Note: Descriptions are shown in the official language in which they were submitted.
~~2~~~~
PATENT APPLICATION of
For
Y-Chromosome Specific Polynucleotide Probes For
Prenatal Sexing
Field of the Invention
The present invention relates to determination of genetic
sex of mammals, more particularly to ruminant sex determinatian
using Y-specific polynucleotide probes.
BACKGROUND OF THE INVENTION
The ability to determine the sex of an embryo soon after
fertilization would provide numerous advantages in the live-
stock and dairy industries as well as in veterinary medicine.
In the dairy and beef cattle industry advances in embryo trans-
fer has resulted in a great demand for a method of quickly
determining the sex of embryos and cells taken from embryos
at early stages of development. The commercial efficiency of
livestock and dairy operations would be greatly improved by
allowing gestation to be established with embryos of the
desired sex. Given the advantage to the dairy industry of a
preponderance of female progeny, it would be advantageous if
the sex of embryos could be routinely determined prior to embryo
transplant into a maternal host. The advantages of sexed embryos
are numerous including the selection of replacement of stock based
~~1~~~~
2
on desired characteristics, such as size, weight. increased milk
production. etc. In addition, certain diseases. such as X-chromo-
some-linked diseases in humans and similar diseases in other
mammals, affect individuals of only one sex, Early determina-
tion of the sex of an embryo which, if carried to term, would
likely be an individual with such a disease would be particularly
advantageous and provide valuable information on which to base a
decision to allow further development.
Efficient determination of the sex of a conceptus in vivo is
also of significant economic importance, and would have important
commercial applications. In the dairy and livestock industries,
in pregnancies which arise via artificial insemination or natural
mating, early determination of the sex of an embryo or fetus would
allow for termination of the pregnancy if an embryo or fetus of
the desired sex was not obtained.
In situations where, for health or economic reasons. a
determination of the sex of an embryo or fetus is indicated, it
is important to determine the sex as soon as possible after fer-
tilization. There is a substantial increase in risk to the life
and health of a female if abortion is induced late in gestation.
Wfth livestock, it is commercially inefficient, both because of
reduced reproductive efficiency and dangers to the life and health
of the female, to carry an embryo longer than necessary.
With advances in reproductive biology, it would be feasible
to avoid all risks and costs associated with pregnancy and abor-
5
tion if it were possible to determine the sex of an embryo,
whether produced in vivo or in vitro prior to or at the time of
transfer, and also to determine as early as possible with cer-
tainty the sex of an embryo or fetus in vivo.
The sex of a mammal is determined by the presence or
absence of the entire Y-chromosome or some functional portion
thereof. Genes present on the Y-chromosome govern formation
and the development of the male phenotype. The sex of an
individual mammal is therefore dependent upon whether or not
its genome contains particular DNA sequences, especially those
sequences comprising that part of the Y-chromosome which encode
genes responsible for sex determination.
The sex or presumptive sex of a mammal can therefore be
determined by analysis for Y-specific genes in the DNA of the
individual mammal. Alternatively, sex can be determined by
unrelated but genetically linked sequences which are associated
specifically with the Y-chromosome, preferably on sequences
linked closely to the male-determining genes to reduce possible
errors in analysis due to genetic recombination.
Prior to the present invention a number of investigators
have identified DNA sequences which hybridize preferentially
or exclusively to male DNA. See Kunkel et al., Science 191,
1189-1190 (1976); Bishop et al., Nature 303. 831 (1983);
vergnaud et al., Brit. Med. J. 289. 73-76 (1984); Lau et al.,
The Lancet, Jan. 7, 1984, pp 14-16; Golden et al., The Lancet,
~~~~~2~
Dec. 25, 1982, pp 1416-1919; Bostick et al., Nature 272, 324
(1978). These DNA sequences have not been functionally char-
acterized, and it is unknown whether these sequences are capable
of hybridization to non-human species.
The isolation of sperm separated according to the sex
chromosome they contain, and using these sperm to fertilize ova
is one method currently used to control embryo-sex. Such sperm
isolation methods are significantly limited due to the diffi-
culty of obtaining preparations of sperm in which more than 99~
of the sperm carry the sex chromosome of only one of the sexes.
