimated size of the TcaC peptide (165
kDa), and the derived amino acid sequence matches exactly that
disclosed for the TcaC N-terminal sequence [SEQ ID NO:2].
The lack of an amino acid sequence corresponding to SEQ ID
NO:17; used to design the degenerate oligonucleotide primer pool
in the discovered sequence indicates that the generation of the
PCR products found in isolates GZ4 and HB14, which were used as
probes in the initial library screen, were fortuitously generated
by reverse-strand priming by one of the primers in the degenerate
pool. Further, the derived protein sequence does not include the
internal fragment disclosed herein as SEQ ID NO:18. These
sequences reveal that plasmid pDAB2004 contains the complete
coding region for the TcaC peptide.
Example 9
Screening of the Photorhabdus genomic library
for genes encoding the TcbAii peptide
This example describes a method used to identify DNA clones
that contain the TcbAii peptide-encoding genes, the isolation of
the gene, and the determination of its partial DNA base sequence.
Primers and PCR reactions
The TcbAii polypeptide of the insect active preparation is
-206 kDa. The amino acid sequence of the N-terminus of this peptide
is disclosed as SEQ ID NO:l. Four pools of degenerate
oligonucleotide primers ("Forward primers": TH-4, TH-5, TH-6, and
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TH-7) were synthesized to encode a portion of this amino acid
sequence, as described in Example 8, and are shown below.
Table 11
Amino
Acid Phe Ile Gln Gly Tyr Ser Asp Leu Phe
TH-4 5'-TT(T/C) ATI CA(A/G) GGI TA(T/C) TCI GA(T/C) CTI TT-3'
TH-5 5'-TT(T/C) ATI CA(A/G) GGI TA(T/C) AG(T/C) GA(T/C) CTI TT-3' }
TH-6 5'-TT(T/C) ATI CA(A/G) GGI TA(T/C) TCI GA(T/C) TT(A/G) TT-3'
TH-7 5'-7P(T/C) ATI CA(A/G) GGI TA(T/C) AG(T/C) GA(T/C) TT(A/G) TT-3'
In addition, a primary ("a") and a secondary ("b") sequence
of an internal peptide preparation (TcbAii-PT81) have been
determined and are disclosed herein as SEQ ID No:23 and SEQ ID
No:24, respectively. Four pools of degenerate oligonucleotides
("Reverse Primers": TH-8, TH-9, TH-10 and TH-11) were similarly
designed and synthesized to encode the reverse complement of
sequences that encode a portion of the peptide of SEQ ID NO:23,
as shown below.
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u, in u~ in
1 1 ,
a [~ [~
F F
!d H H H H
~ U U U U
DD C.J U U U
U U U U
E4 Ei E+ E-4
q '.
C9 CE7 CE7 C9 CE--7
U U U U
E-4 E-4 Ey E-+
U U U U
v U
a c~ t~ t~ t~
F in a a a a
a ~a a~ a~ ~a
a Q
f1 H H ~ ~
4) 0 C.D H V
E-+
.C7 C7 CH7 CH7
E E H E+ Eq
C9 C7
~-~l ta7 Ca7
C7 t~ C7 C~
~ a' RC Q
Q E-, Q
H H H H
C7 C7 C.~ C7
E E+ E E-4
[-1 M M M M
O O
a 'Ll W 01 -i .-~
=rl =~ 1 1
. ~ a F F F F
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Sets of these primers were used in PCR reactions to ampii_y
TcbAii- encoding gene fragments from the genomic Photorhabcius
luminescens W-14 DNA prepared in Example 6. All PCR10 reactions
were run with the "Hot Start" technique using AmpliWaxT'" gems and
other Perkin Elmer reagents and protocols. Typically, a mixture
(total volume 11 l) of MgCl, dNTP's, 10X GeneAmp"0 PCR Buffer II,
and the primers were added to tubes containing a single wax beaa.
[lOX GeneAmp9 PCR Buffer II is composed of 100 mM Tris-HCI, pH
8.3; and 500 mM KC1.] The tubes were heated to 80 C for 2
minutes and allowed to cool. To the top of the wax seals, a
solution containing lOX GeneAmp'0 PCR Buffer II, DNA template, and
AmpliTaql DNA polymerase were added. Following melting of the wax
seal and mixing of components by thermal cycling, finai reactioii
conditions (volume of 50 l) were: 10 mM Tris-HC1, pH 8.3; 50 iiit-1
KC1; 2.5 mM MgC12; 200 M each in dATP, dCTP, dGTP, dTTP; 1.25 mM
in a single Forward primer pool; 1.25 M in a single Reverse
primer pool, 1.25 units of AmpliTaq' DNA polymerase, and 170 ng of
template DNA.
The reactions were placed in a thermocycler (as in
Example 8) and run with the following program:
Table 13
Temperature Time Cycle
Repetition
94 C 2 minutes 1X
94 C 15 seconds
55-65 C 30 seconds 30X
72 C 1 minute
72 C 7 minutes ix 15 C Constant
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A series of amplifications was run at three different
annealing temperatures (55 , 600, 65 C) using the degenerate
primer pools. Reactions with annealing at 65 C had no
amplification products visible following agarose gel
electrophoresis. Reactions having a 60 C annealing regime and
containing primers TH-5+TH-10 produced an amplification product
that had a mobility corresponding to 2.9 kbp. A lesser amount of
the 2.9 kbp product was produced under these conditioris with
primers TH-7+TH-10. When reactions were annealed at 55 C, these
primer pairs produced more of the 2.9 kbp product, and this
product was also produced by primer pairs TH-5+TH-8 and TH-5+TH-
11. Additional very faint 2.9 kbp bands were seen in lanes
containing amplification products from primer pairs TH-7 plus TH-
8, TH-9, TH-10, or TH-11.
To obtain sufficient PCR amplification product for cloning
and DNA sequence determination, 10 separate PCR reactions were
set up using the primers TH-5+TH-i0, and were run using the above
conditions with a 55 C annealing temperature. All reactions were
pooled and the 2.9 kbp product was purified by Qiaex extraction
from an agarose gel as described above.
Additional sequences determined for TcbAii internal peptides
are disclosed herein as SEQ ID NO:21 and SEQ ID NO:22. As
before, degenerate oligonucleotides (Reverse primers TH-17 and
TH-18) were made corresponding to the reverse complement of
sequences that encode a portion of the amino acid sequence of
these peptides.
Table 14
From SEQ ID NO:21
Amino
Acid Met Glu Thr Gln Asn Ile Gln Glu Pro
TH-17 3'-TAC CTT/C TGI GTT/C TTA/G TAI GTT/C GTT/C GG-5'
Table 15
From SEQ ID NO:22
= Amino
Acid Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp
TH-18 3'-TT(A/G) GGI TAI TT(A/G) TAI TT(A?G) TGI CCI TAI CT(A/G)-5'
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Deoenerate oliaonucleotides TH-18 and TH-17 were used in an
amplification experiment with Photorhabcius luminescens W-14 DN.y
as template and primers TH-4, TH-5, TH-6, or TH-7 as the 51-
(Forward) primers. These reactions amplified products of
approximately 4 kbp and 4.5 kbp, respectively. These DNAs were
transferred from agarose gels to nylon membranes and hybridized
sx:ith a''P-labeled probe (as described above) prepared from the
2.9 kbp product amplified by the TH-5+TH10 primer pair. Both the
4 kbp and the 4.5 kbp amplification products hybridized strongly
to the 2.9 kbp probe. These results were used to construct a map
ordering the TcbAii internal peptide sequences as shown in
Fig. 3. Approximate distances between the primers are shown in
nucleotides in Fig. 3.
DNA Sequence of the 2.9 kbp TcbAii-encoding fragment
Approximately 200 ng of the purified 2.9 kbp fragment
(prepared above) was precipitated with ethanol and dissolved in
17 ml of water. One-half of this was used as sequencing template
with 25 pmol of the TH-5 pool as primers, the other half was used
as template for TH-10 priming. Sequencing reactions were as
given in Example B. No reliable sequence was produced using the
TH-10 primer pool; however, reactions with TH-5 primer pool
produced the sequence disclosed below:.
1 AATCGTGTTG ATCCCTATGC CGNGCCGGGT TCGGTGGAAT CGATGTCCTC ACCGGGGGTT
61 TATTNGAGGG At1TNGTCCCG TGAGGCCAAA AANTGGAATG AAAGAAGTTC AATTTNTTAC
121 CTAGATAAAC GTCGCCCGGtI TTTAGAAAGN TTANTGNTCA GCCAGAAAAT TTTGGTTGAG
181 GAAATTCCAC CGNTGGTTCT CTCTATTGAT TNGGGCCTGG CCGGGTTCGA ANNP.AAACNA
241 GGAAATI4CAC AAGTTGAGGT GATGGNTTTG TNGCNANCTT NTCGTTTAGG TGGGGAGAAA
301 CCTTNTCANC ACGNTTNTGA AACTGTCCGG GAAATCGTCC ATGANCGTGA fICCAGGNTTPI
361 CGCCATTGG
Based on this sequence, a sequencing primer (TH-21, 5'-
CCGGGCGACGTTTATCTAGG-3') was designed to reverse complement bases
120-139, and initiate polymerization towards the 5' end (i.e.,
TH-5 end) of the gel-purified 2.9 kbp TcbAii-encoding PCR
fraament. The determined sequence is shown below, and is
compared to the biochemically determined N-terminal peptide
sequence of TcbAii SEQ ID NO:1.
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T,::b,4.ii 2.9 kbD PCR fragment Sequence Confirmation
[L'nderlined amino acids = encoded by degenerate oligonucieotides;
SEQ ID NO:l F I Q G Y S D L F G - - A
I I I 1 1 I 1 I I I 1
2.9 kbp seq GC ATG CAG GGG TAT AGT GAC CTG TTT GGT AAT CGT GCT
M Q G Y S D L F G N R A:From the homology of the derived amino acid sequence
to the
= 10 biochemically determined one, it is clear that the 2.9 kbp PCR
fragment represents the TcbA coding region. This 2.9 kbp
fragment was then used as a hybridization probe to screen the
Photorhabdus W-14 genomic library prepared in Example 8 for
cosmids containing the TcbAii-encoding gene.
Screening the Photorhabdus cosmid library
The 2.9 kb gel-purified PCR fragment was labeled with ''P
using the Boehringer Mannheim High Prime labeling kit as
described in Example 8. Filters containing remnants of
approximately 800 colonies from the cosmid library were screened
as described previously (Example 8), and positive clones were
streaked for isolated colonies and rescreened. Three clones
(8A11, 25G8, and 26D1) gave positive results through several
screening and characterization steps. No hybridization of the
TcbAii-specific probe was ever observed with any of the four
cosmids identified in Example 8, and which contain the tcaB and
tcaC genes. DNA from cosmids 8A11, 25G8, and 26D1 was digested
with restriction enzymes Bgl 2, EcoR 1 or Hind 3 (either alone or
in combination with one another), and the fragments were
separated on an agarose gel and transferred to a nylon membrane
as described in Example 8. The membrane was hybridized with 'P-
labeled probe prepared from the 4.5 kbp fragment (generated by
amplification of Photorhabdus genomic DNA with primers TH-5+TH-
17). The patterns generated from cosmid DNAs 8Al1 and 26D1 were
identical to those generated with similarly-cut genomic DNA on
the same membrane. It is concluded that cosmids 8A11 and 26D1
are accurate representations of the genomic TcbAii encoding
locus. However, cosmid 25G8 has a single Bgl 2 fragment which is
slightly larger than the genomic DNA. This may result from
positioning of the insert within the vector.
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DNA sequence of the tcbA-encoding gene
The membrane hybridization analysis of cosmid 26D1 revealed
that the 4.5 kbp probe hybridized to a single large EcoR 1
fragment (greater than 9 kbp). This fragment was gel purified
and ligated into the EcoR 1 site of pBC KS (+) as described in
Example 8, to generate plasmid pBC-S1/R1. The partial DNA
sequence of the insert DNA of this plasmid was determined by
"primer walking" from the flanking vector sequence, using procedures described
in Example 8. Further sequence was
generated by extension from new oligonucleotides designed from
the previously determined sequence. When compared to the
determined DNA sequence for the tcbA gene identified by other
methods (disclosed herein as SEQ ID NO:ll as described in Example
12 below), complete homology was found to nucleotides 1-272, 319-
826, 2578-3036, and 3068-3540 (total bases = 1712). It was
concluded that both approaches can be used to identify DNA
fragments encoding the TcbAii peptide.
Analysis of the derived amino acid sequence of the tcbA gene.
The sequence of the DNA fragment identified as SEQ ID NO:ll
encodes a protein whose derived amino acid sequence is disclosed herein
as SEQ ID NO:12. Several features verify the identity of the gene as
that encoding the TcbAii protein. The TcbAii N-terminal peptide (SEQ
ID NO:1; Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala) is
encoded as amino acids 88-100. The TcbAii internal peptide TcbAii-
PT81(a) (SEQ ID NO:23) is encoded as amino acids 1065-1077, and TcbAii-
PT81(b) (SEQ ID NO:24) is encoded as amino acids 1571-1592. Further,
the internal peptide TcbAii-PT56 (SEQ ID NO:22) is encoded as amino
acids 1474-1488, and the internal peptide TcbAii-PT103 (SEQ ID NO:24)
is encoded as amino acids 1614-1639. It is obvious that this gene is
an authentic clone encoding the TcbAii peptide as isolated from
insecticidal protein preparations of Photorhabdus luminescens strain
w-14.
The protein isolated as peptide TcbAii is derived from cleavage
of a longer peptide. Evidence for this is provided by the fact that
the nucleotides encoding the TcbAii N-terminal peptide SEQ ID NO:1 are
preceded by 261 bases (encoding 87 N-terminal-proximal amino acids) of
a longer open reading frame (SEQ ID NO:11). This reading frame begins
with nucleotides that encode the amino acid sequence Met Gln Asn Ser
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Leu, which corresponds to the N-terminal sequence of the large
peptide TcbA, and is disclosed herein as SEQ ID NO:16. It is
thought that TcbA is the precursor protein for TcbAii'
Relationship of tcbA, tcaB and tcaC genes.
The tcaB and tcaC genes are closely linked and may
be transcribed as a single mRNA (Example 8). The tcbA gene is
borne on cosmids that apparently do not overlap the ones
harboring the tcaB and tcaC cluster, since the respective
genomic library screens identified different cosmids.
However, comparison of the amino sequences encoded by the tcaB
and tcaC genes with the tcbA gene reveals a substantial degree
of homology. The amino acid conservation (Protein Alignment
Mode of MacVectorTM Sequence Analysis Software, scoring matrix
pam250, hash value = 2; Kodak Scientific Imaging Systems,
Rochester, NY) is shown in Fig. 4A and Fig 4B. On the score
line of each panel in Fig. 4A and Fig. 4B, up carats (~)
indicate homology or conservative amino acid changes, and down
carats (v) indicate nonhomology.
This analysis shows that the amino acid sequence of
the TcbA peptide from residues 1739 to 1894 is highly
hornologous to amino acids 441 to 603 of the TcaBi peptide (162
of the total 627 amino acids of P8; SEQ ID NO:28). In
addition, the sequence of TcbA amino acids 1932 to 2459 is
highly homologous to amino acids 12 to 531 of peptide TcaBii
(520 of the total 562 amino acids; SEQ ID NO:30). Considering
that the TcbA peptide (SEQ ID NO:12) comprises 2505 amino
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acids, a total of 684 amino acids (27%) at the C-proximal end
of it is homologous to the TcaBi or TcaBii peptides, and the
homologies are arranged colinear to the arrangement of the
putative TcaB preprotein (SEQ ID NO:26). A sizeable gap in
the TcbA homology coincides with the Junction between the
TcaBi and TcaBii portions of the TcaB preprotein. Clearly the
TcbA and TcaB gene products are evolutionarily related, and it
is proposed that they share some common function(s) in
Photorhabdus.
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Example 10
Characterization of zinc-metalloproteases in Photorhabdus Broth:
Protease Inhibition, Classification, and Purification
Protease Inhibition and Classification Assays: Protease
assays were performed using FITC-casein dissolved in water as
substrate (0.08% final assay concentration). Proteolysis
reactions were performed at 25 C for 1 h in the appropriate
buffer with 25 ul of Photorhabdus broth (150 til total reaction
volume). Samples were also assayed in the presence and absence
of dithiothreitol. After incubation, an equal volume of 12%
trichloroacetic acid was added to precipitate undigested protein.
Following precipitation for 0.5 h and subsequent centrifugation,
100 ul of the supernatant was placed into a 96-well microtiter
plate and the pH of the solution was adjusted by addition of an
equal volume of 4N NaOH. Proteolysis was then quantitated using
a Fluoroskan II fluorometric plate reader at excitation and
emission wavelengths of 485 and 538 nm, respectively. Protease
activity was tested over a range from pH 5.0-10.0 in 0.5 units
increments. The following buffers were used at 50 mM final
concentration: sodium acetate (pH 5.0 - 6.5); Tris-HCL (pH 7.0 -
8.0); and bis-Tris propane (pH 8.5-10.0). To identify the class
of protease(s) observed, crude broth was treated with a variety
of protease inhibitors (0.5 ug/ul final concentration) and then
examined for protease activity at pH 8.0 using the substrate
described above. The protease inhibitors used included E-64 (L-
trans-expoxysaccinylleucylamido[4-,-guanidino]-butane), 3,4
dichloroisocoumarin, Leupeptin, pepstatin, amastatin,
ethylenediaminetetraacetic acid (EDTA) and 1,10 phenanthroline.
Protease assays performed over a pH range revealed that
indeed protease(s) were present which exhibited maximal activity
at - pH 8.0 (Table 16). Addition of DTT did not have any effect
on protease activity. Crude broth was then treated with a
variety of protease inhibitors (Table 17). Treatment of crude
broth with the inhibitors described above revealed that 1,10
phenanthroline caused complete inhibition of all protease
activity when added at a final concentration of 50 pg, with the
IC50 = 5 ug in 100 ul of a 2 mg/ml crude broth solution. These =
data indicate that the most abundant protease(s) found in the
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Photoriiabdus broth are from the zinc-metalloprotease class cf
enzymes.
Table 16
Effect of pH on the protease activity found in a Day 1 production
of Photorhabdus luminescens (strain W-14).
pH Flu. Unitsa Percent
Activityb
5.0 3013 78 17
5.5 7994 448 45
6.0 12965 483 74
6.5 14390 1291 82
7.0 14386 1287 82
7.5 14135 198 80
8.0 17582 831 100
8.5 16183 953 92
9.0 16795 760 96
9.5 16279 1022 93
10.0 15225 210 87
a Flu. Units = Fluorescence Units (Maximum =--28,000;
background = - 2200).
b. Percent activity relative to the maximum at pH 8.0
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Table 17
Effect of different protease inhibitors on the protease activitl
at pH 8 found in a Day 1 production of Photorhabdus lurninescens
(strain W-14).
Inhibitor Corrected Flu. Unitsa Percent Inhibitionb
Control 13053 0
E-64 14259 0
1,10 Phenanthrolinec 15 99 =
3,4 Dichloroisocoumarind 7956 39
Leupeptin 13074 0
Pepstatinc 13441 0
Amastatin 12474 4
DMSO Control 12005 8
Methanol Control 12125 7
a. Corrected Flu. Units = Fluorescence Units -
background(2200 flu. units).
b. Percent Inhibition relative to protease activity at pH
8Ø
c. Inhibitors were dissolved in methanol.
d. Inhibitors were dissolved in DMSO.
The isolation of a zinc-metalloprotease was performed by
applying dialyzed 10-80% ammonium sulfate pellet to a Q Sepharose
column equilibrated at 50 mM Na2PO4, pH 7.0 as described in
Example 5 for Photorhabdus toxin. After extensive washing, a 0
to 0.5 M NaCl gradient was used to elute toxin protein. The
majority of biological activity and protein was eluted from 0.15
- 0.45 M NaCl. However, it was observed that the majority of
proteolytic activity was present in the 0.25-0.35 M NaCl traction
with some activity in the 0.15-0.25 M NaCl fraction. SDS PAGE
analysis of the 0.25-0.35 M NaCl fraction showed a major peptide
band of approximately 60 kDa. The 0.15-0.25 M NaCl fraction
contained a similar 60 kDa band but at lower relative protein
concentration. Subsequent gel filtration of this fraction using
a Superose 12 HR 16/50 column resulted in a major peak migratirig
at 57.5 kDa that contained a predominant (> 90% of total stained
protein) 58.5 kDa band by SDS PAGE analysis. Additional analysis
of this fraction using various protease inhibitors as described
above determined that the protease was a zinc-metalloprotease.
Nearly all of the protease activity present in Photorhabdus broth
at day 1 of fermentation corresponded to the -58 kDa zinc-
metalloprotease.
In yet a second isolation of zinc-metalloprotease(s), W-14
Photorhabdus broth grown for three days was taken and protease
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activity was visualized using sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) laced with gelatin
as described in Schmidt, T.M., Bleakley, B. and Nealson, K.M.
1988. SDS running gels (5.5 x 8 cm) were made with 12.5 %
polyacrylamide (40% stock solution of acrylamide/bis-acr-ylamide;
Sigma Chemical Co., St. Louis, MO) into which 0.1% gelatin final
concentration (Biorad EIA grade reagent; Richmond CA) was
incorporated upon dissolving in water. SDS-stacking gels (1.0 x
8 cm) were made with 5% polyacrylamide, also laced with 0.1%
gelatin. Typically, 2.5 g of protein to be tested was diluted
in 0.03 ml of SDS-PAGE loading buffer without dithiothreitol
(DTT) and loaded onto the gel. Proteins were electrophoresed in
SDS running buffer (Laemmli, U.K. 1970. Nature 227, 680) at 00 C
and at 8 mA. After electrophoresis was complete, the gel was
washed for 2 h in 2.5% (v/v) Triton X-100. Gels were then
incubated for 1 h at 37 C in 0.1 M glycine (pH 8.0). After
incubation, gels were fixed and stained overnight with 0.1% amido
black in methanol-acetic acid- water (30:10:60, vol./vol./vol.;
Sigma Chemical Co.). Protease activity was visualized as light
areas against a dark, amido black stained background due to
proteolysis and subsequent diffusion of incorporated gelatin. At
least three distinct bands produced by proteolytic activity at
58-, 41-, and 38 kDa were observed.
Activity assays of thedifferent proteases in W-14 day three
culture broth were performed using FITC-casein dissolved in water
as substrate (0.02% final assay concentration). Proteolysis
experiments were performed at 37. C for 0-0.5 h in 0.1M Tris-HC1
(pH 8.0) with different protein fractions in a total volume of
0.15 ml. Reactions were terminated by addition of an equal
volume of 12% trichloroacetic acid (TCA) dissolved in water.
After incubation at room temperature for 0.25 h, samples were
centrifuged at 10,000 x g for 0.25 h and 0.10 ml aliquots were
removed and placed into 96-well microtiter plates. The solution
was then neutralized by the addition of an equal volume of 2 21
sodium hydroxide, followed by quantitation using a Fluoroskan II
fluorometric plate reader with excitation and emission
wavelengths of 485 and 538 nm, respectively. Activity
measurements were performed using FITC-Casein with different
protease concentrations at 37 C for 0-10 min. A unit of
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activity was arbitrarily defined as the amount of enzyme needed
to produce 1000 fluorescent units/min and specific activity was
defined as units/mg of protease.
Inhibition studies were performed using two zinc-
metalloprotease inhibitors; 1,10 phenanthroline and N-(a-
rhamnopyranosyloxyhydroxyphosphinyl)-Leu-Trp(phosphoramidon) with
stock solutions of the inhibitors dissolved in 100% ethanol and water,
respectively. Stock concentrations were typically 10
mg/ml and 5 mg/mi for 1,10 phenanthroline and phosphoramidon,
respectively, with final concentrations of inhibitor at 0.5-1.0
mg/ml per reaction. Treatment of three day W-14 crude broth with
1,10 phenanthroline, an inhibitor of all zinc metalloproteases,
resulted in complete elimination of all protease activity while
treatment with phosphoramidon, an inhibitor of thermolysin-like
proteases (Weaver, L.H., Kester, W.R., and Matthews, B.W. 1977.
J. Mol. Biol. 114, 119-132), resulted in -56% reduction of
protease activity. The residual proteolytic activity could not
be further reduced with additional phosphoramidon.
The proteases of three day W-14 Photorhabdus broth were
purified as follows: 4.0 liters of broth were concentrated using
an Amicon spiral ultra filtration cartridge Type SlYlOO attached
to an Amicon M-12 filtration device. The flow-through material
having native proteins less than 100 kDa in size (3.8 L) was
concentrated to 0.375 L using an Amicon spiral ultra filtration
cartridge Type S1Y10 attached to an Amicon M-12 filtration
device. The retentate material contained proteins ranging in
size from 10-100 kDa. This material was loaded onto a Pharmacia
HR16/10 column which had been packed with PerSeptive Biosystem
(Framington, MA) Poros 50 HQ strong anion exchange packing that
had been equilibrated in 10 mM sodium phosphate buffer (pH 7.0).
Proteins were loaded on the column at a flow rate of 5 ml/min,
followed by washing unbound protein with buffer until A280 =
0.00. Afterwards, proteins were eluted using a NaCl gradient of
0-1.0 M NaCl in 40 min at a-flow rate of 7.5 ml/min. Fractions
were assayed for protease activity, supra., and active fractions
were pooled. Proteolytically active fractions were diluted with
50% (v/v) 10 mM sodium phosphate buffer (pH 7.0) and loaded onto
a Pharmacia HR 10/10 Mono Q column equilibrated in 10 mM sodium
phosphate. After washing the column with buffer until A280 -
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0.00, proteins were eluted using a NaCl gradient of 0-0.5 c4 tIaC1
for I h at a flow rate of 2.0 ml/min. Fractions were assayed for
procease activity. Those fractions having the greatest amount of
phosphoramidon-sensitive protease activity, the phosphoramidon
sensitive activity being due to the 41/38 kDa protease, infra.,
were pooled. These fractions were found to elute at a range of
0.15-0.25 M NaCl. Fractions containing a predominance of
phosphoramidon-insensitive protease activity, the 58 kDa
protease, were also pooled. These fractions were found to elute
at a range of 0.25-0.35 M NaC1. The phosphoramidon-sensitive
procease fractions were then concentrated to a final volume of
0.75 ml using a Millipore Ultrafree -15 centrifugal filter device
Bioma:c 5K NMWL membrane. This material was applied at a flow
rate of 0.5 ml/min to a Pharmacia HR 10/30 column that had been
packed with Pharmacia Sephadex G-50 equilibrated in 10 mM sodium
phosphate buffer (pH 7.0)/ 0.1 H NaC1. Fractions having the
maximal phosphoramidon-sensitive protease activity were then
pooled and centrifuged over a Millipore Ultrafree4D-15 centrifugal
filter device Biomax-50K NMWL membrane. Proteolytic activity
analysis, supra., indicated this macerial to have only
phosphoramidon-sensitive protease activity. Pooling of the
phosphoramidon-insensitive protease, the 58 kDa protein, was
followed by concentrating in a Millipore Ultrafree -15
centrifugal filter device Biomax-S0K NMWL membrane and further
separation on a Pharmacia Superdex-75 column. Fractions
containing the protease were pooled.
Analysis of purified 58- and 41/38 kDa purified proteases
re-fealed that, while both types of protease were completely
inhibited with 1,10 phenanthroline, only the 41/38 kDa protease
was inhibited with phosphoramidon. Further analysis of crude
broth indicated that protease activity of day 1 w-14 broth has
23% of the total protease activity due to the 41/38 kDa protease,
increasing to 44% in day three W-14 broth.
Standard SDS-PAGE analysis for examining protein purity and
obtaining amino terminal sequence was performed using 4-20%
gradient MiniPlus SepraGels purchased from Integrated Separation
Systems (Natick, MA). Proteins to be amino-terminal sequenced
were blotted onto PVDF membrane following purification, infra.,
(ProBlottr" Membranes; Applied Siosystems, Foster City, CA),
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:Tisualized with 0.1% amido black, excised, and sent to cambridge
Prochem; Cambridge, b1A, for sequencing.
Deduced amino terminal sequence of the 58- (SEQ ID NO:45)
and 41/38 kDa (SEQ ID NO:44) proteases from three day old W-14
broth were DV-GSEKANEKLK (SEQ ID NO: 45) and DSGDDDKVTNTDIHR (SEQ
ID NO:44), respectively. Sequencing of the 41/38 kDa protease revealed several
amino
termini, each one having an additional amino acid removed by
proteolysis. Examination of the primary, secondary, tertiary and
quartenary sequences for the 38 and 41 kDa polypeptides allowed
for deduction of the sequence shown above and revealed that these
two proteases are homologous.
Example 11, Part A
Screening of Photorhabdus Genomic Library via use of Antibodies
for Genes encoding TcbA Peptide
In parallel to the sequencing described above, suitable
probing and sequencing was done based on the TcbAii peptide (SEQ
ID NO:1). This sequencing was performed by preparing bacterial
culture broths and purifying the toxin as described in Examples 1
and 2 above.
Genomic DNA was isolated from the Photorhabdus luminescens
strain W-14 grown in Grace's insect tissue culture medium. The
bacteria were grown in 5 ml of culture medium in a 250 ml
Erlenmeyer flask at 28 C and 250 rpm for approximately 24 hours.
Bacterial cells from 100 ml of culture medium were pelleted at
5000 x g for 10 minutes. The supernatant was discarded, and the
cell pellets then were used for the genomic DNA isolation.
The genomic DNA was isolated using a modification of the
CTAB method described in Section 2.4.3 of Ausubel (supra.). The
section entitled "Large Scale CsCl prep of bacterial genomic DT1A"
was followed through step 6. At this point, an additional
chloroform/isoamyl alcohol (24:1) extraction was performed
followed by a phenol/chloroform/isoamyl (25:24:1) extraction step
and a final chloroform/isoamyl/alcohol (24:1) extraction. The
DNA was precipitated by the addition of a 0.6 volume of
isopropanol. The precipitated DNA was hooked and wound around
the end of a bent glass rod, dipped briefly into 70% ethariol as a
final wash, and dissolved in 3 ml of TE buffer. -77-
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The DNA concentration, estimated by optical density at
280/260 nm, was approximately 2 mg/ml.
Using this genomic DNA, a library was prepared.
Approximately 50 ug of genomic DNA was partly digested with Sau3
Al. Then NaCl density gradient centrifugation was used to size
' fractionate the partially digested DNA fragments. Fractions
~ containing DtIA fragments with an average size of 12 kb, or
larger, as determined by agarose gel electrophoresis, were
ligated into the plasmid BluScript, Stratagene, La Jolla,
California, and transformed into an E. coli DH5a or DHBIO strain.
Separately, purified aliquots of the protein were sent to
the biotechnology hybridoma center at the University of
Wisconsin, Madison for production of monoclonal antibodies to the
proteins. The material that was sent was the HPLC purified
fraction containing native bands 1 and 2 which had been denatured
at 65 C, and 20 g of which was injected into each of four mice.
Stable monoclonal antibody-producing hybridoma cell lines were
recovered after spleen cells from unimmunized mouse were fused
with a stable myeloma cell line. Monoclonal antibodies were
recovered from the hybridomas.
Separateiy, polyclonal antibodies were created by taking
native agarose gel purified band 1 (see Example 1) protein which
was then used to immunize a New Zealand white rabbit. The
protein was prepared by excising the band from the native agarose
gels, briefly heating the gel pieces to 65 C to melt the agarose,
and immediately emulsifying with adjuvant. Freund's complete
adjuvant was used for the primary immunizations and Freund's
incomplete was used for 3 additional injections at monthly
intervals. For each injection, approximately 0.2 ml of
emulsified band 1, containing 50 to 100 micrograms of protein,
was delivered by multiple subcontaneous injections into the back
of the rabbit. Serum was obtained 10 days after the final
injection and additional bleeds were performed at weekly
intervals for 3 weeks. The serum complement was inactivated by
heating to 56 C for 15 minutes and then stored at -20 C.
The monoclonal and polyclonal antibodies were then used to
screen the genomic library for the expression of antigens which
could be detected by the epitope. Positive clones were detected
on nitrocellulose filter colony lifts. An immunoblot analysis of
the positive clones was undertaken.
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An analysis of the clones as defined by both
immunoblot and Southern analysis resulted in the tentative
identification of five classes of clones.
In the first class of clone was a gene encoding the
peptide designated here as TcbAii. Full DNA sequence of this
gene (TcbA) was obtained. It is set forth as SEQ ID NO:11.
Confirmation that the sequence encodes the internal sequence
of SEQ ID NO:l is demonstrated by the presence of SEQ ID NO:1
at amino acid number 88 from the deduced amino acid sequence
created by the open reading frame of SEQ ID NO:11. This can
be confirmed by referring to SEQ ID NO:12, which is the
deduced amino acid sequence created by SEQ ID NO:11.
The second class of toxin peptides contains the
segments referred to above as TcaBi, TcaBii and TcaC (Fig.
6A). Following the screening of the library with the
polyclonal antisera, this second class of toxin genes was
identified by several clones which produced different size
proteins, all of which cross-reacted with the polyclonal
antibody on an immunoblot and were also found to share DNA
homology on a Southern Blot. Sequence comparison revealed
that they belonged to the gene complex designated TcaB and
TcaC above.
Three other classes of antibody toxin clones were
also isolated in the polyclonal screen. These classes
produced proteins that cross-react with a polyclonal antibody
and also shared DNA homology with the classes as determined by
Southern blotting. The classes have been designated Class
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III, Class IV and Class V. It was also possible to identify
monoclonals that cross-reacted with Class I, II, III, and IV.
This suggests that all have regions of high protein homology.
Thus, it appears that the P. luminescens extracellular protein
genes represent a family of genes which are evolutionarily
related.
To further pursue the concept that there might be
evolutionarily related variations in the toxin peptides
contained within this organism, two approaches have been
undertaken to examine other strains of P. luminescens for the
presence of related proteins. This was done both by PCR
amplification of genomic DNA and by immunoblot analysis using
the polyclonal and monoclonal antibodies.
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The results indicate that related proteins are produced b,=
P. luminescens strains WX-2, WX-3, WX-4, WX-5, WX-6, WX-7, 'r7X-?,
WX-11, WX-12, WX-15 and W-14.
Example 11, Part B
Sequence and anaylsis of C1ass III toxin clones - rcc
Further DNA sequencing was performed on plasmids isolated
from Class III E. coli clones described in Example 11, Part A.
The nucleotide sequence was shown to be three closely linked open
reading frames at this genomic locus. This locus was designated
tcc with the three open reading frames designated tccA SEQ ID
NO:56, tccB SEQ ID NO:58 and tccC SEQ ID NO:60 (Fig. 6B).
The deduced amino acid from the tccA open reading frame
indicates the gene encodes a protein of 105,459 Da. This protein
was designated TccA. The first 12 amino acids of this protein
match the N-terminal sequence obtained from a 108 kDa protein,
SEQ ID NO:7, previously identified as part of the toxin complex.
The deduced amino acid from the tccB open reading frame
indicates this gene encodes a protein of 175,716 Da. This
protein was designated TccB. The first 11 amino acids of this
protein match the N-terminal sequence obtained from a protein
with estimated molecular weight of 185 kDa, SEQ ID NO:8.
The deduced amino acid sequence of tccC indicated that this
open reading frame encodes a protein of 111,694 Da and the
protein product was designated TccC.
Example 12
Characterization of Photorhabdus Strains
In order to establish that the collection described herein
was comprised of Photorhabdus strains, the strains herein were
assessed in terms of recognized microbiological traits that are
characteristic of Photorhabdus and which differentiate it from
other Enterobacteriaceae and Xenorhabdus spp. (Farmer, J.J. 1984.
Bergey's Manual of Systemic Bacteriology, vol 1. pp. 510-511.
(ed. Kreig N.R. and Holt, J.G.). Williams & Wilkins, Baltimore.;
, Akhurst and Boemare, 1988, Boemare et al., 1993). These
characteristic traits are as follows: Gram's stain negative
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roo+=s, .~rganism size of 0.5-2 um in width and 2-10 um in !_ngth,
red;yellow colcny pigmencacicn, presence of cr-/scaliine incluslOri
bodies, presence of cacalase, inability to reduce nitrate,
presence of bioluminescence, abilicy to take up dye Erom growth
media, posicive for procease production, grewth-temceraturs range
below 3% C, survival under anaerobic conditior.s and pesici=:e1v
mocile. (Table 18). Reference Escherichia coli, Xenorrcaodus and
Phocorhabdus strains were included in all tescs for ccmparison.
The overall results are consistent with all strains being part ot
the family Encerobacceriaceae and the genus Phocorhabdus.
A luminometer was used to establish the bioluminescence of
each strain and provide a quantitative and relative measurement
of light production. For measurement of relative light emitting
units, the broths from each strain (cells and media) were
measured at three time intervals after inoculation in liquid
culture (6, 12, and 24 hr) and compared to background luminosity
(uninoculated media and water). Prior to measuring light
emission from the various broths, cell density was established by
measuring light absorbance (560 nM) in a Gilford Systems
(Oberlin, OH) spectrephotometer using a sipper cell. Apprcrriace
dilutions were then made (to normalize optical density to 1.0
unit) before measuring luminosity. Aliquots of the diluted
broths were then placed into cuvettes (300 ul each) and read in a
Bio-Orbit* 1251 Luminometer (Bio-Orbit Oy, Twiku, Finland) . The
integration period for each sample was 45 seconds. The samples
were continuously mixed (spun in baffled cuvettes) while being
read to provide oxygen availability. A positive test was
determined as being >_ 5-fold background luminescence (-5-10
units). In addition, colony luminosity was detected with
photographic film overlays and visually, after adaptation in a
darkroom. The Gram's staining characteristics of each strain
were established with a commercial Gram's stain kit (BBL,
Cockeysville, MD) used in conjunction with Gram's stain control
slides (Fisher Scientific, Pittsburgh, PA). Microscopic
evaluation was then performed using a Zeiss microscope (Carl
Zeiss, Germany) 100X oil immersion objective lens (with lOX
ocular and 2X body magnification). Microscopic examination of
individual scrains for organism size, cellular descripticn and
inclusion bodies (the lacter after logarithmic growth) was
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performed using wet mcunt siides !10j ocular, 2:{ body and 40Z
objec_ive magnification) with oil immersion and phase contrast
microscopy with a micromecer (Akhurst, R.J. and Boemare, N.E.
1990. EncomooachoQenic Nematodes in Biological Control (ed.
Gaugler, R. and Kaya. H.). pp. 75-?0. CRC Press, Boca Raton,
USA.; Bachdiguian S., Boyer-Giglio M.H., Thaler, J.O., Bonnct G.,
Boemare U. 1993. Biol. Cell 7?, 177-185.). Colony pigmentation
was observed after inoculacion on Bacto nutrient agar, (Difco
Laboratories, Detroit, MI) prepared as per label instructions.
Incubation occurred at 28 C and descripcions were produced after
5-7 days. To test for the presence of the enzyme catalase, a
colony of the test organism was removed on a small plug fr::~ a
nutrient agar plate and placed into the bottom of a glass tesc
tube. One ml of a household hydrogen peroxide solution was gently
added down the side of the tube. A positive reaction was
recorded when bubbles of gas (presumptive oxygen) appeared
immediately or within 5 seconds. Controls of uninoculated
nutrient agar and hydrogen peroxide solution were also examined.
To test for nitrate reduction, each culture was inoculated inco
10 ml of Bacto Nitrate Broth (Difco Laboratories, Detroit, :=II).
After 24 hours incubation at 28 C, nitrite production was tested
by the addition of two drops of sulfanilic acid reagent and two
drops of alpha-naphthylamine reagent (see Difco Manual, 10th
edition, Difco Laboratories, Detroit, MI, 1984). The generation
of a distinct pink or red color indicates the formation of
nitrite from nitrate. The ability of each strain to uptake dye
from growth media was tested with Bacto MacConkey agar containiiig
the dye neutral red; Bacto Tergitol-7 agar containing the dye
bromothymol blue and Bacto EMB Agar containing the dye eosin-Y
(agars from Difco Laboratories, Detroit, MI, all prepared
according to label instructions). After inoculation on these
media, dye uptake was recorded after incubation at 28 C for 5
days. Growth on these latter media is characteristic for members
of the family EnceroDacceriaceae. Motility of each strain was
tested using a solution of Bacto Motility Test Medium (Difco
Laboratories, Detroit, MI) prepared as per label inscructions. A
butt-stab inoculation was performed with each strain and motility
was judged macroscopically by a diffuse zone of growth spreading
from the line of inoculum. In many c3ses, motility was also
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observed microscopically from liquid culture under wet mount
slides. Biochemical nutrient evaluation for each strain was
performed using BBL Enterotube II (Benton, Dickinson, Germany).
Product instructions were followed with the exception that
incubation was carried out at 28 C for 5 days. Results were
consistent with previously cited reports for Photorhabdus. The
production of protease was tested by observing hydrolysis of
gelatin using Bacto gelatin (Difco Laboratories, Detroit, MI)
plates made as power label instructions. Cultures were
inoculated and the plates were incubated at 28 C for 5 days. To
assess growth at different temperatures, agar plates [2%
proteose peptone #3 with two percent Bacto-Agar (Difco,
Detroit, MI) in deionized water] were streaked from a common
source of inoculum. Plates were sealed with NescoO film and
incubated at 20, 28 and 37 C for up to three weeks. Plates
showing no growth at 37 C showed no cell viability after
transfer to a 28 C incubator for one week. Oxygen requirements
for Photorhabdus strains were tested in the following manner.
A butt-stab inoculation into fluid thioglycolate broth medium
(Difco, Detroit, MI) was made. The tubes were incubated at
room temperature for one week and cultures were then examined
for type and extent of growth. The indicator resazurin
demonstrates the level of medium oxidation or the aerobiosis
zone (Difco Manual, 10th edition, Difco Laboratories, Detroit,
MI). Growth zone results obtained for the Photorhabdus strains
tested were consistent with those of a facultative anaerobic
microorganism.
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Table 18
Taxonomic Traits of Photorhabdus Strains
Traits Assessed*
Strain A B C D E F G H I J K L M N 0 P Q
W-14 _t + + rd S + - + + + 0 + + + + + + -
wx-i - + + rd S + - + + + 0 + + + + + + -
WX-2 - + + rd S + - + + + 0 + + + + + + -
WX-3 - + + rd S + - + + + YT + + + + + + -
WX-4 - + + rd S + - + + + YT + + + + + +
wX-5 - + + rd S + - + + + LO + + + + + + -
WX-6 - + + rd S + - + + + LY + + + + + + -
WX-7 - + + rd S + - + + + R + + + + + +
WX-8 - + + rd S + - + + + 0 + + + + + +
WX-9 - + + rd S + - + + + YT + + + + + + -
wx-io - + + rd S - + + + Ro + + + + + + -
WX-11 - + + rd S + - + + + Ro + + + + + + -
WX-12 - + + rd S + - + + + 0 + + + + + + -
WX-14 - + + rd S + - + + + LR + + + + + + -
WX-15 - + + rd S + - + + + LR + + + , + +
H9 - + + rd S + - + + + LY + + + + + +
Hb - + + rd S + - + + + YT + + + + + + -
Hm - + + rd S + - + + + TY + + + + + + -
HP88 - + + rd S + - + + + LY + + + + + + -
NC-1 - + + rd S -~ - + + + 0 + + + + + + -
W30 - + + rd S + - + + + YT + + + + +
WIR - + + rd S + - + + + RO + + + + +
B2 - + + rd S + - + + + R + + + + + + -
43948 - + + rd S - + + + 0 + + + + + +
43949 - + + rd S + - + + +
-4 0 + + + + + + -
43950 - + + rd S + - + + + 0 + + + + + + -
43951 - + + rd S + - + + + 0 + + + + + + -
43952 - + + rd S + - + + + 0 + J + + + + + -
* - A Gram's stain, B=Crystaline inclusion bodies,
C=Bioluminescence, D=Cell form, E=Motility, F=Nitrate
reduction, G=Presence of catalase, H=Gelatin hydrolysis, I=Dye
uptake, J=Pigmentation, K=Growth on EMB agar, L=Growth on
MacConkey agar, M=Growth on Tergitol-7 agar, N=Facultative
anaerobe, O=Growth at 20 C, P=Growth at 28 C, Q=Growth at 37 C,
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t: + = positive for trait, -= negative for trait, rd=rod,
S=sized within Genus descriptors, RO=red-orange, LR= light red,
R= red, 0= orange, Y= yellow, T= tan, LY= light yellow, YT=
yellow tan, and LO = light orange.
Cellular fatty acid analysis is a recognized tool for
bacterial characterization at the genus and species level
(Tornabene, T.G. 1985. Lipid Analysis and the Relationship to
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Chemccaxcncr: iz Methods in Microbiolog'r,Vol 13, 20'1-2:4. ,
Goodfellow, t4. and O'Donnell, A.G. 1993. P.oocs of Bacterial
Systemacics in Handbock of t)ew Bacterial Systemacics (ed.
Goodfellow, M. & O'Donnell, A.G.) pp. 3-54. London: acaciemic
Press Ltd.)
and were used to confirm that our collection was
related at the genus level. Cultures were shipped to an
external, contract laboratory for fatty acid methyl ester
analysis (FAME) using a Microbial ID (MIDI, Newark, DE, USa)
Microbial Identification System (MIS). The MIS system consists of
a Hewlett Packard HP5890A gas chromatograph with a 25mm x 0.2mm
5% methylphenyl silicone fused silica capillary column. Hydrogen
is used as the carrier gas and a flame-ionizacion detector
functions in conjunction with an automatic sampler, incegrator
and compucer. The computer compares the sample fatty acid methyl
esters to a microbial fatty acid library and against a
calibration mix of known fatty acids. As selected by the
contract laboratory, strains were grown for 24 hours at 23 C on
trypticase soy agar prior to analysis. Extraction of samples was
performed by the contract lab as per standard FAME methodology.
There was no direct identification of the strains to any
luminescent bacterial group other than Photorhabdus. wh,~n the
cluster analysis was performed, which ccmpares the fatty acid
profiles of a group of isolates, the strain fatty acid profiles
were related at the genus level.
The evolutionary diversity of the Photorhabdus strains in
our collection was measured by analysis of PCR (Polymerase Chain
Reaction) mediated genomic fingerprinting using genamic DNA from
each strain. This technique is based on families of repetitive
DNA sequences present throughout the genome of diverse bacterial
species (reviewed by Versalovic, J., Schneider, M., DE Bruijn,
F.J. and Lupski, J.R. 1994. Methods Mol. Cell. Biol., 5, 25-40.).
Three of these, repetitive extragenic palindromic sequence (REP),
enterobacterial repetitive intergenic consensus (ERIC) arid the
BOX element are thought to play an important role in the
organization of the bacterial genome. Genomic organization is
believed to be shaped by selection and the differential
dispersion of these elemencs within the genome of closely related
bacterial strains can be used to discriminate these scrains (e.g.
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LJ'14iS. F.J. , =~ilbriyht, O.W. . Stephens, C.T. and DE Brui7r.. , .,, .
1994 . Appl. Environ. t4icro. 60, 2286-2295.). Rep-PCR utilizes
oligonucleocide primers :omplemencary to these repecitive
sequences to amplify the variably sized DNA fragments lying
between them. The resulcing products are separated by
eleccrophoresis to establish the DtdA Lingerprint" for each
strain.
To isolace genomic DNA from our strains, cell pellets were
resuspended in TE buffer (10 mM Tris-HCI, 1 mM EDTA, pH 8.0) to a
final volume of 10 ml and 12 ml of 5 M NaC1 was then added. This
mixture was centrifuged 20 min. at 15,000 x g. The resulting
pellet was resuspended in 5.7 ml of TE and 300 ul of 10% SDS and
60 ul 20 mg/ml proteinase K (Gibco BP.L Products, Grand Island,
NY) were added. This mixture was incubated at 37 C for 1 hr,
approximately 10 mg of lysoz=yme was then added and the mixture
was incubaced for an additional 45 min. One milliliter of 5M NaC1
and 800 ul of CTAB/D1aC1 solution (10% w/v CTAB, 0.7 M NaC1) were
then added and the mixture was incubated 10 min. at 65 C, gently
agitated, then incubated and agitated for an additional 20 min.
to aid in clearing of the cellular material. An equal volume of
chloroform/isoamyl alcohol solution (24:1, v/v) was added, mixed
gently then centrifuged. Two extractions were then performed with
an equal volume of phenol /chloroform/ isoamyl alcohol (50:49:1)
Gencmic DNA was precipitated with 0.6 volume of isopropanol.
Precipitated DNA was removed with a glass rod, washed twice with
70% ethanol, dried and dissolved in 2 ml of STE (10 mM Tris-HC1
pH8.0, 10 mM NaCl, 1 mM EDTA). The DNA was then quantitated by
optical density at 260 nm. To perform rep-PCR analysis of
Photorhabdus genomic DNA the following primers were used, REP1P.-
I; 5'-IIIICGICGICATCIGGC-3' and REP2-I; 5'-ICGICTTATCIGGCCTAC-3'.
PCR was performed using the following 25ul reaction: 7.75 ul H20,
2.5 ul lOX LA buffer (PanVera Corp., Madison, WI), 16 ul dNTP mix
(2.5 mM each), 1 ul of each primer at 50 pM/ul, 1 ul DMSO, 1.5 u1
genomic DNA (concentrations ranged from 0.075-0.480 ug/ul) and
0.25 ul TaKaRa EY Taq (PanVera Corp., Madison, WI). The PCR
amplification was performed in a Perkin Elmer DNA Thermal Cycler
(Norwalk, CT) using the following conditions: 95 C/7 min. then 35
cycles of; 94 C/1 min.,44 C/1 min., 65 C/8 min., followed by 15
min. at 65 C. After cycling, the 25 ul reaction was added to 5 ul
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of 6X gel loading buffer (0.25o bromophenol blue, 40o w-r sucrose
in H20). A 15x20cm 1%-agarose gel was then run in TBE buffer
(0.09 M Tris-borate, 0.002 M EDTA) using 8 ul of each reaction.
The gel was run for approximately 16 hours at 45v. Gels were then
stained in 20 ug%'ml ethidium bromide for 1 hour and destained in
TBE buffer for approximately 3 hours. PolaroidO photographs of
i
the gels were then taken under UV illumination.
The presence or absence of bands at specific sizes for each
strain was scored from the photographs and entered as a
similarity matrix in the numerical taxonomy software program,
NTSYS-pc (Exeter Software, Setauket, NY). Controls of E. coli
strain HB101 and Xanthomonas oryzae pv. oryzae assayed at the
same time produced PCR "fingerprints" corresponding to published
reports (Versalovic, J., Koeuth, T. and Lupski, J.R. 1991.
Nucleic Acids Res. 19, 6823-6831; Vera Cruz, C.M., Halda-Alija,
L., Louws, F., Skinner, D.Z., George, M.L., Nelson, R.J., DE
Bruijn, F.J., Rice, C. and Leach, J.E. 1995. Int. Rice Res.
Notes, 20, 23-24.; Vera Cruz, C.M., Ardales, E.Y., Skinner, D.Z.,
Talag, J., Nelson, R.J., Louws, F.J., Leung, H., Mew, T.W. and
Leach, J.E. 1996. Phytopathology (in press, respectively). The
data.from Photorhabdus strains were then analyzed with a series
of programs within NTSYS-pc; SIMQUAL (Similarity for Qualitative
data) to generate a matrix of similarity coefficients (using the
Jaccard coefficient) and SAHN (Sequential, Agglomerative,
Heirarchical and Nested) clustering [using the UPGMA (Unweighted
Pair-Group Method with Arithmetic Averages) method] which groups
related strains and can be expressed as a phenogram (Figure 5).
The COPH (cophenetic values) and MXCOMP (matrix comparison)
programs were used to generate a cophenetic value matrix and
compare the correlation between this and the original matrix upon
which the clustering was based. A resulting normalized Mantel
statistic (r) was generated which is a measure of the goodness of
fit for a cluster analysis (r=0.8-0.9 represents a very good
fit). In our case r = 0.919. Therefore, our collection is
comprised of a diverse group of easily distinguishable strains
representative of the Photorhabdus genus.
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Example 13
Insecticidal Utility of Toxin(s) Produced
by Various Photorhabdus Strains
Initial "seed" cultures of the various Photorhabdus strains
were produced by inoculating 175 ml of 2% Proteose Peptone #3
(PP3) (Difco Laboratories, Detroit, MI) liquid media with a
primary variant subclone in a 500 ml tribaffled flask with a
Delong neck, covered with a Kaput. Inoculum for each seed culture
was derived from oil-overlay agar slant cultures or plate
cultures. After inoculation, these flasks were incubated for 16
hrs at 28 C on a rotary shaker at 150 rpm. These seed cultures
were then used as uniform inoculum sources for a given
fermentation of each strain. Additionally, overlaying the post-
log seed culture with sterile mineral oil, adding a sterile
magnetic stir bar for future resuspension and storing the culture
in the dark, at room temperature provided long-term preservation
of inoculum in a toxin-competent state. The production broths
were inoculated by adding 1% of the actively growing seed culture
to fresh 2% PP3 media (e.g. 1.75 ml per 175 ml fresh media).