At present, known techniques for separating sperm according to
sex are not practical for obtaining mixtures of sperm with more
than about 75~ harboring the same sex chromosome. Therefore
determining sex by segregating sperm is limited by economic and
commercial considerations.
Karyotyping fetal cells obtained after several weeks ges-
tation by amniocentesis, chorion biopsy, and other procedures
is another known method of determining sex. Such procedures
are limited, however. in commercial application due to the
expense, risk of infection, and time required to carry out this
type of analysis.
Other prior art attempts to deal with this problem have
been indirect and incomplete. U.S. Patent No. 4,769,319 issued
to Ellis et al. discloses male specific nucleic acid hybridi-
ration probes which have sequences complementary to sequences of
5
~9~~~~~~
segments in bovine male specific DNA. These nucleic acid
sequences are stated to be useful as hybridization probes for
sexing embryos and fetuses. Australian Patent Application No.
59561/86 discloses bovine DNA probes which hybridize prefer-
entially to male DNA, and are also stated to be useful in sexing
embryos and fetuses. These DNA sequences are indicated to be
species specific. International Patent Application No.
PCT/AU87/00254, discloses a 307 base pair nucleic acid sequence
designated BRY.1 comprising Y-specific DNA which is capable of
hybridizing with male bovine and ovine derived DNA but not with
DNA isolated from female animals. PCT Application No.PCT/AU
89/0029 discloses nucleic acid isolates capable of hybridizing
to Y-specific DNA sequences of ruminants.
Moreover, there is nothing in the prior art to indicate that
any like DNA segments exist which could be used to provide the
basis of a polynucleotide probe to sex by nucleic acid hybridi-
zation. with as few as 2 cells, with virtually 100% accuracy, in
an extremely short time period, a mammalian embryo at a morula
or blastocyst stage, at or before transfer of the embryo for
further development.
SiJMMARY OF THE INVEWTIOid
The present invention arises from the discovery of segments
of Y-chromosome specific DNA sequences, designated SEQ ID N0:1;
BtYl and SEQ ID N0:3; BtY2 and corresponding RNA sequences that
make possible the rapid, virtually 100% accurate sexing of bovine
embryos by nucleic acid hybridization with an amount of DNA
equal to the amount obtained from 2 or fewer embryonic cells.
The sequences are repeated to varying degrees, with a repeat
number differing between unrelated species, and are stably
inherited. Furthermore, with the nucleic acid probes of this
invention embryos may be sexed in less than one day at an early
stage, at or before the time embryo transfer is carried out.
With the present invention, the benefits of early and essen-
tially certain sexing of bovine embryos can be achieved.
The present invention accomplishes this by providing sensi-
tive Y-chromosome probes, designated SEQ ID N0:1: BtYl and SEQ ID
N0:3; BtY2, making possible the rapid, reliable, and economical
sexing of cells obtained from an embryo. Probes of the present
invention, which are sufficiently sensitive to sex a ruminant/
bovine with DNA from as few as 2 of its cells, can also be used to
sex fetuses and embryos. While sexing of fetuses by nucleic acid
hybridization or karyotyping is essentially 100 accurate, it
occurs after several weeks of gestation and involves significant
risks to the fetus and mother. Thus, these known procedures for
sexing fetuses have significant disadvantages compared with early
embryo sexing made possible by the present invention.
The polynucleotide probes of the present invention were
described through their association with male bovine DNA. Cer-
tain of these sequences are more efficacious than the prior art
for determining the genetic sex of ruminants due to their prefer-
7
ential binding to male, in comparison to female, DNA of species
of the genus Bos (bovine). Their superiority also results because
they exist in higher copy number, axe present in multiple copies
in males but not in females, and show stronger sequence similar-
ity between individual elements than have been sequenced.
In addition, the present invention encompasses methods for
applying such DNA sequences in determining the sex of ruminant/
bovine and isolating, from male DNA of such species, nucleic
acid sequences which hybridize to significantly greater extent
with the nucleic acids of the male rather than the female of the
species. Such sequences provide for nucleic acid hybridization
probes for sexing embryos and cells.
Definitions and Abreviations
DNA - deoxyribonucleic acid
RNA - ribonucleic acid
A-adenine
T-thymine ,
G-guanine
C-cytosine
U-uracil
Polynucleotide - single or double-stranded DNA or
RNA
EDTA Ethylenediamine tetra acetate
Tris Tris (hydroxymethyl) amino methane
~v~~~2~
rpm rotations per minute
mPi millimolar, Molar
mm millimetre
O.D. Optical density measured at X nanometers
DEAE Diethyl amino ethane
TE 10 mM Tris HC1 pH 8.0 1 mM EDTA
DTT Dithiathreitol
M9 minimal plates, see Maniatis et. al. (1982) Molecular
Cloning, A Lab Manual for formula
LB Luria Bertani broth
ATP Adenosine triphosphate
amp ampicillin cased at 150 mg/ml
SSPE See Maniatis et. al.
Denhardts (100 X formula in Maniatis et. al)
SDS Sodium dodecyl sulphate
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF INVENTION
The present invention includes nucleic acids which are use-
ful as hybridization probes for prenatally sexing mammals; the
probes may be labeled sa as to be detectable in a hybridiza-
tion methodology or utilized in unlabeled form; methods of
isolating and identifying such nucleic acids and probes; and
procedures for using the probes in prenatal sexing of mammals axe
also disclosed.
The nucleic acid sequences of this invention can be single-
stranded or double stranded DNA or RNA, or hybrids between DNA
and RNA. The sequences may be labeled or unlabeled. The
sequence of the labeled nucleic acid is the sequence the nucleic
acid would have if each labeled nucleotide in the sequence were
replaced with the corresponding unlabeled nucleotide. For
example, if a DNA is labeled with a non-radioactive marker such
as biotin on the 5-position of deoxyuridylate residues, the
sequence of the labeled DNA is the same as that of the DNA where
all of the biotin labeled deoxyuridylates are replaced with
thymidylates. The sequences of a DNA and a RNA are the same
if every deoxyribonucleotide, except thymidylate in the DNA is
replaced with the corresponding ribonucleotide in the RNA and
every thymidylate in the DNA is substituted in the RNA by
uridylate.
The preferred nucleie acid probes, according to the inven-
tion. hybridize to a significantly greater extent with total
male DNA than total female DNA of a bovine species, when hybridi-
zations are carried out under similar conditions. The probes of
this invention will not hybridize detectably to total female
bovine DNA in an hybridization under stringent conditions over a
hybridization period during which detectable hybridizations with
total male bovine DNA occur.
Preferably the nucleic acid probes, according to the inven-
tion, are hybridized under stringent conditions with chromosomal
DNA derived from cells of an embryo of the species being tested.
The probes correspond to all or part of a DNA sequence found on
to
the Y-chromosome of at least one of bovine, ovine, and caprine
animals.
The fundamental feature of the nucleic acids of the in~ren-
tion, both unlabeled and labeled, is that, when in single
stranded form, they hybridize with a probability greater than
0.99 preferentially with total male DNA rather than total female
DNA of a bovine species under substantially the same hybridiza-
tion conditions.
In particular, there are provided and defined two nueleic acid
isolates from male bovine that are capable of hybridizing only
to sequences of nucleic acid from bovine which contain the
Y-chromosomal DNA sequences. The nucleic acid isolates corres-
pond to DNA sequences comprising part of tine Y-chromosomal DNA
of bovine mammals, and are referred to as SEQ TD N0:1; BtYl and
SEQ ID N0:3; BtY2, respectively.