Production of broths occurred in either 500 ml tribaffled flasks
(see above), or 2800 ml baffled, convex bottom flasks (500 ml
volume) covered by a silicon foam closure. Production flasks
were incubated for 24-48 hrs under the above mentioned
conditions. Following incubation, the broths were dispensed into
sterile 1 L polyethylene bottles, spun at 2600 x g for 1 hr at
10 C and decanted from the cell and debris pellet. The liquid
broth was then vacuum filtered through Whatman GF/D (2.7 uM
retention) and GF/B (1.0 i1M retention) glass filters to remove
debris. Further broth clarification was achieved with a
tangential flow microfiltration device (Pall Filtron,
Northborough, MA) using a 0.5 pM open-channel filter. When
necessary, additional clarification could be obtained by chilling
the broth (to 4 C) and centrifuging for several hours at 2600 x
g. Following these procedures, the broth was filter sterilized
using a 0.2 uM nitrocellulose membrane filter. Sterile broths
were then used directly for biological assay, biochemical
analysis or concentrated (up to 15-fold) using a 10,000 tnJ cut-
off, M12 ultra-filtration device (Amicon, Beverly MA) or
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centrifugal concentrators (Millipore, Bedford, MA and Pall
Filtron, Northborougii, MA) with a 10,000 MW pore size. In the
case of centrifugal concentrators, the broth was spun at 2000 x g
for approximately 2 hr. The 10,000 MW permeate was added to the '
corresponding retentate to achieve the desired concentration of
components greater than 10,000 MW. Heat inactivation of
processed broth samples was acheived by heating the samples at 100 C in a sand-
filled heat block for 10 minutes.
The broth(s) and toxin complex(es) from different
Photorhabdus strains are useful for reducing populations of
insects and were used in a method of inhibiting an insect
population which comprises applying to a locus of the insect an
effective insect inactivating amount of the active described. A
demonstration of the breadth of insecticidal activity observed
from broths of a selected group of Photorhabdus strains fermented
as described above is shown in Table 19. It is possible that
additional insecticidal activities could be detected with these
strains through increased concentration of the broth or by
employing different fermentation methods. Consistent with the
activity being associated with a protein, the insecticidal
activity of all strains tested was heat labile (see above).
Culture broth(s) from diverse Photorhabdus strains show
differential insecticidal activity (mortality and/or growth
inhibition, reduced adult emergence) against a number of insects.
More specifically, the activity is seen against corn rootworm
larvae and boll weevil larvae which are members of the insect
order Coleoptera. Other members of the Coleoptera include
wireworms, pollen beetles, flea beetles, seed beetles and
Colorado potato beetle. Activity is also observed against aster
leafhopper and corn plant hopper, which are members of the order
Homoptera. Other members of the Homoptera include planthoppers,
pear psylla, apple sucker, scale insects, whiteflies, spittle
bugs as well as numerous host specific aphid species. The broths
and purified toxin complex(es) are also active against tobacco
budworm, tobacco hornworm and European corn borer which are
members of the order Lepidoptera. Other typical members of this
order are beet armyworm, cabbage looper, black cutworm, corn
earworm, codling moth, clothes moth, Indian mealmoth, leaf
rollers, cabbage worm, cotton bollworm, bagworm, Eastern tent
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caterpillar, sod webworm and fall armyworm. Activit_v is also
seen against fruitfly and mosquito larvae which are members of
the order Diptera. Other members of the order Diptera are, pea
midge, carrot fly, cabbage root fly, turnip root fly, onion fly,
crane fly and house fly and various mosquito species. Activity
with broth(s) and toxin complex(es) is also seen against two-
spotted spider mite which is a member of the order Acarina which
includes strawberry spider mites, broad mites, citrus red mite,
European red mite, pear rust mite and tomato russet mite.
Activity against corn rootworm larvae was tested as follows.
Photorhabdus culture broth(s) (0-15 fold concentrated, filter
sterilized), 2% Proteose Peptone #3, purified toxin complex(es)
[0.23 mg/mll or 10 mM sodium phosphate buffer , pH 7.0 were
applied directly to the surface (about 1.5 cm2) of artificial
diet (Rose, R. I. and McCabe, J. M. (1973). J. Econ. Entomol. 66,
(398-400) in 40 ul aliquots. Toxin complex was diluted in 10 mM
sodium phosphate buffer, pH 7Ø The diet plates were allowed to
air-dry in a sterile flow-hood and the wells were infested with
single, neonate Diabrotica undecimpunctata howardi (Southern corn
rootworm, SCR) hatched from surface sterilized eggs. The plates
were sealed, placed in a humidified growth chamber and maintained
at 27 C for the appropriate period (3-5 days). Mortality and
larval weight determinations were then scored. Generally, 16
insects per treatment were used in all studies. Control
mortality was generally less than 5%.
Activity against boll weevil (Anthomonas grandis) was tested
as follows. Concentrated (1-10 fold) Photorhabdus broths,
control medium (2% Proteose Peptone #3), purified toxin
complex(es) [0.23 mg/mll or 10 mM sodium phosphate buffer, pH 7.0
were applied in 60 ul aliquots to the surface of 0.35 g of
artificial diet (Stoneville Yellow lepidopteran diet) and allowed
to dry. A single, 12-24 hr boll weevil larva was placed on the
diet, and the wells were sealed and held at 25 C, 50% RH for 5
days. Mortality and larval weights were then assessed. Control
mortality ranged between 0-13%.
Activity against mosquito larvae was tested as follows. The
assay was conducted in a 96-well microtiter plate. Each well
= contained 200 ul of aqueous solution (10-fold concentrated
Photorhabdus culture broth(s), control medium (2% Proteose
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Peptone #3), 10 mM sodium phosphate buffer, toxin complex(es) q
0.23 mg/ml or H20) and approximately 20, 1-day old larvae (Aedes
aegypti). There were 6 wells per treatment. The results were
read at 3-4 days after infestation. Control mortality was
between 0-20%.
Activity against fruitflies was tested as follows.
Purchased Drosophila melanogaster medium was prepared using 50% dry medium and
a 50% liquid of either water, control medium (2%
Proteose Peptone #3), 10-fold concentrated Photorhabdus culture
broth(s), purified toxin complex(es) [0.23 mg/ml] or 10 mM sodium
phosphate buffer , pH 7Ø This was accomplished by placing 4.0
ml of dry medium in each of 3 rearing vials per treatment and
adding 4.0 ml of the appropriate liquid. Ten late instar
Drosophila melanogaster maggots were then added to each 25 ml
vial. The vials were held on a laboratory bench, at room
temperature, under fluorescent ceiling lights. Pupal or adult
counts were made after 15 days of exposure. Adult emergence as
compared to water and control medium (0-16% reduction).
Activity against aster leafhopper adults (Macrosteles
severirni) and corn planthopper nymphs (Peregrinus maidis) was
tested with an ingestion assay designed to allow ingestion of the
active without other external contact. The reservoir for the
active/"food" solution is made by making 2 holes in the center of
the bottom portion of a 35X10 mm Petri dish. A 2 inch Parafilm
M square is placed across the top of the dish and secured with
an "0" ring. A 1 oz. plastic cup is then infested with
approximately 7 hoppers and the reservoir is placed on top of the
cup, Parafilm down. The test solution is then added to the
reservoir through the holes. In tests using 10-fold concentrated
Photorhabdus culture broth(s), the broth and control medium (2%
Proteose Peptone #3) were dialyzed against 10 mM sodium phosphate
buffer, pH 7.0 and sucrose (to 5%) was added to the resulting
solution to reduce control mortality. Purified toxin complex(es)
[0.23 mg/ml] or 10 mM sodium phosphate buffer, pH 7.0 was also
tested. Mortality is reported at day 3. The assay was held in
an incubator at 28 C, 70% RH with a 16/8 photoperiod. The assays
were graded for mortality at 72 hours. Control mortality was
less than 6%.
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Activity against lepidopteran larvae was tested as follo=Ns.
Concentrated (10-fold) Photorhabdus culture broth(s), control
medium (2% Proteose Peptone #3), purified toxin complex(es) [0.23
mgimi] or 10 mM sodium phosphate buffer, pH 7.0 were applied
directly to the surface (-1.5 cm2) of standard artificial
lepidopteran diet (Stoneville Yellow diet) in 40 tll aliquots.
The diet plates were allowed to air-dry in a sterile flow-hood
and each well was infested with a single, neonate larva. European
corn borer (Ostrinia nubilalis) and tobacco hornworm (Manduca
sexta) eggs were obtained from commercial sources and hatched in-
house, whereas tobacco budworm (Heliothis virescens) larvae were
supplied internally. Following infestation with larvae, the diet
plates were sealed, placed in a humidified growth chamber and
maintained in the dark at 270C for the appropriate period.
P4ortality and weight determinations were scored at day 5.
Generally, 16 insects per treatment were used in all studies.
Control mortality generally ranged from 4-12.5% for control
medium and was less than 10% for phosphate buffer.
Activity against two-spotted spider mite (Tetranychus
urticae) was determined as follows. Young squash plants were
trimmed to a single cotyledon and sprayed to run-off with 10-fold
concentrated broth(s), control medium (2% Proteose Peptone #3),
purified toxin complex(es) [0.23 mg/ml] or 10 mM sodium phosphate
buffer, pH 7Ø After drying, the plants were infested with a
mixed population of spider mites and held at lab temperature and
humidity for 72 hr. Live mites were then counted to determine
levels of control.
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Table 19
Observed Insecticidal Spectrum of Broths From Different
Photorhabdus Strains
Phocorhabdus Strain Sensitive* Insect Species
wx-1 3**, 4, 5, 6, 7, 8
WX-2 2, 4 =
WX-3 1, 4
WX-4 1, 4
WX-5 4
WX-6 4
WX-7 3, 4, 5, 6, 7, 8
WX-8 1, 2, 4
WX-9 1, 2, 4
wx-1o 4
WX-11 1, 2, 4
WX-12 2, 4, 5, 6, 7, 8
WX-14 1, 2, 4
WX-15 1, 2, 4
W30 3, 4, 5, 8
NC=1 1, 2, 3, 4, 5, 6, 7, 8, 9
WIR 2, 3, 5, 6, 7, 8
HP88 1, 3, 4, 5, 7, 8
Hb 3, 4, 5, 7, 8
Hm 1, 2, 3, 4, 5, 7, 8
H9 1, 2, 3, 4, 5, 6, 7, 8
W-14 1, 2, 3, 4, 5, 6, 7, 8, 10
ATCC 43948 4
ATCC 43949 4
ATCC 43950 4
ATCC 43951 4
ATCC 43952 4
>_ 25% mortality and/or growth inhibition vs. control
** = 1; Tobacco budworm, 2; European corn borer, 3;
Tobacco hornworm, 4; Southern corn rootworm, 5;
Boll weevil, 6; Mosquito, 7; Fruit Fly, 8;
Aster Leafhopper, 9; Corn planthopper, 10;
Two-spotted spider mite.
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Example 14
Non ,,1-14 Photorhabdus Strains:
Purification, Characteri?at-ion and Activity Spectrum
Purification
The protocol, as follows, is similar to that developed for
+ the purification of W-14 and was established based on purifying
those fractions having the most activity against Southern corn
root worm (SCR), as determined in bioassays (see Example 13).
Typically, 4-20 L of broth that had been filtered, as described
in Example 13, were received and concentrated using an Amicon
spiral ultra filtration cartridge Type S1Y100 attached to an
Amicon M-12 filtration device. The retentate contained native
proteins consisting of molecular sizes greater than 100 kDa,
whereas the flow through material contained native proteins less
than 100 kDa in size. The majority of the activity against SCR
was contained in the 100 kDa retentate. The retentate was then
continually diafiltered with 10 mM sodium phosphate (pH = 7.0)
until the filtrate reached an A280 < 0.100. Unless otherwise
stated, all procedures from this point were performed in buffer
as defined by 10 mM sodium phosphate (pH 7.0). The retentate was
then concentrated to a final volume of approximately 0.20 L and
filtered using a 0.45 mm NalgeneT" Filterware sterile filtration
unit. The filtered material was loaded at 7.5 ml/min onto a
Pharmacia HR16/10 column which had been packed with PerSeptive
Biosystem PorosO 50 HQ strong anion exchange matrix equilibrated
in buffer using a PerSeptive Biosystem Sprint HPLC system.
After loading, the column was washed with buffer until an A280
0.100 was achieved. Proteins were then eluted from the column at
2.5 ml/min using buffer with 0.4 M NaCl for 20 min for a total
volume of 50 ml. The column was then washed using buffer with
1.0 M NaCl at the same flow rate for an additional 20 min (final
volume = 50 ml). Proteins eluted with 0.4 M and 1.0 M NaCl were
placed in separate dialysis bags (Spectra/Por Membrane MWCO:
2,000) and allowed to dialyze overnight at 4 C in 12 L buffer.
The majority of the activity against SCR was contained in the 0.4
M fraction. The 0.4 M fraction was further purified by
application of 20 ml to a Pharmacia XK 26/100 column that had
been prepacked with Sepharose CL4B (Pharmacia) using a flow rate
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of 0.75 ml/min. Fractions were pooled based cn A280 peak profiie
and concentrated to a final volume of 0.75 ml using a Millipore
Ultrafree -15 centrifugal filter device Biomax-50K LiMWL membrane.
Protein concentrations were determined using a Biorad Protein 5 Assay Kit with
bovine gamma globulin as a standard.
Characterization
The native molecular weight of the SCR toxin complex was
determined using a Pharmacia HR 16/50 that had been prepacked
with Sepharose CL4B in buffer. The column was then calibrated
using proteins of known molecular size thereby allowing for
calculation of the toxin approximate native molecular size. As
shown in Table 20, the molecular size of the toxin complex ranged
from 777 kDa with strain Hb to 1,900 kDa with strain WX-14. The
yield of toxin complex also varied, from strain WX-12 producing
0.8 mg/L to strain Hb, which produced 7.0 mg/L.
Proteins found in the toxin complex were examined for
individual polypeptide size using SDS-PAGE analysis. Typically,
mg protein of the toxin complex from each strain was loaded
20 onto a 2-15% polyacrylamide gel (Integrated Separation Systems)
and electrophoresed at 20 mA in Biorad SDS-PAGE buffer. After
completion of electrophoresis, the gels were stained overnight in
Biorad Coomassie blue R-250 (0.2% in methanol: acetic acid:
water; 40:10:40 v/v/v). Subsequently, gels were destained in
methanol:acetic acid: water; 40:10:40 (v/v/v). The gels were
then rinsed with water for 15 min and scanned using a Molecular
Dynamics Personal Laser Densitometer . Lanes were quantitated
and molecular sizes were calculated as compared to Biorad high
molecular weight standards, which ranged from 200-45 kDa.
Sizes of the individual polypeptides comprising the SCR
toxin complex from each strain are listed in Table 21. The sizes
of the individual polypeptides ranged from 230 kDa with strain
WX-1 to a size of 16 kDa, as seen with strain WX-7. Every
strain, with the exception of strain Hb, had polypeptides
comprising the toxin complex that were in the 160-230 kDa range,
the 100-160 kDa range, and the 50-80 kDa range. These data
indicate that the toxin complex may vary in peptide composition
and components from strain to strain, however, in all cases the
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toxin attributes appears to consist of a large, oligomeric
protein complex.
Table 20
Characterization of a Toxin Complex From
Non W-14 Photorhabdus Strains
Strain Approx. Yield
Native Active
Molecular Wt.a Fraction
(mg/L)b
H9 972,000 1.8
Hb 777,000 7.0
Hm 1,400,000 1.1
HP88 813,000 2.5
NC1 1,092,000 3.3
WIR 979,000 1.0
wx-1 973,000 0.8
WX-2 951,000 2.2
WX-7 1,000,000 1.5
WX-12 898,000 0.4
WX-14 1,900,000 1.9
W-14 860,000 7.5
a Native molecular weight determined using a Pharmacia HP.
16/50 column packed with Sepharose CL4B
b Amount of toxin complex recovered from culture broth.
Activity Spectrum
As shown in Table 21, the toxin complexes purified from
strains Hm and H9 were tested for activity against a variety of
insects, with the toxin complex from strain W-14 for comparison.
The assays were performed as described in Example 13. The toxin
complex from all three strains exhibited activity against tobacco
bud worm, European corn borer, Southern corn root worm, and aster
leafhopper. Furthermore, the toxin complex from strains Hm and
W-14 also exhibited activity against two-spotted spider mite. In
addition, the toxin complex from W-14 exhibited activity against
mosquito larvae. These data indicate that the toxin complex,
while having similarities in activities between certain orders of
insects, can also exhibit differential activities against other
orders of insects.
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Table 21
The Approximate Sizes (in kDa) of Peptides in a Purified
Toxin Complex From Non W-14 Photorhabdus
H9 Hb Hm HP NC-1 WIR WX-1 WX-2 WX-7 WX-12 WX-14 W-11
88
180 150 170 170 180 170 230 200 200 180 210 190
170 140 140 160 170 160 190 170 180 160 180 180
160 139 100 140 140 120 170 150 110 140 160 170
140 130 81 130 110 110 160 120 87 139 120 160
120 120 72 129 44 89 110 110 75 130 110 150
98 100 68 110 16 79 98 82 43 110 100 130
87 98 49 100 74 76 64 33 92 95 120
84 88 46 86 62 58 37 28 87 80 111-)
79 81 30 81 51 53 30 26 80 69 93
72 75 22 77 40 41 23 73 49 90
68 69 20 73 39 35 22 59 41 77
60 60 19 60 37 31 21 56 33 69
57 57 58 33 28 19 51 65
52 54 45 30 24 18 37 63
46 49 39 28 22 16 33 60
40 44 35 27 32 51
37 39 25 26 46
37 23 40
35 39
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Table 22
Observed Insecticidal Spectrum of a Purified Toxin Complex from
Photorhabdus Strains
Phocorhabdus Strain Sensitive* Insect Species
Hm Toxin Complex 1**, 2, 3, 5, 6, 7, 8
H9 Toxin Complex 1, 2, 3, 6, 7, 8
w-14 Toxin Complex 1, 2, 3, 4, 5, 6, 7, 8
*=> 25% mortality or growth inhibition
*=> 25% mortality or growth inhibition
** = 1; Tobacco bud worm, 2; European corn borer, 3; Southern
corn root worm, 4; Mosquito, 5; Two-spotted spider mite,
6; Aster Leafhopper, 7; Fruit Fly, 8; Boll Weevil
Example 15
Sub-Fractionation of Photorhabdus Protein Toxin Complex
The Photorhabdus protein toxin complex was isolated as
described in Example 14. Next, about 10 mg toxin was applied to
a MonoQ 5/5 column equilibrated with 20 mM Tris-HC1, pH 7.0 at a
flow rate of lml/min. The column was washed with 20 cnM Tris-HC1,
pH 7.0 until the optical density at 280 nm returned to baseline
absorbance. The proteins bound to the column were eluted with a
linear gradient of 0 to 1.0 M NaCl in 20 mM Tris-HC1, pH 7.0 at 1
ml/min for 30 min. One ml fractions were collected and subjected
to Southern corn rootworm (SCR) bioassay (see Example 13). Peaks
of activity were determined by a series of dilutions of each
fraction in SCR bioassays. Two activity peaks against SCR were
observed and were named A (eluted at about 0.2-0.3 M NaCl) and B
(eluted at 0.3-0.4 M NaCl). Activity peaks A and B were pooled
separately and both peaks were further purified using a 3-step
procedure described below.
Solid (NH4)2SO4 was added to the above protein fraction to a
final concentration of 1.7 M. Proteins were then applied to a
phenyl-Superose 5/5 column equilibrated with 1.7 M(NH4)2SO4 in
50 mM potassium phosphate buffer, pH 7 at 1 ml/min. Proteins
bound to the column were eluted with a linear gradient of 1.7 M
(NH4)2SO4, 0% ethylene glycol, 50 mM potassium phosphate, pH 7.0
to 25% ethylene glycol, 25 mM potassium phosphate, pH 7.0 (no
(NH4)2SO4) at 0.5 ml/min. Fractions were dialyzed overnight
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against 10 mM sodium phosphate buffer, pH 7Ø Acti-:ities in
each fraction against SCR were determined by bioassay.
The fractions with the highest activity were pooled and
applied to a MonoQ 5/5 column which was equilibrated with 20 mM
Tris-HC1, pH 7.0 at 1 ml/min. The proteins bound to the column
were eluted at 1 ml/min by a linear gradient of 0 to 1P=I NaCl in
20 mM Tris-HC1, pH 7Ø
For the final step of purification, the most active
fractions above (determined by SCR bioassay) were pooled and
subjected to a second phenyl-Superose 5/5/ column. Solid
(NH4)2SO4 was added to a final concentration of 1.7 M. The
solution was then loaded onto the column equilibrated with 1.7 M
(NH4)2SO4 in 50 mM potassium phosphate buffer, pH 7 at lml/min.
Proteins bound to the column were eluted with a linear gradient
of 1.7 M(NH4)2SO4, 50 mM potassium phosphate, pH 7.0 to 10 mM
potassium phosphate, pH 7.0 at 0.5 ml/min. Fractions were
dialyzed overnight against 10 mM sodium phosphate buffer, pH 7Ø
Activities in each fraction against SCR were determined by
bioassay.
The final purified protein by the above 3-step procedure
from peak A was named toxin A and the final purified protein from
peak B was named toxin B.
Characterization and Amino Acid Sequencing of Toxin A and Toxin B
In SDS-PAGE, both toxin A and toxin B contained two major (>
90% of total Commassie stained protein) peptides: 192 kDa (named
Al and B1, respectively) and 58 kDa (named A2 and B2,
respectively). Both toxin A and toxin B revealed only one major
band in native PAGE, indicating Al and A2 were subunits of one
protein complex, and El and B2 were subunits of one protein
complex. Further, the native molecular weight of both toxin A
and toxin B were determined to be 860 kDa by gel filtration
chromatography. The relative molar concentrations of Al to A2
was judged to be a 1 to 1 equivalence as determined by
densiometric analysis of SDS-PAGE gels. Similarly, Bl and B2
peptides were present at the same molar concentration.
Toxin A and toxin B were electrophoresed in 10% SDS-PAGE and
transblotted to PVDF membranes. Blots were sent for amino acid
analysis and N-terminal amino acid sequencing at Harvard
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amino sequence of B1 was determined to be identical to SEK ID
11O:1, the TcbAii region of the tcbA gene (SEQ ID NO:12, pcsition
87 to 99). A unique N-terminal sequence was obtained for peptide
B2 (SEQ ID NO:40). The 11-terminal amino acid sequence of peptide
B2 was identical to the TcbAiii region of the derived amino acid
sequence for the tcbA gene (SEQ ID NO:12, position 1935 to 1945).
Therefore, the B toxin contained predominantly two peptides,
TcbAii and TcbAiii, that were observed to be derived from the
same gene product, TcbA.
The N-terminal sequence of A2 (SEQ ID NO:41) was unique in
comparison to the TcbAiii peptide and other peptides. The A2
peptide was denoted TcdAiii (see Example 17). SEQ ID NO:6 was
determined to be a mixture of amino acid sequences SEQ ID NO:40
and 41.
Peptides Al and A2 were further subjected to internal amino
acid sequencing. For internal amino acid sequencing, 10 ug of
toxin A was electrophoresized in 10% SDS-PAGE and transblotted to
PVDF membrane. After the blot was stained with amido black,
peptides Al and A2, denoted TcdAii and TcdAiii, respectively,
were excised from the blot and sent to Harvard MicroChem and
Cambridge ProChem. Peptides were subjected to trypsin digestion
followed by HPLC chromatography to separate individual peptides.
N-terminal amino acid analysis was performed on selected tryptic
peptide fragments. Two internal amino acid sequences of peptide
Al (TcdAii-PK71, SEQ ID NO:38 and TcdAii-PK44, SEQ ID NO:39) were
found to have significant homologies with deduced amino acid
sequences of the TcbAii region of the tcbA gene (SEQ ID 11O:12).
Similarly, the N-terminal sequence (SEQ ID NO:41) and two
internal sequences of peptides A2 (TcdAiii-PK57, SEQ ID NO:42 and
TcdAiii-PK20, SEQ ID NO.43) also showed significant homology with
deduced amino acid sequences of TcbAiii region of the tcbA gene
(SEQ ID NO:12).
In summary of above results, the toxin complex has at least
two active protein toxin complexes against SCR; toxin A and toxin
B. Toxin A and toxin B are similar in their native and subunits
molecular weight, however, their peptide compositions are
different. Toxin A contained peptides TcdAii and TcdAiii as the
major peptides and the toxin B contains TcbAii and TcbAiii as the
major peptides.
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Example 16
Cleavage and Activation of TcbA Peptide
In the toxin B complex, peptide TcbAii and TcbAiii originate
from the single gene product TcbA (Example 15). The processing of
TcbA peptide to TcbAii and TcbAiii is presumably by the action of
Photorhabdus protease(s), and most likely, the metalloproteases
described in Example 10. In some cases, it was noted that when
Photorhabdus W-14 broth was processed, TcbA peptide was present in
toxin B complex as a major component, in addition to peptides
TcbAii and TcbAiii= Identical procedures, described for the
purification of toxin B complex (Example 15), were used to enrich
peptide TcbA from toxin complex fraction of W-14 broth. The final
purified material was analyzed in a 4-20% gradient SDS-PAGE and
major peptides were quantified by densitometry. It was determined
that TcbA, TcbAii and TcbAiii comprised 58%, 36%, and 6%,
respectively, of total protein. The identities of these peptides
were confirmed by their respective molecular sizes in SDS-PAGE and
Western blot analysis using monospecific antibodies. The native
molecular weight of this fraction was determined to be 860 kDa.
The cleavage of TcbA was evaluated by treating the above
purified material with purified 38 kDa and 58 kDa W-14
.25 Photorhabdus metalloproteases (Example 10), and Trypsin as a
control enzyme (Sigma, MO). The standard reaction consisted 17.5
ug the above purified fraction, 1.5 unit protease, and 0.1 M Tris
buffer, pH 8.0 in a total volume of 100 ul. For the control
reaction, protease was omitted. The reaction mixtures were
incubated at 37 C for 90 min. At the end of the reaction, 20 ul
was taken and boiled with SDS-PAGE sample buffer immediately for
electrophoresis analysis in a 4-20% gradient SDS-PAGE. It was
determined from SDS-PAGE that in both 38 kDa and 58 kDa protease
treatments, the amount of peptides TcbAii and TcbAiii increased
about 3-fold while the amount of TcbA peptide decreased
proportionally (Table 23). The relative reduction and
augmentation of selected peptides was confirmed by Western blot
analyses. Furthermore, gel filtration of the cleaved material
revealed that the native molecular size of the complex remained
the same. Upon trypsin treatment, peptides TcbA and TcbAii were
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nonspecifically digested into small peptides. This indicated that
38 kDa and 58 kDa Photorhabdus proteases can specifically process
peptide TcbA into peptides TcbAii and TcbAiii= Protease treated
and untreated control of the remaining 80 jil reaction mixture were
serial diluted with 10 mP4 sodium phosphate buffer, pH 7.0 and
analyzed by SCR bioassay. By comparing activity in several
dilution, it was determined that the 38 kDa protease treatment
increased SCR insecticidal activity-approximately 3 to 4 fold.
The growth inhibition of remaining insects in the protease
treatment was also more severe than control (Table 23).
Table 23
Conversion and activation of peptide TcbA into peptides TcbAii and
TcbAiii by protease treatment.
Control 38 kDa protease treatment
SO (% of totai protein) 58 18
Sl (% of total protein) 36 64
S9 (% of total protein) 6 18
LD50 (ug protein) 2.1 0.52
SCR Weight (mg/insect)* 0.2 0.1
*: an indication of growth inhibition by measuring the average
weight of live insect after 5 days on diet in the assay.
Example 17
Screening of the library for a gene encoding the TcdAii Peptide
The cloning and characterization of a gene encoding the
TcdAii peptide, described as SEQ ID NO:17 (internal peptide
TcdAii-PT111 N-terminal sequence) and SEQ ID NO:18 (internal
peptide TcdAii-PT79 N-terminal sequence) was completed. Two
pools of degenerate oligonucleotides, designed to encode the
amino acid sequences of SEQ ID NO:17 (Table 24) and SEQ ID NO:18
(Table 25), and the reverse complements of those sequences, were
synthesized as described in Example B. The DNA sequence of the
oligonucleotides is given below:
-102-
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WO 97/17432 PCTIUS96/18003
~ M
co 0
~ .
U
c- +~0 E rn ch M M rn c*~ '
> Mz r~ ~ 1-4 ri 04
U
E 1- U E"
ÃLE 1 o
H U U U U U U
Z 1D E E E 4 H E Z '4 t4 U U U
9 C9 ~ s ~ A =~-~ ~ ~
a OI
vwj ~~ u u Ecn rn~ x x E E E U
E E C7 0
a E. 4 > c~ts 0 0
'~ a 0
>s z>+>+ E-~E U~
N E N 'b a) it-4 0 u u
U' C7 U' R: OG 11 II
0
(1) y '+
~ -~
E C9 .Q ,. i ~ m E-+ E+ 4
In G
E E"' u >+>+ >+U rort1
m ~ U U
trl c~ 0 ~ ~ U
+ v~ E1 Q a a a~ s4
u- 0
O 11 U
E E' 0 {a z >+ 7+ U U
E
ro E ~~ ~4
$4 0 x
(1) N c~ >+EE 'W
cEi E Ea-E Q N E
0
~ N E E EE 4 C7 N 'O Ia
Q E E E~ E~ a ri td E E~-, 0 00 V O
> C7 C7 C7 >4 >+ pq C~
E E a n
\7 C\7 N r+ E E E~ E+ E U
uu ~ ~a d QQ 4
E-1
U U U U U U U U U >4, '14, 0
d C9 C7 C9 ~~ U a) ~i C7
r+ -- - - - - a
r+ rC ~n in ~n ~n - - - - -
en in in tn Ln C U
V rt
r1 (7 U N~ 01 i e1 f'''1
n
~~ m ~o ut vai uai E+ A+b q a- o- a nG 0
0 ~
i i rn rn rai ~ b ~ a, a'
a q ~of a r
;t' ~ z 4 4 4 a1 ou a a a a 4 a U a a a a
-103-
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Polymerase Chain Reactions (PCR) were performed essentially
as described in Example 8, using as forward primers P2.3.5.CB or
P2.3.5, and as reverse primers P2.79.R.1 or P2.79R.CB, in all
forward/reverse combinations, using Photorhabdus W-14 genomic DtIA
as template. In another set of reactions, primers P2.79.2 or
P2.79.3 were used as forward primers, and P2.3.5R, P2.3.5RI, and
P2.3R.CB were used as reverse primers in all forward/reverse
combinations. Only in the reactions containing P2.3.6.CB as the
forward primers combined with P2.79.R.1 or P2.79R.CB as the
reverse primers was a non-artifactual amplified product seen, of
estimated size (mobility on agarose gels) of 2500 base pairs.
The order of the primers used to obtain this amplification
product indicates that the peptide fragment TcdAii-PT111 lies
amino-proximal to the peptide fragment TcdAii-PT79.
The 2500 bp PCR products were ligated to the plasmid vector
pCRTMII (Invitrogen, San Diego, CA) according to the supplier's
instructions, and the DNA sequences across the ends of the insert
fragments of two isolates (HS24 and HS27) were determined using
the supplier's recommended primers and the sequencing methods
described previously. The sequence of both isolates was the
same. New primers were synthesized based on the determined
sequence, and used to prime additional sequencing reactions to
obtain a total of 2557 bases of the insert [SEQ ID NO:361.
Translation of the partial peptide encoded by SEQ ID No: 36
yields the 845 amino acid sequence disclosed as SEQ ID NO:37.
Protein homology analysis of this portion of the TcdAii peptide
fragment reveals substantial amino acid homology (68% similarity;
53% identity) to residues 542 to 1390 of protein TcbA [SEQ ID
NO:12]. It is therefore apparent that the gene represented in
part by SEQ ID NO:36 produces a protein of similar, but not
identical, amino acid sequence as the TcbA protein, and which
likely has similar, but not identical biological activity as the
TcbA protein.
In yet another instance, a gene encoding the peptides
TcdAii-PK44 and the TcdAiii 58 kDa N-terminal peptide, described
as SEQ ID NO:9 (internal peptide TcdAii-PK44 sequence), and SEQ ID
NO:41(TcdAiii 58 kDa N-terminal peptide sequence) was isolated.
Two pools of degenerate oligonucleotides, designed to encode the
amino acid sequences described as SEQ ID NO:39 (Table 27) and SEQ
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ID NO:41 !Table 26), and the reverse complements ot those
sequences, were synthesized as described in Example 8, and their
DNA sequences.
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MM
pj ~ G1 M M M M
ft HE~ UE+U
z 2 a uau
a H (1.' H H
A A ao rl U 0 U 0
c~uc~
a a
W W
En
H u ?4 H H
FC7+ W H Q Q a
' ~EE
rts
E+ N C! ?+
~ U
E-4
0 0 -4 nHRC aaQ
=~ r-
0 0
ro
M>tE 7 CE7t E &4
tn
tr- tri
Oa a HH
cv N U U U U
~n W U C7 U C7
>+ H H H
U1 Lf1 lf1 ifl
H N N
= . .
O 0
_ ~ ,~ t = = ~C ~ 4
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Polymerase Chain Reactions (PCR) were performed essentially
as described in Example 8, using as forward primers A1.44.1 or
A1.44.2, and reverse primers A2.3R or A2.4R, in all
forward/reverse combinations, using Photorhabdus W-14 genomic DNA
as template. In another set of reactions, primers A2.1 or A2.2
were used as forward primers, and A1.44.1R, and A1.44.2R were
used as reverse primers in all forward/reverse combinations. Only in the
reactions containing A1.44.1 or A1.44.2 as the
forward primers combined with A2.3R as the reverse primer was a
non-artifactual amplified product seen, of estimated size
(mobility on agarose gels) of 1400 base pairs. The order of the
primers used to obtain this amplification product indicates that
the peptide fragment TcdAii-PK44 lies amino-proximal to the 58
kDa peptide fragment of TcdAiii-
The 1400 bp PCR products were ligated to the plasmid vector
pCRT"II according to the supplier's instructions. The DNA
sequences across the ends of the insert fragments of four
isolates were determined using primers similar in sequence to the
supplier's recommended primers and using sequencing methods
described previously. The nucleic acid sequence of all isolates
differed as expected in the regions corresponding to the
degenerate primer sequences, but the amino acid sequences deduced
from these data were the same as the actual amino acid sequences
for the peptides determined previously, (SEQ ID NOS:41 and 39).
Screening of the W-14 genomic cosmid library as described in
Example 8 with a radiolabeled probe comprised of the DNA
prepared above (SEQ ID NO:36) identified five hybridizing cosmid
isolates, namely 17D9, 20B10, 21D2, 27B10, and 26D1. These
cosmids were distinct from those previously identified with
probes corresponding to the genes described as SEQ ID NO:11 or
SEQ ID NO:25. Restriction enzyme analysis and DNA blot
hybridizations identified three EcoR I fragments, of approximate
sizes 3.7, 3.7, and 1.1 kbp, that span the region comprising the
DNA of SEQ ID NO:36. Screening of the W-14 genomic cosmid
library using as probe the radiolabeled 1.4 kbp DNA fragment
prepared in this example identified the same five cosmids (17D9,
20B10, 21D2, 27B10, and 26D1). DNA blot hybridization to EcoR I-
digested cosmid DNAs also showed hybridization to the same subset -107-
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of EcoR I fragments as seen with the 2.5 kbp TcdAii gene probe,
indicating that both fragments are encoded on the genomic DNA.
DNA sequence determination of the cloned EcoR I fragments
revealed an uninterrupted reading frame of 7551 base pairs (SEQ
ID NO:46), encoding a 282.9 kDa protein of 2516 amino acids (SEQ
ID NO:47). Analysis of the amino acid sequence of this protein
revealed all expected internal fraaments of peptides TcdAii(SEQ
ID NOS:17, 18, 37, 38 and 39) and the TcdAiii peptide N-terminus
(SEQ ID NO:41) and all TcdAiii internal peptides (SEQ ID NOS:42
and 43). The peptides isolated and identified as TcdAii and
TcdAiii are each products of the open reading frame, denoted
tcdA, disclosed as SEQ ID NO:46. Further, SEQ ID NO:47 shows,
starting at position 89, the sequence disclosed as SEQ ID NO:13,
which is the N-terminal sequence of a peptide of size
approximately 201 kDa, indicating that the initial protein
produced from SEQ ID No: 46 is processed in a manner similar to
that previously disclosed for SEQ ID NO:12. In addition, the
protein is further cleaved to generate a product of size 209.2
kDa, encoded by SEQ ID NO:48 and disclosed as SEQ ID NO:49
(TcdAii peptide), and a product of size 63.6 kDa, encoded by SEQ
ID NO:50 and disclosed as SEQ ID NO:51 (TcdAiii peptide). Thus,
it is thought that the insecticidal activity identified as toxin
A (Example 15) derived from the products of SEQ ID NO:46, as
exemplified by the full-length protein of 282.9 kDa disclosed as
SEQ ID N0:47, is processed to produce the peptides disclosed as
SEQ ID NOS:49 and 51. It is thought that the insecticidal
activity identified as toxin B (Example 15) derives from the
products of SEQ ID NO:11, as exemplified by the 280.6 kDa protein
disclosed as SEQ ID NO:12. This protein is proteolytically
processed to yield the 207.6 kDa peptide disclosed as SEQ ID
NO:53, which is encoded by SEQ ID NO:52, and the 62.9 kDa peptide
having N-terminal sequence disclosed as SEQ ID NO:40, and further
disclosed as SEQ ID NO:55, which is encoded by SEQ ID NO:54.
Amino acid sequence comparisons between the proteins
disclosed as SEQ ID NO:12 and SEQ ID NO:47 reveal that they have
69% similarity and 54% identity. This high degree of
evolutionary relationship is not uniform throughout the entire
amino acid sequence of these peptides, but is higher towards the
carboxy-terminal end of the proteins, since the peptides
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disclosed as SEQ ID NO:51 (derived from SEQ ID r1O:47) and SEQ 1D
NO:55 (derived from SEQ ID N0:12) have 76% similarity and 64%
identity.
Example 18
Control of European Cornborer-Induced Leaf Damage on Maize Plants
by Spray Application of Photorhabdus (Strain W-14) Broth
The ability of Photorhabdus toxin(s) to reduce plant damage
caused by insect larvae was demonstrated by measuring leaf damage
caused by European corn borer (Ostrinia nubilalis) infested onto
maize plants treated with Photorhabdus broth. Fermentation broth
from Photorhabdus strain W-14 was produced and concentrated
approximately 10-fold using ultrafiltration (10,000 MW pore-size)
as described in Example 13. The resulting concentrated broth was
then filter sterilized using 0.2 micron nitrocellulose membrane
filters. A similarly prepared sample of uninoculated 2% proteose
peptone #3 was used for control purposes. Maize plants (a
DowElanco proprietary inbred line) were grown from seed to
vegetative stage 7 or 8 in pots containing a soilless mixture in
a greenhouse (27 C day; 22 C night, about 50%RH, 14 hr day-
length, watered/fertilized as needed). The test plants were
arranged in a randomized complete block design (3 reps/treatment,
6 plants/treatment) in a greenhouse with temperature about 22 C
day; 18 C night, no artificial light and with partial shading,
about 50%RH and watered/fertilized as needed. Treatments
(uninoculated media and concentrated Photorhabdus broth) were
applied with a syringe sprayer, 2.0 mis applied from directly
(about 6 inches) over the whorl and 2.0 additional mis applied in
a circular motion from approximately one foot above the whorl.
In addition, one group of plants received no treatment. After
the treatments had dried (approximately 30 minutes), twelve
neonate European corn borer larvae (eggs obtained from commercial
sources and hatched in-house) were applied directly to the whorl.
After one week, the plants were scored for damage to the leaves using a
modified Guthrie Scale (Koziel, M. G., Beland, G. L.,
Bowman, C., Carozzi, N. B., Crenshaw, R., Crossland, L., Dawson, J., Desai,
N., Hill, M., Kadwell, S., Launis, K., Lewis, K.,
Maddox, D., McPherson, K., Meghji, M. Z., Merlin, E., Rhodes, R.,
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Warren, G. W., Wright, M. and EVola, S. V. 1993).
Bio/Technology, 11, 194-195.) and the scores were compared
statistically [T-test (LSD) p<0.05 and Tukey's Studentized Range
(HSD) Test p<0.1]. The results are shown in Table 28. For
reference, a score of 1 represents no damage, a score of 2
represents fine "window pane" damage on the unfurled leaf with no
pinhole penetration and a score of 5 represents leaf penetration
with elongated lesions and/or mid rib feeding evident on more
than three leaves (lesions < 1 inch). These data indicate that
broth or other protein containing fractions may confer protection
against specific insect pests when delivered in a sprayable
formulation or when the gene or derivative thereof, encoding the
protein or part thereof, is delivered via a transgenic plant or
microbe.
Table 28
Effect of Photorhabdus Culture Broth on
European Corn Borer-Induced Leaf Damage on Maize
Treatment Average Guthrie Score
No Treatment 5.02a
Uninoculated medium 5.15a
Photorhabdus Broth 2.24b
Means with different letters are statistically different
(p<0.05 or p<0.1).
Example 19
Genetic Engineering of Genes for Expression in E. coli
summary of constructions
A series of plasmids were constructed to express the tcbA
gene of Photorhabdus W-14 in Escherichia coli. A list of the
plasmids is shown in Table 29. A brief description of each
construction follows as well as a summary of the E. coli
expression data obtained.
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Table 29
Expression plasmids for the tcbA gene.
Plasmid Gene Vector/Selection Compartment
pDAB634 tcbA pBC/Chl Intracellular
pAcGP67B/ tcbA tcbA pAcGP67B/Amp Baculovirus,
secreted
pDAB635 tcbA pET27b/Kan Periplasm
pET15-tcbA tcbA pET15-tcbA Intracelluiar
Abbreviations: Kan=kanamycin, Chl=chloramphenicol, Amp=ampicillin
Construction of pDAB634
In Example 9, a large EcoR I fragment which hybridizes to
the TcbAii probe is described. This fragment was subcloned into
pBC (Stratagene, La Jolla CA). Sequence analysis indicates that
this fragment is 8816 base pairs. The fragment encodes the tcbA
gene with the initiating ATG at position 571 and the terminating
TAA at position 8086. The fragment therefore carries 570 base
pairs of Photorhabdus DNA upstream of the ATG and 730 base pairs
downstream of the TAA.
Construction of Plasmid pAcGP67B/tcbA
The tcbA gene was PCR amplified using the following primers;
5' primer (S1Ac51) 5' TTT AAA CCA TGG GAA ACT CAT TAT CAA GCA CTA
TC 3' and 31 primer (S1Ac31) 5' TTT AAA GCG GCC GCT TAA CGG ATG
GTA TAA CGA ATA TG 3'. PCR was performed using a TaKaRa LA PCR
kit from PanVera (Madison, Wisconsin) in the following reaction:
57.5 ml water, 10 ml lOX LA buffer, 16 ml dNTPs (2.5 mM each
stock solution), 20 ml each primer at 10 pmoles/ml, 300 ng of the
plasmid pDAB634 containing the W-14 tcbA gene and one ml of
TaKaRa LA Taq polymerase. The cycling conditions were 98 C/20
sec, 68 C/5 min, 72 C/10 min for 30 cycles. A PCR product of the
expected about 7526bp was isolated in a 0.8% agarose gel in TBE
(100 mM Tris, 90 mM boric acid, 1 mM EDTA) buffer and purified
using a Qiaex II kit from Qiagen (Chatsworth, California). The
purified tcbA gene was digested with Nco I and Not I and ligated
into the baculovirus transfer vector pAcGP67B (PharMingen (San Diego,
California)) and transformed into DH5a E. coli. The tcbA
gene was then cut from pAcGP67B and transferred to pET27b to create plasmid
pDAB635. A missense mutation in the tcbA gene was
repaired in pDAB635.
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The repaired tcbA gene contains two changes from the
sequence shown in Sequence ID NO:11; an A>G at 212 changing an
asparagine 71 to serine 71 and a G.-A at 229 changing an alanine
77 to threonine 77. These changes are both upstream of the
proposed TcbAii N-term3.nus.
Construction of pET15-tcbA
The tcbA coding region of pDAB635 was transferred to vector
pET15b. This was accomplished using shotgun ligations, the DNAs
were cut with restriction enzymes Nco I and Xho I. The resulting
recombinant is called pET15-tcbA.
Expression of TcbA in E. coli from plasmid pET15-tcbA
Expression of tcbA in E. co1.i was obtained by modification
of the methods previously described by Studier et al. (Studier,
F.W., Rosenberg, A., Dunn, J., and Dubendorff, J., (1990) Use of
T7 RNA polymerase to direct expression of cloned genes. Methods
Enzymol., 185: 60-89.). Competent E. coli cells strain BL21(DE3)
were transformed with plasmid pET15-tcbA and plated on LB agar
containing 100 g/ml ampicillin and 40 mM glucose. The
transformed cells were plated to a density of several hundred
isolated colonies/plate. Following overnight incubation at 37 C
the cells were scraped from the plates and suspended in LB broth
containing 100 g /ml ampicillin. Typical culture volumes were
from 200-500 ml. At time zero, culture densities (OD600) were
from 0.05-0.15 depending on the experiment. Cultures were shaken
at one of three temperatures (22 C, 30 C or 37 C) until a density
of 0.15-0.5 was obtained at which time they were induced with 1
mM isopropylthio-p-galactoside (IPTG). Cultures were incubated
at the designated temperature for 4-5 hours and then were
transferred to 4 C until processing (12-72 hours).
Purification and characterization of TcbA expressed in E.co1i
from Plasmid pET15-tcbA.
E. coli cultures expressing TcbA peptides were processed as
follows. Cells were harvested by centrifugation at 17,000 x G and
the media was decanted and saved in a separate container.
The media was concentrated about 8x using the M12 (Amicon,
Beverly MA) filtration system and a 100 kD molecular mass cut-off
filter. The concentrated media was loaded onto an anion exchange
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column and the bound proteins were eluted with 1.0 M tIaC1. The
1.0 M NaCl elution peak was found to cause mortality against
Southern corn rootworm (SCR) larvae Table 30). The 1.0 P=s NaCl
fraction was dialyzed against 10 mM sodium phosphate buffer pH 5 7.0,
concentrated, and subjected to gel filtration on Sepharose
CL-4B (Pharmacia, Piscataway, New Jersey). The region of the CL-
4B elution profile corresponding to calculated molecular weight
(about 900 kDa) as the native W-14 toxin complex was collected,
concentrated and bioassayed against larvae. The collected 900
kDa fraction was found to have insecticidal activity (see Table
30 below), with symptomology similar to that caused by native w-
14 toxin complex. This fraction was subjected to Proteinase K
and heat treatment, the activity in both cases was either
eliminated or reduced, providing evidence that the activity is
proteinaceous in nature. In addition, the active fraction tested
immunologically positive for the TcbA and TcbAiii peptides in
immunoblot analysis when tested with an anti-TcbAiii monoclonal
antibody (Table 30).
Table 30
Results of Immunoblot and SCR Bioassays.
Fraction SCR Activity Immunoblot Native Size
% % Growth Peptides (CL-4B
Mortality Inhibit. Detected Estimated
Size]
TcbA Media 1.0 M +++ +++ TcbA
Ion Exchange
TcbA Media CL-4B +++ +++ TcbA, -900 kDa
TcbAiii
TcbA Media CL-4B ++ +++ NT
+ Proteinase K
TcbA Media CL-4B - - NT
+ heat treatment
.... .. .:...: :.::...-.:. .
:.z:......,.: _r.w.,._.__.
.::;:.>::._..........._....... . .::::.:;:.::::.~....._...:.:...,
...:........:...: ::: .::.:.... ... :.::........ . ..,.: . . ..._ .._.. -
. ... ,....._ .
TcbA Cell Sup CL-4B - +++ NT --900 kD
PK = Proteinase K treatment 2 hours; Heat treatment = 100 C for 10
minutes; ND = None Detected; NT = Not Tested. Scoring system for
mortality and growth inhibition as compared to control samples; 5-
240="+~, 25-49%="++", 50-100a="+++".
The cell pellet was resuspended in 10 mM sodium phosphate
buffer, pH=7.0, and lysed by passage through a Bio-NebT" cell 30 nebulizer
(Glas-Col Inc., Terra Haute, IN). The pellets were
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treated with Dtlase to remove DNA and centrifuged at 17,000 x g ro
separate the cell pellet from the cell supernatant. The
supernatant fraction was decanted and filtered through a 0.2
micron filter to remove large particles and subjected to anion
exchange chromatography. Bound proteins were eluted with 1.0 M
NaCl, dialyzed and concentrated using BiomaxTM (Millipore Corp,
Bedford, MA) concentrators with a molecular mass cut-off of
50,000 Daltons. The concentrated fraction was subjected to gel
filtration chromatography using Sepharose CL-4B beaded matrix.
Bioassay data for material prepared in this way is shown in Table
30 and is denoted as " TcbA Cell Sup".
In yet another method to handle large amounts of material,
the cell pellets were re-suspended in 10 mM sodium phosphate
buffer, pH = 7.0 and thoroughly homogenized by using a Kontes
Glass Company (Vineland, NJ) 40 ml tissue grinder. The cellular
debris was pelleted by centrifugation at 25,000 x g and the cell
supernatant was decanted, passed through a 0.2 micron filter and
subjected to anion exchange chromatography using a Pharmacia
10/10 column packed with Poros HQ 50 beads. The bound proteins
were eluted by performing a NaCl gradient of 0.0 to 1.0 M.
Fractions containing the TcbA protein were combined and
concentrated using a 50 kDa concentrator and subjected to gel
filtration chromatography using Pharmacia CL-4B beaded matrix.
The fractions containing TcbA oligomer, molecular mass of
approximately 900 kDa, were collected and subjected to anion
exchange chromatography using a Pharmacia Mono Q 10/10 column
equilibrated with 20 mM Tris buffer pH = 7.3. A gradient of 0.0
to 1.0 M NaCl was used to elute recombinant TcbA protein.
Recombinant TcbA eluted from the column at a salt concentration
of approximately 0.3-0.4 M NaCl, the same molarity at which
native TcbA oligomer is eluted from the Mono Q 10/10 column. The
recombinant TcbA fraction was found to cause SCR mortality in
bioassay experiments similar to those in Table 30.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: WISCONSIN ALUMNI RESEARCH FOUNDATION
(ii) TITLE OF INVENTION: INSECTICIDAL PROTEIN TOXINS FROM PHOTORHABDUS
(iii) NUMBER OF SEQUENCES: 61
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: FETHERSTONHAUGH & CO.
(B) STREET: P.O. BOX 2999, STATION D
(C) CITY: OTTAWA
(D) STATE: ONT
(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (text)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,209,659
(B) FILING DATE: 06-NOV-1996
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/007,255
(B) FILING DATE: 06-NOV-1995
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/608,423
(B) FILING DATE: 28-FEB-1996
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/705,484
(B) FILING DATE: 28-AUG-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: FETHERSTONHAUGH & CO.