It is well known in the nucleic acid hybridization probe art
that nucleic acids with different sequences may, under the same
conditions, hybridize detestably to the same
°°target°° nucleic
acid. Two nucleic acids hybridize detestably, under stringent
conditions over a sufficiently long hybridization period because
one comprises a segment of at least about 12 nucleotides in a
sequence complementary or nearly complementary to the sequence
of at least one segment of the target nucleic acid, The physical
basis for hybridization is base-pairing between these comple-
11
mentary or nearly complementary segments. If the time during
which hybridization is allowed to occur is held constant, at a
value which, under stringent conditions, two nucleic acids with
exactly complementary base-pairing segments hybridize detectably
to each other, increasing departures from exact complementarity
can be made into the base-pairing segments, but sufficient base
pairing will still occur to an extent to make hybridization
detectable, as the base-pairing segments of two nucleic acids
becomes larger and as the conditions of the hybridization become
less stringent. Moreover, segments outside of the probing seg-
meat of a probe nucleic acid may be altered significantly in
sequence without substantially diminishing the extent of hybrid-
ization between the probe and its target so long as the altera-
tion does not introduce a probing segment complementary or nearly
complementary to a target segment in a different target present
in samples to be probed. If segments outside the probing seg-
ment are changed substantially in length. the rate of hybridi-
zation may also be altered. 'the term "substantially the same
sequence" is used within the meaning of the present specifica-
tion to mean that two single stranded nucleic acid segments
(a) both form a base-paired duplex with the same segment, and
(b) the melting temperatures of said two duplexes in a solution
of 0.5 x SSPE differ by less than 10 degrees Celsius. mwo
double-stranded nucleic acid segments have "substantially the
same sequence" if either strand of one of the segments has
12
"substantially the same sequence" as one of the strands of the
other segment. Any labeling method known in the art would be
suitable in the practice of the present invention.
Application of a nucleic acid as a hybridization probe in
accordance to the invention may be made by the labeling of the
probe in order to facilitate detection. Preferably the nucleic
acids of the invention are detestably labeled with a non-
radioactive label such as biotin. Other non-radioactive labels
such as bromodeoxyuridine may also be used.
Radioactive isotopes may also be used for labeling the
nucleic acid probes of this invention. Preferably the probes
are labeled with 32P. However. other radioactive labels may
also be conveniently employed such as 3H, 14C. 35S, or 125I.
Labeling may be easily accomplished by nick-translating a sample
of the DNA for example, in the presence of one or more deoxy-
nueleoside-5-triphosphates which are labeled with the isotope.
An alternative to the use of non-radioactive or radio-
active labels is the chemical labeling of the nucleic acid of
this invention. Fox example. conventional nick-translation of the
nucleic acid in the presence of deoxyuridylate biotinylated at
the 5-position of the uracil moiety to replace thymidylate resi-
dues is suitable. The resulting labeled probe will include the
biotinylated uridylate in place of thymidylate residuals. The
resulting labeled probe will include biotinylated uridylate in
~3
place of thymidylate residues and is detectable via the biotin
moieties by any of a number of commercially available detection
systems utilizing the binding of streptavidin to the biotin.
See. for example, Singer and Ward, Proc. Natl. Acad. Sci. U.S.A.
79, 7331-7335 (1982). Detection systems are commercially avail-
able from, e.g., Bethesda Research Laboratories, Inc., Gaithers-
burg, MD., U.S.A. and Enzo Biochemicals, Inc., New York, NY, U.S.A.
The present invention also includes any contiguous portion
of 12 or more nucleotides or any and all of the sequences
illustrated in SEQ ID NOS: 1-4. Such nucleic acids may be used
as hybridization probes to detect any of the illustrated
sequences or similar sequences in bovine and non-bovine species.
Such nucleic acid sequences may also be constructed syntheti-
cally using commercially available DNA synthesizers, such as
the Applied Biosystems 380A DNA Synthesizer, obtained from
Applied Biosystems, Inc., Foster City, CA, U.S.A. Such nucleic
acid probes which comprise less than about 12 nucleotides have
limited usefulness as hybridization probes. Therefore the
preferred probes, according to the invention, are those nucleic
acid isolates comprising any contiguous portion of 12 or more
nucleotides of any and all of the sequences described herein.