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 29355-1
- 115 -
29355-1
CA 02209659 1998-09-15
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613)-235-4373
(B) TELEFAX: (613)-232-8440
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn
1 5 10
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Gln Asp Ser Pro Glu Val Ser Ile Thr Thr Trp
1 5 10
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: N-terminal
- 116 -
29355-1
CA 02209659 1998-09-15
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp Ala
1 5 10 15
Leu Val Ala
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Ala Ser Pro Leu Ser Thr Ser Glu Leu Thr Ser Lys Leu Asn
1 5 10
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Ala Gly Asp Thr Ala Asn Ile Gly Asp
1 5
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Leu Gly Gly Ala Ala Thr Leu Leu Asp Leu Leu Leu Pro Gln Ile
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Met Leu Ser Thr Met Glu Lys Gln Leu Asn Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Asn Leu Ala Ser Pro Leu Ile Ser
1 5
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met Ile Asn Leu Asp Ile Asn Glu Gln Asn Lys Ile Met Val Val Ser
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ala Ala Lys Asp Val Lys Phe Gly Ser Asp Ala Arg Val Lys Met Leu
1 5 10 15
Arg Gly Val Asn
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7515 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..7515
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
ATG CAA AAC TCA TTA TCA AGC ACT ATC GAT ACT ATT TGT CAG AAA CTG 48
Met Gln Asn Ser Leu Ser Ser Thr Ile Asp Thr Ile Cys Gln Lys Leu
1 5 10 15
CAA TTA ACT TGT CCG GCG GAA ATT GCT TTG TAT CCC TTT GAT ACT TTC 96
Gln Leu Thr Cys Pro Ala Glu Ile Ala Leu Tyr Pro Phe Asp Thr Phe
20 25 30
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CGG GAA AAA ACT CGG GGA ATG GTT AAT TGG GGG GAA GCA AAA CGG ATT 144
Arg Glu Lys Thr Arg Gly Met Val Asn Trp Gly Glu Ala Lys Arg Ile
35 40 45
TAT GAA ATT GCA CAA GCG GAA CAG GAT AGA AAC CTA CTT CAT GAA AAA 192
Tyr Glu Ile Ala Gln Ala Glu Gln Asp Arg Asn Leu Leu His Glu Lys
50 55 60
CGT ATT TTT GCC TAT GCT AAT CCG CTG CTG AAA AAC GCT GTT CGG TTG 240
Arg Ile Phe Ala Tyr Ala Asn Pro Leu Leu Lys Asn Ala Val Arg Leu
65 70 75 80
GGT ACC CGG CAA ATG TTG GGT TTT ATA CAA GGT TAT AGT GAT CTG TTT 288
Gly Thr Arg Gln Met Leu Gly Phe Ile Gln Gly Tyr Ser Asp Leu Phe
85 90 95
GGT AAT CGT GCT GAT AAC TAT GCC GCG CCG GGC TCG GTT GCA TCG ATG 336
Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gly Ser Val Ala Ser Met
100 105 110
TTC TCA CCG GCG GCT TAT TTG ACG GAA TTG TAC CGT GAA GCC AAA AAC 384
Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Lys Asn
115 120 125
TTG CAT GAC AGC AGC TCA ATT TAT TAC CTA GAT AAA CGT CGC CCG GAT 432
Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu Asp Lys Arg Arg Pro Asp
130 135 140
TTA GCA AGC TTA ATG CTC AGC CAG AAA AAT ATG GAT GAG GAA ATT TCA 480
Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Met Asp Glu Glu Ile Ser
145 150 155 160
ACG CTG GCT CTC TCT AAT GAA TTG TGC CTT GCC GGG ATC GAA ACA AAA 528
Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Ala Gly Ile Glu Thr Lys
165 170 175
ACA GGA AAA TCA CAA GAT GAA GTG ATG GAT ATG TTG TCA ACT TAT CGT 576
Thr Gly Lys Ser Gln Asp Glu Val Met Asp Met Leu Ser Thr Tyr Arg
180 185 190
TTA AGT GGA GAG ACA CCT TAT CAT CAC GCT TAT GAA ACT GTT CGT GAA 624
Leu Ser Gly Glu Thr Pro Tyr His His Ala Tyr Glu Thr Val Arg Glu
195 200 205
ATC GTT CAT GAA CGT GAT CCA GGA TTT CGT CAT TTG TCA CAG GCA CCC 672
Ile Val His Glu Arg Asp Pro Gly Phe Arg His Leu Ser Gln Ala Pro
210 215 220
ATT GTT GCT GCT AAG CTC GAT CCT GTG ACT TTG TTG GGT ATT AGC TCC 720
Ile Val Ala Ala Lys Leu Asp Pro Val Thr Leu Leu Gly Ile Ser Ser
225 230 235 240
CAT ATT TCG CCA GAA CTG TAT AAC TTG CTG ATT GAG GAG ATC CCG GAA 768
His Ile Ser Pro Glu Leu Tyr Asn Leu Leu Ile Glu Glu Ile Pro Glu
245 250 255
AAA GAT GAA GCC GCG CTT GAT ACG CTT TAT AAA ACA AAC TTT GGC GAT 816
Lys Asp Glu Ala Ala Leu Asp Thr Leu Tyr Lys Thr Asn Phe Gly Asp
260 265 270
ATT ACT ACT GCT CAG TTA ATG TCC CCA AGT TAT CTG GCC CGG TAT TAT 864
Ile Thr Thr Ala Gln Leu Met Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr
275 280 285
GGC GTC TCA CCG GAA GAT ATT GCC TAC GTG ACG ACT TCA TTA TCA CAT 912
Gly Val Ser Pro Glu Asp Ile Ala Tyr Val Thr Thr Ser Leu Ser His
290 295 300
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GTT GGA TAT AGC AGT GAT ATT CTG GTT ATT CCG TTG GTC GAT GGT GTG 960
Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pro Leu Val Asp Gly Val
305 310 315 320
GGT AAG ATG GAA GTA GTT CGT GTT ACC CGA ACA CCA TCG GAT AAT TAT 1008
Gly Lys Met Glu Val Val Arg Val Thr Arg Thr Pro Ser Asp Asn Tyr
325 330 335
ACC AGT CAG ACG AAT TAT ATT GAG CTG TAT CCA CAG GGT GGC GAC AAT 1056
Thr Ser Gln Thr Asn Tyr Ile Glu Leu Tyr Pro Gln Gly Gly Asp Asn
340 345 350
TAT TTG ATC AAA TAC AAT CTA AGC AAT AGT TTT GGT TTG GAT GAT TTT 1104
Tyr Leu Ile Lys Tyr Asn Leu Ser Asn Ser Phe Gly Leu Asp Asp Phe
355 360 365
TAT CTG CAA TAT AAA GAT GGT TCC GCT GAT TGG ACT GAG ATT GCC CAT 1152
Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp Trp Thr Glu Ile Ala His
370 375 380
AAT CCC TAT CCT GAT ATG GTC ATA AAT CAA AAG TAT GAA TCA CAG GCG 1200
Asn Pro Tyr Pro Asp Met Val Ile Asn Gln Lys Tyr Glu Ser Gln Ala
385 390 395 400
ACA ATC AAA CGT AGT GAC TCT GAC AAT ATA CTC AGT ATA GGG TTA CAA 1248
Thr Ile Lys Arg Ser Asp Ser Asp Asn Ile Leu Ser Ile Gly Leu Gln
405 410 415
AGA TGG CAT AGC GGT AGT TAT AAT TTT GCC GCC GCC AAT TTT AAA ATT 1296
Arg Trp His Ser Gly Ser Tyr Asn Phe Ala Ala Ala Asn Phe Lys Ile
420 425 430
GAC CAA TAC TCC CCG AAA GCT TTC CTG CTT AAA ATG AAT AAG GCT ATT 1344
Asp Gln Tyr Ser Pro Lys Ala Phe Leu Leu Lys Met Asn Lys Ala Ile
435 440 445
CGG TTG CTC AAA GCT ACC GGC CTC TCT TTT GCT ACG TTG GAG CGT ATT 1392
Arg Leu Leu Lys Ala Thr Gly Leu Ser Phe Ala Thr Leu Glu Arg Ile
450 455 460
GTT GAT AGT GTT AAT AGC ACC AAA TCC ATC ACG GTT GAG GTA TTA AAC 1440
Val Asp Ser Val Asn Ser Thr Lys Ser Ile Thr Val Glu Val Leu Asn
465 470 475 480
AAG GTT TAT CGG GTA AAA TTC TAT ATT GAT CGT TAT GGC ATC AGT GAA 1488
Lys Val Tyr Arg Val Lys Phe Tyr Ile Asp Arg Tyr Gly Ile Ser Glu
485 490 495
GAG ACA GCC GCT ATT TTG GCT AAT ATT AAT ATC TCT CAG CAA GCT GTT.1536
Glu Thr Ala Ala Ile Leu Ala Asn Ile Asn Ile Ser Gln Gln Ala Val
500 505 510
GGC AAT CAG CTT AGC CAG TTT GAG CAA CTA TTT AAT CAC CCG CCG CTC 1584
Gly Asn Gln Leu Ser Gln Phe Glu Gln Leu Phe Asn His Pro Pro Leu
515 520 525
AAT GGT ATT CGC TAT GAA ATC AGT GAG GAC AAC TCC AAA CAT CTT CCT 1632
Asn Gly Ile Arg Tyr Glu Ile Ser Glu Asp Asn Ser Lys His Leu Pro
530 535 540
AAT CCT GAT CTG AAC CTT AAA CCA GAC AGT ACC GGT GAT GAT CAA CGC 1680
Asn Pro Asp Leu Asn Leu Lys Pro Asp Ser Thr Gly Asp Asp Gln Arg
545 550 555 560
AAG GCG GTT TTA AAA CGC GCG TTT CAG GTT AAC GCC AGT GAG TTG TAT 1728
Lys Ala Val Leu Lys Arg Ala Phe Gln Val Asn Ala Ser Glu Leu Tyr
565 570 575
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CAG ATG TTA TTG ATC ACT GAT CGT AAA GAA GAC GGT GTT ATC AAA AAT 1776
Gln Met Leu Leu Ile Thr Asp Arg Lys Glu Asp Gly Val Ile Lys Asn
580 585 590
AAC TTA GAG AAT TTG TCT GAT CTG TAT TTG GTT AGT TTG CTG GCC CAG 1824
Asn Leu Glu Asn Leu Ser Asp Leu Tyr Leu Val Ser Leu Leu Ala Gln
595 600 605
ATT CAT AAC CTG ACT ATT GCT GAA TTG AAC ATT TTG TTG GTG ATT TGT 1872
Ile His Asn Leu Thr Ile Ala Glu Leu Asn Ile Leu Leu Val Ile Cys
610 615 620
GGC TAT GGC GAC ACC AAC ATT TAT CAG ATT ACC GAC GAT AAT TTA GCC 1920
Gly Tyr Gly Asp Thr Asn Ile Tyr Gln Ile Thr Asp Asp Asn Leu Ala
625 630 635 640
AAA ATA GTG GAA ACA TTG TTG TGG ATC ACT CAA TGG TTG AAG ACC CAA 1968
Lys Ile Val Glu Thr Leu Leu Trp Ile Thr Gln Trp Leu Lys Thr Gln
645 650 655
AAA TGG ACA GTT ACC GAC CTG TTT CTG ATG ACC ACG GCC ACT TAC AGC 2016
Lys Trp Thr Val Thr Asp Leu Phe Leu Met Thr Thr Ala Thr Tyr Ser
660 665 670
ACC ACT TTA ACG CCA GAA ATT AGC AAT CTG ACG GCT ACG TTG TCT TCA 2064
Thr Thr Leu Thr Pro Glu Ile Ser Asn Leu Thr Ala Thr Leu Ser Ser
675 680 685
ACT TTG CAT GGC AAA GAG AGT CTG ATT GGG GAA GAT CTG AAA AGA GCA 2112
Thr Leu His Gly Lys Glu Ser Leu Ile Gly Glu Asp Leu Lys Arg Ala
690 695 700
ATG GCG CCT TGC TTC ACT TCG GCT TTG CAT TTG ACT TCT CAA GAA GTT 2160
Met Ala Pro Cys Phe Thr Ser Ala Leu His Leu Thr Ser Gln Glu Val
705 710 715 720
GCG TAT GAC CTG CTG TTG TGG ATA GAC CAG ATT CAA CCG GCA CAA ATA 2208
Ala Tyr Asp Leu Leu Leu Trp Ile Asp Gln Ile Gln Pro Ala Gln Ile
725 730 735
ACT GTT GAT GGG TTT TGG GAA GAA GTG CAA ACA ACA CCA ACC AGC TTG 2256
Thr Val Asp Gly Phe Trp Glu Glu Val Gln Thr Thr Pro Thr Ser Leu
740 745 750
AAG GTG ATT ACC TTT GCT CAG GTG CTG GCA CAA TTG AGC CTG ATC TAT 2304
Lys Val Ile Thr Phe Ala Gln Val Leu Ala Gln Leu Ser Leu Ile Tyr
755 760 765
CGT CGT ATT GGG TTA AGT GAA ACG GAA CTG TCA CTG ATC GTG ACT CAA 2352
Arg Arg Ile Gly Leu Ser Glu Thr Glu Leu Ser Leu Ile Val Thr Gln
770 775 780
TCT TCT CTG CTA GTG GCA GGC AAA AGC ATA CTG GAT CAC GGT CTG TTA 2400
Ser Ser Leu Leu Val Ala Gly Lys Ser Ile Leu Asp His Gly Leu Leu
785 790 795 800
ACC CTG ATG GCC TTG GAA GGT TTT CAT ACC TGG GTT AAT GGC TTG GGG 2448
Thr Leu Met Ala Leu Glu Gly Phe His Thr Trp Val Asn Gly Leu Gly
805 810 815
CAA CAT GCC TCC TTG ATA TTG GCG GCG TTG AAA GAC GGA GCC TTG ACA 2496
Gln His Ala Ser Leu Ile Leu Ala Ala Leu Lys Asp Gly Ala Leu Thr
820 825 830
GTT ACC GAT GTA GCA CAA GCT ATG AAT AAG GAG GAA TCT CTC CTA CAA 2544
Val Thr Asp Val Ala Gln Ala Met Asn Lys Glu Glu Ser Leu Leu Gln
835 840 845
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ATG GCA GCT AAT CAG GTG GAG AAG GAT CTA ACA AAA CTG ACC AGT TGG 2592
Met Ala Ala Asn Gln Val Glu Lys Asp Leu Thr Lys Leu Thr Ser Trp
850 855 860
ACA CAG ATT GAC GCT ATT CTG CAA TGG TTA CAG ATG TCT TCG GCC TTG 2640
Thr Gln Ile Asp Ala Ile Leu Gln Trp Leu Gln Met Ser Ser Ala Leu
865 870 875 880
GCG GTT TCT CCA CTG GAT CTG GCA GGG ATG ATG GCC CTG AAA TAT GGG 2688
Ala Val Ser Pro Leu Asp Leu Ala Gly Met Met Ala Leu Lys Tyr Gly
885 890 895
ATA GAT CAT AAC TAT GCT GCC TGG CAA GCT GCG GCG GCT GCG CTG ATG 2736
Ile Asp His Asn Tyr Ala Ala Trp Gln Ala Ala Ala Ala Ala Leu Met
900 905 910
GCT GAT CAT GCT AAT CAG GCA CAG AAA AAA CTG GAT GAG ACG TTC AGT 2784
Ala Asp His Ala Asn Gln Ala Gln Lys Lys Leu Asp Glu Thr Phe Ser
915 920 925
AAG GCA TTA TGT AAC TAT TAT ATT AAT GCT GTT GTC GAT AGT GCT GCT 2832
Lys Ala Leu Cys Asn Tyr Tyr Ile Asn Ala Val Val Asp Ser Ala Ala
930 935 940
GGA GTA CGT GAT CGT AAC GGT TTA TAT ACC TAT TTG CTG ATT GAT AAT 2880
Gly Val Arg Asp Arg Asn Gly Leu Tyr Thr Tyr Leu Leu Ile Asp Asn
945 950 955 960
CAG GTT TCT GCC GAT GTG ATC ACT TCA CGT ATT GCA GAA GCT ATC GCC 2928
Gln Val Ser Ala Asp Val Ile Thr Ser Arg Ile Ala Glu Ala Ile Ala
965 970 975
GGT ATT CAA CTG TAC GTT AAC CGG GCT TTA AAC CGA GAT GAA GGT CAG 2976
Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu Gly Gln
980 985 990
CTT GCA TCG GAC GTT AGT ACC CGT CAG TTC TTC ACT GAC TGG GAA CGT 3024
Leu Ala Ser Asp Val Ser Thr Arg Gln Phe Phe Thr Asp Trp Glu Arg
995 1000 1005
TAC AAT AAA CGT TAC AGT ACT TGG GCT GGT GTC TCT GAA CTG GTC TAT 3072
Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu Val Tyr
1010 1015 1020
TAT CCA GAA AAC TAT GTT GAT CCC ACT CAG CGC ATT GGG CAA ACC AAA 3120
Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln Thr Lys
1025 1030 1035 1040
ATG ATG GAT GCG CTG TTG CAA TCC ATC AAC CAG AGC CAG CTA AAT GCG 3168
Met Met Asp Ala Leu Leu Gln Ser Ile Asn Gln Ser Gln Leu Asn Ala
1045 1050 1055
GAT ACG GTG GAA GAT GCT TTC AAA ACT TAT TTG ACC AGC TTT GAG CAG 3216
Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Leu Thr Ser Phe Glu Gln
1060 1065 1070
GTA GCA AAT CTG AAA GTA ATT AGT GCT TAC CAC GAT AAT GTG AAT GTG 3264
Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Val Asn Val
1075 1080 1085
GAT CAA GGA TTA ACT TAT TTT ATC GGT ATC GAC CAA GCA GCT CCG GGT 3312
Asp Gln Gly Leu Thr Tyr Phe Ile Gly Ile Asp Gln Ala Ala Pro Gly
1090 1095 1100
ACG TAT TAC TGG CGT AGT GTT GAT CAC AGC AAA TGT GAA AAT GGC AAG 3360
Thr Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Cys Glu Asn Gly Lys
1105 1110 1115 1120
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TTT GCC GCT AAT GCT TGG GGT GAG TGG AAT AAA ATT ACC TGT GCT GTC 3408
Phe Ala Ala Asn Ala Trp Gly Glu Trp Asn Lys Ile Thr Cys Ala Val
1125 1130 1135
AAT CCT TGG AAA AAT ATC ATC CGT CCG GTT GTT TAT ATG TCC CGC TTA 3456
Asn Pro Trp Lys Asn Ile Ile Arg Pro Val Val Tyr Met Ser Arg Leu
1140 1145 1150
TAT CTG CTA TGG CTG GAG CAG CAA TCA AAG AAA AGT GAT GAT GGT AAA 3504
Tyr Leu Leu Trp Leu Glu Gln Gln Ser Lys Lys Ser Asp Asp Gly Lys
1155 1160 1165
ACC ACG ATT TAT CAA TAT AAC TTA AAA CTG GCT CAT ATT CGT TAC GAC 3552
Thr Thr Ile Tyr Gln Tyr Asn Leu Lys Leu Ala His Ile Arg Tyr Asp
1170 1175 1180
GGT AGT TGG AAT ACA CCA TTT ACT TTT GAT GTG ACA GAA AAG GTA AAA 3600
Gly Ser Trp Asn Thr Pro Phe Thr Phe Asp Val Thr Glu Lys Val Lys
1185 1190 1195 1200
AAT TAC ACG TCG AGT ACT GAT GCT GCT GAA TCT TTA GGG TTG TAT TGT 3648
Asn Tyr Thr Ser Ser Thr Asp Ala Ala Glu Ser Leu Gly Leu Tyr Cys
1205 1210 1215
ACT GGT TAT CAA GGG GAA GAC ACT CTA TTA GTT ATG TTC TAT TCG ATG 3696
Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Ser Met
1220 1225 1230
CAG AGT AGT TAT AGC TCC TAT ACC GAT AAT AAT GCG CCG GTC ACT GGG 3744
Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val Thr Gly
1235 1240 1245
CTA TAT ATT TTC GCT GAT ATG TCA TCA GAC AAT ATG ACG AAT GCA CAA 3792
Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr Asn Ala Gln
1250 1255 1260
GCA ACT AAC TAT TGG AAT AAC AGT TAT CCG CAA TTT GAT ACT GTG ATG 3840
Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gln Phe Asp Thr Val Met
1265 1270 1275 1280
GCA GAT CCG GAT AGC GAC AAT AAA AAA GTC ATA ACC AGA AGA GTT AAT 3888
Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Ile Thr Arg Arg Val Asn
1285 1290 1295
AAC CGT TAT GCG GAG GAT TAT GAA ATT CCT TCC TCT GTG ACA AGT AAC 3936
Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Thr Ser Asn
1300 1305 1310
AGT AAT TAT TCT TGG GGT GAT CAC AGT TTA ACC ATG CTT TAT GGT GGT 3984
Ser Asn Tyr Ser Trp Gly Asp His Ser Leu Thr Met Leu Tyr Gly Gly
1315 1320 1325
AGT GTT CCT AAT ATT ACT TTT GAA TCG GCG GCA GAA GAT TTA AGG CTA 4032
Ser Val Pro Asn Ile Thr Phe Glu Ser Ala Ala Glu Asp Leu Arg Leu
1330 1335 1340
TCT ACC AAT ATG GCA TTG AGT ATT ATT CAT AAT GGA TAT GCG GGA ACC 4080
Ser Thr Asn Met Ala Leu Ser Ile Ile His Asn Gly Tyr Ala Gly Thr
1345 1350 1355 1360
CGC CGT ATA CAA TGT AAT CTT ATG AAA CAA TAC GCT TCA TTA GGT GAT 4128
Arg Arg Ile Gln Cys Asn Leu Met Lys Gln Tyr Ala Ser Leu Gly Asp
1365 1370 1375
AAA TTT ATA ATT TAT GAT TCA TCA TTT GAT GAT GCA AAC CGT TTT AAT 4176
Lys Phe Ile Ile Tyr Asp Ser Ser Phe Asp Asp Ala Asn Arg Phe Asn
1380 1385 1390
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CTG GTG CCA TTG TTT AAA TTC GGA AAA GAC GAG AAC TCA GAT GAT AGT 4224
Leu Val Pro Leu Phe Lys Phe Gly Lys Asp Glu Asn Ser Asp Asp Ser
1395 1400 1405
ATT TGT ATA TAT AAT GAA AAC CCT TCC TCT GAA GAT AAG AAG TGG TAT 4272
Ile Cys Ile Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys Trp Tyr
1410 1415 1420
TTT TCT TCG AAA GAT GAC AAT AAA ACA GCG GAT TAT AAT GGT GGA ACT 4320
Phe Ser Ser Lys Asp Asp Asn Lys Thr Ala Asp Tyr Asn Gly Gly Thr
1425 1430 1435 1440
CAA TGT ATA GAT GCT GGA ACC AGT AAC AAA GAT TTT TAT TAT AAT CTC 4368
Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr Asn Leu
1445 1450 1455
CAG GAG ATT GAA GTA ATT AGT GTT ACT GGT GGG TAT TGG TCG AGT TAT 4416
Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser Ser Tyr
1460 1465 1470
AAA ATA TCC AAC CCG ATT AAT ATC AAT ACG GGC ATT GAT AGT GCT AAA 4464
Lys Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys
1475 1480 1485
GTA AAA GTC ACC GTA AAA GCG GGT GGT GAC GAT CAA ATC TTT ACT GCT 4512
Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln Ile Phe Thr Ala
1490 1495 1500
GAT AAT AGT ACC TAT GTT CCT CAG CAA CCG GCA CCC AGT TTT GAG GAG 4560
Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Ala Pro Ser Phe Glu Glu
1505 1510 1515 1520
ATG ATT TAT CAG TTC AAT AAC CTG ACA ATA GAT TGT AAG AAT TTA AAT 4608
Met Ile Tyr Gln Phe Asn Asn Leu Thr Ile Asp Cys Lys Asn Leu Asn
1525 1530 1535
TTC ATC GAC AAT CAG GCA CAT ATT GAG ATT GAT TTC ACC GCT ACG GCA 4656
Phe Ile Asp Asn Gln Ala His Ile Glu Ile Asp Phe Thr Ala Thr Ala
1540 1545 1550
CAA GAT GGC CGA TTC TTG GGT GCA GAA ACT TTT ATT ATC CCG GTA ACT 4704
Gln Asp Gly Arg Phe Leu Gly Ala Glu Thr Phe Ile Ile Pro Val Thr
1555 1560 1565
AAA AAA GTT CTC GGT ACT GAG AAC GTG ATT GCG TTA TAT AGC GAA AAT 4752
Lys Lys Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn
1570 1575 1580
AAC GGT GTT CAA TAT ATG CAA ATT GGC GCA TAT CGT ACC CGT TTG AAT 4800
Asn Gly Val Gln Tyr Met Gln Ile Gly Ala Tyr Arg Thr Arg Leu Asn
1585 1590 1595 1600
ACG TTA TTC GCT CAA CAG TTG GTT AGC CGT GCT AAT CGT GGC ATT GAT 4848
Thr Leu Phe Ala Gln Gln Leu Val Ser Arg Ala Asn Arg Gly Ile Asp
1605 1610 1615
GCA GTG CTC AGT ATG GAA ACT CAG AAT ATT CAG GAA CCG CAA TTA GGA 4896
Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly
1620 1625 1630
GCG GGC ACA TAT GTG CAG CTT GTG TTG GAT AAA TAT GAT GAG TCT ATT 4944
Ala Gly Thr Tyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu Ser Ile
1635 1640 1645
CAT GGC ACT AAT AAA AGC TTT GCT ATT GAA TAT GTT GAT ATA TTT AAA 4992
His Gly Thr Asn Lys Ser Phe Ala Ile Glu Tyr Val Asp Ile Phe Lys
1650 1655 1660
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GAG AAC GAT AGT TTT GTG ATT TAT CAA GGA GAA CTT AGC GAA ACA AGT 5040
Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu Thr Ser
1665 1670 1675 1680
CAA ACT GTT GTG AAA GTT TTC TTA TCC TAT TTT ATA GAG GCG ACT GGA 5088
Gln Thr Val Val Lys Val Phe Leu Ser Tyr Phe Ile Glu Ala Thr Gly
1685 1690 1695
AAT AAG AAC CAC TTA TGG GTA CGT GCT AAA TAC CAA AAG GAA ACG ACT 5136
Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln Lys Glu Thr Thr
1700 1705 1710
GAT AAG ATC TTG TTC GAC CGT ACT GAT GAG AAA GAT CCG CAC GGT TGG 5184
Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys Asp Pro His Gly Trp
1715 1720 1725
TTT CTC AGC GAC GAT CAC AAG ACC TTT AGT GGT CTC TCT TCC GCA CAG 5232
Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gly Leu Ser Ser Ala Gln
1730 1735 1740
GCA TTA AAG AAC GAC AGT GAA CCG ATG GAT TTC TCT GGC GCC AAT GCT 5280
Ala Leu Lys Asn Asp Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ala
1745 1750 1755 1760
CTC TAT TTC TGG GAA CTG TTC TAT TAC ACG CCG ATG ATG ATG GCT CAT 5328
Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Met Met Ala His
1765 1770 1775
CGT TTG TTG CAG GAA CAG AAT TTT GAT GCG GCG AAC CAT TGG TTC CGT 5376
Arg Leu Leu Gln Glu Gln Asn Phe Asp Ala Ala Asn His Trp Phe Arg
1780 1785 1790
TAT GTC TGG AGT CCA TCC GGT TAT ATC GTT GAT GGT AAA ATT GCT ATC 5424
Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val Asp Gly Lys Ile Ala Ile
1795 1800 1805
TAC CAC TGG AAC GTG CGA CCG CTG GAA GAA GAC ACC AGT TGG AAT GCA 5472
Tyr His Trp Asn Val Arg Pro Leu Glu Glu Asp Thr Ser Trp Asn Ala
1810 1815 1820
CAA CAA CTG GAC TCC ACC GAT CCA GAT GCT GTA GCC CAA GAT GAT CCG 5520
Gln Gln Leu Asp Ser Thr Asp Pro Asp Ala Val Ala Gln Asp Asp Pro
1825 1830 1835 1840
ATG CAC TAC AAG GTG GCT ACC TTT ATG GCG ACG TTG GAT CTG CTA ATG 5568
Met His Tyr Lys Val Ala Thr Phe Met Ala Thr Leu Asp Leu Leu Met
1845 1850 1855
GCC CGT GGT GAT GCT GCT TAC CGC CAG TTA GAG CGT GAT ACG TTG GCT 5616
Ala Arg Gly Asp Ala Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Ala
1860 1865 1870
GAA GCT AAA ATG TGG TAT ACA CAG GCG CTT AAT CTG TTG GGT GAT GAG 5664
Glu Ala Lys Met Trp Tyr Thr Gln Ala Leu Asn Leu Leu Gly Asp Glu
1875 1880 1885
CCA CAA GTG ATG CTG AGT ACG ACT TGG GCT AAT CCA ACA TTG GGT AAT 5712
Pro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu Gly Asn
1890 1895 1900
GCT GCT TCA AAA ACC ACA CAG CAG GTT CGT CAG CAA GTG CTT ACC CAG 5760
Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val Leu Thr Gln
1905 1910 1915 1920
TTG CGT CTC AAT AGC AGG GTA AAA ACC CCG TTG CTA GGA ACA GCC AAT 5808
Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu Leu Gly Thr Ala Asn
1925 1930 1935
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TCC CTG ACC GCT TTA TTC CTG CCG CAG GAA AAT AGC AAG CTC AAA GGC 5856
Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn Ser Lys Leu Lys Gly
1940 1945 1950
TAC TGG CGG ACA CTG GCG CAG CGT ATG TTT AAT TTA CGT CAT AAT CTG 5904
Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn Leu Arg His Asn Leu
1955 1960 1965
TCG ATT GAC GGC CAG CCG CTC TCC TTG CCG CTG TAT GCT AAA CCG GCT 5952
Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu Tyr Ala Lys Pro Ala
1970 1975 1980
GAT CCA AAA GCT TTA CTG AGT GCG GCG GTT TCA GCT TCT CAA GGG GGA 6000
Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser Ala Ser Gln Gly Gly
1985 1990 1995 2000
GCC GAC TTG CCG AAG GCG CCG CTG ACT ATT CAC CGC TTC CCT CAA ATG 6048
Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His Arg Phe Pro Gln Met
2005 2010 2015
CTA GAA GGG GCA CGG GGC TTG GTT AAC CAG CTT ATA CAG TTC GGT AGT 6096
Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu Ile Gln Phe Gly Ser
2020 2025 2030
TCA CTA TTG GGG TAC AGT GAG CGT CAG GAT GCG GAA GCT ATG AGT CAA 6144
Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala Glu Ala Met Ser Gln
2035 2040 2045
CTA CTG CAA ACC CAA GCC AGC GAG TTA ATA CTG ACC AGT ATT CGT ATG 6192
Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu Thr Ser Ile Arg Met
2050 2055 2060
CAG GAT AAC CAA TTG GCA GAG CTG GAT TCG GAA AAA ACC GCC TTG CAA 6240
Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu Lys Thr Ala Leu Gln
2065 2070 2075 2080
GTC TCT TTA GCT GGA GTG CAA CAA CGG TTT GAC AGC TAT AGC CAA CTG 6288
Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp Ser Tyr Ser Gln Leu
2085 2090 2095
TAT GAG GAG AAC ATC AAC GCA GGT GAG CAG CGA GCG CTG GCG TTA CGC 6336
Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg Ala Leu Ala Leu Arg
2100 2105 2110
TCA GAA TCT GCT ATT GAG TCT CAG GGA GCG CAG ATT TCC CGT ATG GCA 6384
Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln Ile Ser Arg Met Ala
2115 2120 2125
GGC GCG GGT GTT GAT ATG GCA CCA AAT ATC TTC GGC CTG GCT GAT GGC 6432
Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly Leu Ala Asp Gly
2130 2135 2140
GGC ATG CAT TAT GGT GCT ATT GCC TAT GCC ATC GCT GAC GGT ATT GAG 6480
Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile Ala Asp Gly Ile Glu
2145 2150 2155 2160
TTG AGT GCT TCT GCC AAG ATG GTT GAT GCG GAG AAA GTT GCT CAG TCG 6528
Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu Lys Val Ala Gln Ser
2165 2170 2175
GAA ATA TAT CGC CGT CGC CGT CAA GAA TGG AAA ATT CAG CGT GAC AAC 6576
Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys Ile Gln Arg Asp Asn
2180 2185 2190
GCA CAA GCG GAG ATT AAC CAG TTA AAC GCG CAA CTG GAA TCA CTG TCT 6624
Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln Leu Glu Ser Leu Ser
2195 2200 2205
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ATT CGC CGT GAA GCC GCT GAA ATG CAA AAA GAG TAC CTG AAA ACC CAG 6672
Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu Tyr Leu Lys Thr Gln
2210 2215 2220
CAA GCT CAG GCG CAG GCA CAA CTT ACT TTC TTA AGA AGC AAA TTC AGT 6720
Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu Arg Ser Lys Phe Ser
2225 2230 2235 2240
AAT CAA GCG TTA TAT AGT TGG TTA CGA GGG CGT TTG TCA GGT ATT TAT 6768
Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg Leu Ser Gly Ile Tyr
2245 2250 2255
TTC CAG TTC TAT GAC TTG GCC GTA TCA CGT TGC CTG ATG GCA GAG CAA 6816
Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys Leu Met Ala Glu Gln
2260 2265 2270
TCC TAT CAA TGG GAA GCT AAT GAT AAT TCC ATT AGC TTT GTC AAA CCG 6864
Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile Ser Phe Val Lys Pro
2275 2280 2285
GGT GCA TGG CAA GGA ACT TAC GCC GGC TTA TTG TGT GGA GAA GCT TTG 6912
Gly Ala Trp Gin Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu Ala Leu
2290 2295 2300
ATA CAA AAT CTG GCA CAA ATG GAA GAG GCA TAT CTG AAA TGG GAA TCT 6960
Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp Glu Ser
2305 2310 2315 2320
CGC GCT TTG GAA GTA GAA CGC ACG GTT TCA TTG GCA GTG GTT TAT GAT 7008
Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val Val Tyr Asp
2325 2330 2335
TCA CTG GAA GGT AAT GAT CGT TTT AAT TTA GCG GAA CAA ATA CCT GCA 7056
Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu Gln Ile Pro Ala
2340 2345 2350
TTA TTG GAT AAG GGG GAG GGA ACA GCA GGA ACT AAA GAA AAT GGG TTA 7104
Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr Lys Glu Asn Gly Leu
2355 2360 2365
TCA TTG GCT AAT GCT ATC CTG TCA GCT TCG GTC AAA TTG TCC GAC TTG 7152
Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val Lys Leu Ser Asp Leu
2370 2375 2380
AAA CTG GGA ACG GAT TAT CCA GAC AGT ATC GTT GGT AGC AAC AAG GTT 7200
Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val Gly Ser Asn Lys Val
2385 2390 2395 2400
CGT CGT ATT AAG CAA ATC AGT GTT TCG CTA CCT GCA TTG GTT GGG CCT 7248
Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro Ala Leu Val Gly Pro
2405 2410 2415
TAT CAG GAT GTT CAG GCT ATG CTC AGC TAT GGT GGC AGT ACT CAA TTG 7296
Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly Gly Ser Thr Gln Leu
2420 2425 2430
CCG AAA GGT TGT TCA GCG TTG GCT GTG TCT CAT GGT ACC AAT GAT AGT 7344
Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His Gly Thr Asn Asp Ser
2435 2440 2445
GGT CAG TTC CAG TTG GAT TTC AAT GAC GGC AAA TAC CTG CCA TTT GAA 7392
Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Tyr Leu Pro Phe Glu
2450 2455 2460
GGT ATT GCT CTT GAT GAT CAG GGT ACA CTG AAT CTT CAA TTT CCG AAT 7440
Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn Leu Gln Phe Pro Asn
2465 2470 2475 2480
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GCT ACC GAC AAG CAG AAA GCA ATA TTG CAA ACT ATG AGC GAT ATT ATT 7488
Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr Met Ser Asp Ile Ile
2485 2490 2495
TTG CAT ATT CGT TAT ACC ATC CGT TAA 7515
Leu His Ile Arg Tyr Thr Ile Arg
2500 2504
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2504 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Gln Asn Ser Leu Ser Ser Thr Ile Asp Thr Ile Cys Gln Lys Leu
1 5 10 15
Gln Leu Thr Cys Pro Ala Glu Ile Ala Leu Tyr Pro Phe Asp Thr Phe
20 25 30
Arg Glu Lys Thr Arg Gly Met Val Asn Trp Gly Glu Ala Lys Arg Ile
35 40 45
Tyr Glu Ile Ala Gln Ala Glu Gln Asp Arg Asn Leu Leu His Glu Lys
50 55 60
Arg Ile Phe Ala Tyr Ala Asn Pro Leu Leu Lys Asn Ala Val Arg Leu
65 70 75 80
Gly Thr Arg Gln Met Leu Gly Phe Ile Gln Gly Tyr Ser Asp Leu Phe
85 90 95
Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gly Ser Val Ala Ser Met
100 105 110
Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Lys Asn
115 120 125
Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu Asp Lys Arg Arg Pro Asp
130 135 140
Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Met Asp Glu Glu Ile Ser
145 150 155 160
Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Ala Gly Ile Glu Thr Lys
165 170 175
Thr Gly Lys Ser Gln Asp Glu Val Met Asp Met Leu Ser Thr Tyr Arg
180 185 190
Leu Ser Gly Glu Thr Pro Tyr His His Ala Tyr Glu Thr Val Arg Glu
195 200 205
Ile Val His Glu Arg Asp Pro Gly Phe Arg His Leu Ser Gln Ala Pro
210 215 220
Ile Val Ala Ala Lys Leu Asp Pro Val Thr Leu Leu Gly Ile Ser Ser
225 230 235 240
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His Ile Ser Pro Glu Leu Tyr Asn Leu Leu Ile Glu Glu Ile Pro Glu
245 250 255
Lys Asp Glu Ala Ala Leu Asp Thr Leu Tyr Lys Thr Asn Phe Gly Asp
260 265 270
Ile Thr Thr Ala Gln Leu Met Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr
275 280 285
Gly Val Ser Pro Glu Asp Ile Ala Tyr Val Thr Thr Ser Leu Ser His
290 295 300
Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pro Leu Val Asp Gly Val
305 310 315 320
Gly Lys Met Glu Val Val Arg Val Thr Arg Thr Pro Ser Asp Asn Tyr
325 330 335
Thr Ser Gln Thr Asn Tyr Ile Glu Leu Tyr Pro Gln Gly Gly Asp Asn
340 345 350
Tyr Leu Ile Lys Tyr Asn Leu Ser Asn Ser Phe Gly Leu Asp Asp Phe
355 360 365
Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp Trp Thr Glu Ile Ala His
370 375 380
Asn Pro Tyr Pro Asp Met Val Ile Asn Gln Lys Tyr Glu Ser Gln Ala
385 390 395 400
Thr Ile Lys Arg Ser Asp Ser Asp Asn Ile Leu Ser Ile Gly Leu Gln
405 410 415
Arg Trp His Ser Gly Ser Tyr Asn Phe Ala Ala Ala Asn Phe Lys Ile
420 425 430
Asp Gln Tyr Ser Pro Lys Ala Phe Leu Leu Lys Met Asn Lys Ala Ile
435 440 445
Arg Leu Leu Lys Ala Thr Gly Leu Ser Phe Ala Thr Leu Glu Arg Ile
450 455 460
Val Asp Ser Val Asn Ser Thr Lys Ser Ile Thr Val Glu Val Leu Asn
465 470 475 480
Lys Val Tyr Arg Val Lys Phe Tyr Ile Asp Arg Tyr Gly Ile Ser Glu
485 490 495
Glu Thr Ala Ala Ile Leu Ala Asn Ile Asn Ile Ser Gln Gln Ala Val
500 505 510
Gly Asn Gln Leu Ser Gln Phe Glu Gln Leu Phe Asn His Pro Pro Leu
515 520 525
Asn Gly Ile Arg Tyr Glu Ile Ser Glu Asp Asn Ser Lys His Leu Pro
530 535 540
Asn Pro Asp Leu Asn Leu Lys Pro Asp Ser Thr Gly Asp Asp Gln Arg
545 550 555 560
Lys Ala Val Leu Lys Arg Ala Phe Gln Val Asn Ala Ser Glu Leu Tyr
565 570 575
Gln Met Leu Leu Ile Thr Asp Arg Lys Glu Asp Gly Val Ile Lys Asn
580 585 590
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Asn Leu Glu Asn Leu Ser Asp Leu Tyr Leu Val Ser Leu Leu Ala Gln
595 600 605
Ile His Asn Leu Thr Ile Ala Glu Leu Asn Ile Leu Leu Val Ile Cys
610 615 620
Gly Tyr Gly Asp Thr Asn Ile Tyr Gln Ile Thr Asp Asp Asn Leu Ala
625 630 635 640
Lys Ile Val Glu Thr Leu Leu Trp Ile Thr Gln Trp Leu Lys Thr Gln
645 650 655
Lys Trp Thr Val Thr Asp Leu Phe Leu Met Thr Thr Ala Thr Tyr Ser
660 665 670
Thr Thr Leu Thr Pro Glu Ile Ser Asn Leu Thr Ala Thr Leu Ser Ser
675 680 685
Thr Leu His Gly Lys Glu Ser Leu Ile Gly Glu Asp Leu Lys Arg Ala
690 695 700
Met Ala Pro Cys Phe Thr Ser Ala Leu His Leu Thr Ser Gln Glu Val
705 710 715 720
Ala Tyr Asp Leu Leu Leu Trp Ile Asp Gln Ile Gln Pro Ala Gln Ile
725 730 735
Thr Val Asp Gly Phe Trp Glu Glu Val Gln Thr Thr Pro Thr Ser Leu
740 745 750
Lys Val Ile Thr Phe Ala Gln Val Leu Ala Gln Leu Ser Leu Ile Tyr
755 760 765
Arg Arg Ile Gly Leu Ser Glu Thr Glu Leu Ser Leu Ile Val Thr Gln
770 775 780
Ser Ser Leu Leu Val Ala Gly Lys Ser Ile Leu Asp His Gly Leu Leu
785 790 795 800
Thr Leu Met Ala Leu Glu Gly Phe His Thr Trp Val Asn Gly Leu Gly
805 810 815
Gln His Ala Ser Leu Ile Leu Ala Ala Leu Lys Asp Gly Ala Leu Thr
820 825 830
Val Thr Asp Val Ala Gln Ala Met Asn Lys Glu Glu Ser Leu Leu Gln
835 840 845
Met Ala Ala Asn Gln Val Glu Lys Asp Leu Thr Lys Leu Thr Ser Trp
850 855 860
Thr Gln Ile Asp Ala Ile Leu Gln Trp Leu Gln Met Ser Ser Ala Leu
865 870 875 880
Ala Val Ser Pro Leu Asp Leu Ala Gly Met Met Ala Leu Lys Tyr Gly
885 890 895
Ile Asp His Asn Tyr Ala Ala Trp Gln Ala Ala Ala Ala Ala Leu Met
900 905 910
Ala Asp His Ala Asn Gln Ala Gln Lys Lys Leu Asp Glu Thr Phe Ser
915 920 925
Lys Ala Leu Cys Asn Tyr Tyr Ile Asn Ala Val Val Asp Ser Ala Ala
930 935 940
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Gly Val Arg Asp Arg Asn Gly Leu Tyr Thr Tyr Leu Leu Ile Asp Asn
945 950 955 960
Gln Val Ser Ala Asp Val Ile Thr Ser Arg Ile Ala Glu Ala Ile Ala
965 970 975
Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu Gly Gln
980 985 990
Leu Ala Ser Asp Val Ser Thr Arg Gln Phe Phe Thr Asp Trp Glu Arg
995 1000 1005
Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu Val Tyr
1010 1015 1020
Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln Thr Lys
1025 1030 1035 1040
Met Met Asp Ala Leu Leu Gln Ser Ile Asn Gln Ser Gln Leu Asn Ala
1045 1050 1055
Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Leu Thr Ser Phe Glu Gln
1060 1065 1070
Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Val Asn Val
1075 1080 1085
Asp Gln Gly Leu Thr Tyr Phe Ile Gly Ile Asp Gln Ala Ala Pro Gly
1090 1095 1100
Thr Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Cys Glu Asn Gly Lys
1105 1110 1115 1120
Phe Ala Ala Asn Ala Trp Gly Glu Trp Asn Lys Ile Thr Cys Ala Val
1125 1130 1135
Asn Pro Trp Lys Asn Ile Ile Arg Pro Val Val Tyr Met Ser Arg Leu
1140 1145 1150
Tyr Leu Leu Trp Leu Glu Gln Gln Ser Lys Lys Ser Asp Asp Gly Lys
1155 1160 1165
Thr Thr Ile Tyr Gln Tyr Asn Leu Lys Leu Ala His Ile Arg Tyr Asp
1170 1175 1180
Gly Ser Trp Asn Thr Pro Phe Thr Phe Asp Val Thr Glu Lys Val Lys
1185 1190 1195 1200
Asn Tyr Thr Ser Ser Thr Asp Ala Ala Glu Ser Leu Gly Leu Tyr Cys
1205 1210 1215
Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Ser Met
1220 1225 1230
Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val Thr Gly
1235 1240 1245
Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr Asn Ala Gln
1250 1255 1260
Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gln Phe Asp Thr Val Met
1265 1270 1275 1280
Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Ile Thr Arg Arg Val Asn
1285 1290 1295
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Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Thr Ser Asn
1300 1305 1310
Ser Asn Tyr Ser Trp Gly Asp His Ser Leu Thr Met Leu Tyr Gly Gly
1315 1320 1325
Ser Val Pro Asn Ile Thr Phe Glu Ser Ala Ala Glu Asp Leu Arg Leu
1330 1335 1340
Ser Thr Asn Met Ala Leu Ser Ile Ile His Asn Gly Tyr Ala Gly Thr
1345 1350 1355 1360
Arg Arg Ile Gln Cys Asn Leu Met Lys Gln Tyr Ala Ser Leu Gly Asp
1365 1370 1375
Lys Phe Ile Ile Tyr Asp Ser Ser Phe Asp Asp Ala Asn Arg Phe Asn
1380 1385 1390
Leu Val Pro Leu Phe Lys Phe Gly Lys Asp Glu Asn Ser Asp Asp Ser
1395 1400 1405
Ile Cys Ile Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys Trp Tyr
1410 1415 1420
Phe Ser Ser Lys Asp Asp Asn Lys Thr Ala Asp Tyr Asn Gly Gly Thr
1425 1430 1435 1440
Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr Asn Leu
1445 1450 1455
Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser Ser Tyr
1460 1465 1470
Lys Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys
1475 1480 1485
Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln Ile Phe Thr Ala
1490 1495 1500
Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Ala Pro Ser Phe Glu Glu
1505 1510 1515 1520
Met Ile Tyr Gln Phe Asn Asn Leu Thr Ile Asp Cys Lys Asn Leu Asn
1525 1530 1535
Phe Ile Asp Asn Gln Ala His Ile Glu Ile Asp Phe Thr Ala Thr Ala
1540 1545 1550
Gln Asp Gly Arg Phe Leu Gly Ala Glu Thr Phe Ile Ile Pro Val Thr
1555 1560 1565
Lys Lys Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn
1570 1575 1580
Asn Gly Val Gln Tyr Met Gln Ile Gly Ala Tyr Arg Thr Arg Leu Asn
1585 1590 1595 1600
Thr Leu Phe Ala Gln Gln Leu Val Ser Arg Ala Asn Arg Gly Ile Asp
1605 1610 1615
Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly
1620 1625 1630
Ala Gly Thr Tyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu Ser Ile
1635 1640 1645
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His Gly Thr Asn Lys Ser Phe Ala Ile Glu Tyr Val Asp Ile Phe Lys
1650 1655 1660
Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu Thr Ser
1665 1670 1675 1680
Gin Thr Val Val Lys Val Phe Leu Ser Tyr Phe Ile Glu Ala Thr Gly
1685 1690 1695
Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln Lys Glu Thr Thr
1700 1705 1710
Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys Asp Pro His Gly Trp
1715 1720 1725
Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gly Leu Ser Ser Ala Gln
1730 1735 1740
Ala Leu Lys Asn Asp Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ala
1745 1750 1755 1760
Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Met Met Ala His
1765 1770 1775
Arg Leu Leu Gln Glu Gln Asn Phe Asp Ala Ala Asn His Trp Phe Arg
1780 1785 1790
Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val Asp Gly Lys Ile Ala Ile
1795 1800 1805
Tyr His Trp Asn Val Arg Pro Leu Glu Glu Asp Thr Ser Trp Asn Ala
1810 1815 1820
Gln Gln Leu Asp Ser Thr Asp Pro Asp Ala Val Ala Gln Asp Asp Pro
1825 1830 1835 1840
Met His Tyr Lys Val Ala Thr Phe Met Ala Thr Leu Asp Leu Leu Met
1845 1850 1855
Ala Arg Gly Asp Ala Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Ala
1860 1865 1870
Glu Ala Lys Met Trp Tyr Thr Gln Ala Leu Asn Leu Leu Gly Asp Glu
1875 1880 1885
Pro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu Gly Asn
1890 1895 1900
Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val Leu Thr Gln
1905 1910 1915 1920
Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu Leu Gly Thr Ala Asn
1925 1930 1935
Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn Ser Lys Leu Lys Gly
1940 1945 1950
Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn Leu Arg His Asn Leu
1955 1960 1965
Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu Tyr Ala Lys Pro Ala
1970 1975 1980
Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser Ala Ser Gln Gly Gly
1985 1990 1995 2000
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Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His Arg Phe Pro Gln Met
2005 2010 2015
Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu Ile Gln Phe Gly Ser
2020 2025 2030
Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala Glu Ala Met Ser Gln
2035 2040 2045
Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu Thr Ser Ile Arg Met
2050 2055 2060
Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu Lys Thr Ala Leu Gln
2065 2070 2075 2080
Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp Ser Tyr Ser Gln Leu
2085 2090 2095
Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg Ala Leu Ala Leu Arg
2100 2105 2110
Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln Ile Ser Arg Met Ala
2115 2120 2125
Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly Leu Ala Asp Gly
2130 2135 2140
Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile Ala Asp Gly Ile Glu
2145 2150 2155 2160
Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu Lys Val Ala Gln Ser
2165 2170 2175
Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys Ile Gln Arg Asp Asn
2180 2185 2190
Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln Leu Glu Ser Leu Ser
2195 2200 2205
Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu Tyr Leu Lys Thr Gln
2210 2215 2220
Gin Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu Arg Ser Lys Phe Ser
2225 2230 2235 2240
Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg Leu Ser Gly Ile Tyr
2245 2250 2255
Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys Leu Met Ala Glu Gln
2260 2265 2270
Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile Ser Phe Val Lys Pro
2275 2280 2285
Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu Ala Leu
2290 2295 2300
Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp Glu Ser
2305 2310 2315 2320
Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val Val Tyr Asp
2325 2330 2335
Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu Gln Ile Pro Ala
2340 2345 2350
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Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr Lys Glu Asn Gly Leu
2355 2360 2365
Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val Lys Leu Ser Asp Leu
2370 2375 2380
Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val Gly Ser Asn Lys Val
2385 2390 2395 2400
Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro Ala Leu Val Gly Pro
2405 2410 2415
Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly Gly Ser Thr Gln Leu
2420 2425 2430
Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His Gly Thr Asn Asp Ser
2435 2440 2445
Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Tyr Leu Pro Phe Glu
2450 2455 2460
Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn Leu Gln Phe Pro Asn
2465 2470 2475 2480
Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr Met Ser Asp Ile Ile
2485 2490 2495
Leu His Ile Arg Tyr Thr Ile Arg
2500 2504
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Xaa Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
- 136 -
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Met Gln Asn Ser Gln Thr Phe Ser Val Gly Glu Leu
1 5 10
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Ala Gln Asp Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr
1 5 10
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Met Gln Asn Ser Leu
1 5
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Ala Phe Asn Ile Asp Asp Val Ser Leu Phe
1 5 10
- 137 -
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CA 02209659 1998-09-15
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile Gly Ser
1 5 10 15
Leu Gln Leu Phe Ile
30
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
40 Met Tyr Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro
1 5 10
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
- 138 -
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CA 02209659 1998-09-15
(A) LENGTH: 26 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Gly Ile Asp Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro
1 5 10 15
Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu
25
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
20 (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys
1 5 10
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
- 139 -
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CA 02209659 1998-09-15
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly
1 5 10 15
Val Gln Tyr Met Gln Ile
20
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6005 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: RBS
(B) LOCATION: 1..9
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 16..3585
(D) OTHER INFORMATION: /product= "P8"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
AAGAAGGAAT TGATT ATG TCT GAA TCT TTA TTT ACA CAA ACG TTG AAA GAA 51
Met Ser Glu Ser Leu Phe Thr Gin Thr Leu Lys Glu
1 5 10
GCG CGC CGT GAT GCA TTG GTT GCT CAT TAT ATT GCT ACT CAG GTG CCC 99
Ala Arg Arg Asp Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro
15 20 25
GCA GAT TTA AAA GAG AGT ATC CAG ACC GCG GAT GAT CTG TAC GAA TAT 147
Ala Asp Leu Lys Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr
30 35 40
CTG TTG CTG GAT ACC AAA ATT AGC GAT CTG GTT ACT ACT TCA CCG CTG 195
Leu Leu Leu Asp Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu
50 55 60
TCC GAA GCG ATT GGC AGT CTG CAA TTG TTT ATT CAT CGT GCG ATA GAG 243
Ser Glu Ala Ile Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu
65 70 75
- 140 -
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CA 02209659 1998-09-15
GGC TAT GAC GGC ACG CTG GCA GAC TCA GCA AAA CCC TAT TTT GCC GAT 291
Gly Tyr Asp Gly Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp
80 85 90
GAA CAG TTT TTA TAT AAC TGG GAT AGT TTT AAC CAC CGT TAT AGC ACT 339
Glu Gln Phe Leu Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr
95 100 105
TGG GCT GGC AAG GAA CGG TTG AAA TTC TAT GCC GGG GAT TAT ATT GAT 387
Trp Ala Gly Lys Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp
110 115 120
CCA ACA TTG CGA TTG AAT AAG ACC GAG ATA TTT ACC GCA TTT GAA CAA 435
Pro Thr Leu Arg Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln
125 130 135 140
GGT ATT TCT CAA GGG AAA TTA AAA AGT GAA TTA GTC GAA TCT AAA TTA 483
Gly Ile Ser Gln Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu
145 150 155
CGT GAT TAT CTA ATT AGT TAT GAC ACT TTA GCC ACC CTT GAT TAT ATT 531
Arg Asp Tyr Leu Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile
160 165 170
ACT GCC TGC CAA GGC AAA GAT AAT AAA ACC ATC TTC TTT ATT GGC CGT 579
Thr Ala Cys Gln Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg
175 180 185
ACA CAG AAT GCA CCC TAT GCA TTT TAT TGG CGA AAA TTA ACT TTA GTC 627
Thr Gln Asn Ala Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val
190 195 200
ACT GAT GGC GGT AAG TTG AAA CCA GAT CAA TGG TCA GAG TGG CGA GCA 675
Thr Asp Gly Gly Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala
205 210 215 220
ATT AAT GCC GGG ATT AGT GAG GCA TAT TCA GGG CAT GTC GAG CCT TTC 723
Ile Asn Ala Gly Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe
225 230 235
TGG GAA AAT AAC AAG CTG CAC ATC CGT TGG TTT ACT ATC TCG AAA GAA 771
Trp Glu Asn Asn Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu
240 245 250
GAT AAA ATA GAT TTT GTT TAT AAA AAC ATC TGG GTG ATG AGT AGC GAT 819
Asp Lys Ile Asp Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp
255 260 265
TAT AGC TGG GCA TCA AAG AAA AAA ATC TTG GAA CTT TCT TTT ACT GAC 867
Tyr Ser Trp Ala Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp
270 275 280
TAC AAT AGA GTT GGA GCA ACA GGA TCA TCA AGC CCG ACT GAA GTA GCT 915
Tyr Asn Arg Val Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala
285 290 295 300
TCA CAA TAT GGT TCT GAT GCT CAG ATG AAT ATT TCT GAT GAT GGG ACT 963
Ser Gln Tyr Gly Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr
305 310 315
GTA CTT ATT TTT CAG AAT GCC GGC GGA GCT ACT CCC AGT ACT GGA GTG 1011
Val Leu Ile Phe Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val
320 325 330
ACG TTA TGT TAT GAC TCT GGC AAC GTG ATT AAG AAC CTA TCT AGT ACA 1059
Thr Leu Cys Tyr Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr
335 340 345
- 141 -
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CA 02209659 1998-09-15
GGA AGT GCA AAT TTA TCG TCA AAG GAT TAT GCC ACA ACT AAA TTA CGC 1107
Gly Ser Ala Asn Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg
350 355 360
ATG TGT CAT GGA CAA AGT TAC AAT GAT AAT AAC TAC TGC AAT TTT ACA 1155
Met Cys His Gly Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr
365 370 375 380
CTC TCT ATT AAT ACA ATA GAA TTC ACC TCC TAC GGC ACA TTC TCA TCA 1203
Leu Ser Ile Asn Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser
385 390 395
GAT GGA AAA CAA TTT ACA CCA CCT TCT GGT TCT GCC ATT GAT TTA CAC 1251
Asp Gly Lys Gln Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His
400 405 410
CTC CCT AAT TAT GTA GAT CTC AAC GCG CTA TTA GAT ATT AGC CTC GAT 1299
Leu Pro Asn Tyr Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp
415 420 425
TCA CTA CTT AAT TAT GAC GTT CAG GGG CAG TTT GGC GGA TCT AAT CCG 1347
Ser Leu Leu Asn Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro
430 435 440
GTT GAT AAT TTC AGT GGT CCC TAT GGT ATT TAT CTA TGG GAA ATC TTC 1395
Val Asp Asn Phe Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe
445 450 455 460
TTC CAT ATT CCG TTC CTT GTT ACG GTC CGT ATG CAA ACC GAA CAA CGT 1443
Phe His Ile Pro Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg
465 470 475
TAC GAA GAC GCG GAC ACT TGG TAC AAA TAT ATT TTC CGC AGC GCC GGT 1491
Tyr Glu Asp Ala Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly
480 485 490
TAT CGC GAT GCT AAT GGC CAG CTC ATT ATG GAT GGC AGT AAA CCA CGT 1539
Tyr Arg Asp Ala Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg
495 500 505
TAT TGG AAT GTG ATG CCA TTG CAA CTG GAT ACC GCA TGG GAT ACC ACA 1587
Tyr Trp Asn Val Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr
510 515 520
CAG CCC GCC ACC ACT GAT CCA GAT GTG ATC GCT ATG GCG GAC CCG ATG 1635
Gln Pro Ala Thr Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met
525 530 535 540
CAT TAC AAG CTG GCG ATA TTC CTG CAT ACC CTT GAT CTA TTG ATT GCC 1683
His Tyr Lys Leu Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala
545 550 555
CGA GGC GAC AGC GCT TAC CGT CAA CTT GAA CGC GAT ACT CTA GTC GAA 1731
Arg Gly Asp Ser Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu
560 565 570
GCC AAA ATG TAC TAC ATT CAG GCA CAA CAG CTA CTG GGA CCG CGC CCT 1779
Ala Lys Met Tyr Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro
575 580 585
GAT ATC CAT ACC ACC AAT ACT TGG CCA AAT CCC ACC TTG AGT AAA GAA 1827
Asp Ile His Thr Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu
590 595 600
GCT GGC GCT ATT GCC ACA CCG ACA TTC CTC AGT TCA CCG GAG GTG ATG 1875
Ala Gly Ala Ile Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met
605 610 615 620
- 142 -
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CA 02209659 1998-09-15
ACG TTC GCT GCC TGG CTA AGC GCA GGC GAT ACC GCA AAT ATT GGC GAC 1923
Thr Phe Ala Ala Trp Leu Ser Ala Gly Asp Thr Ala Asn Ile Gly Asp
625 630 635
GGT GAT TTC TTG CCA CCG TAC AAC GAT GTA CTA CTC GGT TAC TGG GAT 1971
Gly Asp Phe Leu Pro Pro Tyr Asn Asp Val Leu Leu Gly Tyr Trp Asp
640 645 650
AAA CTT GAG TTA CGC CTA TAC AAC CTG CGC CAC AAT CTG AGT CTG GAT 2019
Lys Leu Glu Leu Arg Leu Tyr Asn Leu Arg His Asn Leu Ser Leu Asp
655 660 665
GGT CAA CCG CTA AAT CTG CCA CTG TAT GCC ACG CCG GTA GAC CCG AAA 2067
Gly Gln Pro Leu Asn Leu Pro Leu Tyr Ala Thr Pro Val Asp Pro Lys
670 675 680
ACC CTG CAA CGC CAG CAA GCC GGA GGG GAC GGT ACA GGC AGT AGT CCG 2115
Thr Leu Gln Arg Gln Gln Ala Gly Gly Asp Gly Thr Gly Ser Ser Pro
685 690 695 700
GCT GGT GGT CAA GGC AGT GTT CAG GGC TGG CGC TAT CCG TTA TTG GTA 2163
Ala Gly Gly Gln Gly Ser Val Gln Gly Trp Arg Tyr Pro Leu Leu Val
705 710 715
GAA CGC GCC CGC TCT GCC GTG AGT TTG TTG ACT CAG TTC GGC AAC AGC 2211
Glu Arg Ala Arg Ser Ala Val Ser Leu Leu Thr Gln Phe Gly Asn Ser
720 725 730
TTA CAA ACA ACG TTA GAA CAT CAG GAT AAT GAA AAA ATG ACG ATA CTG 2259
Leu Gln Thr Thr Leu Glu His Gln Asp Asn Glu Lys Met Thr Ile Leu
735 740 745
TTG CAG ACT CAA CAG GAA GCC ATC CTG AAA CAT CAG CAC GAT ATA CAA 2307
Leu Gln Thr Gln Gln Glu Ala Ile Leu Lys His Gln His Asp Ile Gln
750 755 760
CAA AAT AAT CTA AAA GGA TTA CAA CAC AGC CTG ACC GCA TTA CAG GCT 2355
Gln Asn Asn Leu Lys Gly Leu Gln His Ser Leu Thr Ala Leu Gln Ala
765 770 775 780
AGC CGT GAT GGC GAC ACA TTG CGG CAA AAA CAT TAC AGC GAC CTG ATT 2403
Ser Arg Asp Gly Asp Thr Leu Arg Gln Lys His Tyr Ser Asp Leu Ile
785 790 795
AAC GGT GGT CTA TCT GCG GCA GAA ATC GCC GGT CTG ACA CTA CGC AGC 2451
Asn Gly Gly Leu Ser Ala Ala Glu Ile Ala Gly Leu Thr Leu Arg Ser
800 805 810
ACC GCC ATG ATT ACC AAT GGC GTT GCA ACG GGA TTG CTG ATT GCC GGC 2499
Thr Ala Met Ile Thr Asn Gly Val Ala Thr Gly Leu Leu Ile Ala Gly
815 820 825
GGA ATC GCC AAC GCG GTA CCT AAC GTC TTC GGG CTG GCT AAC GGT GGA 2547
Gly Ile Ala Asn Ala Val Pro Asn Val Phe Gly Leu Ala Asn Gly Gly
830 835 840
TCG GAA TGG GGA GCG CCA TTA ATT GGC TCC GGG CAA GCA ACC CAA GTT 2595
Ser Glu Trp Gly Ala Pro Leu Ile Gly Ser Gly Gln Ala Thr Gln Val
845 850 855 860
GGC GCC GGC ATC CAG GAT CAG AGC GCG GGC ATT TCA GAA GTG ACA GCA 2643
Gly Ala Gly Ile Gln Asp Gln Ser Ala Gly Ile Ser Glu Val Thr Ala
865 870 875
GGC TAT CAG CGT CGT CAG GAA GAA TGG GCA TTG CAA CGG GAT ATT GCT 2691
Gly Tyr Gln Arg Arg Gln Glu Glu Trp Ala Leu Gln Arg Asp Ile Ala
880 885 890
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CA 02209659 1998-09-15
GAT AAC GAA ATA ACC CAA CTG GAT GCC CAG ATA CAA AGC CTG CAA GAG 2739
Asp Asn Glu Ile Thr Gln Leu Asp Ala Gln Ile Gln Ser Leu Gln Glu
895 900 905
CAA ATC ACG ATG GCA CAA AAA CAG ATC ACG CTC TCT GAA ACC GAA CAA 2787
Gln Ile Thr Met Ala Gln Lys Gln Ile Thr Leu Ser Glu Thr Glu Gln
910 915 920
GCG AAT GCC CAA GCG ATT TAT GAC CTG CAA ACC ACT CGT TTT ACC GGG 2835
Ala Asn Ala Gln Ala Ile Tyr Asp Leu Gln Thr Thr Arg Phe Thr Gly
925 930 935 940
CAG GCA CTG TAT AAC TGG ATG GCC GGT CGT CTC TCC GCG CTC TAT TAC 2883
Gln Ala Leu Tyr Asn Trp Met Ala Gly Arg Leu Ser Ala Leu Tyr Tyr
945 950 955
CAA ATG TAT GAT TCC ACT CTG CCA ATC TGT CTC CAG CCA AAA GCC GCA 2931
Gln Met Tyr Asp Ser Thr Leu Pro Ile Cys Leu Gln Pro Lys Ala Ala
960 965 970
TTA GTA CAG GAA TTA GGC GAG AAA GAG AGC GAC AGT CTT TTC CAG GTT 2979
Leu Val Gln Glu Leu Gly Glu Lys Glu Ser Asp Ser Leu Phe Gln Val
975 980 985
CCG GTG TGG AAT GAT CTG TGG CAA GGG CTG TTA GCA GGA GAA GGT TTA 3027
Pro Val Trp Asn Asp Leu Trp Gln Gly Leu Leu Ala Gly Glu Gly Leu
990 995 1000
AGT TCA GAG CTA CAG AAA CTG GAT GCC ATC TGG CTT GCA CGT GGT GGT 3075
Ser Ser Glu Leu Gln Lys Leu Asp Ala Ile Trp Leu Ala Arg Gly Gly
1005 1010 1015 1020
ATT GGG CTA GAA GCC ATC CGC ACC GTG TCG CTG GAT ACC CTG TTT GGC 3123
Ile Gly Leu Glu Ala Ile Arg Thr Val Ser Leu Asp Thr Leu Phe Gly
1025 1030 1035
ACA GGG ACG TTA AGT GAA AAT ATC AAT AAA GTG CTT AAC GGG GAA ACG 3171
Thr Gly Thr Leu Ser Glu Asn Ile Asn Lys Val Leu Asn Gly Glu Thr
1040 1045 1050
GTA TCT CCA TCC GGT GGC GTC ACT CTG GCG CTG ACA GGG GAT ATC TTC 3219
Val Ser Pro Ser Gly Gly Val Thr Leu Ala Leu Thr Gly Asp Ile Phe
1055 1060 1065
CAA GCA ACA CTG GAT TTG AGT CAG CTA GGT TTG GAT AAC TCT TAC AAC 3267
Gln Ala Thr Leu Asp Leu Ser Gln Leu Gly Leu Asp Asn Ser Tyr Asn
1070 1075 1080
TTG GGT AAC GAG AAG AAA CGT CGT ATT AAA CGT ATC GCC GTC ACC CTG 3315
Leu Gly Asn Glu Lys Lys Arg Arg Ile Lys Arg Ile Ala Val Thr Leu
1085 1090 1095 1100
CCA ACA CTT CTG GGG CCA TAT CAA GAT CTT GAA GCC ACA CTG GTA ATG 3363
Pro Thr Leu Leu Gly Pro Tyr Gin Asp Leu Glu Ala Thr Leu Val Met
1105 1110 1115
GGT GCG GAA ATC GCC GCC TTA TCA CAC GGT GTG AAT GAC GGA GGC CGG 3411
Gly Ala Glu Ile Ala Ala Leu Ser His Gly Val Asn Asp Gly Gly Arg
1120 1125 1130
TTT GTT ACC GAC TTT AAC GAC AGC CGT TTT CTG CCT TTT GAA GGT CGA 3459
Phe Val Thr Asp Phe Asn Asp Ser Arg Phe Leu Pro Phe Glu Gly Arg
1135 1140 1145
GAT GCA ACA ACC GGC ACA CTG GAG CTC AAT ATT TTC CAT GCG GGT AAA 3507
Asp Ala Thr Thr Gly Thr Leu Glu Leu Asn Ile Phe His Ala Gly Lys
1150 1155 1160
- 144 -
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CA 02209659 1998-09-15
GAG GGA ACG CAA CAC GAG TTG GTC GCG AAT CTG AGT GAC ATC ATT GTG 3555
Glu Gly Thr Gln His Glu Leu Val Ala Asn Leu Ser Asp Ile Ile Val
1165 1170 1175 1180
CAT CTG AAT TAC ATC ATT CGA GAC GCG TAA ATTTCTTTTC TTTGTCGATT 3605
His Leu Asn Tyr Ile Ile Arg Asp Ala
1185 1189
ACAGGTCCCT ATCAGGGGCC TGTTATTAAG GAGTACTTTA TGCAGGATTC ACCAGAAGTA 3665
TCGATTACAA CGCTGTCACT TCCCAAAGGT GGCGGTGCTA TCAATGGCAT GGGAGAAGCA 3725
CTGAATGCTG CCGGCCCTGA TGGAATGGCC TCCCTATCTC TGCCATTACC CCTTTCGACC 3785
GGCAGAGGGA CGGCTCCTGG ATTATCGCTG ATTTACAGCA ACAGTGCAGG TAATGGGCCT 3845
TTCGGCATCG GCTGGCAATG CGGTGTTATG TCCATTAGCC GACGCACCCA ACATGGCATT 3905
CCACAATACG GTAATGACGA CACGTTCCTA TCCCCACAAG GCGAGGTCAT GAATATCGCC 3965
CTGAATGACC AAGGGCAACC TGATATCCGT CAAGACGTTA AAACGCTGCA AGGCGTTACC 4025
TTGCCAATTT CCTATACCGT GACCCGCTAT CAAGCCCGCC AGATCCTGGA TTTCAGTAAA 4085
ATCGAATACT GGCAACCTGC CTCCGGTCAA GAAGGACGCG CTTTCTGGCT GATATCGACA 4145
CCGGACGGGC ATCTACACAT CTTAGGGAAA ACCGCGCAGG CTTGTCTGGC AAATCCGCAA 4205
AATGACCAAC AAATCGCCCA GTGGTTGCTG GAAGAAACTG TGACGCCAGC CGGTGAACAT 4265
GTCAGCTATC AATATCGAGC CGAAGATGAA GCCCATTGTG ACGACAATGA AAAAACCGCT 4325
CATCCCAATG TTACCGCACA GCGCTATCTG GTACAGGTGA ACTACAGGCA ACATCAAACC 4385
ACAAGCCAGC CTGTTCGTAC TGGATAACGC ACCTCCCGCA CCGGAAGAGT GGCTGTTTCA 4445
TCTGGTCTTT GACCACGGTG AGCGCGTACC TCACTTCATA CCGTGCCAAC ATGGGATGCA 4505
GGTACAGCGC AATGGTCTGT ACGCCCGGAT ATCTTCTCTC GCTATGAATA TGGTTTTGAA 4565
GTGCGTACTC GCCGCTTATG TCAACAAGTG CTGATGTTTC ACCGCACCGC GCTCATGGCC 4625
GGAGAAGCCA GTACCAATGA CGCCCCGGAA CTGGTTGGAC GCTTAATACT GGAATATGAC 4685
AAAAACGCCA GCGTCACCAC GTTGATTACC ATCCGTCAAT TAAGCCATGA ATCGGACGGG 4745
AGGCCAGTCA CCCAGCCACC ACTAGAACTA GCCTGGCAAC GGTTTGATCT GGAGAAAATC 4805
CCGACATGGC AACGCTTTGA CGCACTAGAT AATTTTAACT CGCAGCAACG TTATCAACTG 4865
GTTGATCTGC GGGGAGAAGG GTTGCCAGGT ATGCTGTATC AAGATCGAGG CGCTTGGTGG 4925
TATAAAGCTC CGCAACGTCA GGAAGACGGA GACAGCAATG CCGTCACTTA CGACAAAATC 4985
GCCCCACTGC CTACCCTACC CAATTTGCAG GATAATGCCT CATTGATGGA TATCAACGGA 5045
GACGGCCAAC TGGATTGGGT TGTTACCGCC TCCGGTATTC GCGGATACCA TAGTCAGCAA 5105
CCCGATGGAA AGTGGACGCA CTTTACGCCA ATCAATGCCT TGCCCGTGGA ATATTTTCAT 5165
CCAAGCATCC AGTTCGCTGA CCTTACCGGG GCAGGCTTAT CTGATTTAGT GTTGATCGGG 5225
CCGAAAAGCG TGCGTCTATA TGCCAACCAG CGAAACGGCT GGCGTAAAGG AGAAGATGTC 5285
CCCCAATCCA CAGGTATCAC CCTGCCTGTC ACAGGGACCG ATGCCCGCAA ACTGGTGGCT 5345
TTCAGTGATA TGCTCGGTTC CGGTCAACAA CATCTGGTGG AAATCAAGGG TAATCGCGTC 5405
- 145 -
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CA 02209659 1998-09-15
ACCTGTTGGC CGAATCTAGG GCATGGCCGT TTCGGTCAAC CACTAACTCT GTCAGGATTT 5465
AGCCAGCCCG AAAATAGCTT CAATCCCGAA CGGCTGTTTC TGGCGGATAT CGACGGCTCC 5525
GGCACCACCG ACCTTATCTA TGCGCAATCC GGCTCTTTGC TCATTTATCT CAACCAAAGT 5585
GGTAATCAGT TTGATGCCCC GTTGACATTA GCGTTGCCAG AAGGCGTACA ATTTGACAAC 5645
ACTTGCCAAC TTCAAGTCGC CGATATTCAG GGATTAGGGA TAGCCAGCTT GATTCTGACT 5705
GTGCCACATA TCGCGCCACA TCACTGGCGT TGTGACCTGT CACTGACCAA ACCCTGGTTG 5765
TTGAATGTAA TGAACAATAA CCGGGGCGCA CATCACACGC TACATTATCG TAGTTCCGCG 5825
CAATTCTGGT TGGATGAAAA ATTACAGCTC ACCAAAGCAG GCAAATCTCC GGCTTGTTAT 5885
CTGCCGTTTC CAATGCATTT GCTATGGTAT ACCGAAATTC AGGATGAAAT CAGCGGCAAC 5945
CGGCTCACCA GTGAAGTCAA CTACAGCCAC GGCGTCTGGG ATGGTAAAGA GCGGGAATTC 6005
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1189 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Met Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp
1 5 10 15
Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro Ala Asp Leu Lys
20 25 30
Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr Leu Leu Leu Asp
40 45
Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile
50 55 60
Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu Gly Tyr Asp Gly
65 70 75 80
Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp Glu Gln Phe Leu
85 90 95
Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr Trp Ala Gly Lys
100 105 110
Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp Pro Thr Leu Arg
115 120 125
Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln Gly Ile Ser Gln
130 135 140
Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu Arg Asp Tyr Leu
145 150 155 160
Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile Thr Ala Cys Gln
165 170 175
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Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg Thr Gln Asn Ala
180 185 190
Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val Thr Asp Gly Gly
195 200 205
Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala Ile Asn Ala Gly
210 215 220
Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe Trp Glu Asn Asn
225 230 235 240
Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu Asp Lys Ile Asp
245 250 255
Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp Tyr Ser Trp Ala
260 265 270
Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp Tyr Asn Arg Val
275 280 285
Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala Ser Gln Tyr Gly
290 295 300
Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr Val Leu Ile Phe
305 310 315 320
Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val Thr Leu Cys Tyr
325 330 335
Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr Gly Ser Ala Asn
340 345 350
Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg Met Cys His Gly
355 360 365
Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser Ile Asn
370 375 380
Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser Asp Gly Lys Gln
385 390 395 400
Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His Leu Pro Asn Tyr
405 410 415
Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp Ser Leu Leu Asn
420 425 430
Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro Val Asp Asn Phe
435 440 445
Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe Phe His Ile Pro
450 455 460
Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg Tyr Glu Asp Ala
465 470 475 480
Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly Tyr Arg Asp Ala
485 490 495
Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg Tyr Trp Asn Val
500 505 510
Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr Gln Pro Ala Thr
515 520 525
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Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met His Tyr Lys Leu
530 535 540
Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp Ser
545 550 555 560
Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu Ala Lys Met Tyr
565 570 575
Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro Asp Ile His Thr
580 585 590
Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu Ala Gly Ala Ile
595 600 605
Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met Thr Phe Ala Ala
610 615 620
Trp Leu Ser Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp Phe Leu
625 630 635 640
Pro Pro Tyr Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu Glu Leu
645 650 655
Arg Leu Tyr Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln Pro Leu
660 665 670
Asn Leu Pro Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu Gln Arg
675 680 685
Gln Gln Ala Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly Gly Gln
690 695 700
Gly Ser Val Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg Ala Arg
705 710 715 720
Ser Ala Val Ser Leu Leu Thr Gln Phe Gly Asn Ser Leu Gln Thr Thr
725 730 735
Leu Glu His Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln Thr Gln
740 745 750
Gln Glu Ala Ile Leu Lys His Gln His Asp Ile Gln Gin Asn Asn Leu
755 760 765
Lys Gly Leu Gln His Ser Leu Thr Ala Leu Gln Ala Ser Arg Asp Gly
770 775 780
Asp Thr Leu Arg Gln Lys His Tyr Ser Asp Leu Ile Asn Gly Gly Leu
785 790 795 800
Ser Ala Ala Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala Met Ile
805 810 815
Thr Asn Gly Val Ala Thr Gly Leu Leu Ile Ala Gly Gly Ile Ala Asn
820 825 830
Ala Val Pro Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu Trp Gly
835 840 845
Ala Pro Leu Ile Gly Ser Gly Gln Ala Thr Gln Val Gly Ala Gly Ile
850 855 860
Gln Asp Gln Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr Gln Arg
865 870 875 880
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Arg Gln Glu Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn Glu Ile
885 890 895
Thr Gln Leu Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile Thr Met
900 905 910
Ala Gln Lys Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn Ala Gln
915 920 925
Ala Ile Tyr Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala Leu Tyr
930 935 940
Asn Trp Met Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met Tyr Asp
945 950 955 960
Ser Thr Leu Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val Gln Glu
965 970 975
Leu Gly Glu Lys Glu Ser Asp Ser Leu Phe Gln Val Pro Val Trp Asn
980 985 990
Asp Leu Trp Gln Gly Leu Leu Ala Gly Glu Gly Leu Ser Ser Glu Leu
995 1000 1005
Gln Lys Leu Asp Ala Ile Trp Leu Ala Arg Gly Gly Ile Gly Leu Glu
1010 1015 1020
Ala Ile Arg Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly Thr Leu
1025 1030 1035 1040
Ser Glu Asn Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser Pro Ser
1045 1050 1055
Gly Gly Val Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala Thr Leu
1060 1065 1070
Asp Leu Ser Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly Asn Glu
1075 1080 1085
Lys Lys Arg Arg Ile Lys Arg Ile Ala Val Thr Leu Pro Thr Leu Leu
1090 1095 1100
Gly Pro Tyr Gln Asp Leu Glu Ala Thr Leu Val Met Gly Ala Glu Ile
1105 1110 1115 1120
Ala Ala Leu Ser His Gly Val Asn Asp Gly Gly Arg Phe Val Thr Asp
1125 1130 1135
Phe Asn Asp Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala Thr Thr
1140 1145 1150
Gly Thr Leu Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly Thr Gln
1155 1160 1165
His Glu Leu Val Ala Asn Leu Ser Asp Ile Ile Val His Leu Asn Tyr
1170 1175 1180
Ile Ile Arg Asp Ala
1185 1189
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1881 base pairs
- 149 -
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1881
(D) OTHER INFORMATION: /product= "P8"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
ATG TCT GAA TCT TTA TTT ACA CAA ACG TTG AAA GAA GCG CGC CGT GAT 48
Met Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp
1 5 10 15
GCA TTG GTT GCT CAT TAT ATT GCT ACT CAG GTG CCC GCA GAT TTA AAA 96
Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro Ala Asp Leu Lys
25 30
GAG AGT ATC CAG ACC GCG GAT GAT CTG TAC GAA TAT CTG TTG CTG GAT 144
Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr Leu Leu Leu Asp
20 35 40 45
ACC AAA ATT AGC GAT CTG GTT ACT ACT TCA CCG CTG TCC GAA GCG ATT 192
Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile
50 55 60
GGC AGT CTG CAA TTG TTT ATT CAT CGT GCG ATA GAG GGC TAT GAC GGC 240
Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu Gly Tyr Asp Gly
65 70 75 80
ACG CTG GCA GAC TCA GCA AAA CCC TAT TTT GCC GAT GAA CAG TTT TTA 288
Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp Glu Gln Phe Leu
85 90 95
TAT AAC TGG GAT AGT TTT AAC CAC CGT TAT AGC ACT TGG GCT GGC AAG 336
Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr Trp Ala Gly Lys
100 105 110
GAA CGG TTG AAA TTC TAT GCC GGG GAT TAT ATT GAT CCA ACA TTG CGA 384
Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp Pro Thr Leu Arg
115 120 125
TTG AAT AAG ACC GAG ATA TTT ACC GCA TTT GAA CAA GGT ATT TCT CAA 432
Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln Gly Ile Ser Gln
130 135 140
GGG AAA TTA AAA AGT GAA TTA GTC GAA TCT AAA TTA CGT GAT TAT CTA 480
Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu Arg Asp Tyr Leu
145 150 155 160
ATT AGT TAT GAC ACT TTA GCC ACC CTT GAT TAT ATT ACT GCC TGC CAA 528
Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile Thr Ala Cys Gln
165 170 175
GGC AAA GAT AAT AAA ACC ATC TTC TTT ATT GGC CGT ACA CAG AAT GCA 576
Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg Thr Gln Asn Ala
180 185 190
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CCC TAT GCA TTT TAT TGG CGA AAA TTA ACT TTA GTC ACT GAT GGC GGT 624
Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val Thr Asp Gly Gly
195 200 205
AAG TTG AAA CCA GAT CAA TGG TCA GAG TGG CGA GCA ATT AAT GCC GGG 672
Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala Ile Asn Ala Gly
210 215 220
ATT AGT GAG GCA TAT TCA GGG CAT GTC GAG CCT TTC TGG GAA AAT AAC 720
Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe Trp Glu Asn Asn
225 230 235 240
AAG CTG CAC ATC CGT TGG TTT ACT ATC TCG AAA GAA GAT AAA ATA GAT 768
Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu Asp Lys Ile Asp
245 250 255
TTT GTT TAT AAA AAC ATC TGG GTG ATG AGT AGC GAT TAT AGC TGG GCA 816
Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp Tyr Ser Trp Ala
260 265 270
TCA AAG AAA AAA ATC TTG GAA CTT TCT TTT ACT GAC TAC AAT AGA GTT 864
Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp Tyr Asn Arg Val
275 280 285
GGA GCA ACA GGA TCA TCA AGC CCG ACT GAA GTA GCT TCA CAA TAT GGT 912
Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala Ser Gln Tyr Gly
290 295 300
TCT GAT GCT CAG ATG AAT ATT TCT GAT GAT GGG ACT GTA CTT ATT TTT 960
Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr Val Leu Ile Phe
305 310 315 320
CAG AAT GCC GGC GGA GCT ACT CCC AGT ACT GGA GTG ACG TTA TGT TAT 1008
Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val Thr Leu Cys Tyr
325 330 335
GAC TCT GGC AAC GTG ATT AAG AAC CTA TCT AGT ACA GGA AGT GCA AAT 1056
Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr Gly Ser Ala Asn
340 345 350
TTA TCG TCA AAG GAT TAT GCC ACA ACT AAA TTA CGC ATG TGT CAT GGA 1104
Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg Met Cys His Gly
355 360 365
CAA AGT TAC AAT GAT AAT AAC TAC TGC AAT TTT ACA CTC TCT ATT AAT 1152
Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser Ile Asn
370 375 380
ACA ATA GAA TTC ACC TCC TAC GGC ACA TTC TCA TCA GAT GGA AAA CAA 1200
Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser Asp Gly Lys Gln
385 390 395 400
TTT ACA CCA CCT TCT GGT TCT GCC ATT GAT TTA CAC CTC CCT AAT TAT 1248
Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His Leu Pro Asn Tyr
405 410 415
GTA GAT CTC AAC GCG CTA TTA GAT ATT AGC CTC GAT TCA CTA CTT AAT 1296
Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp Ser Leu Leu Asn
420 425 430
TAT GAC GTT CAG GGG CAG TTT GGC GGA TCT AAT CCG GTT GAT AAT TTC 1344
Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro Val Asp Asn Phe
435 440 445
AGT GGT CCC TAT GGT ATT TAT CTA TGG GAA ATC TTC TTC CAT ATT CCG 1392
Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe Phe His Ile Pro
450 455 460
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TTC CTT GTT ACG GTC CGT ATG CAA ACC GAA CAA CGT TAC GAA GAC GCG 1440
Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg Tyr Glu Asp Ala
465 470 475 480
GAC ACT TGG TAC AAA TAT ATT TTC CGC AGC GCC GGT TAT CGC GAT GCT 1488
Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly Tyr Arg Asp Ala
485 490 495
AAT GGC CAG CTC ATT ATG GAT GGC AGT AAA CCA CGT TAT TGG AAT GTG 1536
Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg Tyr Trp Asn Val
500 505 510
ATG CCA TTG CAA CTG GAT ACC GCA TGG GAT ACC ACA CAG CCC GCC ACC 1584
Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr Gln Pro Ala Thr
515 520 525
ACT GAT CCA GAT GTG ATC GCT ATG GCG GAC CCG ATG CAT TAC AAG CTG 1632
Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met His Tyr Lys Leu
530 535 540
GCG ATA TTC CTG CAT ACC CTT GAT CTA TTG ATT GCC CGA GGC GAC AGC 1680
Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp Ser
545 550 555 560
GCT TAC CGT CAA CTT GAA CGC GAT ACT CTA GTC GAA GCC AAA ATG TAC 1728
Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu Ala Lys Met Tyr
565 570 575
TAC ATT CAG GCA CAA CAG CTA CTG GGA CCG CGC CCT GAT ATC CAT ACC 1776
Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro Asp Ile His Thr
580 585 590
ACC AAT ACT TGG CCA AAT CCC ACC TTG AGT AAA GAA GCT GGC GCT ATT 1824
Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu Ala Gly Ala Ile
595 600 605
GCC ACA CCG ACA TTC CTC AGT TCA CCG GAG GTG ATG ACG TTC GCT GCC 1872
Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met Thr Phe Ala Ala
610 615 620
TGG CTA AGC 1881
Trp Leu Ser
625
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 627 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Met Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp
1 5 10 15
Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro Ala Asp Leu Lys
20 25 30
Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr Leu Leu Leu Asp
35 40 45
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Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile
50 55 60
Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu Gly Tyr Asp Gly
65 70 75 80
Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp Glu Gln Phe Leu
85 90 95
Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr Trp Ala Gly Lys
100 105 110
Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp Pro Thr Leu Arg
115 120 125
Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln Gly Ile Ser Gln
130 135 140
Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu Arg Asp Tyr Leu
145 150 155 160
Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile Thr Ala Cys Gln
165 170 175
Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg Thr Gln Asn Ala
180 185 190
Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val Thr Asp Gly Gly
195 200 205
Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala Ile Asn Ala Gly
210 215 220
Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe Trp Glu Asn Asn
225 230 235 240
Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu Asp Lys Ile Asp
245 250 255
Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp Tyr Ser Trp Ala
260 265 270
Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp Tyr Asn Arg Val
275 280 285
Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala Ser Gln Tyr Gly
290 295 300
Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr Val Leu Ile Phe
305 310 315 320
Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val Thr Leu Cys Tyr
325 330 335
Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr Gly Ser Ala Asn
340 345 350
Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg Met Cys His Gly
355 360 365
Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser Ile Asn
370 375 380
Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser Asp Gly Lys Gln
385 390 395 400
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Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His Leu Pro Asn Tyr
405 410 415
Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp Ser Leu Leu Asn
420 425 430
Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro Val Asp Asn Phe
435 440 445
Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe Phe His Ile Pro
450 455 460
Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg Tyr Glu Asp Ala
465 470 475 480
Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly Tyr Arg Asp Ala
485 490 495
Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg Tyr Trp Asn Val
500 505 510
Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr Gln Pro Ala Thr
515 520 525
Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met His Tyr Lys Leu
530 535 540
Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp Ser
545 550 555 560
Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu Ala Lys Met Tyr
565 570 575
Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro Asp Ile His Thr
580 585 590
Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu Ala Gly Ala Ile
595 600 605
Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met Thr Phe Ala Ala
610 615 620
Trp Leu Ser
625
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1689 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1689
(D) OTHER INFORMATION: /product= "S8"
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
GCA GGC GAT ACC GCA AAT ATT GGC GAC GGT GAT TTC TTG CCA CCG TAC 48
Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp Phe Leu Pro Pro Tyr
1 5 10 15
AAC GAT GTA CTA CTC GGT TAC TGG GAT AAA CTT GAG TTA CGC CTA TAC 96
Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu Glu Leu Arg Leu Tyr
20 25 30
AAC CTG CGC CAC AAT CTG AGT CTG GAT GGT CAA CCG CTA AAT CTG CCA 144
Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln Pro Leu Asn Leu Pro
35 40 45
CTG TAT GCC ACG CCG GTA GAC CCG AAA ACC CTG CAA CGC CAG CAA GCC 192
Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu Gln Arg Gln Gln Ala
50 55 - 60
GGA GGG GAC GGT ACA GGC AGT AGT CCG GCT GGT GGT CAA GGC AGT GTT 240
Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly Gly Gln Gly Ser Val
65 70 75 80
CAG GGC TGG CGC TAT CCG TTA TTG GTA GAA CGC GCC CGC TCT GCC GTG 288
Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg Ala Arg Ser Ala Val
85 90 95
AGT TTG TTG ACT CAG TTC GGC AAC AGC TTA CAA ACA ACG TTA GAA CAT 336
Ser Leu Leu Thr Gln Phe Gly Asn Ser Leu Gln Thr Thr Leu Glu His
100 105 110
CAG GAT AAT GAA AAA ATG ACG ATA CTG TTG CAG ACT CAA CAG GAA GCC 384
Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln Thr Gln Gln Glu Ala
115 120 125
ATC CTG AAA CAT CAG CAC GAT ATA CAA CAA AAT AAT CTA AAA GGA TTA 432
Ile Leu Lys His Gln His Asp Ile Gln Gln Asn Asn Leu Lys Gly Leu
130 135 140
CAA CAC AGC CTG ACC GCA TTA CAG GCT AGC CGT GAT GGC GAC ACA TTG 480
Gln His Ser Leu Thr Ala Leu Gln Ala Ser Arg Asp Gly Asp Thr Leu
145 150 155 160
CGG CAA AAA CAT TAC AGC GAC CTG ATT AAC GGT GGT CTA TCT GCG GCA 528
Arg Gln Lys His Tyr Ser Asp Leu Ile Asn Gly Gly Leu Ser Ala Ala
165 170 175
GAA ATC GCC GGT CTG ACA CTA CGC AGC ACC GCC ATG ATT ACC AAT GGC 576
Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala Met Ile Thr Asn Gly
180 185 190
GTT GCA ACG GGA TTG CTG ATT GCC GGC GGA ATC GCC AAC GCG GTA CCT 624
Val Ala Thr Gly Leu Leu Ile Ala Gly Gly Ile Ala Asn Ala Val Pro
195 200 205
AAC GTC TTC GGG CTG GCT AAC GGT GGA TCG GAA TGG GGA GCG CCA TTA 672
Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu Trp Gly Ala Pro Leu
210 215 220
ATT GGC TCC GGG CAA GCA ACC CAA GTT GGC GCC GGC ATC CAG GAT CAG 720
Ile Gly Ser Gly Gln Ala Thr Gln Val Gly Ala Gly Ile Gln Asp Gln
225 230 235 240
AGC GCG GGC ATT TCA GAA GTG ACA GCA GGC TAT CAG CGT CGT CAG GAA 768
Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr Gln Arg Arg Gln Glu
245 250 255
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GAA TGG GCA TTG CAA CGG GAT ATT GCT GAT AAC GAA ATA ACC CAA CTG 816
Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn Glu Ile Thr Gln Leu
260 265 270
GAT GCC CAG ATA CAA AGC CTG CAA GAG CAA ATC ACG ATG GCA CAA AAA 864
Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile Thr Met Ala Gln Lys
275 280 285
CAG ATC ACG CTC TCT GAA ACC GAA CAA GCG AAT GCC CAA GCG ATT TAT 912
Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn Ala Gln Ala Ile Tyr
290 295 300
GAC CTG CAA ACC ACT CGT TTT ACC GGG CAG GCA CTG TAT AAC TGG ATG 960
Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala Leu Tyr Asn Trp Met
305 310 315 320
GCC GGT CGT CTC TCC GCG CTC TAT TAC CAA ATG TAT GAT TCC ACT CTG 1008
Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met Tyr Asp Ser Thr Leu
325 330 335
CCA ATC TGT CTC CAG CCA AAA GCC GCA TTA GTA CAG GAA TTA GGC GAG 1056
Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val Gln Glu Leu Gly Glu
340 345 350
AAA GAG AGC GAC AGT CTT TTC CAG GTT CCG GTG TGG AAT GAT CTG TGG 1104
Lys Glu Ser Asp Ser Leu Phe Gln Val Pro Val Trp Asn Asp Leu Trp
355 360 365
CAA GGG CTG TTA GCA GGA GAA GGT TTA AGT TCA GAG CTA CAG AAA CTG 1152
Gln Gly Leu Leu Ala Gly Glu Gly Leu Ser Ser Glu Leu Gln Lys Leu
370 375 380
GAT GCC ATC TGG CTT GCA CGT GGT GGT ATT GGG CTA GAA GCC ATC CGC 1200
Asp Ala Ile Trp Leu Ala Arg Gly Gly Ile Gly Leu Glu Ala Ile Arg
385 390 395 400
ACC GTG TCG CTG GAT ACC CTG TTT GGC ACA GGG ACG TTA AGT GAA AAT 1248
Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly Thr Leu Ser Glu Asn
405 410 415
ATC AAT AAA GTG CTT AAC GGG GAA ACG GTA TCT CCA TCC GGT GGC GTC 1296
Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser Pro Ser Gly Gly Val
420 425 430
ACT CTG GCG CTG ACA GGG GAT ATC TTC CAA GCA ACA CTG GAT TTG AGT 1344
Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala Thr Leu Asp Leu Ser
435 440 445
CAG CTA GGT TTG GAT AAC TCT TAC AAC TTG GGT AAC GAG AAG AAA CGT 1392
Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly Asn Glu Lys Lys Arg
450 455 460
CGT ATT AAA CGT ATC GCC GTC ACC CTG CCA ACA CTT CTG GGG CCA TAT 1440
Arg Ile Lys Arg Ile Ala Val Thr Leu Pro Thr Leu Leu Gly Pro Tyr
465 470 475 480
CAA GAT CTT GAA GCC ACA CTG GTA ATG GGT GCG GAA ATC GCC GCC TTA 1488
Gln Asp Leu Glu Ala Thr Leu Val Met Gly Ala Glu Ile Ala Ala Leu
485 490 495
TCA CAC GGT GTG AAT GAC GGA GGC CGG TTT GTT ACC GAC TTT AAC GAC 1536
Ser His Gly Val Asn Asp Gly Gly Arg Phe Val Thr Asp Phe Asn Asp
500 505 510
AGC CGT TTT CTG CCT TTT GAA GGT CGA GAT GCA ACA ACC GGC ACA CTG 1584
Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala Thr Thr Gly Thr Leu
515 520 525
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GAG CTC AAT ATT TTC CAT GCG GGT AAA GAG GGA ACG CAA CAC GAG TTG 1632
Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly Thr Gln His Glu Leu
530 535 540
GTC GCG AAT CTG AGT GAC ATC ATT GTG CAT CTG AAT TAC ATC ATT CGA 1680
Val Ala Asn Leu Ser Asp Ile Ile Val His Leu Asn Tyr Ile Ile Arg
545 550 555 560
GAC GCG TAA 1689
Asp Ala
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 562 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp Phe Leu Pro Pro Tyr
1 5 10 15
Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu Glu Leu Arg Leu Tyr
20 25 30
Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln Pro Leu Asn Leu Pro
35 40 45
Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu Gln Arg Gln Gln Ala
50 55 60
Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly Gly Gln Gly Ser Val
65 70 75 80
Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg Ala Arg Ser Ala Val
85 90 95
Ser Leu Leu Thr Gln Phe Gly Asn Ser Leu Gln Thr Thr Leu Glu His
100 105 110
Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln Thr Gln Gln Glu Ala
115 120 125
Ile Leu Lys His Gln His Asp Ile Gln Gln Asn Asn Leu Lys Gly Leu
130 135 140
Gln His Ser Leu Thr Ala Leu Gln Ala Ser Arg Asp Gly Asp Thr Leu
145 150 155 160
Arg Gln Lys His Tyr Ser Asp Leu Ile Asn Gly Gly Leu Ser Ala Ala
165 170 175
Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala Met Ile Thr Asn Gly
180 185 190
Val Ala Thr Gly Leu Leu Ile Ala Gly Gly Ile Ala Asn Ala Val Pro
195 200 205
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Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu Trp Gly Ala Pro Leu
210 215 220
Ile Gly Ser Gly Gln Ala Thr Gln Val Gly Ala Gly Ile Gln Asp Gln
225 230 235 240
Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr Gln Arg Arg Gln Glu
245 250 255
Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn Glu Ile Thr Gln Leu
260 265 270
Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile Thr Met Ala Gln Lys
275 280 285
Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn Ala Gln Ala Ile Tyr
290 295 300
Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala Leu Tyr Asn Trp Met
305 310 315 320
Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met Tyr Asp Ser Thr Leu
325 330 335
Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val Gln Glu Leu Gly Glu
340 345 350
Lys Glu Ser Asp Ser Leu Phe Gln Val Pro Val Trp Asn Asp Leu Trp
355 360 365
Gln Gly Leu Leu Ala Gly Glu Gly Leu Ser Ser Glu Leu Gln Lys Leu
370 375 380
Asp Ala Ile Trp Leu Ala Arg Gly Gly Ile Gly Leu Glu Ala Ile Arg
385 390 395 400
Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly Thr Leu Ser Glu Asn
405 410 415
Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser Pro Ser Gly Gly Val
420 425 430
Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala Thr Leu Asp Leu Ser
435 440 445
Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly Asn Glu Lys Lys Arg
450 455 460
Arg Ile Lys Arg Ile Ala Val Thr Leu Pro Thr Leu Leu Gly Pro Tyr
465 470 475 480
Gln Asp Leu Glu Ala Thr Leu Val Met Gly Ala Glu Ile Ala Ala Leu
485 490 495
Ser His Gly Val Asn Asp Gly Gly Arg Phe Val Thr Asp Phe Asn Asp
500 505 510
Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala Thr Thr Gly Thr Leu
515 520 525
Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly Thr Gln His Glu Leu
530 535 540
Val Ala Asn Leu Ser Asp Ile Ile Val His Leu Asn Tyr Ile Ile Arg
545 550 555 560
Asp Ala
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(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4458 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..4458
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
ATG CAG GAT TCA CCA GAA GTA TCG ATT ACA ACG CTG TCA CTT CCC AAA 48
Met Gln Asp Ser Pro Glu Val Ser Ile Thr Thr Leu Ser Leu Pro Lys
1 5 10 15
GGT GGC GGT GCT ATC AAT GGC ATG GGA GAA GCA CTG AAT GCT GCC GGC 96
Gly Gly Gly Ala Ile Asn Gly Met Gly Glu Ala Leu Asn Ala Ala Gly
25 30
CCT GAT GGA ATG GCC TCC CTA TCT CTG CCA TTA CCC CTT TCG ACC GGC 144
Pro Asp Gly Met Ala Ser Leu Ser Leu Pro Leu Pro Leu Ser Thr Gly
35 40 45
AGA GGG ACG GCT CCT GGA TTA TCG CTG ATT TAC AGC AAC AGT GCA GGT 192
Arg Gly Thr Ala Pro Gly Leu Ser Leu Ile Tyr Ser Asn Ser Ala Gly
50 55 60
AAT GGG CCT TTC GGC ATC GGC TGG CAA TGC GGT GTT ATG TCC ATT AGC 240
Asn Gly Pro Phe Gly Ile Gly Trp Gin Cys Gly Val Met Ser Ile Ser
65 70 75 80
CGA CGC ACC CAA CAT GGC ATT CCA CAA TAC GGT AAT GAC GAC ACG TTC 288
Arg Arg Thr Gln His Gly Ile Pro Gln Tyr Gly Asn Asp Asp Thr Phe
85 90 95
CTA TCC CCA CAA GGC GAG GTC ATG AAT ATC GCC CTG AAT GAC CAA GGG 336
Leu Ser Pro Gln Gly Glu Val Met Asn Ile Ala Leu Asn Asp Gln Gly
100 105 110
CAA CCT GAT ATC CGT CAA GAC GTT AAA ACG CTG CAA GGC GTT ACC TTG 384
Gln Pro Asp Ile Arg Gln Asp Val Lys Thr Leu Gln Gly Val Thr Leu
115 120 125
CCA ATT TCC TAT ACC GTG ACC CGC TAT CAA GCC CGC CAG ATC CTG GAT 432
Pro Ile Ser Tyr Thr Val Thr Arg Tyr Gln Ala Arg Gln Ile Leu Asp
130 135 140
TTC AGT AAA ATC GAA TAC TGG CAA CCT GCC TCC GGT CAA GAA GGA CGC 480
Phe Ser Lys Ile Glu Tyr Trp Gln Pro Ala Ser Gly Gln Glu Gly Arg
145 150 155 160
GCT TTC TGG CTG ATA TCG ACA CCG GAC GGG CAT CTA CAC ATC TTA GGG 528
Ala Phe Trp Leu Ile Ser Thr Pro Asp Gly His Leu His Ile Leu Gly
165 170 175
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AAA ACC GCG CAG GCT TGT CTG GCA AAT CCG CAA AAT GAC CAA CAA ATC 576
Lys Thr Ala Gln Ala Cys Leu Ala Asn Pro Gln Asn Asp Gln Gln Ile