Various embodiments include the use of recombinant DNA molecules
constructed from all or part of the shown sequences and include
a vector capable of propagation in host prokaryotic or eukarylotic
cells for the purpose of cloning, amplification and/or express-
14 ~~~~~~6
ion of the claimed sequences. Any number of a wide range of
vector molecules may be used depending on the intended use of
the nucleic acid. Examples of such vectors include molecules
such as pT218u or pTZl9u, and Bluescript (Stratagene), however,
vector molecules include both eukaryotic and prokaryotic vectors,
plasmids, phagemids, shuttle vectors, bacteriophage, and the like.
RNA corresponding to all or part of the sequences as
described may be produced using any method well known in the art
including but not limited to in vitro transcription systems,
utilizing for example, the RNA polymerases of bacteriophage.
Numerous commercially available polymerases are suitable such
as T3, T7 or SP6. See Melton, et al. Nuc. Acid. Res., 12,
7035-7056 (1984), and Taylor, et al. Biochem. Biophys. Acta
442, 324-330. Therefore, in various embodiments, the nucleic
acid isolates of the invention include both DNA and RNA seq -
uences which preferentially hybridize to Y-chromosome-specific
DNA and RNA sequences and hence are useful in the determina-
tion of the genetic sex of a mammal. ,
SEQ ID N0:1; BtYl is a 1.859 kb Pst I fragment cloned from
bovine DNA. The restriction fragment SEQ ID NO:le BtYI was
sub-cloned and subsequently shown by hybridization to Southern
blots of genomic DNA from individual male and female mammals
to be generally conserved, male-specific, arid repeated in
bovines and other ruminants. SEQ ID N0:2 shows double-stranded
RNA corresponding to the DNA sequence of SEQ ID N0:1; BtYl where
''~~~~~~
is
thymidine of the corresponding DNA in SEQ ID N0:1; BtYl is
replaced by uracil.
SEQ ID N0:3; BtY2 is a 3.71 kb SacI fragment cloned bovine
DNA. The SEQ ID N0:1; BtYl fragment was used to isolate SEQ ID
N0:3; BtY2 and is contained within the SEQ ID N0:3 BtY2 fragment.
The SacI restriction fragment of SEQ ID N0:3; BtY2 was sub-
cloned and subsequently shown by hybridization to Southern blots
of genomic DNA from individual male and female mammals to be
generally conserved. SEQ ID N0:9 shows double-stranded RNA
corresponding to the DNA sequence of SEQ ID N0:3; BtY2 where
thymidine of the corresponding DNA in SEQ ID N0:3 BtY2 is
replaced by uracil, and repeated in bovines.
The terms SEQ ID N0:1 BtYl and SEQ ID N0:3; BtY2 refer to.
where provided, the specific sequences set forth in SEQ ID
NOS:1 and 3. These terms also include closed circularized and
linearized SEQ ID N0:1; BtYl and SEQ ID N0:3; BtY2 and variants
where nucleotides have been substituted, added to, or deleted
from. the relevant sequences shown, so long as the variants
hybridize with all or part of any of the sequences SEQ ID
NOS:1-4. Such variants may be naturally occurring allelic
and/or eis variants which may arise within a population of
individuals by virtue of insertions, deletions, or point muta-
tions of DNA sequences, by recombination or by rearrangement.
Alternatively, such variants may be artificially produced, for
example, by deletion of fragments of DNA by exonuclease, by
16
site-directed mutagenesis, or by the addition of DNA sequences
by ligating portions of DNA together, or template-independent
or template-dependent DNA polymerase.
Making and using the nucleic acids of the invention axe
described in detail in the following examples.
The nucleic acid isolates, according to the preferred
embodiment, are used as hybridization probes to detect
Y-chromosome specific DNA and RNA sequences and therefore the
sex of, for example, embryo or fetal cells of bovine or other
ruminants. In a related application. the nucleic acid probes
of this invention may be used to detect variations in amounts
and/or variations in sequence of corresponding sequences in
individual mammals. Such applications are useful in, for
example. paternity testing of male offspring. Various types
of cells may be analyzed using the nucleic acid isolates and
methods described herein, a useful example being their applica-
tion to fractionated sperm where various fractions may be tested
for sperm bearing a Y-chromosome using the nucleic acid isolates
of the present invention.