180 185 190
GCC CAG TGG TTG CTG GAA GAA ACT GTG ACG CCA GCC GGT GAA CAT GTC 624
Ala Gln Trp Leu Leu Glu Glu Thr Val Thr Pro Ala Gly Glu His Val
195 200 205
AGC TAT CAA TAT CGA GCC GAA GAT GAA GCC CAT TGT GAC GAC AAT GAA 672
Ser Tyr Gln Tyr Arg Ala Glu Asp Glu Ala His Cys Asp Asp Asn Glu
210 215 220
AAA ACC GCT CAT CCC AAT GTT ACC GCA CAG CGC TAT CTG GTA CAG GTG 720
Lys Thr Ala His Pro Asn Val Thr Ala Gln Arg Tyr Leu Val Gln Val
225 230 235 240
AAC TAC GGC AAC ATC AAA CCA CAA GCC AGC CTG TTC GTA CTG GAT AAC 768
Asn Tyr Gly Asn Ile Lys Pro Gln Ala Ser Leu Phe Val Leu Asp Asn
245 250 255
GCA CCT CCC GCA CCG GAA GAG TGG CTG TTT CAT CTG GTC TTT GAC CAC 816
Ala Pro Pro Ala Pro Glu Glu Trp Leu Phe His Leu Val Phe Asp His
260 265 270
GGT GAG CGC GAT ACC TCA CTT CAT ACC GTG CCA ACA TGG GAT GCA GGT 864
Gly Glu Arg Asp Thr Ser Leu His Thr Val Pro Thr Trp Asp Ala Gly
275 280 285
ACA GCG CAA TGG TCT GTA CGC CCG GAT ATC TTC TCT CGC TAT GAA TAT 912
Thr Ala Gln Trp Ser Val Arg Pro Asp Ile Phe Ser Arg Tyr Glu Tyr
290 295 300
GGT TTT GAA GTG CGT ACT CGC CGC TTA TGT CAA CAA GTG CTG ATG TTT 960
Gly Phe Glu Val Arg Thr Arg Arg Leu Cys Gln Gln Val Leu Met Phe
305 310 315 320
CAC CGC ACC GCG CTC ATG GCC GGA GAA GCC AGT ACC AAT GAC GCC CCG 1008
His Arg Thr Ala Leu Met Ala Gly Glu Ala Ser Thr Asn Asp Ala Pro
325 330 335
GAA CTG GTT GGA CGC TTA ATA CTG GAA TAT GAC AAA AAC GCC AGC GTC 1056
Glu Leu Val Gly Arg Leu Ile Leu Glu Tyr Asp Lys Asn Ala Ser Val
340 345 350
ACC ACG TTG ATT ACC ATC CGT CAA TTA AGC CAT GAA TCG GAC GGG AGG 1104
Thr Thr Leu Ile Thr Ile Arg Gln Leu Ser His Glu Ser Asp Gly Arg
355 360 365
CCA GTC ACC CAG CCA CCA CTA GAA CTA GCC TGG CAA CGG TTT GAT CTG 1152
Pro Val Thr Gln Pro Pro Leu Glu Leu Ala Trp Gln Arg Phe Asp Leu
370 375 380
GAG AAA ATC CCG ACA TGG CAA CGC TTT GAC GCA CTA GAT AAT TTT AAC 1200
Glu Lys Ile Pro Thr Trp Gln Arg Phe Asp Ala Leu Asp Asn Phe Asn
385 390 395 400
TCG CAG CAA CGT TAT CAA CTG GTT GAT CTG CGG GGA GAA GGG TTG CCA 1248
Ser Gln Gln Arg Tyr Gln Leu Val Asp Leu Arg Gly Glu Gly Leu Pro
405 410 415
GGT ATG CTG TAT CAA GAT CGA GGC GCT TGG TGG TAT AAA GCT CCG CAA 1296
Gly Met Leu Tyr Gln Asp Arg Gly Ala Trp Trp Tyr Lys Ala Pro Gln
420 425 430
CGT CAG GAA GAC GGA GAC AGC AAT GCC GTC ACT TAC GAC AAA ATC GCC 1344
Arg Gln Glu Asp Gly Asp Ser Asn Ala Val Thr Tyr Asp Lys Ile Ala
435 440 445
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CCA CTG CCT ACC CTA CCC AAT TTG CAG GAT AAT GCC TCA TTG ATG GAT 1392
Pro Leu Pro Thr Leu Pro Asn Leu Gln Asp Asn Ala Ser Leu Met Asp
450 455 460
ATC AAC GGA GAC GGC CAA CTG GAT TGG GTT GTT ACC GCC TCC GGT ATT 1440
Ile Asn Gly Asp Gly Gln Leu Asp Trp Val Val Thr Ala Ser Gly Ile
465 470 475 480
CGC GGA TAC CAT AGT CAG CAA CCC GAT GGA AAG TGG ACG CAC TTT ACG 1488
Arg Gly Tyr His Ser Gln Gln Pro Asp Gly Lys Trp Thr His Phe Thr
485 490 495
CCA ATC AAT GCC TTG CCC GTG GAA TAT TTT CAT CCA AGC ATC CAG TTC 1536
Pro Ile Asn Ala Leu Pro Val Glu Tyr Phe His Pro Ser Ile Gln Phe
500 505 510
GCT GAC CTT ACC GGG GCA GGC TTA TCT GAT TTA GTG TTG ATC GGG CCG 1584
Ala Asp Leu Thr Gly Ala Gly Leu Ser Asp Leu Val Leu Ile Gly Pro
515 520 525
AAA AGC GTG CGT CTA TAT GCC AAC CAG CGA AAC GGC TGG CGT AAA GGA 1632
Lys Ser Val Arg Leu Tyr Ala Asn Gln Arg Asn Gly Trp Arg Lys Gly
530 535 540
GAA GAT GTC CCC CAA TCC ACA GGT ATC ACC CTG CCT GTC ACA GGG ACC 1680
Glu Asp Val Pro Gln Ser Thr Gly Ile Thr Leu Pro Val Thr Gly Thr
545 550 555 560
GAT GCC CGC AAA CTG GTG GCT TTC AGT GAT ATG CTC GGT TCC GGT CAA 1728
Asp Ala Arg Lys Leu Val Ala Phe Ser Asp Met Leu Gly Ser Gly Gln
565 570 575
CAA CAT CTG GTG GAA ATC AAG GGT AAT CGC GTC ACC TGT TGG CCG AAT 1776
Gln His Leu Val Glu Ile Lys Gly Asn Arg Val Thr Cys Trp Pro Asn
580 585 590
CTA GGG CAT GGC CGT TTC GGT CAA CCA CTA ACT CTG TCA GGA TTT AGC 1824
Leu Gly His Gly Arg Phe Gly Gln Pro Leu Thr Leu Ser Gly Phe Ser
595 600 605
CAG CCC GAA AAT AGC TTC AAT CCC GAA CGG CTG TTT CTG GCG GAT ATC 1872
Gln Pro Glu Asn Ser Phe Asn Pro Glu Arg Leu Phe Leu Ala Asp Ile
610 615 620
GAC GGC TCC GGC ACC ACC GAC CTT ATC TAT GCG CAA TCC GGC TCT TTG 1920
Asp Gly Ser Gly Thr Thr Asp Leu Ile Tyr Ala Gln Ser Gly Ser Leu
625 630 635 640
CTC ATT TAT CTC AAC CAA AGT GGT AAT CAG TTT GAT GCC CCG TTG ACA 1968
Leu Ile Tyr Leu Asn Gln Ser Gly Asn Gln Phe Asp Ala Pro Leu Thr
645 650 655
TTA GCG TTG CCA GAA GGC GTA CAA TTT GAC AAC ACT TGC CAA CTT CAA 2016
Leu Ala Leu Pro Glu Gly Val Gln Phe Asp Asn Thr Cys Gln Leu Gln
660 665 670
GTC GCC GAT ATT CAG GGA TTA GGG ATA GCC AGC TTG ATT CTG ACT GTG 2064
Val Ala Asp Ile Gln Gly Leu Gly Ile Ala Ser Leu Ile Leu Thr Val
675 680 685
CCA CAT ATC GCG CCA CAT CAC TGG CGT TGT GAC CTG TCA CTG ACC AAA 2112
Pro His Ile Ala Pro His His Trp Arg Cys Asp Leu Ser Leu Thr Lys
690 695 700
CCC TGG TTG TTG AAT GTA ATG AAC AAT AAC CGG GGC GCA CAT CAC ACG 2160
Pro Trp Leu Leu Asn Val Met Asn Asn Asn Arg Gly Ala His His Thr
705 710 715 720
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CTA CAT TAT CGT AGT TCC GCG CAA TTC TGG TTG GAT GAA AAA TTA CAG 2208
Leu His Tyr Arg Ser Ser Ala Gln Phe Trp Leu Asp Glu Lys Leu Gln
725 730 735
CTC ACC AAA GCA GGC AAA TCT CCG GCT TGT TAT CTG CCG TTT CCA ATG 2256
Leu Thr Lys Ala Gly Lys Ser Pro Ala Cys Tyr Leu Pro Phe Pro Met
740 745 750
CAT TTG CTA TGG TAT ACC GAA ATT CAG GAT GAA ATC AGC GGC AAC CGG 2304
His Leu Leu Trp Tyr Thr Glu Ile Gln Asp Glu Ile Ser Gly Asn Arg
755 760 765
CTC ACC AGT GAA GTC AAC TAC AGC CAC GGC GTC TGG GAT GGT AAA GAG 2352
Leu Thr Ser Glu Val Asn Tyr Ser His Gly Val Trp Asp Gly Lys Glu
770 775 780
CGG GAA TTC AGA GGA TTT GGC TGC ATC AAA CAG ACA GAT ACC ACA ACG 2400
Arg Glu Phe Arg Gly Phe Gly Cys Ile Lys Gln Thr Asp Thr Thr Thr
785 790 795 800
TTT TCT CAC GGC ACC GCC CCC GAA CAG GCG GCA CCG TCG CTG AGT ATT 2448
Phe Ser His Gly Thr Ala Pro Glu Gln Ala Ala Pro Ser Leu Ser Ile
805 810 815
AGC TGG TTT GCC ACC GGC ATG GAT GAA GTA GAC AGC CAA TTA GCT ACG 2496
Ser Trp Phe Ala Thr Gly Met Asp Glu Val Asp Ser Gln Leu Ala Thr
820 825 830
GAA TAT TGG CAG GCA GAC ACG CAA GCT TAT AGC GGA TTT GAA ACC CGT 2544
Glu Tyr Trp Gln Ala Asp Thr Gln Ala Tyr Ser Gly Phe Glu Thr Arg
835 840 845
TAT ACC GTC TGG GAT CAC ACC AAC CAG ACA GAC CAA GCA TTT ACC CCC 2592
Tyr Thr Val Trp Asp His Thr Asn Gln Thr Asp Gln Ala Phe Thr Pro
850 855 860
AAT GAG ACA CAA CGT AAC TGG CTG ACG CGA GCG CTT AAA GGC CAA CTG 2640
Asn Glu Thr Gln Arg Asn Trp Leu Thr Arg Ala Leu Lys Gly Gln Leu
865 870 875 880
CTA CGC ACT GAG CTC TAC GGT CTG GAC GGA ACA GAT AAG CAA ACA GTG 2688
Leu Arg Thr Glu Leu Tyr Gly Leu Asp Gly Thr Asp Lys Gln Thr Val
885 890 895
CCT TAT ACC GTC AGT GAA TCG CGC TAT CAG GTA CGC TCT ATT CCC GTA 2736
Pro Tyr Thr Val Ser Glu Ser Arg Tyr Gln Val Arg Ser Ile Pro Val
900 905 910
AAT AAA GAA ACT GAA TTA TCT GCC TGG GTG ACT GCT ATT GAA AAT CGC 2784
Asn Lys Glu Thr Glu Leu Ser Ala Trp Val Thr Ala Ile Glu Asn Arg
915 920 925
AGC TAC CAC TAT GAA CGT ATC ATC ACT GAC CCA CAG TTC AGC CAG AGT 2832
Ser Tyr His Tyr Glu Arg Ile Ile Thr Asp Pro Gin Phe Ser Gln Ser
930 935 940
ATC AAG TTG CAA CAC GAT ATC TTT GGT CAA TCA CTG CAA AGT GTC GAT 2880
Ile Lys Leu Gln His Asp Ile Phe Gly Gln Ser Leu Gln Ser Val Asp
945 950 955 960
ATT GCC TGG CCG CGC CGC GAA AAA CCA GCA GTG AAT CCC TAC CCG CCT 2928
Ile Ala Trp Pro Arg Arg Glu Lys Pro Ala Val Asn Pro Tyr Pro Pro
965 970 975
ACC CTG CCG GAA ACG CTA TTT GAC AGC AGC TAT GAT GAT CAA CAA CAA 2976
Thr Leu Pro Glu Thr Leu Phe Asp Ser Ser Tyr Asp Asp Gln Gln Gln
980 985 990
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CTA TTA CGT CTG GTG AGA CAA AAA AAT AGC TGG CAT CAC CTG ACT GAT 3024
Leu Leu Arg Leu Val Arg Gln Lys Asn Ser Trp His His Leu Thr Asp
995 1000 1005
GGG GAA AAC TGG CGA TTA GGT TTA CCG AAT GCA CAA CGC CGT GAT GTT 3072
Gly Glu Asn Trp Arg Leu Gly Leu Pro Asn Ala Gln Arg Arg Asp Val
1010 1015 1020
TAT ACT TAT GAC CGG AGC AAA ATT CCA ACC GAA GGG ATT TCC CTT GAA 3120
Tyr Thr Tyr Asp Arg Ser Lys Ile Pro Thr Glu Gly Ile Ser Leu Glu
1025 1030 1035 1040
ATC TTG CTG AAA GAT GAT GGC CTG CTA GCA GAT GAA AAA GCG GCC GTT 3168
Ile Leu Leu Lys Asp Asp Gly Leu Leu Ala Asp Glu Lys Ala Ala Val
1045 1050 1055
TAT CTG GGA CAA CAA CAG ACG TTT TAC ACC GCC GGT CAA GCG GAA GTC 3216
Tyr Leu Gly Gln Gln Gln Thr Phe Tyr Thr Ala Gly Gln Ala Glu Val
1060 1065 1070
ACT CTA GAA AAA CCC ACG TTA CAA GCA CTG GTC GCG TTC CAA GAA ACC 3264
Thr Leu Glu Lys Pro Thr Leu Gln Ala Leu Val Ala Phe Gln Glu Thr
1075 1080 1085
GCC ATG ATG GAC GAT ACC TCA TTA CAG GCG TAT GAA GGC GTG ATT GAA 3312
Ala Met Met Asp Asp Thr Ser Leu Gln Ala Tyr Glu Gly Val Ile Glu
1090 1095 1100
GAG CAA GAG TTG AAT ACC GCG CTG ACA CAG GCC GGT TAT CAG CAA GTC 3360
Glu Gln Glu Leu Asn Thr Ala Leu Thr Gln Ala Gly Tyr Gln Gln Val
1105 1110 1115 1120
GCG CGG TTG TTT AAT ACC AGA TCA GAA AGC CCG GTA TGG GCG GCA CGG 3408
Ala Arg Leu Phe Asn Thr Arg Ser Glu Ser Pro Val Trp Ala Ala Arg
1125 1130 1135
CAA GGT TAT ACC GAT TAC GGT GAC GCC GCA CAG TTC TGG CGG CCT CAG 3456
Gln Gly Tyr Thr Asp Tyr Gly Asp Ala Ala Gln Phe Trp Arg Pro Gln
1140 1145 1150
GCT CAG CGT AAC TCG TTG CTG ACA GGG AAA ACC ACA CTG ACC TGG GAT 3504
Ala Gln Arg Asn Ser Leu Leu Thr Gly Lys Thr Thr Leu Thr Trp Asp
1155 1160 1165
ACC CAT CAT TGT GTA ATA ATA CAG ACT CAA GAT GCC GCT GGA TTA ACG 3552
Thr His His Cys Val Ile Ile Gln Thr Gln Asp Ala Ala Gly Leu Thr
1170 1175 1180
ACG CAA GCC CAT TAC GAT TAT CGT TTC CTT ACA CCG GTA CAA CTG ACA 3600
Thr Gln Ala His Tyr Asp Tyr Arg Phe Leu Thr Pro Val Gln Leu Thr
1185 1190 1195 1200
GAT ATT AAT GAT AAT CAA CAT ATT GTG ACT CTG GAC GCG CTA GGT CGC 3648
Asp Ile Asn Asp Asn Gln His Ile Val Thr Leu Asp Ala Leu Gly Arg
1205 1210 1215
GTA ACC ACC AGC CGG TTC TGG GGC ACA GAG GCA GGA CAA GCC GCA GGC 3696
Val Thr Thr Ser Arg Phe Trp Gly Thr Glu Ala Gly Gln Ala Ala Gly
1220 1225 1230
TAT TCC AAC CAG CCC TTC ACA CCA CCG GAC TCC GTA GAT AAA GCG CTG 3744
Tyr Ser Asn Gln Pro Phe Thr Pro Pro Asp Ser Val Asp Lys Ala Leu
1235 1240 1245
GCA TTA ACC GGC GCA CTC CCT GTT GCC CAA TGT TTA GTC TAT GCC GTT 3792
Ala Leu Thr Gly Ala Leu Pro Val Ala Gln Cys Leu Val Tyr Ala Val
1250 1255 1260
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GAT AGC TGG ATG CCG TCG TTA TCT TTG TCT CAG CTT TCT CAG TCA CAA 3840
Asp Ser Trp Met Pro Ser Leu Ser Leu Ser Gln Leu Ser Gln Ser Gln
1265 1270 1275 1280
GAA GAG GCA GAA GCG CTA TGG GCG CAA CTG CGT GCC GCT CAT ATG ATT 3888
Glu Glu Ala Glu Ala Leu Trp Ala Gln Leu Arg Ala Ala His Met Ile
1285 1290 1295
ACC GAA GAT GGG AAA GTG TGT GCG TTA AGC GGG AAA CGA GGA ACA AGC 3936
Thr Glu Asp Gly Lys Val Cys Ala Leu Ser Gly Lys Arg Gly Thr Ser
1300 1305 1310
CAT CAG AAC CTG ACG ATT CAA CTT ATT TCG CTA TTG GCA AGT ATT CCC 3984
His Gln Asn Leu Thr Ile Gln Leu Ile Ser Leu Leu Ala Ser Ile Pro
1315 1320 1325
CGT TTA CCG CCA CAT GTA CTG GGG ATC ACC ACT GAT CGC TAT GAT AGC 4032
Arg Leu Pro Pro His Val Leu Gly Ile Thr Thr Asp Arg Tyr Asp Ser
1330 1335 1340
GAT CCG CAA CAG CAG CAC CAA CAG ACG GTG AGC TTT AGT GAC GGT TTT 4080
Asp Pro Gln Gln Gln His Gln Gln Thr Val Ser Phe Ser Asp Gly Phe
1345 1350 1355 1360
GGC CGG TTA CTC CAG AGT TCA GCT CGT CAT GAG TCA GGT GAT GCC TGG 4128
Gly Arg Leu Leu Gln Ser Ser Ala Arg His Glu Ser Gly Asp Ala Trp
1365 1370 1375
CAA CGT AAA GAG GAT GGC GGG CTG GTC GTG GAT GCA AAT GGC GTT CTG 4176
Gln Arg Lys Glu Asp Gly Gly Leu Val Val Asp Ala Asn Gly Val Leu
1380 1385 1390
GTC AGT GCC CCT ACA GAC ACC CGA TGG GCC GTT TCC GGT CGC ACA GAA 4224
Val Ser Ala Pro Thr Asp Thr Arg Trp Ala Val Ser Gly Arg Thr Glu
1395 1400 1405
TAT GAC GAC AAA GGC CAA CCT GTG CGT ACT TAT CAA CCC TAT TTT CTA 4272
Tyr Asp Asp Lys Gly Gln Pro Val Arg Thr Tyr Gln Pro Tyr Phe Leu
1410 1415 1420
AAT GAC TGG CGT TAC GTT AGT GAT GAC AGC GCA CGA GAT GAC CTG TTT 4320
Asn Asp Trp Arg Tyr Val Ser Asp Asp Ser Ala Arg Asp Asp Leu Phe
1425 1430 1435 1440
GCC GAT ACC CAC CTT TAT GAT CCA TTG GGA CGG GAA TAC AAA GTC ATC 4368
Ala Asp Thr His Leu Tyr Asp Pro Leu Gly Arg Glu Tyr Lys Val Ile
1445 1450 1455
ACT GCT AAG AAA TAT TTG CGA GAA AAG CTG TAC ACC CCG TGG TTT ATT 4416
Thr Ala Lys Lys Tyr Leu Arg Glu Lys Leu Tyr Thr Pro Trp Phe Ile
1460 1465 1470
GTC AGT GAG GAT GAA AAC GAT ACA GCA TCA AGA ACC CCA TAG 4458
Val Ser Glu Asp Glu Asn Asp Thr Ala Ser Arg Thr Pro
1475 1480 1485
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1485 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Met Gln Asp Ser Pro Glu Val Ser Ile Thr Thr Leu Ser Leu Pro Lys
1 5 10 15
Gly Gly Gly Ala Ile Asn Gly Met Gly Glu Ala Leu Asn Ala Ala Gly
20 25 30
Pro Asp Gly Met Ala Ser Leu Ser Leu Pro Leu Pro Leu Ser Thr Gly
35 40 45
Arg Gly Thr Ala Pro Gly Leu Ser Leu Ile Tyr Ser Asn Ser Ala Gly
50 55 60
Asn Gly Pro Phe Gly Ile Gly Trp Gln Cys Gly Val Met Ser Ile Ser
65 70 75 80
Arg Arg Thr Gln His Gly Ile Pro Gln Tyr Gly Asn Asp Asp Thr Phe
85 90 95
Leu Ser Pro Gln Gly Glu Val Met Asn Ile Ala Leu Asn Asp Gln Gly
100 105 110
Gln Pro Asp Ile Arg Gin Asp Val Lys Thr Leu Gln Gly Val Thr Leu
115 120 125
Pro Ile Ser Tyr Thr Val Thr Arg Tyr Gln Ala Arg Gln Ile Leu Asp
130 135 140
Phe Ser Lys Ile Glu Tyr Trp Gln Pro Ala Ser Gly Gln Glu Gly Arg
145 150 155 160
Ala Phe Trp Leu Ile Ser Thr Pro Asp Gly His Leu His Ile Leu Gly
165 170 175
Lys Thr Ala Gln Ala Cys Leu Ala Asn Pro Gln Asn Asp Gln Gln Ile
180 185 190
Ala Gln Trp Leu Leu Glu Glu Thr Val Thr Pro Ala Gly Glu His Val
195 200 205
Ser Tyr Gln Tyr Arg Ala Glu Asp Glu Ala His Cys Asp Asp Asn Glu
210 215 220
Lys Thr Ala His Pro Asn Val Thr Ala Gln Arg Tyr Leu Val Gln Val
225 230 235 240
Asn Tyr Gly Asn Ile Lys Pro Gln Ala Ser Leu Phe Val Leu Asp Asn
245 250 255
Ala Pro Pro Ala Pro Glu Glu Trp Leu Phe His Leu Val Phe Asp His
260 265 270
Gly Glu Arg Asp Thr Ser Leu His Thr Val Pro Thr Trp Asp Ala Gly
275 280 285
Thr Ala Gln Trp Ser Val Arg Pro Asp Ile Phe Ser Arg Tyr Glu Tyr
290 295 300
Gly Phe Glu Val Arg Thr Arg Arg Leu Cys Gln Gln Val Leu Met Phe
305 310 315 320
His Arg Thr Ala Leu Met Ala Gly Glu Ala Ser Thr Asn Asp Ala Pro
325 330 335
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Glu Leu Val Gly Arg Leu Ile Leu Glu Tyr Asp Lys Asn Ala Ser Val
340 345 350
Thr Thr Leu Ile Thr Ile Arg Gln Leu Ser His Glu Ser Asp Gly Arg
355 360 365
Pro Val Thr Gln Pro Pro Leu Glu Leu Ala Trp Gln Arg Phe Asp Leu
370 375 380
Glu Lys Ile Pro Thr Trp Gln Arg Phe Asp Ala Leu Asp Asn Phe Asn
385 390 395 400
Ser Gln Gln Arg Tyr Gln Leu Val Asp Leu Arg Gly Glu Gly Leu Pro
405 410 415
Gly Met Leu Tyr Gln Asp Arg Gly Ala Trp Trp Tyr Lys Ala Pro Gln
420 425 430
Arg Gln Glu Asp Gly Asp Ser Asn Ala Val Thr Tyr Asp Lys Ile Ala
435 440 445
Pro Leu Pro Thr Leu Pro Asn Leu Gln Asp Asn Ala Ser Leu Met Asp
450 455 460
Ile Asn Gly Asp Gly Gln Leu Asp Trp Val Val Thr Ala Ser Gly Ile
465 470 475 480
Arg Gly Tyr His Ser Gln Gln Pro Asp Gly Lys Trp Thr His Phe Thr
485 490 495
Pro Ile Asn Ala Leu Pro Val Glu Tyr Phe His Pro Ser Ile Gln Phe
500 505 510
Ala Asp Leu Thr Gly Ala Gly Leu Ser Asp Leu Val Leu Ile Gly Pro
515 520 525
Lys Ser Val Arg Leu Tyr Ala Asn Gln Arg Asn Gly Trp Arg Lys Gly
530 535 540
Glu Asp Val Pro Gln Ser Thr Gly Ile Thr Leu Pro Val Thr Gly Thr
545 550 555 560
Asp Ala Arg Lys Leu Val Ala Phe Ser Asp Met Leu Gly Ser Gly Gln
565 570 575
Gln His Leu Val Glu Ile Lys Gly Asn Arg Val Thr Cys Trp Pro Asn
580 585 590
Leu Gly His Gly Arg Phe Gly Gln Pro Leu Thr Leu Ser Gly Phe Ser
595 600 605
Gln Pro Glu Asn Ser Phe Asn Pro Glu Arg Leu Phe Leu Ala Asp Ile
610 615 620
Asp Gly Ser Gly Thr Thr Asp Leu Ile Tyr Ala Gln Ser Gly Ser Leu
625 630 635 640
Leu Ile Tyr Leu Asn Gln Ser Gly Asn Gln Phe Asp Ala Pro Leu Thr
645 650 655
Leu Ala Leu Pro Glu Gly Val Gln Phe Asp Asn Thr Cys Gln Leu Gln
660 665 670
Val Ala Asp Ile Gln Gly Leu Gly Ile Ala Ser Leu Ile Leu Thr Val
675 680 685
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Pro His Ile Ala Pro His His Trp Arg Cys Asp Leu Ser Leu Thr Lys
690 695 700
Pro Trp Leu Leu Asn Val Met Asn Asn Asn Arg Gly Ala His His Thr
705 710 715 720
Leu His Tyr Arg Ser Ser Ala Gln Phe Trp Leu Asp Glu Lys Leu Gln
725 730 735
Leu Thr Lys Ala Gly Lys Ser Pro Ala Cys Tyr Leu Pro Phe Pro Met
740 745 750
His Leu Leu Trp Tyr Thr Glu Ile Gln Asp Glu Ile Ser Gly Asn Arg
755 760 765
Leu Thr Ser Glu Val Asn Tyr Ser His Gly Val Trp Asp Gly Lys Glu
770 775 780
Arg Glu Phe Arg Gly Phe Gly Cys Ile Lys Gln Thr Asp Thr Thr Thr
785 790 795 800
Phe Ser His Gly Thr Ala Pro Glu Gln Ala Ala Pro Ser Leu Ser Ile
805 810 815
Ser Trp Phe Ala Thr Gly Met Asp Glu Val Asp Ser Gln Leu Ala Thr
820 825 830
Glu Tyr Trp Gln Ala Asp Thr Gln Ala Tyr Ser Gly Phe Glu Thr Arg
835 840 845
Tyr Thr Val Trp Asp His Thr Asn Gln Thr Asp Gln Ala Phe Thr Pro
850 855 860
Asn Glu Thr Gln Arg Asn Trp Leu Thr Arg Ala Leu Lys Gly Gln Leu
865 870 875 880
Leu Arg Thr Glu Leu Tyr Gly Leu Asp Gly Thr Asp Lys Gln Thr Val
885 890 895
Pro Tyr Thr Val Ser Glu Ser Arg Tyr Gln Val Arg Ser Ile Pro Val
900 905 910
Asn Lys Glu Thr Glu Leu Ser Ala Trp Val Thr Ala Ile Glu Asn Arg
915 920 925
Ser Tyr His Tyr Glu Arg Ile Ile Thr Asp Pro Gln Phe Ser Gln Ser
930 935 940
Ile Lys Leu Gln His Asp Ile Phe Gly Gln Ser Leu Gln Ser Val Asp
945 950 955 960
Ile Ala Trp Pro Arg Arg Glu Lys Pro Ala Val Asn Pro Tyr Pro Pro
965 970 975
Thr Leu Pro Glu Thr Leu Phe Asp Ser Ser Tyr Asp Asp Gln Gln Gln
980 985 990
Leu Leu Arg Leu Val Arg Gln Lys Asn Ser Trp His His Leu Thr Asp
995 1000 1005
Gly Glu Asn Trp Arg Leu Gly Leu Pro Asn Ala Gln Arg Arg Asp Val
1010 1015 1020
Tyr Thr Tyr Asp Arg Ser Lys Ile Pro Thr Glu Gly Ile Ser Leu Glu
1025 1030 1035 1040
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Ile Leu Leu Lys Asp Asp Gly Leu Leu Ala Asp Glu Lys Ala Ala Val
1045 1050 1055
Tyr Leu Gly Gln Gln Gln Thr Phe Tyr Thr Ala Gly Gln Ala Glu Val
1060 1065 1070
Thr Leu Glu Lys Pro Thr Leu Gln Ala Leu Val Ala Phe Gln Glu Thr
1075 1080 1085
Ala Met Met Asp Asp Thr Ser Leu Gin Ala Tyr Glu Gly Val Ile Glu
1090 1095 1100
Glu Gln Glu Leu Asn Thr Ala Leu Thr Gln Ala Gly Tyr Gln Gln Val
1105 1110 1115 1120
Ala Arg Leu Phe Asn Thr Arg Ser Glu Ser Pro Val Trp Ala Ala Arg
1125 1130 1135
Gln Gly Tyr Thr Asp Tyr Gly Asp Ala Ala Gln Phe Trp Arg Pro Gln
1140 1145 1150
Ala Gln Arg Asn Ser Leu Leu Thr Gly Lys Thr Thr Leu Thr Trp Asp
1155 1160 1165
Thr His His Cys Val Ile Ile Gln Thr Gln Asp Ala Ala Gly Leu Thr
1170 1175 1180
Thr Gln Ala His Tyr Asp Tyr Arg Phe Leu Thr Pro Val Gln Leu Thr
1185 1190 1195 1200
Asp Ile Asn Asp Asn Gln His Ile Val Thr Leu Asp Ala Leu Gly Arg
1205 1210 1215
Val Thr Thr Ser Arg Phe Trp Gly Thr Glu Ala Gly Gln Ala Ala Gly
1220 1225 1230
Tyr Ser Asn Gln Pro Phe Thr Pro Pro Asp Ser Val Asp Lys Ala Leu
1235 1240 1245
Ala Leu Thr Gly Ala Leu Pro Val Ala Gln Cys Leu Val Tyr Ala Val
1250 1255 1260
Asp Ser Trp Met Pro Ser Leu Ser Leu Ser Gln Leu Ser Gln Ser Gln
1265 1270 1275 1280
Glu Glu Ala Glu Ala Leu Trp Ala Gln Leu Arg Ala Ala His Met Ile
1285 1290 1295
Thr Glu Asp Gly Lys Val Cys Ala Leu Ser Gly Lys Arg Gly Thr Ser
1300 1305 1310
His Gln Asn Leu Thr Ile Gln Leu Ile Ser Leu Leu Ala Ser Ile Pro
1315 1320 1325
Arg Leu Pro Pro His Val Leu Gly Ile Thr Thr Asp Arg Tyr Asp Ser
1330 1335 1340
Asp Pro Gln Gln Gln His Gln Gln Thr Val Ser Phe Ser Asp Gly Phe
1345 1350 1355 1360
Gly Arg Leu Leu Gln Ser Ser Ala Arg His Glu Ser Gly Asp Ala Trp
1365 1370 1375
Gln Arg Lys Glu Asp Gly Gly Leu Val Val Asp Ala Asn Gly Val Leu
1380 1385 1390
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Val Ser Ala Pro Thr Asp Thr Arg Trp Ala Val Ser Gly Arg Thr Glu
1395 1400 1405
Tyr Asp Asp Lys Gly Gln Pro Val Arg Thr Tyr Gin Pro Tyr Phe Leu
1410 1415 1420
Asn Asp Trp Arg Tyr Val Ser Asp Asp Ser Ala Arg Asp Asp Leu Phe
1425 1430 1435 1440
Ala Asp Thr His Leu Tyr Asp Pro Leu Gly Arg Glu Tyr Lys Val Ile
1445 1450 1455
Thr Ala Lys Lys Tyr Leu Arg Glu Lys Leu Tyr Thr Pro Trp Phe Ile
1460 1465 1470
Val Ser Glu Asp Glu Asn Asp Thr Ala Ser Arg Thr Pro
1475 1480 1485
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3288 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
ATG GTG ACT GTT ATG CAA AAT AAA ATA TCA TTT TTA TCA GGT ACA TCC 48
Met Val Thr Val Met Gln Asn Lys Ile Ser Phe Leu Ser Gly Thr Ser
1 5 10 15
GAA CAG CCC CTG CTT GAC GCC GGT TAT CAA AAC GTA TTT GAT ATC GCA 96
Glu Gln Pro Leu Leu Asp Ala Gly Tyr Gln Asn Val Phe Asp Ile Ala
20 25 30
TCA ATC AGC CGG GCT ACT TTC GTT CAA TCC GTT CCC ACC CTG CCC GTT 144
Ser Ile Ser Arg Ala Thr Phe Val Gln Ser Val Pro Thr Leu Pro Val
40 45
AAA GAG GCT CAT ACC GTC TAT CGT CAG GCG CGG CAA CGT GCG GAA AAT 192
Lys Glu Ala His Thr Val Tyr Arg Gln Ala Arg Gln Arg Ala Glu Asn
55 60
CTG AAA TCC CTC TAC CGA GCC TGG CAA TTG CGT CAG GAG CCG GTT ATT 240
Leu Lys Ser Leu Tyr Arg Ala Trp Gln Leu Arg Gln Glu Pro Val Ile
65 70 75 80
AAA GGG CTG GCT AAA CTT AAC CTA CAA TCC AAC GTT TCT GTG CTT CAA 288
Lys Gly Leu Ala Lys Leu Asn Leu Gln Ser Asn Val Ser Val Leu Gln
50 85 90 95
GAT GCT TTG GTA GAG AAT ATT GGC GGT GAT GGG GAT TTC AGC GAT TTA 336
Asp Ala Leu Val Glu Asn Ile Gly Gly Asp Gly Asp Phe Ser Asp Leu
100 105 110
ATG AAC CGT GCC AGT CAA TAT GCT GAC GCT GCC TCT ATT CAA TCC CTA 384
Met Asn Arg Ala Ser Gln Tyr Ala Asp Ala Ala Ser Ile Gln Ser Leu
115 120 125
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TTT TCA CCG GGC CGT TAT GCT TCC GCA CTC TAC AGA GTT GCT AAA GAT 432
Phe Ser Pro Gly Arg Tyr Ala Ser Ala Leu Tyr Arg Val Ala Lys Asp
130 135 140
CTG CAT AAA TCA GAT TCC AGT TTG CAT ATT GAT AAT CGC CGC GCT GAT 480
Leu His Lys Ser Asp Ser Ser Leu His Ile Asp Asn Arg Arg Ala Asp
145 150 155 160
CTG AAG GAT CTG ATA TTA AGC GAA ACG ACG ATG AAT AAA GAG GTC ACT 528
Leu Lys Asp Leu Ile Leu Ser Glu Thr Thr Met Asn Lys Glu Val Thr
165 170 175
TCC CTT GAT ATC TTG TTG GAT GTG CTA CAA AAA GGC GGT AAA GAT ATT 576
Ser Leu Asp Ile Leu Leu Asp Val Leu Gln Lys Gly Gly Lys Asp Ile
180 185 190
ACT GAG CTG TCC GGC GCA TTC TTC CCA ATG ACG TTA CCT TAT GAC GAT 624
Thr Glu Leu Ser Gly Ala Phe Phe Pro Met Thr Leu Pro Tyr Asp Asp
195 200 205
CAT CTG TCG CAA ATC GAT TCC GCT TTA TCG GCA CAA GCC AGA ACG CTG 672
His Leu Ser Gln Ile Asp Ser Ala Leu Ser Ala Gln Ala Arg Thr Leu
210 215 220
AAC GGT GTG TGG AAT ACT TTG ACA GAT ACC ACG GCA CAA GCG GTT TCA 720
Asn Gly Val Trp Asn Thr Leu Thr Asp Thr Thr Ala Gln Ala Val Ser
225 230 235 240
GAA CAA ACC AGT AAT ACG AAT ACA CGC AAA CTG TTC GCT GCC CAA GAT 768
Glu Gln Thr Ser Asn Thr Asn Thr Arg Lys Leu Phe Ala Ala Gln Asp
245 250 255
GGT AAT CAA GAT ACA TTT TTT TCC GGA AAC ACT TTT TAT TTC AAA GCG 816
Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr Phe Tyr Phe Lys Ala
260 265 270
GTG GGA TTC AGC GGG CAA CCT ATG GTT TAC CTG TCA CAG TAC ACC AGC 864
Val Gly Phe Ser Gly Gln Pro Met Val Tyr Leu Ser Gln Tyr Thr Ser
275 280 285
GGG AAC GGC ATT GTC GGC GCA CAA TTG ATT GCA GGT AAT CCA GAC CAA 912
Gly Asn Gly Ile Val Gly Ala Gln Leu Ile Ala Gly Asn Pro Asp Gln
290 295 300
GCC GCC GCC GCA ATA GTC GCA CCG TTG AAA CTC ACT TGG TCA ATG GCA 960
Ala Ala Ala Ala Ile Val Ala Pro Leu Lys Leu Thr Trp Ser Met Ala
305 310 315 320
AAA CAG TGT TAC TAC CTC GTC GCT CCC GAT GGT ACA ACG ATG GGA GAC 1008
Lys Gln Cys Tyr Tyr Leu Val Ala Pro Asp Gly Thr Thr Met Gly Asp
325 330 335
GGT AAT GTT CTG ACC GGC TGT TTC TTA AGA GGC AAC AGC CCA ACT AAC 1056
Gly Asn Val Leu Thr Gly Cys Phe Leu Arg Gly Asn Ser Pro Thr Asn
340 345 350
CCG GAT AAA GAC GGT ATT TTT GCT CAG GTA GCC AAC AAA TCA GGC AGT 1104
Pro Asp Lys Asp Gly Ile Phe Ala Gln Val Ala Asn Lys Ser Gly Ser
355 360 365
ACT CAG CCT TTG CCA AGC TTC CAT CTG CCG GTC ACA CTG GAA CAC AGC 1152
Thr Gln Pro Leu Pro Ser Phe His Leu Pro Val Thr Leu Glu His Ser
370 375 380
GAG AAT AAA GAT CAG TAC TAT CTG AAA ACA GAG CAG GGT TAT ATC ACG 1200
Glu Asn Lys Asp Gln Tyr Tyr Leu Lys Thr Glu Gln Gly Tyr Ile Thr
385 390 395 400
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GTA GAT AGT TCC GGA CAG TCA AAT TGG AAA AAC GCG CTG GTT ATC AAT 1248
Val Asp Ser Ser Gly Gln Ser Asn Trp Lys Asn Ala Leu Val Ile Asn
405 410 415
GGG ACA AAA GAC AAG GGG CTG TTA TTA ACC TTT TGC AGC GAT AGC TCA 1296
Gly Thr Lys Asp Lys Gly Leu Leu Leu Thr Phe Cys Ser Asp Ser Ser
420 425 430
GGC ACT CCG ACA AAC CCT GAT GAT GTG ATT CCT CCC GCT ATC AAT GAT 1344
Gly Thr Pro Thr Asn Pro Asp Asp Val Ile Pro Pro Ala Ile Asn Asp
435 440 445
ATT CCA TCG CCG CCA GCC CGC GAA ACA CTG TCA CTG ACG CCG GTC AGT 1392
Ile Pro Ser Pro Pro Ala Arg Glu Thr Leu Ser Leu Thr Pro Val Ser
450 455 460
TAT CAA TTG ATG ACC AAT CCG GCA CCG ACA GAA GAT GAT ATT ACC AAC 1440
Tyr Gln Leu Met Thr Asn Pro Ala Pro Thr Glu Asp Asp Ile Thr Asn
465 470 475 480
CAT TAT GGT TTT AAC GGC GCT AGC TTA CGG GCT TCT CCA TTG TCA ACC 1488
His Tyr Gly Phe Asn Gly Ala Ser Leu Arg Ala Ser Pro Leu Ser Thr
485 490 495
AGC GAG TTG ACC AGC AAA CTG AAT TCT ATC GAT ACT TTC TGT GAG AAG 1536
Ser Glu Leu Thr Ser Lys Leu Asn Ser Ile Asp Thr Phe Cys Glu Lys
500 505 510
ACC CGG TTA AGC TTC AAT CAG TTA ATG GAT TTG ACC GCT CAG CAA TCT 1584
Thr Arg Leu Ser Phe Asn Gln Leu Met Asp Leu Thr Ala Gln Gln Ser
515 520 525
TAC AGT CAA AGC AGC ATT GAT GCG AAA GCA GCC AGC CGC TAT GTT CGT 1632
Tyr Ser Gln Ser Ser Ile Asp Ala Lys Ala Ala Ser Arg Tyr Val Arg
530 535 540
TTT GGG GAA ACC ACC CCA ACC CGC GTC AAT GTC TAC GGT GCC GCT TAT 1680
Phe Gly Glu Thr Thr Pro Thr Arg Val Asn Val Tyr Gly Ala Ala Tyr
545 550 555 560
CTG AAC AGC ACA CTG GCA GAC GCG GCT GAT GGT CAA TAT CTG TGG ATT 1728
Leu Asn Ser Thr Leu Ala Asp Ala Ala Asp Gly Gln Tyr Leu Trp Ile
565 570 575
CAG ACT GAT GGC AAG AGC CTA AAT TTC ACT GAC GAT ACG GTA GTC GCC 1776
Gln Thr Asp Gly Lys Ser Leu Asn Phe Thr Asp Asp Thr Val Val Ala
580 585 590
TTA GCC GGT CGC GCT GAA AAG CTG GTA CGT TTA TCA TCC CAG ACC GGG 1824
Leu Ala Gly Arg Ala Glu Lys Leu Val Arg Leu Ser Ser Gln Thr Gly
595 600 605
CTA TCA TTT GAA GAA TTG GAC TGG CTG ATT GCC AAT GCC AGT CGT AGT 1872
Leu Ser Phe Glu Glu Leu Asp Trp Leu Ile Ala Asn Ala Ser Arg Ser
610 615 620
GTG CCG GAC CAC CAC GAC AAA ATT GTG CTG GAT AAG CCG GTC CTT GAA 1920
Val Pro Asp His His Asp Lys Ile Val Leu Asp Lys Pro Val Leu Glu
625 630 635 640
GCA CTG GCA GAG TAT GTC AGC CTA AAA CAG CGC TAT GGG CTT GAT GCC 1968
Ala Leu Ala Glu Tyr Val Ser Leu Lys Gln Arg Tyr Gly Leu Asp Ala
645 650 655
AAT ACC TTT GCG ACC TTC ATT AGT GCA GTA AAT CCT TAT ACG CCA GAT 2016
Asn Thr Phe Ala Thr Phe Ile Ser Ala Val Asn Pro Tyr Thr Pro Asp
660 665 670
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CAG ACA CCC AGT TTC TAT GAA ACC GCT TTC CGC TCT GCC GAC GGT AAT 2064
Gln Thr Pro Ser Phe Tyr Glu Thr Ala Phe Arg Ser Ala Asp Gly Asn
675 680 685
CAT GTC ATT GCG CTA GGT ACA GAG GTG AAA TAT GCA GAA AAT GAG CAG 2112
His Val Ile Ala Leu Gly Thr Glu Val Lys Tyr Ala Glu Asn Glu Gln
690 695 700
GAT GAG TTA GCC GCC ATA TGC TGC AAA GCA TTG GGT GTC ACC AGT GAT 2160
Asp Glu Leu Ala Ala Ile Cys Cys Lys Ala Leu Gly Val Thr Ser Asp
705 710 715 720
GAA CTG CTC CGT ATT GGT CGC TAT TGC TTC GGT AAT GCA GGC AGT TTT 2208
Glu Leu Leu Arg Ile Gly Arg Tyr Cys Phe Gly Asn Ala Gly Ser Phe
725 730 735
ACC TTG GAT GAA TAT ACC GCC AGT CAG TTG TAT CGC TTC GGC GCC ATT 2256
Thr Leu Asp Glu Tyr Thr Ala Ser Gln Leu Tyr Arg Phe Gly Ala Ile
740 745 750
CCC CGT TTG TTT GGG CTG ACA TTT GCC CAA GCC GAA ATT TTA TGG CGT 2304
Pro Arg Leu Phe Gly Leu Thr Phe Ala Gln Ala Glu Ile Leu Trp Arg
755 760 765
CTG ATG GAA GGC GGA AAA GAT ATC TTA TTG CAA CAG TTA GGT CAG GCA 2352
Leu Met Glu Gly Gly Lys Asp Ile Leu Leu Gln Gln Leu Gly Gln Ala
770 775 780
AAA TCC CTG CAA CCA CTG GCT ATT TTA CGC CGT ACC GAG CAG GTG CTG 2400
Lys Ser Leu Gln Pro Leu Ala Ile Leu Arg Arg Thr Glu Gln Val Leu
785 790 795 800
GAT TGG ATG TCG TCC GTA AAT CTA AGT CTG ACT TAT CTG CAA GGG ATG 2448
Asp Trp Met Ser Ser Val Asn Leu Ser Leu Thr Tyr Leu Gln Gly Met
805 810 815
GTA AGT ACG CAA TGG AGC GGT ACC GCC ACC GCT GAG ATG TTC AAT TTC 2496
Val Ser Thr Gln Trp Ser Gly Thr Ala Thr Ala Glu Met Phe Asn Phe
820 825 830
TTG GAA AAC GTT TGT GAC AGC GTG AAT AGT CAA GCT GCC ACT AAA GAA 2544
Leu Glu Asn Val Cys Asp Ser Val Asn Ser Gln Ala Ala Thr Lys Glu
835 840 845
ACA ATG GAT TCG GCG TTA CAG CAG AAA GTG CTG CGG GCG CTA AGC GCC 2592
Thr Met Asp Ser Ala Leu Gln Gln Lys Val Leu Arg Ala Leu Ser Ala
850 855 860
GGT TTC GGC ATT AAG AGC AAT GTG ATG GGT ATC GTC ACC TTC TGG CTG 2640
Gly Phe Gly Ile Lys Ser Asn Val Met Gly Ile Val Thr Phe Trp Leu
865 870 875 880
GAG AAA ATC ACA ATC GGT AGT GAT AAT CCT TTT ACA TTG GCA AAC TAC 2688
Glu Lys Ile Thr Ile Gly Ser Asp Asn Pro Phe Thr Leu Ala Asn Tyr
885 890 895
TGG CAT GAT ATT CAA ACC CTG TTT AGC CAT GAC AAT GCC ACG TTA GAG 2736
Trp His Asp Ile Gln Thr Leu Phe Ser His Asp Asn Ala Thr Leu Glu
900 905 910
TCC TTA CAA ACC GAC ACT TCT CTG GTA ATT GCT ACT CAG CAA CTT AGC 2784
Ser Leu Gln Thr Asp Thr Ser Leu Val Ile Ala Thr Gln Gln Leu Ser
915 920 925
CAG CTA GTG TTA ATT GTG AAA TGG CTG AGC CTG ACC GAG CAG GAT CTG 2832
Gin Leu Val Leu Ile Val Lys Trp Leu Ser Leu Thr Glu Gln Asp Leu
930 935 940
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CAA TTA CTG ACA ACC TAT CCC GAA CGT TTA ATC AAC GGC ATC ACG AAT 2880
Gln Leu Leu Thr Thr Tyr Pro Glu Arg Leu Ile Asn Gly Ile Thr Asn
945 950 955 960
GTT CCT GTA CCC AAT CCG GAG CTA TTA CTC ACG CTA TCA CGT TTT AAG 2928
Val Pro Val Pro Asn Pro Glu Leu Leu Leu Thr Leu Ser Arg Phe Lys
965 970 975
CAG TGG GAA ACT CAA GTC ACC GTT TCC CGT GAT GAA GCG ATG CGC TGT 2976
Gln Trp Glu Thr Gln Val Thr Val Ser Arg Asp Glu Ala Met Arg Cys
980 985 990
TTC GAT CAA TTA AAT GCC AAT GAT ATG ACG ACT GAA AAT GCA GGT TCA 3024
Phe Asp Gin Leu Asn Ala Asn Asp Met Thr Thr Glu Asn Ala Gly Ser
995 1000 1005
CTG ATC GCC ACA TTG TAT GAG ATG GAT AAA GGT ACG GGA GCG CAA GTT 3072
Leu Ile Ala Thr Leu Tyr Glu Met Asp Lys Gly Thr Gly Ala Gln Val
1010 1015 1020
AAT ACC TTG CTA TTA GGT GAA AAT AAC TGG CCG AAA AGT TTT ACC TCT 3120
Asn Thr Leu Leu Leu Gly Glu Asn Asn Trp Pro Lys Ser Phe Thr Ser
1025 1030 1035 1040
CTC TGG CAA CTT CTG ACC TGG TTA CGC GTC GGG CAA AGA CTG AAT GTC 3168
Leu Trp Gln Leu Leu Thr Trp Leu Arg Val Gly Gln Arg Leu Asn Val
1045 1050 1055
GGT AGT ACC ACT CTG GGC AAT CTG TTG TCC ATG ATG CAA GCA GAC CCT 3216
Gly Ser Thr Thr Leu Gly Asn Leu Leu Ser Met Met Gln Ala Asp Pro
1060 1065 1070
GCT GCC GAG AGT AGC GCT TTA TTG GCA TCA GTA GCC CAA AAC TTA AGT 3264
Ala Ala Glu Ser Ser Ala Leu Leu Ala Ser Val Ala Gln Asn Leu Ser
1075 1080 1085
GCC GCA ATC AGC AAT CGT CAG TAA 3288
Ala Ala Ile Ser Asn Arg Gln
1090 1095
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1095 amino acids
(B) TYPE: amino acids
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
Met Val Thr Val Met Gln Asn Lys Ile Ser Phe Leu Ser Gly Thr Ser
1 5 10 15
Glu Gln Pro Leu Leu Asp Ala Gly Tyr Gln Asn Val Phe Asp Ile Ala
20 25 30
Ser Ile Ser Arg Ala Thr Phe Val Gln Ser Val Pro Thr Leu Pro Val
35 40 45
Lys Glu Ala His Thr Val Tyr Arg Gln Ala Arg Gln Arg Ala Glu Asn
50 55 60
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Leu Lys Ser Leu Tyr Arg Ala Trp Gln Leu Arg Gln Glu Pro Val Ile
65 70 75 80
Lys Gly Leu Ala Lys Leu Asn Leu Gln Ser Asn Val Ser Val Leu Gln
85 90 95
Asp Ala Leu Val Glu Asn Ile Gly Gly Asp Gly Asp Phe Ser Asp Leu
100 105 110
Met Asn Arg Ala Ser Gln Tyr Ala Asp Ala Ala Ser Ile Gln Ser Leu
115 120 125
Phe Ser Pro Gly Arg Tyr Ala Ser Ala Leu Tyr Arg Val Ala Lys Asp
130 135 140
Leu His Lys Ser Asp Ser Ser Leu His Ile Asp Asn Arg Arg Ala Asp
145 150 155 160
Leu Lys Asp Leu Ile Leu Ser Glu Thr Thr Met Asn Lys Glu Val Thr
165 170 175
Ser Leu Asp Ile Leu Leu Asp Val Leu Gln Lys Gly Gly Lys Asp Ile
180 185 190
Thr Glu Leu Ser Gly Ala Phe Phe Pro Met Thr Leu Pro Tyr Asp Asp
195 200 205
His Leu Ser Gln Ile Asp Ser Ala Leu Ser Ala Gln Ala Arg Thr Leu
210 215 220
Asn Gly Val Trp Asn Thr Leu Thr Asp Thr Thr Ala Gln Ala Val Ser
225 230 235 240
Glu Gln Thr Ser Asn Thr Asn Thr Arg Lys Leu Phe Ala Ala Gln Asp
245 250 255
Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr Phe Tyr Phe Lys Ala
260 265 270
Val Gly Phe Ser Gly Gln Pro Met Val Tyr Leu Ser Gln Tyr Thr Ser
275 280 285
Gly Asn Gly Ile Val Gly Ala Gln Leu Ile Ala Gly Asn Pro Asp Gln
290 295 300
Ala Ala Ala Ala Ile Val Ala Pro Leu Lys Leu Thr Trp Ser Met Ala
305 310 315 320
Lys Gln Cys Tyr Tyr Leu Val Ala Pro Asp Gly Thr Thr Met Gly Asp
325 330 335
Gly Asn Val Leu Thr Gly Cys Phe Leu Arg Gly Asn Ser Pro Thr Asn
340 345 350
Pro Asp Lys Asp Gly Ile Phe Ala Gln Val Ala Asn Lys Ser Gly Ser
355 360 365
Thr Gln Pro Leu Pro Ser Phe His Leu Pro Val Thr Leu Glu His Ser
370 375 380
Glu Asn Lys Asp Gln Tyr Tyr Leu Lys Thr Glu Gln Gly Tyr Ile Thr
385 390 395 400
Val Asp Ser Ser Gly Gln Ser Asn Trp Lys Asn Ala Leu Val Ile Asn
405 410 415
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~ . -
Gly Thr Lys Asp Lys Gly Leu Leu Leu Thr Phe Cys Ser Asp Ser Ser
420 425 430
Gly Thr Pro Thr Asn Pro Asp Asp Val Ile Pro Pro Ala Ile Asn Asp
435 440 445
Ile Pro Ser Pro Pro Ala Arg Glu Thr Leu Ser Leu Thr Pro Val Ser
450 455 460
Tyr Gln Leu Met Thr Asn Pro Ala Pro Thr Glu Asp Asp Ile Thr Asn
465 470 475 480
His Tyr Gly Phe Asn Gly Ala Ser Leu Arg Ala Ser Pro Leu Ser Thr
485 490 495
Ser Glu Leu Thr Ser Lys Leu Asn Ser Ile Asp Thr Phe Cys Glu Lys
500 505 510
Thr Arg Leu Ser Phe Asn Gln Leu Met Asp Leu Thr Ala Gln Gln Ser
515 520 525
Tyr Ser Gln Ser Ser Ile Asp Ala Lys Ala Ala Ser Arg Tyr Val Arg
530 535 540
Phe Gly Glu Thr Thr Pro Thr Arg Val Asn Val Tyr Gly Ala Ala Tyr
545 550 555 560
Leu Asn Ser Thr Leu Ala Asp Ala Ala Asp Gly Gln Tyr Leu Trp Ile
565 570 575
Gln Thr Asp Gly Lys Ser Leu Asn Phe Thr Asp Asp Thr Val Val Ala
580 585 590
Leu Ala Gly Arg Ala Glu Lys Leu Val Arg Leu Ser Ser Gln Thr Gly
595 600 605
Leu Ser Phe Glu Glu Leu Asp Trp Leu Ile Ala Asn Ala Ser Arg Ser
610 615 620
Val Pro Asp His His Asp Lys Ile Val Leu Asp Lys Pro Val Leu Glu
625 630 635 640
Ala Leu Ala Glu Tyr Val Ser Leu Lys Gln Arg Tyr Gly Leu Asp Ala
645 650 655
Asn Thr Phe Ala Thr Phe Ile Ser Ala Val Asn Pro Tyr Thr Pro Asp
660 665 670
Gln Thr Pro Ser Phe Tyr Glu Thr Ala Phe Arg Ser Ala Asp Gly Asn
675 680 685
His Val Ile Ala Leu Gly Thr Glu Val Lys Tyr Ala Glu Asn Glu Gln
690 695 700
Asp Glu Leu Ala Ala Ile Cys Cys Lys Ala Leu Gly Val Thr Ser Asp
705 710 715 720
Glu Leu Leu Arg Ile Gly Arg Tyr Cys Phe Gly Asn Ala Gly Ser Phe
725 730 735
Thr Leu Asp Glu Tyr Thr Ala Ser Gln Leu Tyr Arg Phe Gly Ala Ile
740 745 750
Pro Arg Leu Phe Gly Leu Thr Phe Ala Gln Ala Glu Ile Leu Trp Arg
755 760 765
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Leu Met Glu Gly Gly Lys Asp Ile Leu Leu Gln Gln Leu Gly Gln Ala
770 775 780
Lys Ser Leu Gln Pro Leu Ala'Ile Leu Arg Arg Thr Glu Gln Val Leu
785 790 795 800
Asp Trp Met Ser Ser Val Asn Leu Ser Leu Thr Tyr Leu Gln Gly Met
805 810 815
Val Ser Thr Gln Trp Ser Gly Thr Ala Thr Ala Glu Met Phe Asn Phe
820 825 830
Leu Glu Asn Val Cys Asp Ser Val Asn Ser Gln Ala Ala Thr Lys Glu
835 840 845
Thr Met Asp Ser Ala Leu Gln Gln Lys Val Leu Arg Ala Leu Ser Ala
850 855 860
Gly Phe Gly Ile Lys Ser Asn Val Met Gly Ile Val Thr Phe Trp Leu
865 870 875 880
Glu Lys Ile Thr Ile Gly Ser Asp Asn Pro Phe Thr Leu Ala Asn Tyr
885 890 895
Trp His Asp Ile Gln Thr Leu Phe Ser His Asp Asn Ala Thr Leu Glu
900 905 910
Ser Leu Gln Thr Asp Thr Ser Leu Val Ile Ala Thr Gln Gin Leu Ser
915 920 925
Gln Leu Val Leu Ile Val Lys Trp Leu Ser Leu Thr Glu Gln Asp Leu
930 935 940
Gln Leu Leu Thr Thr Tyr Pro Glu Arg Leu Ile Asn Gly Ile Thr Asn
945 950 955 960
Val Pro Val Pro Asn Pro Glu Leu Leu Leu Thr Leu Ser Arg Phe Lys
965 970 975
Gln Trp Glu Thr Gln Val Thr Val Ser Arg Asp Glu Ala Met Arg Cys
980 985 990
Phe Asp Gln Leu Asn Ala Asn Asp Met Thr Thr Glu Asn Ala Gly Ser
995 1000 1005
Leu Ile Ala Thr Leu Tyr Glu Met Asp Lys Gly Thr Gly Ala Gln Val
1010 1015 1020
Asn Thr Leu Leu Leu Gly Glu Asn Asn Trp Pro Lys Ser Phe Thr Ser
1025 1030 1035 1040
Leu Trp Gln Leu Leu Thr Trp Leu Arg Val Gly Gln Arg Leu Asn Val
1045 1050 1055
Gly Ser Thr Thr Leu Gly Asn Leu Leu Ser Met Met Gln Ala Asp Pro
1060 1065 1070
Ala Ala Glu Ser Ser Ala Leu Leu Ala Ser Val Ala Gln Asn Leu Ser
1075 1080 1085
Ala Ala Ile Ser Asn Arg Gln
1090 1095
(2) INFORMATION FOR SEQ ID NO:35
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 603 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Pro Leu Ser Thr Ser Glu Leu Thr Ser Lys Leu Asn Ser Ile Asp Thr
1 5 10 15
Phe Cys Glu Lys Thr Arg Leu Ser Phe Asn Gln Leu Met Asp Leu Thr
20 25 30
Ala Gln Gln Ser Tyr Ser Gln Ser Ser Ile Asp Ala Lys Ala Ala Ser
35 40 45
Arg Tyr Val Arg Phe Gly Glu Thr Thr Pro Thr Arg Val Asn Val Tyr
50 55 60
Gly Ala Ala Tyr Leu Asn Ser Thr Leu Ala Asp Ala Ala Asp Gly Gln
65 70 75 80
Tyr Leu Trp Ile Gln Thr Asp Gly Lys Ser Leu Asn Phe Thr Asp Asp
85 90 95
Thr Val Val Ala Leu Ala Gly Arg Ala Glu Lys Leu Val Arg Leu Ser
100 105 110
Ser Gln Thr Gly Leu Ser Phe Glu Glu Leu Asp Trp Leu Ile Ala Asn
115 120 125
Ala Ser Arg Ser Val Pro Asp His His Asp Lys Ile Val Leu Asp Lys
130 135 140
Pro Val Leu Glu Ala Leu Ala Glu Tyr Val Ser Leu Lys Gln Arg Tyr
145 150 155 160
Gly Leu Asp Ala Asn Thr Phe Ala Thr Phe Ile Ser Ala Val Asn Pro
165 170 175
Tyr Thr Pro Asp Gln Thr Pro Ser Phe Tyr Glu Thr Ala Phe Arg Ser
180 185 190
Ala Asp Gly Asn His Val Ile Ala Leu Gly Thr Glu Val Lys Tyr Ala
195 200 205
Glu Asn Glu Gln Asp Glu Leu Ala Ala Ile Cys Cys Lys Ala Leu Gly
210 215 220
Val Thr Ser Asp Glu Leu Leu Arg Ile Gly Arg Tyr Cys Phe Gly Asn
225 230 235 240
Ala Gly Arg Phe Thr Leu Asp Glu Tyr Thr Ala Ser Gln Leu Tyr Arg
245 250 255
Phe Gly Ala Ile Pro Arg Leu Phe Gly Leu Thr Phe Ala Gln Ala Glu
260 265 270
Ile Leu Trp Arg Leu Met Glu Gly Gly Lys Asp Ile Leu Leu Gln Gln
275 280 285
Xaa Gly Gln Ala Lys Ser Leu Gln Pro Leu Ala Ile Leu Arg Arg Thr
290 295 300
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Glu Gln Val Leu Asp Trp Met Ser Pro Val Asn Leu Ser Leu Thr Tyr
305 310 315 320
Leu Gln Gly Met Val Ser Thr Gln Trp Ser Gly Thr Ala Thr Ala Glu
325 330 335
Met Phe Asn Phe Leu Glu Asn Val Cys Asp Ser Val Asn Ser Gln Ala
340 345 350
Xaa Thr Lys Glu Thr Met Asp Ser Ala Leu Gln Gln Lys Val Leu Arg
355 360 365
Ala Leu Ser Ala Gly Phe Gly Ile Lys Ser Asn Val Met Gly Ile Val
370 375 380
Thr Phe Trp Leu Glu Lys Ile Thr Ile Gly Arg Asp Asn Pro Phe Thr
385 390 395 400
Leu Ala Asn Tyr Trp His Asp Ile Gln Thr Leu Phe Ser His Asp Asn
405 410 415
Ala Thr Leu Glu Ser Leu Gln Thr Asp Thr Ser Leu Val Ile Ala Thr
420 425 430
Gln Gln Leu Ser Gln Leu Val Leu Ile Val Lys Trp Val Ser Leu Thr
435 440 445
Glu Gln Asp Leu Gln Leu Leu Thr Thr Tyr Pro Glu Arg Leu Ile Asn
450 455 460
Gly Ile Thr Asn Val Pro Val Pro Asn Pro Glu Leu Leu Leu Thr Leu
465 470 475 480
Ser Arg Phe Lys Gln Trp Glu Thr Gln Val Thr Val Ser Arg Asp Glu
485 490 495
Ala Met Arg Cys Phe Asp Gln Leu Asn Ala Asn Asp Met Thr Thr Glu
500 505 510
Asn Ala Gly Ser Leu Ile Ala Thr Leu Tyr Glu Met Asp Lys Gly Thr
515 520 525
Gly Ala Gln Val Asn Thr Leu Leu Leu Gly Glu Asn Asn Trp Pro Lys
530 535 540
Ser Phe Thr Ser Leu Trp Gln Leu Leu Thr Trp Leu Arg Val Gly Gln
545 550 555 560
Arg Leu Asn Val Gly Ser Thr Thr Leu Gly Asn Leu Leu Ser Met Met
565 570 575
Gin Ala Asp Pro Ala Ala Glu Ser Ser Ala Leu Leu Ala Ser Val Ala
580 585 590
Gln Asn Leu Ser Ala Ala Ile Ser Asn Arg Gln
595 600
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2557 base pairs
(B) TYPE: nucleic acid
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(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
GAATTCGGCT TGCGTTTAAT ATTGATGATG TCTCGCTCTT CCGCCTGCTT AAAATTACCG 60
ACCATGATAA TAAAGATGGA AAAATTAAAA ATAACCTAAA GAATCTTTCC AATTTATATA 120
TTGGAAAATT ACTGGCAGAT ATTCATCAAT TAACCATTGA TGAACTGGAT TTATTACTGA 180
TTGCCGTAGG TGAAGGAAAA ACTAATTTAT CCGCTATCAG TGATAAGCAA TTGGCTACCC 240
TGATCAGAAA ACTCAATACT ATTACCAGCT GGCTACATAC ACAGAAGTGG AGTGTATTCC 300
AGCTATTTAT CATGACCTCC ACCAGCTATA ACAAAACGCT AACGCCTGAA ATTAAGAATT 360
TGCTGGATAC CGTCTACCAC GGTTTACAAG GTTTTGATAA AGACAAAGCA GATTTGCTAC 420
ATGTCATGGC GCCCTATATT GCGGCCACCT TGCAATTATC ATCGGAAAAT GTCGCCCACT 480
CGGTACTCCT TTGGGCAGAT AAGTTACAGC CCGGCGACGG CGCAATGACA GCAGAGGGAN 540
TCTGGGACTG GTTGAATACT AAGTATACGC CGGGTTCATC GGAAGCCGTA GAAACGCAGG 600
AACATATCGT TCAGTATTGT CAGGCTCTGG CACAATTGGA AATGGTTTAC CATTCCACCG 660
GCATCAACGA AAACGCCTTC CGTCTATTTG TGACAAAACC AGAGATGTTT GGCGCTGCAA 720
CTGGAGCAGC GCCCGCGCAT GATGCCCTTT CACTGATTAT GCTGACACGT TTTGCGGATT 780
GGGTGAACGC ACTAGGCGAA AAAGCGTCCT CGGTGCTAGC GGCATTTGAA GCTAACTCGT 840
TAACGGCAGA ACAACTGGCT GATGCCATGA ATCTTGATGC TAATTTGCTG TTGCAAGCCA 900
GTATTCAAGC ACAAAATCAT CAACATCTTC CCCCAGTAAC TCCAGAAAAT GCGTTCTCCT 960
GTTGGACATC TATCAATACT ATCCTGCAAT GGGTTAATGT CGCACAACAA TTGAAATGTC 1020
GCCCCACAGG GCGTTTCCGC TTTGGTCGGG CTGGATTATA TTCAATCAAT GAAAGAGACA 1080
CCGACCTATG CCCAGTGGGA AAACGCGGCA GGCGTATTAA CCGCCGGGTT GAATTCAACA 1140
ACAGGCTAAT ACATTACAAC GCTTTTCTGG ATGAATCTCG CAGTGCCGCA TTAAGCACCT 1200
ACTATATCCG TCAAGTCGCC AAGGCAGCGG CGGCTATTAA AAGCCGTGAT GACTTGTATC 1260
AATACTTACT GATTGATAAT CAGGTTTCTG CGGCAATAAA AACCACCCGG ATCGCCGAAG 1320
CCATTGCCAG TATTCAACTG TACGTCAACC GGGCATTGGA AAATGTGGAA GAAAATGCCA 1380
ATTCGGGGGT TATCAGCCGC CAATTCTTTA TCGACTGGGA CAAATACAAT AAACGCTACA 1440
GCACTTGGGC GGGTGTTTCT CAATTAGTTT ACTACCCGGA AAACTATATT GATCCGACCA 1500
TGCGTATCGG ACAAACCAAA ATGATGGACG CATTACTGCA ATCCGTCAGC CAAAGCCAAT 1560
TAAACGCCGA TACCGTCGAA GATGCCTTTA TGTCTTATCT GACATCGTTT GAACAAGTGG 1620
CTAATCTTAA AGTTATTAGC GCATATCACG ATAATATTAA TAACGATCAA GGGCTGACCT 1680
ATTTTATCGG ACTCAGTGAA ACTGATGCCG GTGAATATTA TTGGCGCAGT GTCGATCACA 1740
GTAAATTCAA CGACGGTAAA TTCGCGGCTA ATGCCTGGAG TGAATGGCAT AAAATTGATT 1800
GTCCAATTAA CCCTTATAAA AGCACTATCC GTCCAGTGAT ATATAAATCC CGCCTGTATC 1860
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TGCTCTGGTT GGAACAAAAG GAGATCACCA AACAGACAGG AAATAGTAAA GATGGCTATC 1920
AAACTGAAAC GGATTATCGT TATGAACTAA AATTGGCGCA TATCCGCTAT GATGGCACTT 1980