In a preferred method of determining the sex of an embryo,
fetus. or the sex chromosome content of sperm or other cells using
the nucleic acid probes of the invention, a sample of cells is
removed for assay. DNA and/or RNA may be extracted therefrom
using known methods. See Maniatis, et al. (1980 Molecular
1~ ~~~~~2~
Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. The
isolated DNA and/or RNA may then be applied and fixed directly
to a membrane such as nitrocellulose, or a derivative thereof.
Alternatively, the DNA and/or RNA may be electrophoresed through
a gel matrix and then transferred and fixed to a similar mem-
brine.
The nucleic acids so bound to the membrane are then hybrid-
ized with any or all of the nucleic acid isolates of the inven-
tion which are labeled with a detectable marker as previously
described. The labeled nucleic acid isolate which binds to
nucleic acid on the membrane is detected by conventional tech-
niques well known in the art, for example. by autoradiography.
If the labeled isolate hybridizes to similar sequences in the
target sample, sex can be conclusively designated as male.
Using this method, amplified target DNA may be used. See Saiki,
et al., Science 230. 1350-1354 (1986), and Saiki, et al., Nature
324, 163-166 (1986).
An alternative method using the nucleic acid probes of the
invention utilizes nucleic acids which are not extracted from
the sample of cells removed for assay. Such cells are heated
in alkaline solution and the resultant solution is filtered onto
a charge-modified nylon membrane such as a Zeta-Probe membrane
(trademark of Bio-Rad). DNA fixed to the membrane is hybrid-
ized with nucleic acid isolates) of the invention as described
in the method described above.
~~l=~~~~
18
Another method, according to an embodiment of the inven-
Lion, is useful for the determination of the sex chromosome
constitution of a tissue or cell sample comprising, isolating
DNA from the tissue or cell sample, immobilizing the isolated
DNA onto a support matrix, hybridizing the immobilized DNA with
a nucleic acid isolate of this invention, washing the unbound
nucleic acid isolate from the support matrix, and then detecting
the binding of the nucleic acid isolate to the bound DNA by con-
ventional methods. If determining the Y-chromosome presence
or absence in interphase or metaphase chromosome, or in fixed
cells, a preferred methodology comprises hybridizing chromosome
spreads of such cells with the Y-chromosome specific nucleic
acid isolates of the invention under conditions enabling the
nucleic acid isolate to bind to complementary DNA sequences.
Unbound nucleic acid is washed away, and detecting binding of
the nucleic acid isolates using conventional techniques of in
situ hybridization, such as those described in Saiki, et al.,
Science 230. 324, 163-166 (1986), may be applied.
Conventional methods useful for amplifying the levels of
target DNA may also be utilized in combination with the nucleic
acid probes of the invention. For example, DNA isolated from
the tissue or cell sample is denatured to separate the respec-
tive coding and non-coding strands, annealing the denatured DNA
with a synthetic polynucleotide corresponding to 12 or more
19 ~~~'i~~~~
nucleotides from any of the nucleic acid probe sequences of the
invention. incubating the annealed DNA with DNA polymerase to
to extend the polynucleotide through the sequences, and repeat-
ing this sequence as many times as desired to amplify levels
of target DNA. Subsequent detection of the target DNA i,n the
amplified sample may be made by any number of conventional
methods well known in the art. For example, immobilizing the
DNA onto a support matrix, hybridizing the immobilized DNA with
a nucleic acid of the invention under conditions permitting the
labeled nucleic acid probe to bind to complementary sequences,
washing unbound probe from the support matrix, and then detect-
ing binding of the nucleic acid probe to the bound DNA.
Alternatively, if labeled nucleotide precursors are used in
the incubation with the DNA polymerase, the sample may be frac-
tionated by electrophoresis in a gel matrix with subsequent
detection of the labeled target DNA sequences. Hybridizations
are carried out under standard conditions well known in the
art. See Maniatis et al., (1982), Molecular Cloning, A Labora-
tory Manual. Cold Spring Harbor Laboratory.