GGAATACGCC AATCACCTTT GATGTCAATA AAAAAATATC CGAGCTAAAA CTGGAP,AAAA 2040
ATAGAGCGCC CGGACTCTAT TGTGCCGGTT ATCAAGGTGA AGATACGTTG CTGGTGATGT 2100
TTTATAACCA ACAAGACACA CTAGATAGTT ATAAAAACGC TTCAATGCAA GGACTATATA 2160
TCTTTGCTGA TATGGCATCC AAAGATATGA CCCCAGAACA GAGCAATGTT TATCGGGATA 2220
ATAGCTATCA ACAATTTGAT ACCAATAATG TCAGAAGAGT GAATAACCGC TATGCAGAGG 2280
ATTATGAGAT TCCTTCTTCG GTAAGTAGCC GTAAAGACTA TGGTTGGGGA GATTATTACC 2340
TCAGCATGGT ATATAACGGA GATATTCCAA CTATCAATTA CAAAGCCGCA TCAAGTGATT 2400
TAAAAATTTA TATTTCACCA AAATTAAGAA TTATTCATAA TGGATATGAA GGACAGAAGC 2460
GCAATCAATG CAATTTGATG AATAAATATG GCAAACTAGG TGATAAATTT ATTGTGTATA 2520
CCAGCCTGGG CGTTAATCCG AATAATAAGC CGAATTC 2557
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 845 amino acids
(B) TYPE: amino acids
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (partial)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile Thr
1 5 10 15
Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn Leu
20 25 30
Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu Thr
35 40 45
Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys Thr
50 55 60
Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg Lys
65 70 75 80
Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val Phe
85 90 95
Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr Pro
100 105 110
Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly Phe
115 120 125
Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile Ala
130 135 140
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Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu Leu
145 150 155 160
Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu Gly
165 170 175
Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu Ala
180 185 190
Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala Gln
195 200 205
Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe Arg
210 215 220
Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala Ala
225 230 235 240
Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala Asp
245 250 255
Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala Phe
260 265 270
Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn Leu
275 280 285
Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His Gln
290 295 300
His Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr Ser
305 310 315 320
Ile Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Lys Cys
325 330 335
Arg Pro Thr Gly Arg Phe Arg Phe Gly Arg Ala Gly Leu Tyr Ser Ile
340 345 350
Asn Glu Arg Asp Thr Asp Leu Cys Pro Val Gly Lys Arg Gly Arg Arg
355 360 365
Ile Asn Arg Arg Val Glu Phe Asn Asn Arg Leu Ile His Tyr Asn Ala
370 375 380
Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg
385 390 395 400
Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr
405 410 415
Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr
420 425 430
Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala
435 440 445
Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln
450 455 460
Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala
465 470 475 480
Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr
485 490 495
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Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val
500 505 510
Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser
515 520 525
Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala
530 535 540
Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly
545 550 555 560
Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His
565 570 575
Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp
580 585 590
His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro
595 600 605
Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu
610 615 620
Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr
625 630 635 640
Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr
645 650 655
Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu
660 665 670
Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln
675 680 685
Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu
690 695 700
Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp
705 710 715 720
Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp
725 730 735
Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn
740 745 750
Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys
755 760 765
Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp
770 775 780
Ile Pro Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr
785 790 795 800
Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys
805 810 815
Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys
820 825 830
Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn
835 840 845
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(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULAR TYPE: protein
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly
1 5 10 15
Lys
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULAR TYPE: protein
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala
1 5 10 15
Ile Ser Pro Ala Lys
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDNESS: single
40 (D) TOPOLOGY: linear
(ii) MOLECULAR TYPE: protein
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
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Ala Asn Ser Leu Tyr Ala Leu Phe Leu Pro Gln
1 5 10
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULAR TYPE: protein
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln
1 5 10
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULAR TYPE: protein
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Glu Val Tyr
1 5 10 15
Ala Gly Leu Glu
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULAR TYPE: protein
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
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Ile Arg Glu Asp Tyr Pro Ala Ser Leu Gly Lys
1 5 10
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULAR TYPE: protein
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
Asp Asp Ser Gly Asp Asp Asp Lys Val Thr Asn Thr Asp Ile His
1 5 10 15
Arg
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULAR TYPE: protein
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
Asp Val Xaa Gly Ser Glu Lys Ala Asn Glu Lys Leu Lys
1 5 10
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7551 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46
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ATG AAC GAG TCT GTA AAA GAG ATA CCT GAT GTA TTA AAA AGC CAG TGT 48
Met Asn Glu Ser Val Lys Glu Ile Pro Asp Val Leu Lys Ser Gln Cys
1 5 10 15
GGT TTT AAT TGT CTG ACA GAT ATT AGC CAC AGC TCT TTT AAT GAA TTT 96
Gly Phe Asn Cys Leu Thr Asp Ile Ser His Ser Ser Phe Asn Glu Phe
20 25 30
CGC CAG CAA GTA TCT GAG CAC CTC TCC TGG TCC GAA ACA CAC GAC TTA 144
Arg Gln Gln Val Ser Glu His Leu Ser Trp Ser Glu Thr His Asp Leu
35 40 45
TAT CAT GAT GCA CAA CAG GCA CAA AAG GAT AAT CGC CTG TAT GAA GCG 192
Tyr His Asp Ala Gln Gln Ala Gln Lys Asp Asn Arg Leu Tyr Glu Ala
50 55 60
CGT ATT CTC AAA CGC GCC AAT CCC CAA TTA CAA AAT GCG GTG CAT CTT 240
Arg Ile Leu Lys Arg Ala Asn Pro Gln Leu Gin Asn Ala Val His Leu
65 70 75 80
GCC ATT CTC GCT CCC AAT GCT GAA CTG ATA GGC TAT AAC AAT CAA TTT 288
Ala Ile Leu Ala Pro Asn Ala Glu Leu Ile Gly Tyr Asn Asn Gln Phe
85 90 95
AGC GGT AGA GCC AGT CAA TAT GTT GCG CCG GGT ACC GTT TCT TCC ATG 336
Ser Gly Arg Ala Ser Gin Tyr Val Ala Pro Gly Thr Val Ser Ser Met
100 105 110
TTC TCC CCC GCC GCT TAT TTG ACT GAA CTT TAT CGT GAA GCA CGC AAT 384
Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Arg Asn
115 120 125
TTA CAC GCA AGT GAC TCC GTT TAT TAT CTG GAT ACC CGC CGC CCA GAT 432
Leu His Ala Ser Asp Ser Val Tyr Tyr Leu Asp Thr Arg Arg Pro Asp
130 135 140
CTC AAA TCA ATG GCG CTC AGT CAG CAA AAT ATG GAT ATA GAA TTA TCC 480
Leu Lys Ser Met Ala Leu Ser Gln Gln Asn Met Asp Ile Glu Leu Ser
145 150 155 160
ACA CTC TCT TTG TCC AAT GAG CTG TTA TTG GAA AGC ATT AAA ACT GAA 528
Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu Glu Ser Ile Lys Thr Glu
165 170 175
TCT AAA CTG GAA AAC TAT ACT AAA GTG ATG GAA ATG CTC TCC ACT TTC 576
Ser Lys Leu Glu Asn Tyr Thr Lys Val Met Glu Met Leu Ser Thr Phe
180 185 190
CGT CCT TCC GGC GCA ACG CCT TAT CAT GAT GCT TAT GAA AAT GTG CGT 624
Arg Pro Ser Gly Ala Thr Pro Tyr His Asp Ala Tyr Glu Asn Val Arg
195 200 205
GAA GTT ATC CAG CTA CAA GAT CCT GGA CTT GAG CAA CTC AAT GCA TCA 672
Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser
210 215 220
CCG GCA ATT GCC GGG TTG ATG CAT CAA GCC TCC CTA TTG GGT ATT AAC 720
Pro Ala Ile Ala Gly Leu Met His Gln Ala Ser Leu Leu Gly Ile Asn
225 230 235 240
GCT TCA ATC TCG CCT GAG CTA TTT AAT ATT CTG ACG GAG GAG ATT ACC 768
Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile Leu Thr Glu Glu Ile Thr
245 250 255
GAA GGT AAT GCT GAG GAA CTT TAT AAG AAA AAT TTT GGT AAT ATC GAA 816
Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys Asn Phe Gly Asn Ile Glu
260 265 270
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CCG GCC TCA TTG GCT ATG CCG GAA TAC CTT AAA CGT TAT TAT AAT TTA 864
Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu Lys Arg Tyr Tyr Asn Leu
275 280 285
AGC GAT GAA GAA CTT AGT CAG TTT ATT GGT AAA GCC AGC AAT TTT GGT 912
Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Lys Ala Ser Asn Phe Gly
290 295 300
CAA CAG GAA TAT AGT AAT AAC CAA CTT ATT ACT CCG GTA GTC AAC AGC 960
Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile Thr Pro Val Val Asn Ser
305 310 315 320
AGT GAT GGC ACG GTT AAG GTA TAT CGG ATC ACC CGC GAA TAT ACA ACC 1008
Ser Asp Gly Thr Val Lys Val Tyr Arg Ile Thr Arg Glu Tyr Thr Thr
325 330 335
AAT GCT TAT CAA ATG GAT GTG GAG CTA TTT CCC TTC GGT GGT GAG AAT 1056
Asn Ala Tyr Gln Met Asp Val Glu Leu Phe Pro Phe Gly Gly Glu Asn
340 345 350
TAT CGG TTA GAT TAT AAA TTC AAA AAT TTT TAT AAT GCC TCT TAT TTA 1104
Tyr Arg Leu Asp Tyr Lys Phe Lys Asn Phe Tyr Asn Ala Ser Tyr Leu
355 360 365
TCC ATC AAG TTA AAT GAT AAA AGA GAA CTT GTT CGA ACT GAA GGC GCT 1152
Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu Val Arg Thr Glu Gly Ala
370 375 380
CCT CAA GTC AAT ATA GAA TAC TCC GCA AAT ATC ACA TTA AAT ACC GCT 1200
Pro Gln Val Asn Ile Glu Tyr Ser Ala Asn Ile Thr Leu Asn Thr Ala
385 390 395 400
GAT ATC AGT CAA CCT TTT GAA ATT GGC CTG ACA CGA GTA CTT CCT TCC 1248
Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu Thr Arg Val Leu Pro Ser
405 410 415
GGT TCT TGG GCA TAT GCC GCC GCA AAA TTT ACC GTT GAA GAG TAT AAC 1296
Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe Thr Val Glu Glu Tyr Asn
420 425 430
CAA TAC TCT TTT CTG CTA AAA CTT AAC AAG GCT ATT CGT CTA TCA CGT 1344
Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg
435 440 445
GCG ACA GAA TTG TCA CCC ACG ATT CTG GAA GGC ATT GTG CGC AGT GTT 1392
Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu Gly Ile Val Arg Ser Val
450 455 460
AAT CTA CAA CTG GAT ATC AAC ACA GAC GTA TTA GGT AAA GTT TTT CTG 1440
Asn Leu Gln Leu Asp Ile Asn Thr Asp Val Leu Gly Lys Val Phe Leu
465 470 475 480
ACT AAA TAT TAT ATG CAG CGT TAT GCT ATT CAT GCT GAA ACT GCC CTG 1488
Thr Lys Tyr Tyr Met Gln Arg Tyr Ala Ile His Ala Glu Thr Ala Leu
485 490 495
ATA CTA TGC AAC GCG CCT ATT TCA CAA CGT TCA TAT GAT AAT CAA CCT 1536
Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro
500 505 510
AGC CAA TTT GAT CGC CTG TTT AAT ACG CCA TTA CTG AAC GGA CAA TAT 1584
Ser Gln Phe Asp Arg Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln Tyr
515 520 525
TTT TCT ACC GGC GAT GAG GAG ATT GAT TTA AAT TCA GGT AGC ACC GGC 1632
Phe Ser Thr Gly Asp Glu Glu Ile Asp Leu Asn Ser Gly Ser Thr Gly
530 535 540
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GAT TGG CGA AAA ACC ATA CTT AAG CGT GCA TTT AAT ATT GAT GAT GTC 1680
Asp Trp Arg Lys Thr Ile Leu Lys Arg Ala Phe Asn Ile Asp Asp Val
545 550 555 560
TCG CTC TTC CGC CTG CTT AAA ATT ACC GAC CAT GAT AAT AAA GAT GGA 1728
Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys Asp Gly
565 570 575
AAA ATT AAA AAT AAC CTA AAG AAT CTT TCC AAT TTA TAT ATT GGA AAA 1776
Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile Gly Lys
580 585 590
TTA CTG GCA GAT ATT CAT CAA TTA ACC ATT GAT GAA CTG GAT TTA TTA 1824
Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu
595 600 605
CTG ATT GCC GTA GGT GAA GGA AAA ACT AAT TTA TCC GCT ATC AGT GAT 1872
Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile Ser Asp
610 615 620
AAG CAA TTG GCT ACC CTG ATC AGA AAA CTC AAT ACT ATT ACC AGC TGG 1920
Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp
625 630 635 640
CTA CAT ACA CAG AAG TGG AGT GTA TTC CAG CTA TTT ATC ATG ACC TCC 1968
Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser
645 650 655
ACC AGC TAT AAC AAA ACG CTA ACG CCT GAA ATT AAG AAT TTG CTG GAT 2016
Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp
660 665 670
ACC GTC TAC CAC GGT TTA CAA GGT TTT GAT AAA GAC AAA GCA GAT TTG 2064
Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu
675 680 685
CTA CAT GTC ATG GCG CCC TAT ATT GCG GCC ACC TTG CAA TTA TCA TCG 2112
Leu His Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser
690 695 700
GAA AAT GTC GCC CAC TCG GTA CTC CTT TGG GCA GAT AAG TTA CAG CCC 2160
Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro
705 710 715 720
GGC GAC GGC GCA ATG ACA GCA GAA AAA TTC TGG GAC TGG TTG AAT ACT 2208
Gly Asp Gly Ala Met Thr Ala Glu Lys Phe Trp Asp Trp Leu Asn Thr
725 730 735
AAG TAT ACG CCG GGT TCA TCG GAA GCC GTA GAA ACG CAG GAA CAT ATC 2256
Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile
740 745 750
GTT CAG TAT TGT CAG GCT CTG GCA CAA TTG GAA ATG GTT TAC CAT TCC 2304
Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr His Ser
755 760 765
ACC GGC ATC AAC GAA AAC GCC TTC CGT CTA TTT GTG ACA AAA CCA GAG 2352
Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu
770 775 780
ATG TTT GGC GCT GCA ACT GGA GCA GCG CCC GCG CAT GAT GCC CTT TCA 2400
Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala Leu Ser
785 790 795 800
CTG ATT ATG CTG ACA CGT TTT GCG GAT TGG GTG AAC GCA CTA GGC GAA 2448
Leu Ile Met Leu Thr Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu
805 810 815
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AAA GCG TCC TCG GTG CTA GCG GCA TTT GAA GCT AAC TCG TTA ACG GCA 2496
Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala
820 825 830
GAA CAA CTG GCT GAT GCC ATG AAT CTT GAT GCT AAT TTG CTG TTG CAA 2544
Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln
835 840 845
GCC AGT ATT CAA GCA CAA AAT CAT CAA CAT CTT CCC CCA GTA ACT CCA 2592
Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val Thr Pro
850 855 860
GAA AAT GCG TTC TCC TGT TGG ACA TCT ATC AAT ACT ATC CTG CAA TGG 2640
Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu Gln Trp
865 870 875 880
GTT AAT GTC GCA CAA CAA TTG AAT GTC GCC CCA CAG GGC GTT TCC GCT 2688
Val Asn Val Ala Gln Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala
885 890 895
TTG GTC GGG CTG GAT TAT ATT CAA TCA ATG AAA GAG ACA CCG ACC TAT 2736
Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Lys Glu Thr Pro Thr Tyr
900 905 910
GCC CAG TGG GAA AAC GCG GCA GGC GTA TTA ACC GCC GGG TTG AAT TCA 2784
Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Thr Ala Gly Leu Asn Ser
915 920 925
CAA CAG GCT AAT ACA TTA CAC GCT TTT CTG GAT GAA TCT CGC AGT GCC 2832
Gln Gln Ala Asn Thr Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala
930 935 940
GCA TTA AGC ACC TAC TAT ATC CGT CAA GTC GCC AAG GCA GCG GCG GCT 2880
Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala
945 950 955 960
ATT AAA AGC CGT GAT GAC TTG TAT CAA TAC TTA CTG ATT GAT AAT CAG 2928
Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln
965 970 975
GTT TCT GCG GCA ATA AAA ACC ACC CGG ATC GCC GAA GCC ATT GCC AGT 2976
Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser
980 985 990
ATT CAA CTG TAC GTC AAC CGG GCA TTG GAA AAT GTG GAA GAA AAT GCC 3024
Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu Asn Ala
995 1000 1005
AAT TCG GGG GTT ATC AGC CGC CAA TTC TTT ATC GAC TGG GAC AAA TAC 3072
Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr
1010 1015 1020
AAT AAA CGC TAC AGC ACT TGG GCG GGT GTT TCT CAA TTA GTT TAC TAC 3120
Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr
1025 1030 1035 1040
CCG GAA AAC TAT ATT GAT CCG ACC ATG CGT ATC GGA CAA ACC AAA ATG 3168
Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met
1045 1050 1055
ATG GAC GCA TTA CTG CAA TCC GTC AGC CAA AGC CAA TTA AAC GCC GAT 3216
Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp
1060 1065 1070
ACC GTC GAA GAT GCC TTT ATG TCT TAT CTG ACA TCG TTT GAA CAA GTG 3264
Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val
1075 1080 1085
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GCT AAT CTT AAA GTT ATT AGC GCA TAT CAC GAT AAT ATT AAT AAC GAT 3312
Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Ile Asn Asn Asp
1090 1095 1100
CAA GGG CTG ACC TAT TTT ATC GGA CTC AGT GAA ACT GAT GCC GGT GAA 3360
Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Glu Thr Asp Ala Gly Glu
1105 1110 1115 1120
TAT TAT TGG CGC AGT GTC GAT CAC AGT AAA TTC AAC GAC GGT AAA TTC 3408
Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Phe Asn Asp Gly Lys Phe
1125 1130 1135
GCG GCT AAT GCC TGG AGT GAA TGG CAT AAA ATT GAT TGT CCA ATT AAC 3456
Ala Ala Asn Ala Trp Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn
1140 1145 1150
CCT TAT AAA AGC ACT ATC CGT CCA GTG ATA TAT AAA TCC CGC CTG TAT 3504
Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr
1155 1160 1165
CTG CTC TGG TTG GAA CAA AAG GAG ATC ACC AAA CAG ACA GGA AAT AGT 3552
Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser
1170 1175 1180
AAA GAT GGC TAT CAA ACT GAA ACG GAT TAT CGT TAT GAA CTA AAA TTG 3600
Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu
1185 1190 1195 1200
GCG CAT ATC CGC TAT GAT GGC ACT TGG AAT ACG CCA ATC ACC TTT GAT 3648
Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp
1205 1210 1215
GTC AAT AAA AAA ATA TCC GAG CTA AAA CTG GAA AAA AAT AGA GCG CCC 3696
Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg Ala Pro
1220 1225 1230
GGA CTC TAT TGT GCC GGT TAT CAA GGT GAA GAT ACG TTG CTG GTG ATG 3744
Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met
1235 1240 1245
TTT TAT AAC CAA CAA GAC ACA CTA GAT AGT TAT AAA AAC GCT TCA ATG 3792
Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys Asn Ala Ser Met
1250 1255 1260
CAA GGA CTA TAT ATC TTT GCT GAT ATG GCA TCC AAA GAT ATG ACC CCA 3840
Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met Thr Pro
1265 1270 1275 1280
GAA CAG AGC AAT GTT TAT CGG GAT AAT AGC TAT CAA CAA TTT GAT ACC 3888
Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe Asp Thr
1285 1290 1295
AAT AAT GTC AGA AGA GTG AAT AAC CGC TAT GCA GAG GAT TAT GAG ATT 3936
Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile
1300 1305 1310
CCT TCC TCG GTA AGT AGC CGT AAA GAC TAT GGT TGG GGA GAT TAT TAC 3984
Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gly Trp Gly Asp Tyr Tyr
1315 1320 1325
CTC AGC ATG GTA TAT AAC GGA GAT ATT CCA ACT ATC AAT TAC AAA GCC 4032
Leu Ser Met Val Tyr Asn Gly Asp Ile Pro Thr Ile Asn Tyr Lys Ala
1330 1335 1340
GCA TCA AGT GAT TTA AAA ATC TAT ATC TCA CCA AAA TTA AGA ATT ATT 4080
Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile
1345 1350 1355 1360
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CAT AAT GGA TAT GAA GGA CAG AAG CGC AAT CAA TGC AAT CTG ATG AAT 4128
His Asn Gly Tyr Glu Gly Gln Lys Arg Asn Gln Cys Asn Leu Met Asn
1365 1370 1375
AAA TAT GGC AAA CTA GGT GAT AAA TTT ATT GTT TAT ACT AGC TTG GGG 4176
Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile Val Tyr Thr Ser Leu Gly
1380 1385 1390
GTC AAT CCA AAT AAC TCG TCA AAT AAG CTC ATG TTT TAC CCC GTC TAT 4224
Val Asn Pro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr
1395 1400 1405
CAA TAT AGC GGA AAC ACC AGT GGA CTC AAT CAA GGG AGA CTA CTA TTC 4272
Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn Gln Gly Arg Leu Leu Phe
1410 1415 1420
CAC CGT GAC ACC ACT TAT CCA TCT AAA GTA GAA GCT TGG ATT CCT GGA 4320
His Arg Asp Thr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile Pro Gly
1425 1430 1435 1440
GCA AAA CGT TCT CTA ACC AAC CAA AAT GCC GCC ATT GGT GAT GAT TAT 4368
Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala Ala Ile Gly Asp Asp Tyr
1445 1450 1455
GCT ACA GAC TCT CTG AAT AAA CCG GAT GAT CTT AAG CAA TAT ATC TTT 4416
Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp Leu Lys Gln Tyr Ile Phe
1460 1465 1470
ATG ACT GAC AGT AAA GGG ACT GCT ACT GAT GTC TCA GGC CCA GTA GAG 4464
Met Thr Asp Ser Lys Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu
1475 1480 1485
ATT AAT ACT GCA ATT TCT CCA GCA AAA GTT CAG ATA ATA GTC AAA GCG 4512
Ile Asn Thr Ala Ile Ser Pro Ala Lys Val Gln Ile Ile Val Lys Ala
1490 1495 1500
GGT GGC AAG GAG CAA ACT TTT ACC GCA GAT AAA GAT GTC TCC ATT CAG 4560
Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Lys Asp Val Ser Ile Gln
1505 1510 1515 1520
CCA TCA CCT AGC TTT GAT GAA ATG AAT TAT CAA TTT AAT GCC CTT GAA 4608
Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gln Phe Asn Ala Leu Glu
1525 1530 1535
ATA GAC GGT TCT GGT CTG AAT TTT ATT AAC AAC TCA GCC AGT ATT GAT 4656
Ile Asp Gly Ser Gly Leu Asn Phe Ile Asn Asn Ser Ala Ser Ile Asp
1540 1545 1550
GTT ACT TTT ACC GCA TTT GCG GAG GAT GGC CGC AAA CTG GGT TAT GAA 4704
Val Thr Phe Thr Ala Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu
1555 1560 1565
AGT TTC AGT ATT CCT GTT ACC CTC AAG GTA AGT ACC GAT AAT GCC CTG 4752
Ser Phe Ser Ile Pro Val Thr Leu Lys Val Ser Thr Asp Asn Ala Leu
1570 1575 1580
ACC CTG CAC CAT AAT GAA AAT GGT GCG CAA TAT ATG CAA TGG CAA TCC 4800
Thr Leu His His Asn Glu Asn Gly Ala Gln Tyr Met Gin Trp Gln Ser
1585 1590 1595 1600
TAT CGT ACC CGC CTG AAT ACT CTA TTT GCC CGC CAG TTG GTT GCA CGC 4848
Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg
1605 1610 1615
GCC ACC ACC GGA ATC GAT ACA ATT CTG AGT ATG GAA ACT CAG AAT ATT 4896
Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile
1620 1625 1630
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CA 02209659 1998-09-15
CAG GAA CCG CAG TTA GGC AAA GGT TTC TAT GCT ACG TTC GTG ATA CCT 4944
Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Pro
1635 1640 1645
CCC TAT AAC CTA TCA ACT CAT GGT GAT GAA CGT TGG TTT AAG CTT TAT 4992
Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys Leu Tyr
1650 1655 1660
ATC AAA CAT GTT GTT GAT AAT AAT TCA CAT ATT ATC TAT TCA GGC CAG 5040
Ile Lys His Val Val Asp Asn Asn Ser His Ile Ile Tyr Ser Gly Gln
1665 1670 1675 1680
CTA ACA GAT ACA AAT ATA AAC ATC ACA TTA TTT ATT CCT CTT GAT GAT 5088
Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe Ile Pro Leu Asp Asp
1685 1690 1695
GTC CCA TTG AAT CAA GAT TAT CAC GCC AAG GTT TAT ATG ACC TTC AAG 5136
Val Pro Leu Asn Gln Asp Tyr His Ala Lys Val Tyr Met Thr Phe Lys
1700 1705 1710
AAA TCA CCA TCA GAT GGT ACC TGG TGG GGC CCT CAC TTT GTT AGA GAT 5184
Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pro His Phe Val Arg Asp
1715 1720 1725
GAT AAA GGA ATA GTA ACA ATA AAC CCT AAA TCC ATT TTG ACC CAT TTT 5232
Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe
1730 1735 1740
GAG AGC GTC AAT GTC CTG AAT AAT ATT AGT AGC GAA CCA ATG GAT TTC 5280
Glu Ser Val Asn Val Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe
1745 1750 1755 1760
AGC GGC GCT AAC AGC CTC TAT TTC TGG GAA CTG TTC TAC TAT ACC CCG 5328
Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro
1765 1770 1775
ATG CTG GTT GCT CAA CGT TTG CTG CAT GAA CAG AAC TTC GAT GAA GCC 5376
Met Leu Val Ala Gln Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala
1780 1785 1790
AAC CGT TGG CTG AAA TAT GTC TGG AGT CCA TCC GGT TAT ATT GTC CAC 5424
Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His
1795 1800 1805
GGC CAG ATT CAG AAC TAC CAG TGG AAC GTC CGC CCG TTA CTG GAA GAC 5472
Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp
1810 1815 1820
ACC AGT TGG AAC AGT GAT CCT TTG GAT TCC GTC GAT CCT GAC GCG GTA 5520
Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val
1825 1830 1835 1840
GCA CAG CAC GAT CCA ATG CAC TAC AAA GTT TCA ACT TTT ATG CGT ACC 5568
Ala Gln His Asp Pro Met His Tyr Lys Val Ser Thr Phe Met Arg Thr
1845 1850 1855
TTG GAT CTA TTG ATA GCA CGC GGC GAC CAT GCT TAT CGC CAA CTG GAA 5616
Leu Asp Leu Leu Ile Ala Arg Gly Asp His Ala Tyr Arg Gln Leu Glu
1860 1865 1870
CGA GAT ACA CTC AAC GAA GCG AAG ATG TGG TAT ATG CAA GCG CTG CAT 5664
Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gln Ala Leu His
1875 1880 1885
CTA TTA GGT GAC AAA CCT TAT CTA CCG CTG AGT ACG ACA TGG AGT GAT 5712
Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr Thr Trp Ser Asp
1890 1895 1900
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CCA CGA CTA GAC AGA GCC GCG GAT ATC ACT ACC CAA AAT GCT CAC GAC 5760
Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr Thr Gln Asn Ala His Asp
1905 1910 1915 1920
AGC GCA ATA GTC GCT CTG CGG CAG AAT ATA CCT ACA CCG GCA CCT TTA 5808
Ser Ala Ile Val Ala Leu Arg Gln Asn Ile Pro Thr Pro Ala Pro Leu
1925 1930 1935
TCA TTG CGC AGC GCT AAT ACC CTG ACT GAT CTC TTC CTG CCG CAA ATC 5856
Ser Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile
1940 1945 1950
AAT GAA GTG ATG ATG AAT TAC TGG CAG ACA TTA GCT CAG AGA GTA TAC 5904
Asn Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr
1955 1960 1965
AAT CTG CGT CAT AAC CTC TCT ATC GAC GGC CAG CCG TTA TAT CTG CCA 5952
Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gin Pro Leu Tyr Leu Pro
1970 1975 1980
ATC TAT GCC ACA CCG GCC GAT CCG AAA GCG TTA CTC AGC GCC GCC GTT 6000
Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val
1985 1990 1995 2000
GCC ACT TCT CAA GGT GGA GGC AAG CTA CCG GAA TCA TTT ATG TCC CTG 6048
Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu
2005 2010 2015
TGG CGT TTC CCG CAC ATG CTG GAA AAT GCG CGC GGC ATG GTT AGC CAG 6096
Trp Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln
2020 2025 2030
CTC ACC CAG TTC GGC TCC ACG TTA CAA AAT ATT ATC GAA CGT CAG GAC 6144
Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp
2035 2040 2045
GCG GAA GCG CTC AAT GCG TTA TTA CAA AAT CAG GCC GCC GAG CTG ATA 6192
Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile
2050 2055 2060
TTG ACT AAC CTG AGC ATT CAG GAC AAA ACC ATT GAA GAA TTG GAT GCC 6240
Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala
2065 2070 2075 2080
GAG AAA ACG GTG TTG GAA AAA TCC AAA GCG GGA GCA CAA TCG CGC TTT 6288
Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe
2085 2090 2095
GAT AGC TAC GGC AAA CTG TAC GAT GAG AAT ATC AAC GCC GGT GAA AAC 6336
Asp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn
2100 2105 2110
CAA GCC ATG ACG CTA CGA GCG TCC GCC GCC GGG CTT ACC ACG GCA GTT 6384
Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val
2115 2120 2125
CAG GCA TCC CGT CTG GCC GGT GCG GCG GCT GAT CTG GTG CCT AAC ATC 6432
Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile
2130 2135 2140
TTC GGC TTT GCC GGT GGC GGC AGC CGT TGG GGG GCT ATC GCT GAG GCG 6480
Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala
2145 2150 2155 2160
ACA GGT TAT GTG ATG GAA TTC TCC GCG AAT GTT ATG AAC ACC GAA GCG 6528
Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala
2165 2170 2175
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GAT AAA ATT AGC CAA TCT GAA ACC TAC CGT CGT CGC CGT CAG GAG TGG 6576
Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp
2180 2185 2190
GAG ATC CAG CGG AAT AAT GCC GAA GCG GAA TTG AAG CAA ATC GAT GCT 6624
Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala
2195 2200 2205
CAG CTC AAA TCA CTC GCT GTA CGC CGC GAA GCC GCC GTA TTG CAG AAA 6672
Gln Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys
2210 2215 2220
ACC AGT CTG AAA ACC CAA CAA GAA CAG ACC CAA TCT CAA TTG GCC TTC 6720
Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe
2225 2230 2235 2240
CTG CAA CGT AAG TTC AGC AAT CAG GCG TTA TAC AAC TGG CTG CGT GGT 6768
Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly
2245 2250 2255
CGA CTG GCG GCG ATT TAC TTC CAG TTC TAC GAT TTG GCC GTC GCG CGT 6816
Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg
2260 2265 2270
TGC CTG ATG GCA GAA CAA GCT TAC CGT TGG GAA CTC AAT GAT GAC TCT 6864
Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser
2275 2280 2285
GCC CGC TTC ATT AAA CCG GGC GCC TGG CAG GGA ACC TAT GCC GGT CTG 6912
Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu
2290 2295 2300
CTT GCA GGT GAA ACC TTG ATG CTG AGT CTG GCA CAA ATG GAA GAC GCT 6960
Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala
2305 2310 2315 2320
CAT CTG AAA CGC GAT AAA CGC GCA TTA GAG GTT GAA CGC ACA GTA TCG 7008
His Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser
2325 2330 2335
CTG GCC GAA GTT TAT GCA GGA TTA CCA AAA GAT AAC GGT CCA TTT TCC 7056
Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser
2340 2345 2350
CTG GCT CAG GAA ATT GAC AAG CTG GTG AGT CAA GGT TCA GGC AGT GCC 7104
Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala
2355 2360 2365
GGC AGT GGT AAT AAT AAT TTG GCG TTC GGC GCC GGC ACG GAC ACT AAA 7152
Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys
2370 2375 2380
ACC TCT TTG CAG GCA TCA GTT TCA TTC GCT GAT TTG AAA ATT CGT GAA 7200
Thr Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu
2385 2390 2395 2400
GAT TAC CCG GCA TCG CTT GGC AAA ATT CGA CGT ATC AAA CAG ATC AGC 7248
Asp Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser
2405 2410 2415
GTC ACT TTG CCC GCG CTA CTG GGA CCG TAT CAG GAT GTA CAG GCA ATA 7296
Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile
2420 2425 2430
TTG TCT TAC GGC GAT AAA GCC GGA TTA GCT AAC GGC TGT GAA GCG CTG 7344
Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu
2435 2440 2445
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CA 02209659 1998-09-15
GCA GTT TCT CAC GGT ATG AAT GAC AGC GGC CAA TTC CAG CTC GAT TTC 7392
Ala Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe
2450 2455 2460
AAC GAT GGC AAA TTC CTG CCA TTC GAA GGC ATC GCC ATT GAT CAA GGC 7440
Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly
2465 2470 2475 2480
ACG CTG ACA CTG AGC TTC CCA AAT GCA TCT ATG CCG GAG AAA GGT AAA 7488
Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys
2485 2490 2495
CAA GCC ACT ATG TTA AAA ACC CTG AAC GAT ATC ATT TTG CAT ATT CGC 7536
Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg
2500 2505 2510
TAC ACC ATT AAA TAA 7551
Tyr Thr Ile Lys
2516
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2516 amino acids
(B) TYPE: amino acids
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
Met Asn Glu Ser Val Lys Glu Ile Pro Asp Val Leu Lys Ser Gln Cys
1 5 10 15
Gly Phe Asn Cys Leu Thr Asp Ile Ser His Ser Ser Phe Asn Glu Phe
20 25 30
Arg Gln Gln Val Ser Glu His Leu Ser Trp Ser Glu Thr His Asp Leu
40 45
Tyr His Asp Ala Gln Gln Ala Gln Lys Asp Asn Arg Leu Tyr Glu Ala
50 55 60
Arg Ile Leu Lys Arg Ala Asn Pro Gin Leu Gin Asn Ala Val His Leu
65 70 75 80
Ala Ile Leu Ala Pro Asn Ala Glu Leu Ile Gly Tyr Asn Asn Gln Phe
85 90 95
Ser Gly Arg Ala Ser Gln Tyr Val Ala Pro Gly Thr Val Ser Ser Met
100 105 110
Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Arg Asn
115 120 125
Leu His Ala Ser Asp Ser Val Tyr Tyr Leu Asp Thr Arg Arg Pro Asp
130 135 140
Leu Lys Ser Met Ala Leu Ser Gln Gln Asn Met Asp Ile Glu Leu Ser
145 150 155 160
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Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu Glu Ser Ile Lys Thr Glu
165 170 175
Ser Lys Leu Glu Asn Tyr Thr Lys Val Met Glu Met Leu Ser Thr Phe
180 185 190
Arg Pro Ser Gly Ala Thr Pro Tyr His Asp Ala Tyr Glu Asn Val Arg
195 200 205
Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser
210 215 220
Pro Ala Ile Ala Gly Leu Met His Gln Ala Ser Leu Leu Gly Ile Asn
225 230 235 240
Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile Leu Thr Glu Glu Ile Thr
245 250 255
Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys Asn Phe Gly Asn Ile Glu
260 265 270
Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu Lys Arg Tyr Tyr Asn Leu
275 280 285
Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Lys Ala Ser Asn Phe Gly
290 295 300
Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile Thr Pro Val Val Asn Ser
305 310 315 320
Ser Asp Gly Thr Val Lys Val Tyr Arg Ile Thr Arg Glu Tyr Thr Thr
325 330 335
Asn Ala Tyr Gln Met Asp Val Glu Leu Phe Pro Phe Gly Gly Glu Asn
340 345 350
Tyr Arg Leu Asp Tyr Lys Phe Lys Asn Phe Tyr Asn Ala Ser Tyr Leu
355 360 365
Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu Val Arg Thr Glu Gly Ala
370 375 380
Pro Gln Val Asn Ile Glu Tyr Ser Ala Asn Ile Thr Leu Asn Thr Ala
385 390 395 400
Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu Thr Arg Val Leu Pro Ser
405 410 415
Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe Thr Val Glu Glu Tyr Asn
420 425 430
Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg
435 440 445
Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu Gly Ile Val Arg Ser Val
450 455 460
Asn Leu Gln Leu Asp Ile Asn Thr Asp Val Leu Gly Lys Val Phe Leu
465 470 475 480
Thr Lys Tyr Tyr Met Gln Arg Tyr Ala Ile His Ala Glu Thr Ala Leu
485 490 495
Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro
500 505 510
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Ser Gln Phe Asp Arg Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln Tyr
515 520 525
Phe Ser Thr Gly Asp Glu Glu Ile Asp Leu Asn Ser Gly Ser Thr Gly
530 535 540
Asp Trp Arg Lys Thr Ile Leu Lys Arg Ala Phe Asn Ile Asp Asp Val
545 550 555 560
Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys Asp Gly
565 570 575
Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile Gly Lys
580 585 590
Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu
595 600 605
Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile Ser Asp
610 615 620
Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp
625 630 635 640
Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser
645 650 655
Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp
660 665 670
Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu
675 680 685
Leu His Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser
690 695 700
Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro
705 710 715 720
Gly Asp Gly Ala Met Thr Ala Glu Lys Phe Trp Asp Trp Leu Asn Thr
725 730 735
Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile
740 745 750
Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr His Ser
755 760 765
Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu
770 775 780
Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala Leu Ser
785 790 795 800
Leu Ile Met Leu Thr Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu
805 810 815
Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala
820 825 830
Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln
835 840 845
Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val Thr Pro
850 855 860
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Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu Gln Trp
865 870 875 880
Val Asn Val Ala Gln Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala
885 890 895
Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Lys Glu Thr Pro Thr Tyr
900 905 910
Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Thr Ala Gly Leu Asn Ser
915 920 925
Gln Gln Ala Asn Thr Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala
930 935 940
Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala
945 950 955 960
Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln
965 970 975
Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser
980 985 990
Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu Asn Ala
995 1000 1005
Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr
1010 1015 1020
Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr
1025 1030 1035 1040
Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met
1045 1050 1055
Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp
1060 1065 1070
Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val
1075 1080 1085
Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Ile Asn Asn Asp
1090 1095 1100
Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Glu Thr Asp Ala Gly Glu
1105 1110 1115 1120
Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Phe Asn Asp Gly Lys Phe
1125 1130 1135
Ala Ala Asn Ala Trp Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn
1140 1145 1150
Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr
1155 1160 1165
Leu Leu Trp Leu Glu Gin Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser
1170 1175 1180
Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu
1185 1190 1195 1200
Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp
1205 1210 1215
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Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg Ala Pro
1220 1225 1230
Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met
1235 1240 1245
Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys Asn Ala Ser Met
1250 1255 1260
Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met Thr Pro
1265 1270 1275 1280
Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe Asp Thr
1285 1290 1295
Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile
1300 1305 1310
Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gly Trp Gly Asp Tyr Tyr
1315 1320 1325
Leu Ser Met Val Tyr Asn Gly Asp Ile Pro Thr Ile Asn Tyr Lys Ala
1330 1335 1340
Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile
1345 1350 1355 1360
His Asn Gly Tyr Glu Gly Gln Lys Arg Asn Gln Cys Asn Leu Met Asn
1365 1370 1375
Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile Val Tyr Thr Ser Leu Gly
1380 1385 1390
Val Asn Pro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr
1395 1400 1405
Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn Gln Gly Arg Leu Leu Phe
1410 1415 1420
His Arg Asp Thr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile Pro Gly
1425 1430 1435 1440
Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala Ala Ile Gly Asp Asp Tyr
1445 1450 1455
Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp Leu Lys Gln Tyr Ile Phe
1460 1465 1470
Met Thr Asp Ser Lys Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu
1475 1480 1485
Ile Asn Thr Ala Ile Ser Pro Ala Lys Val Gln Ile Ile Val Lys Ala
1490 1495 1500
Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Lys Asp Val Ser Ile Gln
1505 1510 1515 1520
Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gln Phe Asn Ala Leu Glu
1525 1530 1535
Ile Asp Gly Ser Gly Leu Asn Phe Ile Asn Asn Ser Ala Ser Ile Asp
1540 1545 1550
Val Thr Phe Thr Ala Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu
1555 1560 1565
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Ser Phe Ser Ile Pro Val Thr Leu Lys Val Ser Thr Asp Asn Ala Leu
1570 1575 1580
Thr Leu His His Asn Glu Asn Gly Ala Gln Tyr Met Gln Trp Gln Ser
1585 1590 1595 1600
Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg
1605 1610 1615
Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile
1620 1625 1630
Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Pro
1635 1640 1645
Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys Leu Tyr
1650 1655 1660
Ile Lys His Val Val Asp Asn Asn Ser His Ile Ile Tyr Ser Gly Gln
1665 1670 1675 1680
Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe Ile Pro Leu Asp Asp
1685 1690 1695
Val Pro Leu Asn Gln Asp Tyr His Ala Lys Val Tyr Met Thr Phe Lys
1700 1705 1710
Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pro His Phe Val Arg Asp
1715 1720 1725
Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe
1730 1735 1740
Glu Ser Val Asn Val Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe
1745 1750 1755 1760
Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro
1765 1770 1775
Met Leu Val Ala Gln Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala
1780 1785 1790
Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His
1795 1800 1805
Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp
1810 1815 1820
Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val
1825 1830 1835 1840
Ala Gln His Asp Pro Met His Tyr Lys Val Ser Thr Phe Met Arg Thr
1845 1850 1855
Leu Asp Leu Leu Ile Ala Arg Gly Asp His Ala Tyr Arg Gln Leu Glu
1860 1865 1870
Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gln Ala Leu His
1875 1880 1885
Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr Thr Trp Ser Asp
1890 1895 1900
Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr Thr Gln Asn Ala His Asp
1905 1910 1915 1920
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Ser Ala Ile Val Ala Leu Arg Gln Asn Ile Pro Thr Pro Ala Pro Leu
1925 1930 1935
Ser Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile
1940 1945 1950
Asn Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr
1955 1960 1965
Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro
1970 1975 1980
Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val
1985 1990 1995 2000
Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu
2005 2010 2015
Trp Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln
2020 2025 2030
Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp
2035 2040 2045
Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile
2050 2055 2060
Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala
2065 2070 2075 2080
Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe
2085 2090 2095
Asp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn
2100 2105 2110
Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val
2115 2120 2125
Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile
2130 2135 2140
Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala
2145 2150 2155 2160
Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala
2165 2170 2175
Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp
2180 2185 2190
Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala
2195 2200 2205
Gln Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys
2210 2215 2220
Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe
2225 2230 2235 2240
Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly
2245 2250 2255
Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg
2260 2265 2270
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Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser
2275 2280 2285
Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu
2290 2295 2300
Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala
2305 2310 2315 2320
His Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser
2325 2330 2335
Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser
2340 2345 2350
Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala
2355 2360 2365
Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys
2370 2375 2380
Thr Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu
2385 2390 2395 2400
Asp Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser
2405 2410 2415
Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile
2420 2425 2430
Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu
2435 2440 2445
Ala Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe
2450 2455 2460
Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly
2465 2470 2475 2480
Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys
2485 2490 2495
Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg
2500 2505 2510
Tyr Thr Ile Lys
2516
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5547 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
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CTG ATA GGC TAT AAC AAT CAA TTT AGC GGT AGA GCC AGT CAA TAT GTT 48
Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val
1 5 10 15
GCG CCG GGT ACC GTT TCT TCC ATG TTC TCC CCC GCC GCT TAT TTG ACT 96
Ala Pro Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr
20 25 30
GAA CTT TAT CGT GAA GCA CGC AAT TTA CAC GCA AGT GAC TCC GTT TAT 144
Glu Leu Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val Tyr
35 40 45
TAT CTG GAT ACC CGC CGC CCA GAT CTC AAA TCA ATG GCG CTC AGT CAG 192
Tyr Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu Ser Gln
50 55 60
CAA AAT ATG GAT ATA GAA TTA TCC ACA CTC TCT TTG TCC AAT GAG CTG 240
Gln Asn Met Asp Ile Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu
65 70 75 80
TTA TTG GAA AGC ATT AAA ACT GAA TCT AAA CTG GAA AAC TAT ACT AAA 288
Leu Leu Glu Ser Ile Lys Thr Glu Ser Lys Leu Glu Asn Tyr Thr Lys
85 90 95
GTG ATG GAA ATG CTC TCC ACT TTC CGT CCT TCC GGC GCA ACG CCT TAT 336
Val Met Glu Met Leu Ser Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr
100 105 110
CAT GAT GCT TAT GAA AAT GTG CGT GAA GTT ATC CAG CTA CAA GAT CCT 384
His Asp Ala Tyr Glu Asn Val Arg Glu Val Ile Gln Leu Gln Asp Pro
115 120 125
GGA CTT GAG CAA CTC AAT GCA TCA CCG GCA ATT GCC GGG TTG ATG CAT 432
Gly Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu Met His
130 135 140
CAA GCC TCC CTA TTG GGT ATT AAC GCT TCA ATC TCG CCT GAG CTA TTT 480
Gln Ala Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe
145 150 155 160
AAT ATT CTG ACG GAG GAG ATT ACC GAA GGT AAT GCT GAG GAA CTT TAT 528
Asn Ile Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr
165 170 175
AAG AAA AAT TTT GGT AAT ATC GAA CCG GCC TCA TTG GCT ATG CCG GAA 576
Lys Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met Pro Glu
180 185 190
TAC CTT AAA CGT TAT TAT AAT TTA AGC GAT GAA GAA CTT AGT CAG TTT 624
Tyr Leu Lys Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe
195 200 205
ATT GGT AAA GCC AGC AAT TTT GGT CAA CAG GAA TAT AGT AAT AAC CAA 672
Ile Gly Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln
210 215 220
CTT ATT ACT CCG GTA GTC AAC AGC AGT GAT GGC ACG GTT AAG GTA TAT 720
Leu Ile Thr Pro Val Val Asn Ser Ser Asp Gly Thr Val Lys Val Tyr
225 230 235 240
CGG ATC ACC CGC GAA TAT ACA ACC AAT GCT TAT CAA ATG GAT GTG GAG 768
Arg Ile Thr Arg Glu Tyr Thr Thr Asn Ala Tyr Gln Met Asp Val Glu
245 250 255
CTA TTT CCC TTC GGT GGT GAG AAT TAT CGG TTA GAT TAT AAA TTC AAA 816
Leu Phe Pro Phe Gly Gly Glu Asn Tyr Arg Leu Asp Tyr Lys Phe Lys
260 265 270
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AAT TTT TAT AAT GCC TCT TAT TTA TCC ATC AAG TTA AAT GAT AAA AGA 864
Asn Phe Tyr Asn Ala Ser Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg
275 280 285
GAA CTT GTT CGA ACT GAA GGC GCT CCT CAA GTC AAT ATA GAA TAC TCC 912
Glu Leu Val Arg Thr Glu Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser
290 295 300
GCA AAT ATC ACA TTA AAT ACC GCT GAT ATC AGT CAA CCT TTT GAA ATT 960
Ala Asn Ile Thr Leu Asn Thr Ala Asp Ile Ser Gln Pro Phe Glu Ile
305 310 315 320
GGC CTG ACA CGA GTA CTT CCT TCC GGT TCT TGG GCA TAT GCC GCC GCA 1008
Gly Leu Thr Arg Val Leu Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala
325 330 335
AAA TTT ACC GTT GAA GAG TAT AAC CAA TAC TCT TTT CTG CTA AAA CTT 1056
Lys Phe Thr Val Glu Glu Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu
340 345 350
AAC AAG GCT ATT CGT CTA TCA CGT GCG ACA GAA TTG TCA CCC ACG ATT 1104
Asn Lys Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile
355 360 365
CTG GAA GGC ATT GTG CGC AGT GTT AAT CTA CAA CTG GAT ATC AAC ACA 1152
Leu Glu Gly Ile Val Arg Ser Val Asn Leu Gln Leu Asp Ile Asn Thr
370 375 380
GAC GTA TTA GGT AAA GTT TTT CTG ACT AAA TAT TAT ATG CAG CGT TAT 1200
Asp Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr
385 390 395 400
GCT ATT CAT GCT GAA ACT GCC CTG ATA CTA TGC AAC GCG CCT ATT TCA 1248
Ala Ile His Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala Pro Ile Ser
405 410 415
CAA CGT TCA TAT GAT AAT CAA CCT AGC CAA TTT GAT CGC CTG TTT AAT 1296
Gln Arg Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn
420 425 430
ACG CCA TTA CTG AAC GGA CAA TAT TTT TCT ACC GGC GAT GAG GAG ATT 1344
Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile
435 440 445
GAT TTA AAT TCA GGT AGC ACC GGC GAT TGG CGA AAA ACC ATA CTT AAG 1392
Asp Leu Asn Ser Gly Ser Thr Gly Asp Trp Arg Lys Thr Ile Leu Lys
450 455 460
CGT GCA TTT AAT ATT GAT GAT GTC TCG CTC TTC CGC CTG CTT AAA ATT 1440
Arg Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile
465 470 475 480
ACC GAC CAT GAT AAT AAA GAT GGA AAA ATT AAA AAT AAC CTA AAG AAT 1488
Thr Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn
485 490 495
CTT TCC AAT TTA TAT ATT GGA AAA TTA CTG GCA GAT ATT CAT CAA TTA 1536
Leu Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu
500 505 510
ACC ATT GAT GAA CTG GAT TTA TTA CTG ATT GCC GTA GGT GAA GGA AAA 1584
Thr Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys
515 520 525
ACT AAT TTA TCC GCT ATC AGT GAT AAG CAA TTG GCT ACC CTG ATC AGA 1632
Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg
530 535 540
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AAA CTC AAT ACT ATT ACC AGC TGG CTA CAT ACA CAG AAG TGG AGT GTA 1680
Lys Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val
545 550 555 560
TTC CAG CTA TTT ATC ATG ACC TCC ACC AGC TAT AAC AAA ACG CTA ACG 1728
Phe Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr
565 570 575
CCT GAA ATT AAG AAT TTG CTG GAT ACC GTC TAC CAC GGT TTA CAA GGT 1776
Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly
580 585 590
TTT GAT AAA GAC AAA GCA GAT TTG CTA CAT GTC ATG GCG CCC TAT ATT 1824
Phe Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile
595 600 605
GCG GCC ACC TTG CAA TTA TCA TCG GAA AAT GTC GCC CAC TCG GTA CTC 1872
Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu
610 615 620
CTT TGG GCA GAT AAG TTA CAG CCC GGC GAC GGC GCA ATG ACA GCA GAA 1920
Leu Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu
625 630 635 640
AAA TTC TGG GAC TGG TTG AAT ACT AAG TAT ACG CCG GGT TCA TCG GAA 1968
Lys Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu
645 650 655
GCC GTA GAA ACG CAG GAA CAT ATC GTT CAG TAT TGT CAG GCT CTG GCA 2016
Ala Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala
660 665 670
CAA TTG GAA ATG GTT TAC CAT TCC ACC GGC ATC AAC GAA AAC GCC TTC 2064
Gln Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe
675 680 685
CGT CTA TTT GTG ACA AAA CCA GAG ATG TTT GGC GCT GCA ACT GGA GCA 2112
Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala
690 695 700
GCG CCC GCG CAT GAT GCC CTT TCA CTG ATT ATG CTG ACA CGT TTT GCG 2160
Ala Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala
705 710 715 720
GAT TGG GTG AAC GCA CTA GGC GAA AAA GCG TCC TCG GTG CTA GCG GCA 2208
Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala
725 730 735
TTT GAA GCT AAC TCG TTA ACG GCA GAA CAA CTG GCT GAT GCC ATG AAT 2256
Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn
740 745 750
CTT GAT GCT AAT TTG CTG TTG CAA GCC AGT ATT CAA GCA CAA AAT CAT 2304
Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His
755 760 765
CAA CAT CTT CCC CCA GTA ACT CCA GAA AAT GCG TTC TCC TGT TGG ACA 2352
Gln His Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr
770 775 780
TCT ATC AAT ACT ATC CTG CAA TGG GTT AAT GTC GCA CAA CAA TTG AAT 2400
Ser Ile Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Asn
785 790 795 800
GTC GCC CCA CAG GGC GTT TCC GCT TTG GTC GGG CTG GAT TAT ATT CAA 2448
Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln
805 810 815
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TCA ATG AAA GAG ACA CCG ACC TAT GCC CAG TGG GAA AAC GCG GCA GGC 2496
Ser Met Lys Glu Thr Pro Thr Tyr Ala Gln Trp Glu Asn Ala Ala Gly
820 825 830
GTA TTA ACC GCC GGG TTG AAT TCA CAA CAG GCT AAT ACA TTA CAC GCT 2544
Val Leu Thr Ala Gly Leu Asn Ser Gln Gln Ala Asn Thr Leu His Ala
835 840 845
TTT CTG GAT GAA TCT CGC AGT GCC GCA TTA AGC ACC TAC TAT ATC CGT 2592
Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg
850 855 860
CAA GTC GCC AAG GCA GCG GCG GCT ATT AAA AGC CGT GAT GAC TTG TAT 2640
Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr
865 870 875 880
CAA TAC TTA CTG ATT GAT AAT CAG GTT TCT GCG GCA ATA AAA ACC ACC 2688
Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr
885 890 895
CGG ATC GCC GAA GCC ATT GCC AGT ATT CAA CTG TAC GTC AAC CGG GCA 2736
Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala
900 905 910
TTG GAA AAT GTG GAA GAA AAT GCC AAT TCG GGG GTT ATC AGC CGC CAA 2784
Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln
915 920 925
TTC TTT ATC GAC TGG GAC AAA TAC AAT AAA CGC TAC AGC ACT TGG GCG 2832
Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala
930 935 940
GGT GTT TCT CAA TTA GTT TAC TAC CCG GAA AAC TAT ATT GAT CCG ACC 2880
Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr
945 950 955 960
ATG CGT ATC GGA CAA ACC AAA ATG ATG GAC GCA TTA CTG CAA TCC GTC 2928
Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val
965 970 975
AGC CAA AGC CAA TTA AAC GCC GAT ACC GTC GAA GAT GCC TTT ATG TCT 2976
Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser
980 985 990
TAT CTG ACA TCG TTT GAA CAA GTG GCT AAT CTT AAA GTT ATT AGC GCA 3024
Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala
995 1000 1005
TAT CAC GAT AAT ATT AAT AAC GAT CAA GGG CTG ACC TAT TTT ATC GGA 3072
Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly
1010 1015 1020
CTC AGT GAA ACT GAT GCC GGT GAA TAT TAT TGG CGC AGT GTC GAT CAC 3120
Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His
1025 1030 1035 1040
AGT AAA TTC AAC GAC GGT AAA TTC GCG GCT AAT GCC TGG AGT GAA TGG 3168
Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp
1045 1050 1055
CAT AAA ATT GAT TGT CCA ATT AAC CCT TAT AAA AGC ACT ATC CGT CCA 3216
His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro
1060 1065 1070
GTG ATA TAT AAA TCC CGC CTG TAT CTG CTC TGG TTG GAA CAA AAG GAG 3264
Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu
1075 1080 1085
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ATC ACC AAA CAG ACA GGA AAT AGT AAA GAT GGC TAT CAA ACT GAA ACG 3312
Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr
1090 1095 1100
GAT TAT CGT TAT GAA CTA AAA TTG GCG CAT ATC CGC TAT GAT GGC ACT 3360
Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr
1105 1110 1115 1120
TGG AAT ACG CCA ATC ACC TTT GAT GTC AAT AAA AAA ATA TCC GAG CTA 3408
Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu
1125 1130 1135
AAA CTG GAA AAA AAT AGA GCG CCC GGA CTC TAT TGT GCC GGT TAT CAA 3456
Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln
1140 1145 1150
GGT GAA GAT ACG TTG CTG GTG ATG TTT TAT AAC CAA CAA GAC ACA CTA 3504
Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu
1155 1160 1165
GAT AGT TAT AAA AAC GCT TCA ATG CAA GGA CTA TAT ATC TTT GCT GAT 3552
Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp
1170 1175 1180
ATG GCA TCC AAA GAT ATG ACC CCA GAA CAG AGC AAT GTT TAT CGG GAT 3600
Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp
1185 1190 1195 1200
AAT AGC TAT CAA CAA TTT GAT ACC AAT AAT GTC AGA AGA GTG AAT AAC 3648
Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn
1205 1210 1215
CGC TAT GCA GAG GAT TAT GAG ATT CCT TCC TCG GTA AGT AGC CGT AAA 3696
Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys
1220 1225 1230
GAC TAT GGT TGG GGA GAT TAT TAC CTC AGC ATG GTA TAT AAC GGA GAT 3744
Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp
1235 1240 1245
ATT CCA ACT ATC AAT TAC AAA GCC GCA TCA AGT GAT TTA AAA ATC TAT 3792
Ile Pro Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr
1250 1255 1260
ATC TCA CCA AAA TTA AGA ATT ATT CAT AAT GGA TAT GAA GGA CAG AAG 3840
Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys
1265 1270 1275 1280
CGC AAT CAA TGC AAT CTG ATG AAT AAA TAT GGC AAA CTA GGT GAT AAA 3888
Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys
1285 1290 1295
TTT ATT GTT TAT ACT AGC TTG GGG GTC AAT CCA AAT AAC TCG TCA AAT 3936
Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn
1300 1305 1310
AAG CTC ATG TTT TAC CCC GTC TAT CAA TAT AGC GGA AAC ACC AGT GGA 3984
Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly
1315 1320 1325
CTC AAT CAA GGG AGA CTA CTA TTC CAC CGT GAC ACC ACT TAT CCA TCT 4032
Leu Asn Gln Gly Arg Leu Leu Phe His Arg Asp Thr Thr Tyr Pro Ser
1330 1335 1340
AAA GTA GAA GCT TGG ATT CCT GGA GCA AAA CGT TCT CTA ACC AAC CAA 4080
Lys Val Glu Ala Trp Ile Pro Gly Ala Lys Arg Ser Leu Thr Asn Gln
1345 1350 1355 1360
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AAT GCC GCC ATT GGT GAT GAT TAT GCT ACA GAC TCT CTG AAT AAA CCG 4128
Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro
1365 1370 1375
GAT GAT CTT AAG CAA TAT ATC TTT ATG ACT GAC AGT AAA GGG ACT GCT 4176
Asp Asp Leu Lys Gln Tyr Ile Phe Met Thr Asp Ser Lys Gly Thr Ala
1380 1385 1390
ACT GAT GTC TCA GGC CCA GTA GAG ATT AAT ACT GCA ATT TCT CCA GCA 4224
Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser Pro Ala
1395 1400 1405
AAA GTT CAG ATA ATA GTC AAA GCG GGT GGC AAG GAG CAA ACT TTT ACC 4272
Lys Val Gln Ile Ile Val Lys Ala Gly Gly Lys Glu Gln Thr Phe Thr
1410 1415 1420
GCA GAT AAA GAT GTC TCC ATT CAG CCA TCA CCT AGC TTT GAT GAA ATG 4320
Ala Asp Lys Asp Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met
1425 1430 1435 1440
AAT TAT CAA TTT AAT GCC CTT GAA ATA GAC GGT TCT GGT CTG AAT TTT 4368
Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Gly Leu Asn Phe
1445 1450 1455
ATT AAC AAC TCA GCC AGT ATT GAT GTT ACT TTT ACC GCA TTT GCG GAG 4416
Ile Asn Asn Ser Ala Ser Ile Asp Val Thr Phe Thr Ala Phe Ala Glu
1460 1465 1470
GAT GGC CGC AAA CTG GGT TAT GAA AGT TTC AGT ATT CCT GTT ACC CTC 4464
Asp Gly Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro Val Thr Leu
1475 1480 1485
AAG GTA AGT ACC GAT AAT GCC CTG ACC CTG CAC CAT AAT GAA AAT GGT 4512
Lys Val Ser Thr Asp Asn Ala Leu Thr Leu His His Asn Glu Asn Gly
1490 1495 1500
GCG CAA TAT ATG CAA TGG CAA TCC TAT CGT ACC CGC CTG AAT ACT CTA 4560
Ala Gln Tyr Met Gln Trp Gln Ser Tyr Arg Thr Arg Leu Asn Thr Leu
1505 1510 1515 1520
TTT GCC CGC CAG TTG GTT GCA CGC GCC ACC ACC GGA ATC GAT ACA ATT 4608
Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile
1525 1530 1535
CTG AGT ATG GAA ACT CAG AAT ATT CAG GAA CCG CAG TTA GGC AAA GGT 4656
Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly
1540 1545 1550
TTC TAT GCT ACG TTC GTG ATA CCT CCC TAT AAC CTA TCA ACT CAT GGT 4704
Phe Tyr Ala Thr Phe Val Ile Pro Pro Tyr Asn Leu Ser Thr His Gly
1555 1560 1565
GAT GAA CGT TGG TTT AAG CTT TAT ATC AAA CAT GTT GTT GAT AAT AAT 4752
Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn
1570 1575 1580
TCA CAT ATT ATC TAT TCA GGC CAG CTA ACA GAT ACA AAT ATA AAC ATC 4800
Ser His Ile Ile Tyr Ser Gly Gln Leu Thr Asp Thr Asn Ile Asn Ile
1585 1590 1595 1600
ACA TTA TTT ATT CCT CTT GAT GAT GTC CCA TTG AAT CAA GAT TAT CAC 4848
Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp Tyr His
1605 1610 1615
GCC AAG GTT TAT ATG ACC TTC AAG AAA TCA CCA TCA GAT GGT ACC TGG 4896
Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp
1620 1625 1630
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TGG GGC CCT CAC TTT GTT AGA GAT GAT AAA GGA ATA GTA ACA ATA AAC 4944
Trp Gly Pro His Phe Val Arg Asp Asp Lys Gly Ile Val Thr Ile Asn
1635 1640 1645
CCT AAA TCC ATT TTG ACC CAT TTT GAG AGC GTC AAT GTC CTG AAT AAT 4992
Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val Leu Asn Asn
1650 1655 1660
ATT AGT AGC GAA CCA ATG GAT TTC AGC GGC GCT AAC AGC CTC TAT TTC 5040
Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe
1665 1670 1675 1680
TGG GAA CTG TTC TAC TAT ACC CCG ATG CTG GTT GCT CAA CGT TTG CTG 5088
Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln Arg Leu Leu
1685 1690 1695
CAT GAA CAG AAC TTC GAT GAA GCC AAC CGT TGG CTG AAA TAT GTC TGG 5136
His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp
1700 1705 1710
AGT CCA TCC GGT TAT ATT GTC CAC GGC CAG ATT CAG AAC TAC CAG TGG 5184
Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp
1715 1720 1725
AAC GTC CGC CCG TTA CTG GAA GAC ACC AGT TGG AAC AGT GAT CCT TTG 5232
Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu
1730 1735 1740
GAT TCC GTC GAT CCT GAC GCG GTA GCA CAG CAC GAT CCA ATG CAC TAC 5280
Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr
1745 1750 1755 1760
AAA GTT TCA ACT TTT ATG CGT ACC TTG GAT CTA TTG ATA GCA CGC GGC 5328
Lys Val Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly
1765 1770 1775
GAC CAT GCT TAT CGC CAA CTG GAA CGA GAT ACA CTC AAC GAA GCG AAG 5376
Asp His Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys
1780 1785 1790
ATG TGG TAT ATG CAA GCG CTG CAT CTA TTA GGT GAC AAA CCT TAT CTA 5424
Met Trp Tyr Met Gin Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu
1795 1800 1805
CCG CTG AGT ACG ACA TGG AGT GAT CCA CGA CTA GAC AGA GCC GCG GAT 5472
Pro Leu Ser Thr Thr Trp Ser Asp Pro Arg Leu Asp Arg Ala Ala Asp
1810 1815 1820
ATC ACT ACC CAA AAT GCT CAC GAC AGC GCA ATA GTC GCT CTG CGG CAG 5520
Ile Thr Thr Gln Asn Ala His Asp Ser Ala Ile Val Ala Leu Arg Gln
1825 1830 1835 1840
AAT ATA CCT ACA CCG GCA CCT TTA TCA 5547
Asn Ile Pro Thr Pro Ala Pro Leu Ser
1845 1849
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1849 amino acids
(B) TYPE: amino acids
(C) STRANDEDNESS: single
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val
1 5 10 15
Ala Pro Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr
20 25 30
Glu Leu Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val Tyr
35 40 45
Tyr Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu Ser Gln
50 55 60
Gln Asn Met Asp Ile Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu
65 70 75 80
Leu Leu Glu Ser Ile Lys Thr Glu Ser Lys Leu Glu Asn Tyr Thr Lys
85 90 95
Val Met Glu Met Leu Ser Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr
100 105 110
His Asp Ala Tyr Glu Asn Val Arg Glu Val Ile Gln Leu Gln Asp Pro
115 120 125
Gly Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu Met His
130 135 140
Gln Ala Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe
145 150 155 160
Asn Ile Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr
165 170 175
Lys Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met Pro Glu
180 185 190
Tyr Leu Lys Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe
195 200 205
Ile Gly Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln
210 215 220
Leu Ile Thr Pro Val Val Asn Ser Ser Asp Gly Thr Val Lys Val Tyr
225 230 235 240
Arg Ile Thr Arg Glu Tyr Thr Thr Asn Ala Tyr Gln Met Asp Val Glu
245 250 255
Leu Phe Pro Phe Gly Gly Glu Asn Tyr Arg Leu Asp Tyr Lys Phe Lys
260 265 270
Asn Phe Tyr Asn Ala Ser Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg
275 280 285
Glu Leu Val Arg Thr Glu Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser
290 295 300
Ala Asn Ile Thr Leu Asn Thr Ala Asp Ile Ser Gln Pro Phe Glu Ile
305 310 315 320
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Gly Leu Thr Arg Val Leu Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala
325 330 335
Lys Phe Thr Val Glu Glu Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu
340 345 350
Asn Lys Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile
355 360 365
Leu Glu Gly Ile Val Arg Ser Val Asn Leu Gln Leu Asp Ile Asn Thr
370 375 380
Asp Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr
385 390 395 400
Ala Ile His Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala Pro Ile Ser
405 410 415
Gln Arg Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn
420 425 430
Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile
435 440 445
Asp Leu Asn Ser Gly Ser Thr Gly Asp Trp Arg Lys Thr Ile Leu Lys
450 455 460
Arg Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile
465 470 475 480
Thr Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn
485 490 495
Leu Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu
500 505 510
Thr Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys
515 520 525
Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg
530 535 540
Lys Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val
545 550 555 560
Phe Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr
565 570 575
Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly
580 585 590
Phe Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile
595 600 605
Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu
610 615 620
Leu Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu
625 630 635 640
Lys Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu
645 650 655
Ala Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala
660 665 670
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Gln Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe
675 680 685
Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala
690 695 700
Ala Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala
705 710 715 720
Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala
725 730 735
Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn
740 745 750
Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His
755 760 765
Gln His Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr
770 775 780
Ser Ile Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Asn
785 790 795 800
Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln
805 810 815
Ser Met Lys Glu Thr Pro Thr Tyr Ala Gln Trp Glu Asn Ala Ala Gly
820 825 830
Val Leu Thr Ala Gly Leu Asn Ser Gln Gln Ala Asn Thr Leu His Ala
835 840 845
Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg
850 855 860
Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr
865 870 875 880
Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr
885 890 895
Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala
900 905 910
Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln
915 920 925
Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala
930 935 940
Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr
945 950 955 960
Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val
965 970 975
Ser Gln Ser Gin Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser
980 985 990
Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala
995 1000 1005
Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly
1010 1015 1020
Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His
1025 1030 1035 1040
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Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp
1045 1050 1055
His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro
1060 1065 1070
Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu
1075 1080 1085
Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr
1090 1095 1100
Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr
1105 1110 1115 1120
Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu
1125 1130 1135
Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln
1140 1145 1150
Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu
1155 1160 1165
Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp
1170 1175 1180
Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp
1185 1190 1195 1200
Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn
1205 1210 1215
Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys
1220 1225 1230
Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp
1235 1240 1245
Ile Pro Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr
1250 1255 1260
Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys
1265 1270 1275 1280
Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys
1285 1290 1295
Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn
1300 1305 1310
Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly
1315 1320 1325
Leu Asn Gln Gly Arg Leu Leu Phe His Arg Asp Thr Thr Tyr Pro Ser
1330 1335 1340
Lys Val Glu Ala Trp Ile Pro Gly Ala Lys Arg Ser Leu Thr Asn Gln
1345 1350 1355 1360
Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro
1365 1370 1375
Asp Asp Leu Lys Gln Tyr Ile Phe Met Thr Asp Ser Lys Gly Thr Ala
1380 1385 1390
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Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser Pro Ala
1395 1400 1405
Lys Val Gln Ile Ile Val Lys Ala Gly Gly Lys Glu Gln Thr Phe Thr
1410 1415 1420
Ala Asp Lys Asp Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met
1425 1430 1435 1440
Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Gly Leu Asn Phe
1445 1450 1455
Ile Asn Asn Ser Ala Ser Ile Asp Val Thr Phe Thr Ala Phe Ala Glu
1460 1465 1470
Asp Gly Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro Val Thr Leu
1475 1480 1485
Lys Val Ser Thr Asp Asn Ala Leu Thr Leu His His Asn Glu Asn Gly
1490 1495 1500
Ala Gln Tyr Met Gln Trp Gln Ser Tyr Arg Thr Arg Leu Asn Thr Leu
1505 1510 1515 1520
Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile
1525 1530 1535
Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly
1540 1545 1550
Phe Tyr Ala Thr Phe Val Ile Pro Pro Tyr Asn Leu Ser Thr His Gly
1555 1560 1565
Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn
1570 1575 1580
Ser His Ile Ile Tyr Ser Gly Gln Leu Thr Asp Thr Asn Ile Asn Ile
1585 1590 1595 1600
Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp Tyr His
1605 1610 1615
Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp
1620 1625 1630
Trp Gly Pro His Phe Val Arg Asp Asp Lys Gly Ile Val Thr Ile Asn
1635 1640 1645
Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val Leu Asn Asn
1650 1655 1660
Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe
1665 1670 1675 1680
Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln Arg Leu Leu
1685 1690 1695
His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp
1700 1705 1710
Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp
1715 1720 1725
Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu
1730 1735 1740
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Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr
1745 1750 1755 1760
Lys Val Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly
1765 1770 1775
Asp His Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys
1780 1785 1790
Met Trp Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu
1795 1800 1805
Pro Leu Ser Thr Thr Trp Ser Asp Pro Arg Leu Asp Arg Ala Ala Asp
1810 1815 1820
Ile Thr Thr Gln Asn Ala His Asp Ser Ala Ile Val Ala Leu Arg Gln
1825 1830 1835 1840
Asn Ile Pro Thr Pro Ala Pro Leu Ser
1845 1849
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1740 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
TTG CGC AGC GCT AAT ACC CTG ACT GAT CTC TTC CTG CCG CAA ATC AAT 48
Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile Asn
1 5 10 15
GAA GTG ATG ATG AAT TAC TGG CAG ACA TTA GCT CAG AGA GTA TAC AAT 96
Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr Asn
20 25 30
CTG CGT CAT AAC CTC TCT ATC GAC GGC CAG CCG TTA TAT CTG CCA ATC 144
Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro Ile
35 40 45
TAT GCC ACA CCG GCC GAT CCG AAA GCG TTA CTC AGC GCC GCC GTT GCC 192
Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ala
55 60
ACT TCT CAA GGT GGA GGC AAG CTA CCG GAA TCA TTT ATG TCC CTG TGG 240
Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu Trp
65 70 75 80
CGT TTC CCG CAC ATG CTG GAA AAT GCG CGC GGC ATG GTT AGC CAG CTC 288
Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln Leu
85 90 95
ACC CAG TTC GGC TCC ACG TTA CAA AAT ATT ATC GAA CGT CAG GAC GCG 336
Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp Ala
100 105 110
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GAA GCG CTC AAT GCG TTA TTA CAA AAT CAG GCC GCC GAG CTG ATA TTG 384
Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile Leu
115 120 125
ACT AAC CTG AGC ATT CAG GAC AAA ACC ATT GAA GAA TTG GAT GCC GAG 432
Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala Glu
130 135 140
AAA ACG GTG TTG GAA AAA TCC AAA GCG GGA GCA CAA TCG CGC TTT GAT 480
Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe Asp
145 150 155 160
AGC TAC GGC AAA CTG TAC GAT GAG AAT ATC AAC GCC GGT GAA AAC CAA 528
Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn Gln
165 170 175
GCC ATG ACG CTA CGA GCG TCC GCC GCC GGG CTT ACC ACG GCA GTT CAG 576
Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val Gln
180 185 190
GCA TCC CGT CTG GCC GGT GCG GCG GCT GAT CTG GTG CCT AAC ATC TTC 624
Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile Phe
195 200 205
GGC TTT GCC GGT GGC GGC AGC CGT TGG GGG GCT ATC GCT GAG GCG ACA 672
Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala Thr
210 215 220
GGT TAT GTG ATG GAA TTC TCC GCG AAT GTT ATG AAC ACC GAA GCG GAT 720
Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala Asp
225 230 235 240
AAA ATT AGC CAA TCT GAA ACC TAC CGT CGT CGC CGT CAG GAG TGG GAG 768
Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp Glu
245 250 255
ATC CAG CGG AAT AAT GCC GAA GCG GAA TTG AAG CAA ATC GAT GCT CAG 816
Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala Gln
260 265 270
CTC AAA TCA CTC GCT GTA CGC CGC GAA GCC GCC GTA TTG CAG AAA ACC 864
Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys Thr
275 280 285
AGT CTG AAA ACC CAA CAA GAA CAG ACC CAA TCT CAA TTG GCC TTC CTG 912
Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe Leu
290 295 300
CAA CGT AAG TTC AGC AAT CAG GCG TTA TAC AAC TGG CTG CGT GGT CGA 960
Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly Arg
305 310 315 320
CTG GCG GCG ATT TAC TTC CAG TTC TAC GAT TTG GCC GTC GCG CGT TGC 1008
Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg Cys
325 330 335
CTG ATG GCA GAA CAA GCT TAC CGT TGG GAA CTC AAT GAT GAC TCT GCC 1056
Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser Ala
340 345 350
CGC TTC ATT AAA CCG GGC GCC TGG CAG GGA ACC TAT GCC GGT CTG CTT 1104
Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu
355 360 365
GCA GGT GAA ACC TTG ATG CTG AGT CTG GCA CAA ATG GAA GAC GCT CAT 1152
Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala His
370 375 380
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CTG AAA CGC GAT AAA CGC GCA TTA GAG GTT GAA CGC ACA GTA TCG CTG 1200
Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu
385 390 395 400
GCC GAA GTT TAT GCA GGA TTA CCA AAA GAT AAC GGT CCA TTT TCC CTG 1248
Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser Leu
405 410 415
GCT CAG GAA ATT GAC AAG CTG GTG AGT CAA GGT TCA GGC AGT GCC GGC 1296
Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala Gly
420 425 430
AGT GGT AAT AAT AAT TTG GCG TTC GGC GCC GGC ACG GAC ACT AAA ACC 1344
Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys Thr
435 440 445
TCT TTG CAG GCA TCA GTT TCA TTC GCT GAT TTG AAA ATT CGT GAA GAT 1392
Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu Asp
450 455 460
TAC CCG GCA TCG CTT GGC AAA ATT CGA CGT ATC AAA CAG ATC AGC GTC 1440
Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser Val
465 470 475 480
ACT TTG CCC GCG CTA CTG GGA CCG TAT CAG GAT GTA CAG GCA ATA TTG 1488
Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gin Ala Ile Leu
485 490 495
TCT TAC GGC GAT AAA GCC GGA TTA GCT AAC GGC TGT GAA GCG CTG GCA 1536
Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu Ala
500 505 510
GTT TCT CAC GGT ATG AAT GAC AGC GGC CAA TTC CAG CTC GAT TTC AAC 1584
Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn
515 520 525
GAT GGC AAA TTC CTG CCA TTC GAA GGC ATC GCC ATT GAT CAA GGC ACG 1632
Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly Thr
530 535 540
CTG ACA CTG AGC TTC CCA AAT GCA TCT ATG CCG GAG AAA GGT AAA CAA 1680
Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys Gln
545 550 555 560
GCC ACT ATG TTA AAA ACC CTG AAC GAT ATC ATT TTG CAT ATT CGC TAC 1728
Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg Tyr
565 570 575
ACC ATT AAA TAA 1740
Thr Ile Lys
579
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 579 amino acids
(B) TYPE: amino acids
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51
Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile Asn
1 5 10 15
Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr Asn
20 25 30
Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro Ile
35 40 45
Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ala
50 55 60
Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu Trp
65 70 75 80
Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln Leu
85 90 95
Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp Ala
100 105 110
Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile Leu
115 120 125
Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala Glu
130 135 140
Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe Asp
145 150 155 160
Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn Gln
165 170 175
Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val Gln
180 185 190
Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile Phe
195 200 205
Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala Thr
210 215 220
Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala Asp
225 230 235 240
Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp Glu
245 250 255
Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala Gln
260 265 270
Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys Thr
275 280 285
Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe Leu
290 295 300
Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly Arg
305 310 315 320
Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg Cys
325 330 335
Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser Ala
340 345 350
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Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu
355 360 365
Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala His
370 375 380
Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu
385 390 395 400
Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser Leu
405 410 415
Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala Gly
420 425 430
Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys Thr
435 440 445
Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu Asp
450 455 460
Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser Val
465 470 475 480
Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gin Ala Ile Leu
485 490 495
Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu Ala
500 505 510
Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn
515 520 525
Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly Thr
530 535 540
Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys Gln
545 550 555 560
Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg Tyr
565 570 575
Thr Ile Lys
579
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5532 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
TTT ATA CAA GGT TAT AGT GAT CTG TTT GGT AAT CGT GCT GAT AAC TAT 48
Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala Asp Asn Tyr
1 5 10 15
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GCC GCG CCG GGC TCG GTT GCA TCG ATG TTC TCA CCG GCG GCT TAT TTG 96
Ala Ala Pro Gly Ser Val Ala Ser Met Phe Ser Pro Ala Ala Tyr Leu
20 25 30
ACG GAA TTG TAC CGT GAA GCC AAA AAC TTG CAT GAC AGC AGC TCA ATT 144
Thr Glu Leu Tyr Arg Glu Ala Lys Asn Leu His Asp Ser Ser Ser Ile
35 40 45
TAT TAC CTA GAT AAA CGT CGC CCG GAT TTA GCA AGC TTA ATG CTC AGC 192
Tyr Tyr Leu Asp Lys Arg Arg Pro Asp Leu Ala Ser Leu Met Leu Ser
50 55 60
CAG AAA AAT ATG GAT GAG GAA ATT TCA ACG CTG GCT CTC TCT AAT GAA 240
Gln Lys Asn Met Asp Glu Glu Ile Ser Thr Leu Ala Leu Ser Asn Glu
65 70 75 80
TTG TGC CTT GCC GGG ATC GAA ACA AAA ACA GGA AAA TCA CAA GAT GAA 288
Leu Cys Leu Ala Gly Ile Glu Thr Lys Thr Gly Lys Ser Gln Asp Glu
85 90 95
GTG ATG GAT ATG TTG TCA ACT TAT CGT TTA AGT GGA GAG ACA CCT TAT 336
Val Met Asp Met Leu Ser Thr Tyr Arg Leu Ser Gly Glu Thr Pro Tyr
100 105 110
CAT CAC GCT TAT GAA ACT GTT CGT GAA ATC GTT CAT GAA CGT GAT CCA 384
His His Ala Tyr Glu Thr Val Arg Glu Ile Val His Glu Arg Asp Pro
115 120 125
GGA TTT CGT CAT TTG TCA CAG GCA CCC ATT GTT GCT GCT AAG CTC GAT 432
Gly Phe Arg His Leu Ser Gln Ala Pro Ile Val Ala Ala Lys Leu Asp
130 135 140
CCT GTG ACT TTG TTG GGT ATT AGC TCC CAT ATT TCG CCA GAA CTG TAT 480
Pro Val Thr Leu Leu Gly Ile Ser Ser His Ile Ser Pro Glu Leu Tyr
145 150 155 160
AAC TTG CTG ATT GAG GAG ATC CCG GAA AAA GAT GAA GCC GCG CTT GAT 528
Asn Leu Leu Ile Glu Glu Ile Pro Glu Lys Asp Glu Ala Ala Leu Asp
165 170 175
ACG CTT TAT AAA ACA AAC TTT GGC GAT ATT ACT ACT GCT CAG TTA ATG 576
Thr Leu Tyr Lys Thr Asn Phe Gly Asp Ile Thr Thr Ala Gln Leu Met
180 185 190
TCC CCA AGT TAT CTG GCC CGG TAT TAT GGC GTC TCA CCG GAA GAT ATT 624
Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr Gly Val Ser Pro Glu Asp Ile
195 200 205
GCC TAC GTG ACG ACT TCA TTA TCA CAT GTT GGA TAT AGC AGT GAT ATT 672
Ala Tyr Val Thr Thr Ser Leu Ser His Val Gly Tyr Ser Ser Asp Ile
210 215 220
CTG GTT ATT CCG TTG GTC GAT GGT GTG GGT AAG ATG GAA GTA GTT CGT 720
Leu Val Ile Pro Leu Val Asp Gly Val Gly Lys Met Glu Val Val Arg
225 230 235 240
GTT ACC CGA ACA CCA TCG GAT AAT TAT ACC AGT CAG ACG AAT TAT ATT 768
Val Thr Arg Thr Pro Ser Asp Asn Tyr Thr Ser Gln Thr Asn Tyr Ile
245 250 255
GAG CTG TAT CCA CAG GGT GGC GAC AAT TAT TTG ATC AAA TAC AAT CTA 816
Glu Leu Tyr Pro Gln Gly Gly Asp Asn Tyr Leu Ile Lys Tyr Asn Leu
260 265 270
AGC AAT AGT TTT GGT TTG GAT GAT TTT TAT CTG CAA TAT AAA GAT GGT 864
Ser Asn Ser Phe Gly Leu Asp Asp Phe Tyr Leu Gln Tyr Lys Asp Gly
275 280 285
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TCC GCT GAT TGG ACT GAG ATT GCC CAT AAT CCC TAT CCT GAT ATG GTC 912
Ser Ala Asp Trp Thr Glu Ile Ala His Asn Pro Tyr Pro Asp Met Val
290 295 300
ATA AAT CAA AAG TAT GAA TCA CAG GCG ACA ATC AAA CGT AGT GAC TCT 960
Ile Asn Gln Lys Tyr Glu Ser Gln Ala Thr Ile Lys Arg Ser Asp Ser
305 310 315 320
GAC AAT ATA CTC AGT ATA GGG TTA CAA AGA TGG CAT AGC GGT AGT TAT 1008
Asp Asn Ile Leu Ser Ile Gly Leu Gln Arg Trp His Ser Gly Ser Tyr
325 330 335
AAT TTT GCC GCC GCC AAT TTT AAA ATT GAC CAA TAC TCC CCG AAA GCT 1056
Asn Phe Ala Ala Ala Asn Phe Lys Ile Asp Gln Tyr Ser Pro Lys Ala
340 345 350
TTC CTG CTT AAA ATG AAT AAG GCT ATT CGG TTG CTC AAA GCT ACC GGC 1104
Phe Leu Leu Lys Met Asn Lys Ala Ile Arg Leu Leu Lys Ala Thr Gly
355 360 365
CTC TCT TTT GCT ACG TTG GAG CGT ATT GTT GAT AGT GTT AAT AGC ACC 1152
Leu Ser Phe Ala Thr Leu Glu Arg Ile Val Asp Ser Val Asn Ser Thr
370 375 380
AAA TCC ATC ACG GTT GAG GTA TTA AAC AAG GTT TAT CGG GTA AAA TTC 1200
Lys Ser Ile Thr Val Glu Val Leu Asn Lys Val Tyr Arg Val Lys Phe
385 390 395 400
TAT ATT GAT CGT TAT GGC ATC AGT GAA GAG ACA GCC GCT ATT TTG GCT 1248
Tyr Ile Asp Arg Tyr Gly Ile Ser Glu Glu Thr Ala Ala Ile Leu Ala
405 410 415
AAT ATT AAT ATC TCT CAG CAA GCT GTT GGC AAT CAG CTT AGC CAG TTT 1296
Asn Ile Asn Ile Ser Gln Gln Ala Val Gly Asn Gln Leu Ser Gln Phe
420 425 430
GAG CAA CTA TTT AAT CAC CCG CCG CTC AAT GGT ATT CGC TAT GAA ATC 1344
Glu Gln Leu Phe Asn His Pro Pro Leu Asn Gly Ile Arg Tyr Glu Ile
435 440 445
AGT GAG GAC AAC TCC AAA CAT CTT CCT AAT CCT GAT CTG AAC CTT AAA 1392
Ser Glu Asp Asn Ser Lys His Leu Pro Asn Pro Asp Leu Asn Leu Lys
450 455 460
CCA GAC AGT ACC GGT GAT GAT CAA CGC AAG GCG GTT TTA AAA CGC GCG 1440
Pro Asp Ser Thr Gly Asp Asp Gln Arg Lys Ala Val Leu Lys Arg Ala
465 470 475 480
TTT CAG GTT AAC GCC AGT GAG TTG TAT CAG ATG TTA TTG ATC ACT GAT 1488
Phe Gln Val Asn Ala Ser Glu Leu Tyr Gln Met Leu Leu Ile Thr Asp
485 490 495
CGT AAA GAA GAC GGT GTT ATC AAA AAT AAC TTA GAG AAT TTG TCT GAT 1536
Arg Lys Glu Asp Gly Val Ile Lys Asn Asn Leu Glu Asn Leu Ser Asp
500 505 510
CTG TAT TTG GTT AGT TTG CTG GCC CAG ATT CAT AAC CTG ACT ATT GCT 1584
Leu Tyr Leu Val Ser Leu Leu Ala Gln Ile His Asn Leu Thr Ile Ala
515 520 525
GAA TTG AAC ATT TTG TTG GTG ATT TGT GGC TAT GGC GAC ACC AAC ATT 1632