Amplification of target DNA using polymerase chain reaction
(PCR) prior to hybridization with the nucleic acid probes of
the invention is another method useful in determining the sex of
an embryo. fetus, or the like. Polynucleotide primers from a
target sequence are used to amplify the DNA sequence occurring
between the primer sequence of the target DNA. The amplified
CA 02026926 1999-10-19
target sequences may be detected following their fixation to a
membrane and analyzed using conventional hybridization techniques
as previously described. The amplified target sequences may also
be visualized using electrophoresis where the sequence is immobil-
ized and stained in the gel matrix using standard stains such as
silver reagent or ethidium bromide and subsequent visualization
under ultraviolet light.
The nucleic acid isolates of the invention may be further
used to comprise or form part of a kit for detecting the
presence or absence of Y-chromosome specific sequences in a
wide variety of tissue or cell samples. The nucleic acid
isolates may be labeled with a wide variety of labels including
radioactive or non-radioactive markers. The kits may also com-
prise a well known number of suitable components including but
not limited to buffers for diluting reagents. labeled compounds,
solid support for assays, and the like.
EXPERIMENTAL EXAMPLES
Preparation of Genomic DNA
Blood samples (FML) were collected by accepted veterinary
procedures in sterile containers (Vacutainer Becton-Dickinson)
containing 0.07 ml of 15% potassium EDTA and transferred into
sterile 50 ml culture tubes. Red blood cells were lysed by the
addition of 35 ml of 17 mM Tris HC1. pH 7.65, 140 mM ammonium
chloride solution (prewarmed to 37 deg.C) and incubated at
CA 02026926 1999-10-19
21
37 deg.C for 10 minutes. Samples were centrifuged at 2,000 rpm
in the swinging bucket rotor of an IEC Centra-8R centrifuge at
4 deg.C for 10 minutes. The pellet. consisting mainly of nuc-
leated white cells. was resuspended in saline (0.85% NaCl in
sterile H20) and spun as above. Saline supernatant was
removed and the pellet was resuspended in 2 ml of 100 mM Tris HC1,
pH 8Ø 40 mM sodium EDTA. An equal volume of lysis mixture
(100 mM Tris HC1. pH 8.0, 40 mM EDTA and 0.2% SDS) was added and
the sample was chilled overnight at 4 deg.C. Four ml of 10 mM
Tris HC1. pH 8Ø to which Proteinase K (Boehringer Mannheim)
was added to a concentration of 1 mg per ml was added to the lysed
cell suspension and incubated at 65 deg.C for 2 hours with inter-
mittent mixing. Standard phenol chloroform isoamyl alcohol (PCI)
extraction was performed as follows: Redistilled phenol (BRL)
was saturated with Tris HC1, pH 8Ø until the pH of the phenol
was raised above pH 7.5. Hydroxyquinoline was added to 0.1%.
Phenol was added to an equal volume of chloroform; isoamyl
alcohol (24:1). The mixture (PCI) was added, in equal volume,
to the sample to be extracted: Genomic DNA: PCI mixtures were
mixed by slow rotation (50 rpm on a commercial rotating device)
for 20 minutes. All other DNA's were mixed with PCI by vigorous
vortexing. Samples were spun at 10.000 rpm in a HB4 rotor of
a Sorvall centrifuge for 10-20 minutes at room temperature.
The upper. aqueous phase containing DNA was removed with a wide-
bore pipette and reextracted sequentially as above until no
interphase was detectable between the aqueous DNA phase and
the organic PCI phase. DNA was precipitated by the addition of
22
1/lOth volume of 3M sodium acetate (pH 7.0) and 2-2.5 volumes
95% ethanol. Genomic DNA was pelleted by centrifugation at
5,000 rpm in an HB4 rotor at 4 deg. (all other DNA's were
pelleted at 10,000 rpm). Ethanol was removed and the DNA was
redissolved in 1 ml sterile H20 and reprecipitated as above.
These pellets were washed two times with 70% ethanol, dried in
vacuo and dissolved in sterile H20. DNA concentration was
determined by measuring the O.D. 260 in a spectrophotometer.
Genomic DNA was stored at 4 deg.C until use (all other DNA's
were stored frozen -20 deg.C).