Glu Leu Asn Ile Leu Leu Val Ile Cys Gly Tyr Gly Asp Thr Asn Ile
530 535 540
TAT CAG ATT ACC GAC GAT AAT TTA GCC AAA ATA GTG GAA ACA TTG TTG 1680
Tyr Gln Ile Thr Asp Asp Asn Leu Ala Lys Ile Val Glu Thr Leu Leu
545 550 555 560
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TGG ATC ACT CAA TGG TTG AAG ACC CAA AAA TGG ACA GTT ACC GAC CTG 1728
Trp Ile Thr Gln Trp Leu Lys Thr Gln Lys Trp Thr Val Thr Asp Leu
565 570 575
TTT CTG ATG ACC ACG GCC ACT TAC AGC ACC ACT TTA ACG CCA GAA ATT 1776
Phe Leu Met Thr Thr Ala Thr Tyr Ser Thr Thr Leu Thr Pro Glu Ile
580 585 590
AGC AAT CTG ACG GCT ACG TTG TCT TCA ACT TTG CAT GGC AAA GAG AGT 1824
Ser Asn Leu Thr Ala Thr Leu Ser Ser Thr Leu His Gly Lys Glu Ser
595 600 605
CTG ATT GGG GAA GAT CTG AAA AGA GCA ATG GCG CCT TGC TTC ACT TCG 1872
Leu Ile Gly Glu Asp Leu Lys Arg Ala Met Ala Pro Cys Phe Thr Ser
610 615 620
GCT TTG CAT TTG ACT TCT CAA GAA GTT GCG TAT GAC CTG CTG TTG TGG 1920
Ala Leu His Leu Thr Ser Gln Glu Val Ala Tyr Asp Leu Leu Leu Trp
625 630 635 640
ATA GAC CAG ATT CAA CCG GCA CAA ATA ACT GTT GAT GGG TTT TGG GAA 1968
Ile Asp Gln Ile Gln Pro Ala Gln Ile Thr Val Asp Gly Phe Trp Glu
645 650 655
GAA GTG CAA ACA ACA CCA ACC AGC TTG AAG GTG ATT ACC TTT GCT CAG 2016
Glu Val Gln Thr Thr Pro Thr Ser Leu Lys Val Ile Thr Phe Ala Gln
660 665 670
GTG CTG GCA CAA TTG AGC CTG ATC TAT CGT CGT ATT GGG TTA AGT GAA 2064
Val Leu Ala Gln Leu Ser Leu Ile Tyr Arg Arg Ile Gly Leu Ser Glu
675 680 685
ACG GAA CTG TCA CTG ATC GTG ACT CAA TCT TCT CTG CTA GTG GCA GGC 2112
Thr Glu Leu Ser Leu Ile Val Thr Gln Ser Ser Leu Leu Val Ala Gly
690 695 700
AAA AGC ATA CTG GAT CAC GGT CTG TTA ACC CTG ATG GCC TTG GAA GGT 2160
Lys Ser Ile Leu Asp His Gly Leu Leu Thr Leu Met Ala Leu Glu Gly
705 710 715 720
TTT CAT ACC TGG GTT AAT GGC TTG GGG CAA CAT GCC TCC TTG ATA TTG 2208
Phe His Thr Trp Val Asn Gly Leu Gly Gln His Ala Ser Leu Ile Leu
725 730 735
GCG GCG TTG AAA GAC GGA GCC TTG ACA GTT ACC GAT GTA GCA CAA GCT 2256
Ala Ala Leu Lys Asp Gly Ala Leu Thr Val Thr Asp Val Ala Gln Ala
740 745 750
ATG AAT AAG GAG GAA TCT CTC CTA CAA ATG GCA GCT AAT CAG GTG GAG 2304
Met Asn Lys Glu Glu Ser Leu Leu Gln Met Ala Ala Asn Gln Val Glu
755 760 765
AAG GAT CTA ACA AAA CTG ACC AGT TGG ACA CAG ATT GAC GCT ATT CTG 2352
Lys Asp Leu Thr Lys Leu Thr Ser Trp Thr Gln Ile Asp Ala Ile Leu
770 775 780
CAA TGG TTA CAG ATG TCT TCG GCC TTG GCG GTT TCT CCA CTG GAT CTG 2400
Gln Trp Leu Gln Met Ser Ser Ala Leu Ala Val Ser Pro Leu Asp Leu
785 790 795 800
GCA GGG ATG ATG GCC CTG AAA TAT GGG ATA GAT CAT AAC TAT GCT GCC 2448
Ala Gly Met Met Ala Leu Lys Tyr Gly Ile Asp His Asn Tyr Ala Ala
805 810 815
TGG CAA GCT GCG GCG GCT GCG CTG ATG GCT GAT CAT GCT AAT CAG GCA 2496
Trp Gln Ala Ala Ala Ala Ala Leu Met Ala Asp His Ala Asn Gln Ala
820 825 830
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CAG AAA AAA CTG GAT GAG ACG TTC AGT AAG GCA TTA TGT AAC TAT TAT 2544
Gln Lys Lys Leu Asp Glu Thr Phe Ser Lys Ala Leu Cys Asn Tyr Tyr
835 840 845
ATT AAT GCT GTT GTC GAT AGT GCT GCT GGA GTA CGT GAT CGT AAC GGT 2592
Ile Asn Ala Val Val Asp Ser Ala Ala Gly Val Arg Asp Arg Asn Gly
850 855 860
TTA TAT ACC TAT TTG CTG ATT GAT AAT CAG GTT TCT GCC GAT GTG ATC 2640
Leu Tyr Thr Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Asp Val Ile
865 870 875 880
ACT TCA CGT ATT GCA GAA GCT ATC GCC GGT ATT CAA CTG TAC GTT AAC 2688
Thr Ser Arg Ile Ala Glu Ala Ile Ala Gly Ile Gln Leu Tyr Val Asn
885 890 895
CGG GCT TTA AAC CGA GAT GAA GGT CAG CTT GCA TCG GAC GTT AGT ACC 2736
Arg Ala Leu Asn Arg Asp Glu Gly Gln Leu Ala Ser Asp Val Ser Thr
900 905 910
CGT CAG TTC TTC ACT GAC TGG GAA CGT TAC AAT AAA CGT TAC AGT ACT 2784
Arg Gln Phe Phe Thr Asp Trp Glu Arg Tyr Asn Lys Arg Tyr Ser Thr
915 920 925
TGG GCT GGT GTC TCT GAA CTG GTC TAT TAT CCA GAA AAC TAT GTT GAT 2832
Trp Ala Gly Val Ser Glu Leu Val Tyr Tyr Pro Glu Asn Tyr Val Asp
930 935 940
CCC ACT CAG CGC ATT GGG CAA ACC AAA ATG ATG GAT GCG CTG TTG CAA 2880
Pro Thr Gln Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln
945 950 955 960
TCC ATC AAC CAG AGC CAG CTA AAT GCG GAT ACG GTG GAA GAT GCT TTC 2928
Ser Ile Asn Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe
965 970 975
AAA ACT TAT TTG ACC AGC TTT GAG CAG GTA GCA AAT CTG AAA GTA ATT 2976
Lys Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile
980 985 990
AGT GCT TAC CAC GAT AAT GTG AAT GTG GAT CAA GGA TTA ACT TAT TTT 3024
Ser Ala Tyr His Asp Asn Val Asn Val Asp Gln Gly Leu Thr Tyr Phe
995 1000 1005
ATC GGT ATC GAC CAA GCA GCT CCG GGT ACG TAT TAC TGG CGT AGT GTT 3072
Ile Gly Ile Asp Gln Ala Ala Pro Gly Thr Tyr Tyr Trp Arg Ser Val
1010 1015 1020
GAT CAC AGC AAA TGT GAA AAT GGC AAG TTT GCC GCT AAT GCT TGG GGT 3120
Asp His Ser Lys Cys Glu Asn Gly Lys Phe Ala Ala Asn Ala Trp Gly
1025 1030 1035 1040
GAG TGG AAT AAA ATT ACC TGT GCT GTC AAT CCT TGG AAA AAT ATC ATC 3168
Glu Trp Asn Lys Ile Thr Cys Ala Val Asn Pro Trp Lys Asn Ile Ile
1045 1050 1055
CGT CCG GTT GTT TAT ATG TCC CGC TTA TAT CTG CTA TGG CTG GAG CAG 3216
Arg Pro Val Val Tyr Met Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln
1060 1065 1070
CAA TCA AAG AAA AGT GAT GAT GGT AAA ACC ACG ATT TAT CAA TAT AAC 3264
Gln Ser Lys Lys Ser Asp Asp Gly Lys Thr Thr Ile Tyr Gln Tyr Asn
1075 1080 1085
TTA AAA CTG GCT CAT ATT CGT TAC GAC GGT AGT TGG AAT ACA CCA TTT 3312
Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Ser Trp Asn Thr Pro Phe
1090 1095 1100
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ACT TTT GAT GTG ACA GAA AAG GTA AAA AAT TAC ACG TCG AGT ACT GAT 3360
Thr Phe Asp Val Thr Glu Lys Val Lys Asn Tyr Thr Ser Ser Thr Asp
1105 1110 1115 1120
GCT GCT GAA TCT TTA GGG TTG TAT TGT ACT GGT TAT CAA GGG GAA GAC 3408
Ala Ala Glu Ser Leu Gly Leu Tyr Cys Thr Gly Tyr Gln Gly Glu Asp
1125 1130 1135
ACT CTA TTA GTT ATG TTC TAT TCG ATG CAG AGT AGT TAT AGC TCC TAT 3456
Thr Leu Leu Val Met Phe Tyr Ser Met Gln Ser Ser Tyr Ser Ser Tyr
1140 1145 1150
ACC GAT AAT AAT GCG CCG GTC ACT GGG CTA TAT ATT TTC GCT GAT ATG 3504
Thr Asp Asn Asn Ala Pro Val Thr Gly Leu Tyr Ile Phe Ala Asp Met
1155 1160 1165
TCA TCA GAC AAT ATG ACG AAT GCA CAA GCA ACT AAC TAT TGG AAT AAC 3552
Ser Ser Asp Asn Met Thr Asn Ala Gln Ala Thr Asn Tyr Trp Asn Asn
1170 1175 1180
AGT TAT CCG CAA TTT GAT ACT GTG ATG GCA GAT CCG GAT AGC GAC AAT 3600
Ser Tyr Pro Gln Phe Asp Thr Val Met Ala Asp Pro Asp Ser Asp Asn
1185 1190 1195 1200
AAA AAA GTC ATA ACC AGA AGA GTT AAT AAC CGT TAT GCG GAG GAT TAT 3648
Lys Lys Val Ile Thr Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr
1205 1210 1215
GAA ATT CCT TCC TCT GTG ACA AGT AAC AGT AAT TAT TCT TGG GGT GAT 3696
Glu Ile Pro Ser Ser Val Thr Ser Asn Ser Asn Tyr Ser Trp Gly Asp
1220 1225 1230
CAC AGT TTA ACC ATG CTT TAT GGT GGT AGT GTT CCT AAT ATT ACT TTT 3744
His Ser Leu Thr Met Leu Tyr Gly Gly Ser Val Pro Asn Ile Thr Phe
1235 1240 1245
GAA TCG GCG GCA GAA GAT TTA AGG CTA TCT ACC AAT ATG GCA TTG AGT 3792
Glu Ser Ala Ala Glu Asp Leu Arg Leu Ser Thr Asn Met Ala Leu Ser
1250 1255 1260
ATT ATT CAT AAT GGA TAT GCG GGA ACC CGC CGT ATA CAA TGT AAT CTT 3840
Ile Ile His Asn Gly Tyr Ala Gly Thr Arg Arg Ile Gln Cys Asn Leu
1265 1270 1275 1280
ATG AAA CAA TAC GCT TCA TTA GGT GAT AAA TTT ATA ATT TAT GAT TCA 3888
Met Lys Gln Tyr Ala Ser Leu Gly Asp Lys Phe Ile Ile Tyr Asp Ser
1285 1290 1295
TCA TTT GAT GAT GCA AAC CGT TTT AAT CTG GTG CCA TTG TTT AAA TTC 3936
Ser Phe Asp Asp Ala Asn Arg Phe Asn Leu Val Pro Leu Phe Lys Phe
1300 1305 1310
GGA AAA GAC GAG AAC TCA GAT GAT AGT ATT TGT ATA TAT AAT GAA AAC 3984
Gly Lys Asp Glu Asn Ser Asp Asp Ser Ile Cys Ile Tyr Asn Glu Asn
1315 1320 1325
CCT TCC TCT GAA GAT AAG AAG TGG TAT TTT TCT TCG AAA GAT GAC AAT 4032
Pro Ser Ser Glu Asp Lys Lys Trp Tyr Phe Ser Ser Lys Asp Asp Asn
1330 1335 1340
AAA ACA GCG GAT TAT AAT GGT GGA ACT CAA TGT ATA GAT GCT GGA ACC 4080
Lys Thr Ala Asp Tyr Asn Gly Gly Thr Gln Cys Ile Asp Ala Gly Thr
1345 1350 1355 1360
AGT AAC AAA GAT TTT TAT TAT AAT CTC CAG GAG ATT GAA GTA ATT AGT 4128
Ser Asn Lys Asp Phe Tyr Tyr Asn Leu Gln Glu Ile Glu Val Ile Ser
1365 1370 1375
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GTT ACT GGT GGG TAT TGG TCG AGT TAT AAA ATA TCC AAC CCG ATT AAT 4176
Val Thr Gly Gly Tyr Trp Ser Ser Tyr Lys Ile Ser Asn Pro Ile Asn
1380 1385 1390
ATC AAT ACG GGC ATT GAT AGT GCT AAA GTA AAA GTC ACC GTA AAA GCG 4224
Ile Asn Thr Gly Ile Asp Ser Ala Lys Val Lys Val Thr Val Lys Ala
1395 1400 1405
GGT GGT GAC GAT CAA ATC TTT ACT GCT GAT AAT AGT ACC TAT GTT CCT 4272
Gly Gly Asp Asp Gln Ile Phe Thr Ala Asp Asn Ser Thr Tyr Val Pro
1410 1415 1420
CAG CAA CCG GCA CCC AGT TTT GAG GAG ATG ATT TAT CAG TTC AAT AAC 4320
Gln Gln Pro Ala Pro Ser Phe Glu Glu Met Ile Tyr Gln Phe Asn Asn
1425 1430 1435 1440
CTG ACA ATA GAT TGT AAG AAT TTA AAT TTC ATC GAC AAT CAG GCA CAT 4368
Leu Thr Ile Asp Cys Lys Asn Leu Asn Phe Ile Asp Asn Gln Ala His
1445 1450 1455
ATT GAG ATT GAT TTC ACC GCT ACG GCA CAA GAT GGC CGA TTC TTG GGT 4416
Ile Glu Ile Asp Phe Thr Ala Thr Ala Gln Asp Gly Arg Phe Leu Gly
1460 1465 1470
GCA GAA ACT TTT ATT ATC CCG GTA ACT AAA AAA GTT CTC GGT ACT GAG 4464
Ala Glu Thr Phe Ile Ile Pro Val Thr Lys Lys Val Leu Gly Thr Glu
1475 1480 1485
AAC GTG ATT GCG TTA TAT AGC GAA AAT AAC GGT GTT CAA TAT ATG CAA 4512
Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly Val Gln Tyr Met Gln
1490 1495 1500
ATT GGC GCA TAT CGT ACC CGT TTG AAT ACG TTA TTC GCT CAA CAG TTG 4560
Ile Gly Ala Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Gln Gln Leu
1505 1510 1515 1520
GTT AGC CGT GCT AAT CGT GGC ATT GAT GCA GTG CTC AGT ATG GAA ACT 4608
Val Ser Arg Ala Asn Arg Gly Ile Asp Ala Val Leu Ser Met Glu Thr
1525 1530 1535
CAG AAT ATT CAG GAA CCG CAA TTA GGA GCG GGC ACA TAT GTG CAG CTT 4656
Gln Asn Ile Gln Glu Pro Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu
1540 1545 1550
GTG TTG GAT AAA TAT GAT GAG TCT ATT CAT GGC ACT AAT AAA AGC TTT 4704
Val Leu Asp Lys Tyr Asp Glu Ser Ile His Gly Thr Asn Lys Ser Phe
1555 1560 1565
GCT ATT GAA TAT GTT GAT ATA TTT AAA GAG AAC GAT AGT TTT GTG ATT 4752
Ala Ile Glu Tyr Val Asp Ile Phe Lys Glu Asn Asp Ser Phe Val Ile
1570 1575 1580
TAT CAA GGA GAA CTT AGC GAA ACA AGT CAA ACT GTT GTG AAA GTT TTC 4800
Tyr Gln Gly Glu Leu Ser Glu Thr Ser Gln Thr Val Val Lys Val Phe
1585 1590 1595 1600
TTA TCC TAT TTT ATA GAG GCG ACT GGA AAT AAG AAC CAC TTA TGG GTA 4848
Leu Ser Tyr Phe Ile Glu Ala Thr Gly Asn Lys Asn His Leu Trp Val
1605 1610 1615
CGT GCT AAA TAC CAA AAG GAA ACG ACT GAT AAG ATC TTG TTC GAC CGT 4896
Arg Ala Lys Tyr Gln Lys Glu Thr Thr Asp Lys Ile Leu Phe Asp Arg
1620 1625 1630
ACT GAT GAG AAA GAT CCG CAC GGT TGG TTT CTC AGC GAC GAT CAC AAG 4944
Thr Asp Glu Lys Asp Pro His Gly Trp Phe Leu Ser Asp Asp His Lys
1635 1640 1645
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ACC TTT AGT GGT CTC TCT TCC GCA CAG GCA TTA AAG AAC GAC AGT GAA 4992
Thr Phe Ser Gly Leu Ser Ser Ala Gln Ala Leu Lys Asn Asp Ser Glu
1650 1655 1660
CCG ATG GAT TTC TCT GGC GCC AAT GCT CTC TAT TTC TGG GAA CTG TTC 5040
Pro Met Asp Phe Ser Gly Ala Asn Ala Leu Tyr Phe Trp Glu Leu Phe
1665 1670 1675 1680
TAT TAC ACG CCG ATG ATG ATG GCT CAT CGT TTG TTG CAG GAA CAG AAT 5088
Tyr Tyr Thr Pro Met Met Met Ala His Arg Leu Leu Gln Glu Gln Asn
1685 1690 1695
TTT GAT GCG GCG AAC CAT TGG TTC CGT TAT GTC TGG AGT CCA TCC GGT 5136
Phe Asp Ala Ala Asn His Trp Phe Arg Tyr Val Trp Ser Pro Ser Gly
1700 1705 1710
TAT ATC GTT GAT GGT AAA ATT GCT ATC TAC CAC TGG AAC GTG CGA CCG 5184
Tyr Ile Val Asp Gly Lys Ile Ala Ile Tyr His Trp Asn Val Arg Pro
1715 1720 1725
CTG GAA GAA GAC ACC AGT TGG AAT GCA CAA CAA CTG GAC TCC ACC GAT 5232
Leu Glu Glu Asp Thr Ser Trp Asn Ala Gln Gln Leu Asp Ser Thr Asp
1730 1735 1740
CCA GAT GCT GTA GCC CAA GAT GAT CCG ATG CAC TAC AAG GTG GCT ACC 5280
Pro Asp Ala Val Ala Gln Asp Asp Pro Met His Tyr Lys Val Ala Thr
1745 1750 1755 1760
TTT ATG GCG ACG TTG GAT CTG CTA ATG GCC CGT GGT GAT GCT GCT TAC 5328
Phe Met Ala Thr Leu Asp Leu Leu Met Ala Arg Gly Asp Ala Ala Tyr
1765 1770 1775
CGC CAG TTA GAG CGT GAT ACG TTG GCT GAA GCT AAA ATG TGG TAT ACA 5376
Arg Gln Leu Glu Arg Asp Thr Leu Ala Glu Ala Lys Met Trp Tyr Thr
1780 1785 1790
CAG GCG CTT AAT CTG TTG GGT GAT GAG CCA CAA GTG ATG CTG AGT ACG 5424
Gln Ala Leu Asn Leu Leu Gly Asp Glu Pro Gln Val Met Leu Ser Thr
1795 1800 1805
ACT TGG GCT AAT CCA ACA TTG GGT AAT GCT GCT TCA AAA ACC ACA CAG 5472
Thr Trp Ala Asn Pro Thr Leu Gly Asn Ala Ala Ser Lys Thr Thr Gln
1810 1815 1820
CAG GTT CGT CAG CAA GTG CTT ACC CAG TTG CGT CTC AAT AGC AGG GTA 5520
Gln Val Arg Gln Gln Val Leu Thr Gln Leu Arg Leu Asn Ser Arg Val
1825 1830 1835 1840
AAA ACC CCG TTG 5532
Lys Thr Pro Leu
1844
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1844 amino acids
(B) TYPE: amino acids
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala Asp Asn Tyr
1 5 10 15
Ala Ala Pro Gly Ser Val Ala Ser Met Phe Ser Pro Ala Ala Tyr Leu
20 25 30
Thr Glu Leu Tyr Arg Glu Ala Lys Asn Leu His Asp Ser Ser Ser Ile
35 40 45
Tyr Tyr Leu Asp Lys Arg Arg Pro Asp Leu Ala Ser Leu Met Leu Ser
50 55 60
Gln Lys Asn Met Asp Glu Glu Ile Ser Thr Leu Ala Leu Ser Asn Glu
65 70 75 80
Leu Cys Leu Ala Gly Ile Glu Thr Lys Thr Gly Lys Ser Gln Asp Glu
85 90 95
Val Met Asp Met Leu Ser Thr Tyr Arg Leu Ser Gly Glu Thr Pro Tyr
100 105 110
His His Ala Tyr Glu Thr Val Arg Glu Ile Val His Glu Arg Asp Pro
115 120 125
Gly Phe Arg His Leu Ser Gln Ala Pro Ile Val Ala Ala Lys Leu Asp
130 135 140
Pro Val Thr Leu Leu Gly Ile Ser Ser His Ile Ser Pro Glu Leu Tyr
145 150 155 160
Asn Leu Leu Ile Glu Glu Ile Pro Glu Lys Asp Glu Ala Ala Leu Asp
165 170 175
Thr Leu Tyr Lys Thr Asn Phe Gly Asp Ile Thr Thr Ala Gln Leu Met
180 185 190
Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr Gly Val Ser Pro Glu Asp Ile
195 200 205
Ala Tyr Val Thr Thr Ser Leu Ser His Val Gly Tyr Ser Ser Asp Ile
210 215 220
Leu Val Ile Pro Leu Val Asp Gly Val Gly Lys Met Glu Val Val Arg
225 230 235 240
Val Thr Arg Thr Pro Ser Asp Asn Tyr Thr Ser Gln Thr Asn Tyr Ile
245 250 255
Glu Leu Tyr Pro Gln Gly Gly Asp Asn Tyr Leu Ile Lys Tyr Asn Leu
260 265 270
Ser Asn Ser Phe Gly Leu Asp Asp Phe Tyr Leu Gln Tyr Lys Asp Gly
275 280 285
Ser Ala Asp Trp Thr Glu Ile Ala His Asn Pro Tyr Pro Asp Met Val
290 295 300
Ile Asn Gln Lys Tyr Glu Ser Gln Ala Thr Ile Lys Arg Ser Asp Ser
305 310 315 320
Asp Asn Ile Leu Ser Ile Gly Leu Gln Arg Trp His Ser Gly Ser Tyr
325 330 335
Asn Phe Ala Ala Ala Asn Phe Lys Ile Asp Gln Tyr Ser Pro Lys Ala
340 345 350
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Phe Leu Leu Lys Met Asn Lys Ala Ile Arg Leu Leu Lys Ala Thr Gly
355 360 365
Leu Ser Phe Ala Thr Leu Glu Arg Ile Val Asp Ser Val Asn Ser Thr
370 375 380
Lys Ser Ile Thr Val Glu Val Leu Asn Lys Val Tyr Arg Val Lys Phe
385 390 395 400
Tyr Ile Asp Arg Tyr Gly Ile Ser Glu Glu Thr Ala Ala Ile Leu Ala
405 410 415
Asn Ile Asn Ile Ser Gln Gln Ala Val Gly Asn Gln Leu Ser Gln Phe
420 425 430
Glu Gln Leu Phe Asn His Pro Pro Leu Asn Gly Ile Arg Tyr Glu Ile
435 440 445
Ser Glu Asp Asn Ser Lys His Leu Pro Asn Pro Asp Leu Asn Leu Lys
450 455 460
Pro Asp Ser Thr Gly Asp Asp Gln Arg Lys Ala Val Leu Lys Arg Ala
465 470 475 480
Phe Gln Val Asn Ala Ser Glu Leu Tyr Gln Met Leu Leu Ile Thr Asp
485 490 495
Arg Lys Glu Asp Gly Val Ile Lys Asn Asn Leu Glu Asn Leu Ser Asp
500 505 510
Leu Tyr Leu Val Ser Leu Leu Ala Gln Ile His Asn Leu Thr Ile Ala
515 520 525
Glu Leu Asn Ile Leu Leu Val Ile Cys Gly Tyr Gly Asp Thr Asn Ile
530 535 540
Tyr Gln Ile Thr Asp Asp Asn Leu Ala Lys Ile Val Glu Thr Leu Leu
545 550 555 560
Trp Ile Thr Gln Trp Leu Lys Thr Gln Lys Trp Thr Val Thr Asp Leu
565 570 575
Phe Leu Met Thr Thr Ala Thr Tyr Ser Thr Thr Leu Thr Pro Glu Ile
580 585 590
Ser Asn Leu Thr Ala Thr Leu Ser Ser Thr Leu His Gly Lys Glu Ser
595 600 605
Leu Ile Gly Glu Asp Leu Lys Arg Ala Met Ala Pro Cys Phe Thr Ser
610 615 620
Ala Leu His Leu Thr Ser Gln Glu Val Ala Tyr Asp Leu Leu Leu Trp
625 630 635 640
Ile Asp Gln Ile Gln Pro Ala Gln Ile Thr Val Asp Gly Phe Trp Glu
645 650 655
Glu Val Gln Thr Thr Pro Thr Ser Leu Lys Val Ile Thr Phe Ala Gln
660 665 670
Val Leu Ala Gln Leu Ser Leu Ile Tyr Arg Arg Ile Gly Leu Ser Glu
675 680 685
Thr Glu Leu Ser Leu Ile Val Thr Gln Ser Ser Leu Leu Val Ala Gly
690 695 700
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Lys Ser Ile Leu Asp His Gly Leu Leu Thr Leu Met Ala Leu Glu Gly
705 710 715 720
Phe His Thr Trp Val Asn Gly Leu Gly Gln His Ala Ser Leu Ile Leu
725 730 735
Ala Ala Leu Lys Asp Gly Ala Leu Thr Val Thr Asp Val Ala Gln Ala
740 745 750
Met Asn Lys Glu Glu Ser Leu Leu Gln Met Ala Ala Asn Gln Val Glu
755 760 765
Lys Asp Leu Thr Lys Leu Thr Ser Trp Thr Gln Ile Asp Ala Ile Leu
770 775 780
Gln Trp Leu Gln Met Ser Ser Ala Leu Ala Val Ser Pro Leu Asp Leu
785 790 795 800
Ala Gly Met Met Ala Leu Lys Tyr Gly Ile Asp His Asn Tyr Ala Ala
805 810 815
Trp Gln Ala Ala Ala Ala Ala Leu Met Ala Asp His Ala Asn Gln Ala
820 825 830
Gln Lys Lys Leu Asp Glu Thr Phe Ser Lys Ala Leu Cys Asn Tyr Tyr
835 840 845
Ile Asn Ala Val Val Asp Ser Ala Ala Gly Val Arg Asp Arg Asn Gly
850 855 860
Leu Tyr Thr Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Asp Val Ile
865 870 875 880
Thr Ser Arg Ile Ala Glu Ala Ile Ala Gly Ile Gln Leu Tyr Val Asn
885 890 895
Arg Ala Leu Asn Arg Asp Glu Gly Gln Leu Ala Ser Asp Val Ser Thr
900 905 910
Arg Gln Phe Phe Thr Asp Trp Glu Arg Tyr Asn Lys Arg Tyr Ser Thr
915 920 925
Trp Ala Gly Val Ser Glu Leu Val Tyr Tyr Pro Glu Asn Tyr Val Asp
930 935 940
Pro Thr Gln Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln
945 950 955 960
Ser Ile Asn Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe
965 970 975
Lys Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile
980 985 990
Ser Ala Tyr His Asp Asn Val Asn Val Asp Gln Gly Leu Thr Tyr Phe
995 1000 1005
Ile Gly Ile Asp Gln Ala Ala Pro Gly Thr Tyr Tyr Trp Arg Ser Val
1010 1015 1020
Asp His Ser Lys Cys Glu Asn Gly Lys Phe Ala Ala Asn Ala Trp Gly
1025 1030 1035 1040
Glu Trp Asn Lys Ile Thr Cys Ala Val Asn Pro Trp Lys Asn Ile Ile
1045 1050 1055
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Arg Pro Val Val Tyr Met Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln
1060 1065 1070
Gln Ser Lys Lys Ser Asp Asp Gly Lys Thr Thr Ile Tyr Gln Tyr Asn
1075 1080 1085
Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Ser Trp Asn Thr Pro Phe
1090 1095 1100
Thr Phe Asp Val Thr Glu Lys Val Lys Asn Tyr Thr Ser Ser Thr Asp
1105 1110 1115 1120
Ala Ala Glu Ser Leu Gly Leu Tyr Cys Thr Gly Tyr Gln Gly Glu Asp
1125 1130 1135
Thr Leu Leu Val Met Phe Tyr Ser Met Gln Ser Ser Tyr Ser Ser Tyr
1140 1145 1150
Thr Asp Asn Asn Ala Pro Val Thr Gly Leu Tyr Ile Phe Ala Asp Met
1155 1160 1165
Ser Ser Asp Asn Met Thr Asn Ala Gln Ala Thr Asn Tyr Trp Asn Asn
1170 1175 1180
Ser Tyr Pro Gln Phe Asp Thr Val Met Ala Asp Pro Asp Ser Asp Asn
1185 1190 1195 1200
Lys Lys Val Ile Thr Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr
1205 1210 1215
Glu Ile Pro Ser Ser Val Thr Ser Asn Ser Asn Tyr Ser Trp Gly Asp
1220 1225 1230
His Ser Leu Thr Met Leu Tyr Gly Gly Ser Val Pro Asn Ile Thr Phe
1235 1240 1245
Glu Ser Ala Ala Glu Asp Leu Arg Leu Ser Thr Asn Met Ala Leu Ser
1250 1255 1260
Ile Ile His Asn Gly Tyr Ala Gly Thr Arg Arg Ile Gln Cys Asn Leu
1265 1270 1275 1280
Met Lys Gln Tyr Ala Ser Leu Gly Asp Lys Phe Ile Ile Tyr Asp Ser
1285 1290 1295
Ser Phe Asp Asp Ala Asn Arg Phe Asn Leu Val Pro Leu Phe Lys Phe
1300 1305 1310
Gly Lys Asp Glu Asn Ser Asp Asp Ser Ile Cys Ile Tyr Asn Glu Asn
1315 1320 1325
Pro Ser Ser Glu Asp Lys Lys Trp Tyr Phe Ser Ser Lys Asp Asp Asn
1330 1335 1340
Lys Thr Ala Asp Tyr Asn Gly Gly Thr Gln Cys Ile Asp Ala Gly Thr
1345 1350 1355 1360
Ser Asn Lys Asp Phe Tyr Tyr Asn Leu Gln Glu Ile Glu Val Ile Ser
1365 1370 1375
Val Thr Gly Gly Tyr Trp Ser Ser Tyr Lys Ile Ser Asn Pro Ile Asn
1380 1385 1390
Ile Asn Thr Gly Ile Asp Ser Ala Lys Val Lys Val Thr Val Lys Ala
1395 1400 1405
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Gly Gly Asp Asp Gln Ile Phe Thr Ala Asp Asn Ser Thr Tyr Val Pro
1410 1415 1420
Gln Gln Pro Ala Pro Ser Phe Glu Glu Met Ile Tyr Gln Phe Asn Asn
1425 1430 1435 1440
Leu Thr Ile Asp Cys Lys Asn Leu Asn Phe Ile Asp Asn Gln Ala His
1445 1450 1455
Ile Glu Ile Asp Phe Thr Ala Thr Ala Gln Asp Gly Arg Phe Leu Gly
1460 1465 1470
Ala Glu Thr Phe Ile Ile Pro Val Thr Lys Lys Val Leu Gly Thr Glu
1475 1480 1485
Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly Val Gln Tyr Met Gln
1490 1495 1500
Ile Gly Ala Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Gln Gln Leu
1505 1510 1515 1520
Val Ser Arg Ala Asn Arg Gly Ile Asp Ala Val Leu Ser Met Glu Thr
1525 1530 1535
Gln Asn Ile Gln Glu Pro Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu
1540 1545 1550
Val Leu Asp Lys Tyr Asp Glu Ser Ile His Gly Thr Asn Lys Ser Phe
1555 1560 1565
Ala Ile Glu Tyr Val Asp Ile Phe Lys Glu Asn Asp Ser Phe Val Ile
1570 1575 1580
Tyr Gln Gly Glu Leu Ser Glu Thr Ser Gln Thr Val Val Lys Val Phe
1585 1590 1595 1600
Leu Ser Tyr Phe Ile Glu Ala Thr Gly Asn Lys Asn His Leu Trp Val
1605 1610 1615
Arg Ala Lys Tyr Gln Lys Glu Thr Thr Asp Lys Ile Leu Phe Asp Arg
1620 1625 1630
Thr Asp Glu Lys Asp Pro His Gly Trp Phe Leu Ser Asp Asp His Lys
1635 1640 1645
Thr Phe Ser Gly Leu Ser Ser Ala Gln Ala Leu Lys Asn Asp Ser Glu
1650 1655 1660
Pro Met Asp Phe Ser Gly Ala Asn Ala Leu Tyr Phe Trp Glu Leu Phe
1665 1670 1675 1680
Tyr Tyr Thr Pro Met Met Met Ala His Arg Leu Leu Gln Glu Gln Asn
1685 1690 1695
Phe Asp Ala Ala Asn His Trp Phe Arg Tyr Val Trp Ser Pro Ser Gly
1700 1705 1710
Tyr Ile Val Asp Gly Lys Ile Ala Ile Tyr His Trp Asn Val Arg Pro
1715 1720 1725
Leu Glu Glu Asp Thr Ser Trp Asn Ala Gln Gln Leu Asp Ser Thr Asp
1730 1735 1740
Pro Asp Ala Val Ala Gln Asp Asp Pro Met His Tyr Lys Val Ala Thr
1745 1750 1755 1760
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Phe Met Ala Thr Leu Asp Leu Leu Met Ala Arg Gly Asp Ala Ala Tyr
1765 1770 1775
Arg Gln Leu Glu Arg Asp Thr Leu Ala Glu Ala Lys Met Trp Tyr Thr
1780 1785 1790
Gln Ala Leu Asn Leu Leu Gly Asp Glu Pro Gln Val Met Leu Ser Thr
1795 1800 1805
Thr Trp Ala Asn Pro Thr Leu Gly Asn Ala Ala Ser Lys Thr Thr Gln
1810 1815 1820
Gln Val Arg Gln Gln Val Leu Thr Gln Leu Arg Leu Asn Ser Arg Val
1825 1830 1835 1840
Lys Thr Pro Leu
1844
(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1722 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
CTA GGA ACA GCC AAT TCC CTG ACC GCT TTA TTC CTG CCG CAG GAA AAT 48
Leu Gly Thr Ala Asn Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn
1 5 10 15
AGC AAG CTC AAA GGC TAC TGG CGG ACA CTG GCG CAG CGT ATG TTT AAT 96
Ser Lys Leu Lys Gly Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn
20 25 30
TTA CGT CAT AAT CTG TCG ATT GAC GGC CAG CCG CTC TCC TTG CCG CTG 144
Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu
40 45
TAT GCT AAA CCG GCT GAT CCA AAA GCT TTA CTG AGT GCG GCG GTT TCA 192
Tyr Ala Lys Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser
55 60
GCT TCT CAA GGG GGA GCC GAC TTG CCG AAG GCG CCG CTG ACT ATT CAC 240
Ala Ser Gln Gly Gly Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His
65 70 75 80
CGC TTC CCT CAA ATG CTA GAA GGG GCA CGG GGC TTG GTT AAC CAG CTT 288
50 Arg Phe Pro Gln Met Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu
85 90 95
ATA CAG TTC GGT AGT TCA CTA TTG GGG TAC AGT GAG CGT CAG GAT GCG 336
Ile Gln Phe Gly Ser Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala
100 105 110
GAA GCT ATG AGT CAA CTA CTG CAA ACC CAA GCC AGC GAG TTA ATA CTG 384
Glu Ala Met Ser Gln Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu
115 120 125
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ACC AGT ATT CGT ATG CAG GAT AAC CAA TTG GCA GAG CTG GAT TCG GAA 432
Thr Ser Ile Arg Met Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu
130 135 140
AAA ACC GCC TTG CAA GTC TCT TTA GCT GGA GTG CAA CAA CGG TTT GAC 480
Lys Thr Ala Leu Gln Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp
145 150 155 160
AGC TAT AGC CAA CTG TAT GAG GAG AAC ATC AAC GCA GGT GAG CAG CGA 528
Ser Tyr Ser Gln Leu Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg
165 170 175
GCG CTG GCG TTA CGC TCA GAA TCT GCT ATT GAG TCT CAG GGA GCG CAG 576
Ala Leu Ala Leu Arg Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln
180 185 190
ATT TCC CGT ATG GCA GGC GCG GGT GTT GAT ATG GCA CCA AAT ATC TTC 624
Ile Ser Arg Met Ala Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe
195 200 205
GGC CTG GCT GAT GGC GGC ATG CAT TAT GGT GCT ATT GCC TAT GCC ATC 672
Gly Leu Ala Asp Gly Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile
210 215 220
GCT GAC GGT ATT GAG TTG AGT GCT TCT GCC AAG ATG GTT GAT GCG GAG 720
Ala Asp Gly Ile Glu Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu
225 230 235 240
AAA GTT GCT CAG TCG GAA ATA TAT CGC CGT CGC CGT CAA GAA TGG AAA 768
Lys Val Ala Gln Ser Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys
245 250 255
ATT CAG CGT GAC AAC GCA CAA GCG GAG ATT AAC CAG TTA AAC GCG CAA 816
Ile Gln Arg Asp Asn Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln
260 265 270
CTG GAA TCA CTG TCT ATT CGC CGT GAA GCC GCT GAA ATG CAA AAA GAG 864
Leu Glu Ser Leu Ser Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu
275 280 285
TAC CTG AAA ACC CAG CAA GCT CAG GCG CAG GCA CAA CTT ACT TTC TTA 912
Tyr Leu Lys Thr Gln Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu
290 295 300
AGA AGC AAA TTC AGT AAT CAA GCG TTA TAT AGT TGG TTA CGA GGG CGT 960
Arg Ser Lys Phe Ser Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg
305 310 315 320
TTG TCA GGT ATT TAT TTC CAG TTC TAT GAC TTG GCC GTA TCA CGT TGC 1008
Leu Ser Gly Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys
325 330 335
CTG ATG GCA GAG CAA TCC TAT CAA TGG GAA GCT AAT GAT AAT TCC ATT 1056
Leu Met Ala Glu Gln Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile
340 345 350
AGC TTT GTC AAA CCG GGT GCA TGG CAA GGA ACT TAC GCC GGC TTA TTG 1104
Ser Phe Val Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu
355 360 365
TGT GGA GAA GCT TTG ATA CAA AAT CTG GCA CAA ATG GAA GAG GCA TAT 1152
Cys Gly Glu Ala Leu Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr
370 375 380
CTG AAA TGG GAA TCT CGC GCT TTG GAA GTA GAA CGC ACG GTT TCA TTG 1200
Leu Lys Trp Glu Ser Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu
385 390 395 400
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GCA GTG GTT TAT GAT TCA CTG GAA GGT AAT GAT CGT TTT AAT TTA GCG 1248
Ala Val Val Tyr Asp Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala
405 410 415
GAA CAA ATA CCT GCA TTA TTG GAT AAG GGG GAG GGA ACA GCA GGA ACT 1296
Glu Gln Ile Pro Ala Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr
420 425 430
AAA GAA AAT GGG TTA TCA TTG GCT AAT GCT ATC CTG TCA GCT TCG GTC 1344
Lys Glu Asn Gly Leu Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val
435 440 445
AAA TTG TCC GAC TTG AAA CTG GGA ACG GAT TAT CCA GAC AGT ATC GTT 1392
Lys Leu Ser Asp Leu Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val
450 455 460
GGT AGC AAC AAG GTT CGT CGT ATT AAG CAA ATC AGT GTT TCG CTA CCT 1440
Gly Ser Asn Lys Val Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro
465 470 475 480
GCA TTG GTT GGG CCT TAT CAG GAT GTT CAG GCT ATG CTC AGC TAT GGT 1488
Ala Leu Val Gly Pro Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly
485 490 495
GGC AGT ACT CAA TTG CCG AAA GGT TGT TCA GCG TTG GCT GTG TCT CAT 1536
Gly Ser Thr Gln Leu Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His
500 505 510
GGT ACC AAT GAT AGT GGT CAG TTC CAG TTG GAT TTC AAT GAC GGC AAA 1584
Gly Thr Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys
515 520 525
TAC CTG CCA TTT GAA GGT ATT GCT CTT GAT GAT CAG GGT ACA CTG AAT 1632
Tyr Leu Pro Phe Glu Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn
530 535 540
CTT CAA TTT CCG AAT GCT ACC GAC AAG CAG AAA GCA ATA TTG CAA ACT 1680
Leu Gln Phe Pro Asn Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr
545 550 555 560
ATG AGC GAT ATT ATT TTG CAT ATT CGT TAT ACC ATC CGT TAA 1722
Met Ser Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Arg
565 570 573
(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 573 amino acids
(B) TYPE: amino acids
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
Leu Gly Thr Ala Asn Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn
1 5 10 15
Ser Lys Leu Lys Gly Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn
20 25 30
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Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu
35 40 45
Tyr Ala Lys Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser
50 55 60
Ala Ser Gln Gly Gly Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His
65 70 75 80
Arg Phe Pro Gln Met Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu
85 90 95
Ile Gln Phe Gly Ser Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala
100 105 110
Glu Ala Met Ser Gln Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu
115 120 125
Thr Ser Ile Arg Met Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu
130 135 140
Lys Thr Ala Leu Gln Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp
145 150 155 160
Ser Tyr Ser Gln Leu Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg
165 170 175
Ala Leu Ala Leu Arg Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln
180 185 190
Ile Ser Arg Met Ala Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe
195 200 205
Gly Leu Ala Asp Gly Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile
210 215 220
Ala Asp Gly Ile Glu Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu
225 230 235 240
Lys Val Ala Gln Ser Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys
245 250 255
Ile Gln Arg Asp Asn Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln
260 265 270
Leu Glu Ser Leu Ser Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu
275 280 285
Tyr Leu Lys Thr Gln Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu
290 295 300
Arg Ser Lys Phe Ser Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg
305 310 315 320
Leu Ser Gly Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys
325 330 335
Leu Met Ala Glu Gln Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile
340 345 350
Ser Phe Val Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu
355 360 365
Cys Gly Glu Ala Leu Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr
370 375 380
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Leu Lys Trp Glu Ser Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu
385 390 395 400
Ala Val Val Tyr Asp Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala
405 410 415
Glu Gln Ile Pro Ala Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr
420 425 430
Lys Glu Asn Gly Leu Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val
435 440 445
Lys Leu Ser Asp Leu Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val
450 455 460
Gly Ser Asn Lys Val Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro
465 470 475 480
Ala Leu Val Gly Pro Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly
485 490 495
Gly Ser Thr Gln Leu Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His
500 505 510
Gly Thr Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys
515 520 525
Tyr Leu Pro Phe Glu Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn
530 535 540
Leu Gln Phe Pro Asn Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr
545 550 555 560
Met Ser Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Arg
565 570 573
(2) INFORMATION FOR SEQ ID NO:56
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2898 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
ATG AAT CAA CTC GCC AGT CCC CTG ATT TCC CGC ACC GAA GAG ATC CAC 48
Met Asn Gln Leu Ala Ser Pro Leu Ile Ser Arg Thr Glu Giu Ile His
1 5 10 15
AAC TTA CCC GGT AAA TTG ACC GAT CTT GGT TAT ACC TCA GTG TTT GAT 96
Asn Leu Pro Gly Lys Leu Thr Asp Leu Gly Tyr Thr Ser Val Phe Asp
20 25 30
GTG GTA CGT ATG CCG CGT GAG CGT TTT ATT CGT GAG CAT CGT GCT GAT 144
Val Val Arg Met Pro Arg Glu Arg Phe Ile Arg Glu His Arg Ala Asp
35 40 45
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CTC GGG CGC AGT GCT GAA AAA ATG TAT GAC CTG GCA GTG GGC TAT GCT 192
Leu Gly Arg Ser Ala Glu Lys Met Tyr Asp Leu Ala Val Gly Tyr Ala
50 55 60
CAT CAG GTG TTA CAC CAT TTT CGC CGT AAT TCT CTT AGT GAA GCT GTT 240
His Gln Val Leu His His Phe Arg Arg Asn Ser Leu Ser Glu Ala Val
65 70 75 80
CAG TTT GGC TTG AGA AGT CCG TTC TCC GTA TCA GGC CCG GAT TAC GCC 288
Gln Phe Gly Leu Arg Ser Pro Phe Ser Val Ser Gly Pro Asp Tyr Ala
85 90 95
AAT CAG TTT CTT GAT GCA AAC ACG GGT TGG AAA GAT AAA GCA CCA AGT 336
Asn Gln Phe Leu Asp Ala Asn Thr Gly Trp Lys Asp Lys Ala Pro Ser
100 105 110
GGA TCA CCG GAA GCC AAT GAT GCG CCG GTA GCC TAT CTG ACT CAT ATT 384
Gly Ser Pro Glu Ala Asn Asp Ala Pro Val Ala Tyr Leu Thr His Ile
115 120 125
TAT CAA TTG GCC CTT GAA CAG GAA AAG AAT GGC GCC ACT ACC ATT ATG 432
Tyr Gln Leu Ala Leu Glu Gln Glu Lys Asn Gly Ala Thr Thr Ile Met
130 135 140
AAT ACG CTG GCG GAG CGT CGC CCC GAT CTG GGT GCT TTG TTA ATT AAT 480
Asn Thr Leu Ala Glu Arg Arg Pro Asp Leu Gly Ala Leu Leu Ile Asn
145 150 155 160
GAT AAA GCA ATC AAT GAG GTG ATA CCG CAA TTG CAG TTG GTC AAT GAA 528
Asp Lys Ala Ile Asn Glu Val Ile Pro Gln Leu Gln Leu Val Asn Glu
165 170 175
ATT CTG TCC AAA GCT ATT CAG AAG AAA CTG AGT TTG ACT GAT CTG GAA 576
Ile Leu Ser Lys Ala Ile Gln Lys Lys Leu Ser Leu Thr Asp Leu Glu
180 185 190
GCG GTA AAC GCC AGA CTT TCC ACT ACC CGT TAC CCG AAT AAT CTG CCG 624
Ala Val Asn Ala Arg Leu Ser Thr Thr Arg Tyr Pro Asn Asn Leu Pro
195 200 205
TAT CAT TAT GGT CAT CAG CAG ATT CAG ACA GCT CAA TCG GTA TTG GGT 672
Tyr His Tyr Gly His Gln Gln Ile Gln Thr Ala Gln Ser Val Leu Gly
210 215 220
ACT ACG TTG CAA GAT ATC ACT TTG CCA CAG ACG CTG GAT CTG CCG CAA 720
Thr Thr Leu Gln Asp Ile Thr Leu Pro Gln Thr Leu Asp Leu Pro Gln
225 230 235 240
AAC TTC TGG GCA ACA GCA AAA GGA AAA CTG AGC GAT ACG ACT GCC AGT 768
Asn Phe Trp Ala Thr Ala Lys Gly Lys Leu Ser Asp Thr Thr Ala Ser
245 250 255
GCT TTG ACC CGA CTG CAA ATC ATG GCG AGT CAG TTT TCG CCA GAG CAG 816
Ala Leu Thr Arg Leu Gln Ile Met Ala Ser Gln Phe Ser Pro Glu Gln
260 265 270
CAG AAA ATC ATT ACG GAG ACT GTC GGT CAG GAT TTC TAT CAG CTT AAC 864
Gln Lys Ile Ile Thr Glu Thr Val Gly Gln Asp Phe Tyr Gln Leu Asn
275 280 285
TAT GGT GAC AGT TCG CTT ACT GTG AAT AGT TTC AGC GAC ATG ACC ATA 912
Tyr Gly Asp Ser Ser Leu Thr Val Asn Ser Phe Ser Asp Met Thr Ile
290 295 300
ATG ACT GAT CGA ACA AGT TTG ACT GTA CCC CAG GTA GAA CTG ATG TTG 960
Met Thr Asp Arg Thr Ser Leu Thr Val Pro Gln Val Glu Leu Met Leu
305 310 315 320
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TGT TCA ACT GTC GGA GGT TCT ACG GTT GTT AAG TCT GAT AAT GTG AGT 1008
Cys Ser Thr Val Gly Gly Ser Thr Val Val Lys Ser Asp Asn Val Ser
325 330 335
TCT GGT GAC ACG ACA GCG ACG CCA TTT GCG TAT GGC GCC CGC TTT ATT 1056
Ser Gly Asp Thr Thr Ala Thr Pro Phe Ala Tyr Gly Ala Arg Phe Ile
340 345 350
CAT GCC GGT AAG CCG GAG GCG ATT ACC CTG AGT CGC AGT GGT GCG GAG 1104
His Ala Gly Lys Pro Glu Ala Ile Thr Leu Ser Arg Ser Gly Ala Glu
355 360 365
GCG CAT TTT GCT CTG ACG GTT AAC AAT CTG ACA GAT GAC AAG TTG GAC 1152
Ala His Phe Ala Leu Thr Val Asn Asn Leu Thr Asp Asp Lys Leu Asp
370 375 380
CGT ATT AAC CGC ACA GTG CGC CTG CAA AAA TGG CTG AAT CTG CCT TAT 1200
Arg Ile Asn Arg Thr Val Arg Leu Gln Lys Trp Leu Asn Leu Pro Tyr
385 390 395 400
GAG GAT ATT GAC CTG TTA GTG ACT TCT GCT ATG GAT GCG GAA ACA GGA 1248
Glu Asp Ile Asp Leu Leu Val Thr Ser Ala Met Asp Ala Glu Thr Gly
405 410 415
AAT ACC GCG CTG TCG ATG AAC GAC AAT ACG CTG CGT ATG TTG GGA GTG 1296
Asn Thr Ala Leu Ser Met Asn Asp Asn Thr Leu Arg Met Leu Gly Val
420 425 430
TTC AAA CAT TAT CAG GCG AAG TAT GGT GTT AGC GCT AAA CAA TTT GCT 1344
Phe Lys His Tyr Gln Ala Lys Tyr Gly Val Ser Ala Lys Gln Phe Ala
435 440 445
GGC TGG CTG CGC GTA GTG GCC CCG TTT GCC ATT ACA CCG GCA ACG CCG 1392
Gly Trp Leu Arg Val Val Ala Pro Phe Ala Ile Thr Pro Ala Thr Pro
450 455 460
TTT TTA GAC CAA GTG TTT AAC TCC GTC GGC ACC TTT GAT ACA CCG TTT 1440
Phe Leu Asp Gln Val Phe Asn Ser Val Gly Thr Phe Asp Thr Pro Phe
465 470 475 480
GTG ATA GAT AAT CAG GAT TTT GTC TAT ACA TTG ACC ACC GGG GGC GAT 1488
Val Ile Asp Asn Gln Asp Phe Val Tyr Thr Leu Thr Thr Gly Gly Asp
485 490 495
GGG GCG CGT GTT AAG CAT ATC AGC ACG GCA CTG GGC CTC AAT CAT CGT 1536
Gly Ala Arg Val Lys His Ile Ser Thr Ala Leu Gly Leu Asn His Arg
500 505 510
CAG TTC CTG TTA TTG GCG GAT AAT ATT GCC CGT CAA CAG GGG AAT GTC 1584
Gln Phe Leu Leu Leu Ala Asp Asn Ile Ala Arg Gln Gln Gly Asn Val
515 520 525
ACG CAA AGC ACA CTC AAC TGT AAT CTG TTT GTG GTG TCA GCT TTC TAC 1632
Thr Gln Ser Thr Leu Asn Cys Asn Leu Phe Val Val Ser Ala Phe Tyr
530 535 540
CGT CTG GCT AAT TTG GCG CGC ACA TTG GGG ATA AAT CCA GAG TCT TTC 1680
Arg Leu Ala Asn Leu Ala Arg Thr Leu Gly Ile Asn Pro Glu Ser Phe
545 550 555 560
TGT GCC TTG GTT GAT CGA TTA GAT GCA GGT ACA GGC ATC GTC TGG CAG 1728
Cys Ala Leu Val Asp Arg Leu Asp Ala Gly Thr Gly Ile Val Trp Gln
565 570 575
CAA TTG GCA GGG AAA CCC ACA ATC ACG GTA CCA CAA AAA GAT TCC CCG 1776
Gln Leu Ala Gly Lys Pro Thr Ile Thr Val Pro Gln Lys Asp Ser Pro
580 585 590
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CTG GCG GCG GAT ATT CTG AGT TTG CTG CAA GCG CTA AGT GCG ATT GCT 1824
Leu Ala Ala Asp Ile Leu Ser Leu Leu Gln Ala Leu Ser Ala Ile Ala
595 600 605
CAA TGG CAA CAA CAG CAC GAT TTA GAA TTT TCA GCA CTG CTT TTG CTG 1872
Gln Trp Gln Gln Gln His ASp Leu Glu Phe Ser Ala Leu Leu Leu Leu
610 615 620
TTG AGT GAC AAC CCT ATT TCT ACC TCG CAG GGC ACT GAC GAT CAA TTG 1920
Leu Ser Asp Asn Pro Ile Ser Thr Ser Gin Gly Thr Asp Asp Gln Leu
625 630 635 640
AAC TTT ATC CGT CAA GTG TGG CAG AAC CTA GGC AGT ACG TTT GTG GGT 1968
Asn Phe Ile Arg Gln Val Trp Gln Asn Leu Gly Ser Thr Phe Val Gly
645 650 655
GCA ACA TTG TTG TCC CGC AGT GGG GCA CCA TTA GTC GAT ACC AAC GGC 2016.
Ala Thr Leu Leu Ser Arg Ser Gly Ala Pro Leu Val Asp Thr Asn Gly
660 665 670
CAC GCT ATT GAC TGG TTT GCT CTG CTC TCA GCA GGT AAT AGT CCG CTT 2064
His Ala Ile Asp Trp Phe Ala Leu Leu Ser Ala Gly Asn Ser Pro Leu
675 680 685
ATC GAT AAG GTT GGT CTG GTG ACT GAT GCT GGC ATA CAA AGT GTT ATA 2112
Ile Asp Lys Val Gly Leu Val Thr Asp Ala Gly Ile Gln Ser Val Ile
690 695 700
GCA ACG GTG GTC AAT ACA CAA AGC TTA TCT GAT GAA GAT AAG AAG CTG 2160
Ala Thr Val Val Asn Thr Gln Ser Leu Ser Asp Glu Asp Lys Lys Leu
705 710 715 720
GCA ATC ACT ACT CTG ACT AAT ACG TTG AAT CAG GTA CAG AAA ACT CAA 2208
Ala Ile Thr Thr Leu Thr Asn Thr Leu Asn Gln Val Gln Lys Thr Gln
725 730 735
CAG GGC GTG GCC GTC AGT CTG TTG GCG CAG ACT CTG AAC GTG AGT CAG 2256
Gln Gly Val Ala Val Ser Leu Leu Ala Gln Thr Leu Asn Val Ser Gln
740 745 750
TCA CTG CCT GCG TTA TTG TTG CGC TGG AGT GGA CAA ACA ACC TAC CAG 2304
Ser Leu Pro Ala Leu Leu Leu Arg Trp Ser Gly Gln Thr Thr Tyr Gln
755 760 765
TGG TTG AGT GCG ACT TGG GCA TTG AAG GAT GCC GTT AAG ACT GCC GCC 2352
Trp Leu Ser Ala Thr Trp Ala Leu Lys Asp Ala Val Lys Thr Ala Ala
770 775 780
GAT ATT CCC GCT GAC TAT CTG CGT CAA TTA CGT GAA GTG GTA CGC CGC 2400
Asp Ile Pro Ala Asp Tyr Leu Arg Gln Leu Arg Glu Val Val Arg Arg
785 790 795 800
TCC TTG TTG ACC CAA CAA TTC ACG CTG AGT CCT GCA ATG GTG CAA ACC 2448
Ser Leu Leu Thr Gln Gln Phe Thr Leu Ser Pro Ala Met Val Gln Thr
805 810 815
TTG CTG GAC TAT CCA GCC TAT TTT GGC GCT TCC GCA GAA ACA GTG ACC 2496
Leu Leu Asp Tyr Pro Ala Tyr Phe Gly Ala Ser Ala Glu Thr Val Thr
820 825 830
GAT ATC AGT TTG TGG ATG CTT TAT ACC CTG AGC TGT TAT AGC GAT TTA 2544
Asp Ile Ser Leu Trp Met Leu Tyr Thr Leu Ser Cys Tyr Ser Asp Leu
835 840 845
TTG CTC CAA ATG GGT GAA GCT GGT GGT ACC GAA GAT GAT GTA CTG GCC 2592
Leu Leu Gin Met Gly Glu Ala Gly Gly Thr Glu Asp Asp Val Leu Ala
850 855 860
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TAC TTA CGC ACA GCT AAT GCT ACC ACA CCG TTG AGC CAA TCT GAT GCT 2640
Tyr Leu Arg Thr Ala Asn Ala Thr Thr Pro Leu Ser Gln Ser Asp Ala
865 870 875 880
GCA CAG ACG TTG GCA ACG CTA TTG GGT TGG GAG GTT AAC GAG TTG CAA 2688
Ala Gln Thr Leu Ala Thr Leu Leu Gly Trp Glu Val Asn Glu Leu Gln
885 890 895
GCC GCT TGG TCG GTA TTG GGC GGG ATT GCC AAA ACC ACA CCG CAA CTG 2736
Ala Ala Trp Ser Val Leu Gly Gly Ile Ala Lys Thr Thr Pro Gln Leu
900 905 910
GAT GCG CTT CTG CGT TTG CAA CAG GCA CAG AAC CAA ACT GGT CTT GGC 2784
Asp Ala Leu Leu Arg Leu Gln Gln Ala Gln Asn Gln Thr Gly Leu Gly
915 920 925
GTT ACA CAG CAA CAG CAA GGC TAT CTC CTG AGT CGT GAC AGT GAT TAT 2832
Val Thr Gln Gln Gln Gln Gly Tyr Leu Leu Ser Arg Asp Ser Asp Tyr
930 935 940
ACC CTT TGG CAA AGC ACC GGT CAG GCG CTG GTG GCT GGC GTA TCC CAT 2880
Thr Leu Trp Gln Ser Thr Gly Gln Ala Leu Val Ala Gly Val Ser His
945 950 955 960
GTC AAG GGC AGT AAC TGA 2898
Val Lys Gly Ser Asn
965
(2) INFORMATION FOR SEQ ID NO:57
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 965 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
Met Asn Gln Leu Ala Ser Pro Leu Ile Ser Arg Thr Glu Glu Ile His
1 5 10 15
Asn Leu Pro Gly Lys Leu Thr Asp Leu Gly Tyr Thr Ser Val Phe Asp
20 25 30
Val Val Arg Met Pro Arg Glu Arg Phe Ile Arg Glu His Arg Ala Asp
35 40 45
Leu Gly Arg Ser Ala Glu Lys Met Tyr Asp Leu Ala Val Gly Tyr Ala
55 60
His Gln Val Leu His His Phe Arg Arg Asn Ser Leu Ser Glu Ala Val
65 70 75 80
Gln Phe Gly Leu Arg Ser Pro Phe Ser Val Ser Gly Pro Asp Tyr Ala
85 90 95
Asn Gln Phe Leu Asp Ala Asn Thr Gly Trp Lys Asp Lys Ala Pro Ser
100 105 110
Gly Ser Pro Glu Ala Asn Asp Ala Pro Val Ala Tyr Leu Thr His Ile
115 120 125
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DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
Brevets.
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THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
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NOTE: For additional volumes please contact the Canadian Patent Office.