Genomic DNA (50-100 ug) was digested with a four-fold
excess (i.e. 4U enzyme/ug DNA) of either PstI or SacI restric-
tion endonucleases (New England Biolabs), in buffers supplied
by the manufacturer, for 4 hours at 37 deg.C. Disgested DNA
was purified by standard PCI extraction and ethanol precipita-
tion. DNA was dissolved in sterile H20 at a concentration of
0.5 mg/ml.
Electrophoresis
Genomic DNA was electrophoresed in agarose gels (0.8-1.2%
agarose, Bio Rad Molecular biology-grade) using the Bio Rad
Horizontal DNA Sub Cell. Tank buffers were 40 mM Tris acetate
pH 8.0, 2 mM EDTA and 0.5 ug/ml ethidium biomide. DNA was
visualized by UV transillumination. Size-cuts of digested
genomic DNA were isolated using commercially available molecular
CA 02026926 1999-10-19
23
weight markers as a guide. Slits across the gel lane were intro-
duced. into which DEAE cellulose paper (NA45, Schleicher and
Schuell) was placed. Electrophoresis was continued until all
the fluorescence had absorbed to the paper. By sticking paper
strips at intervals up the gel lane, discreet size fractions
were isolated. A similar method was used to isolate DNA frag-
ments from digested clones. The paper strips were washed 2x by
vortexing in T.E. DNA was eluted by heating strips in TE
supplemented to 1M with respect to NaCl and heating for 20 minutes
at 65 deg.C. The buffer, containing the DNA of interest, was PCI
extracted by standard procedures and ethanol precipitated. DNA
was redissolved in sterile H20 and stored at -20 deg.C until
use.
Preparation of Vector DNA
Plasmid DNA's were prepared by standard procedures using
alkaline lysis and cesium chloride gradient centrifugation (see
Maniatis). All fragments were subcloned into either Bluescript~+
KS or Bluescript~ + SK vectors (Stratagene). Vector DNA (5 ug)
was digested with a four-fold excess (20 units) of appropriate
restriction endonuclease. DNA was PCI extracted by standard
procedures and precipitated with ethanol. DNA was resuspended
in 200 ul 10 mM Tris, pH 8.3, 5 mM MgCl and 0.1 mM ZnCl2
heated to 75 deg. C and quick cooled on ice. Calf intestinal alka
line phophatase (CIAP-Boehringer Mannheim-molecular biology grade)
f
24 C ~?
was added at 0.5 U/ug of DNA and incubated at 42 deg.C for 30 min-
utes, 55 deg.C for 30 minutes and heated to 75 deg.C .for 10 min-
utes before cooling on ice. An additional 2U of phosphatase was
added and incubated as above. SDS was added to 0.1~ and the
mixture was extracted repeatedly with PCI, by standard procedures,
and ethanol precipitated. DNA was dissolved at a concentration
of 0.5 ug/ul in sterile H20, heated to 75 deg.C for 15 minutes
and allowed to cool to room temperature before storage at -20
deg.C prior to use.
Lidation of Inserts to Vector DNA
Recombinant DNA molecules were prepared as follows. Vector
and insert DNA were mixed at a 3:1 molar ratio. For a ZO ul
ligation reaction, 2 ul of a solution containing 25 mM Tris HC1
(pH 7.6), 50 mM MgCl2, 5 mM DTT and 259 polyethylene glycol
8000 was added. One microlitre each of 10 mM ATP and T4 DNA
ligase (6-10 units) were added, the total volume was adjusted
to 10 ul with sterile H20 and the mixture was incubated at
12 deg.C for a minimum of 8 hours. Ligation of blunt ended mole-
rules (typically for subcloning fragments) was performed using
1/lOth the above concentration of ATP. double the amount of T4
DNA ligase and incubation at room temperature overnigh~. TE
(90 ul) was added to ligation reactions, which were then purified
by standard PCI extraction and ethanol precipitation. DNA was
dissolved in 10-20 ul sterile H20 and stored at 4 deg.C until
its use for transformation of competent cells.
CA 02026926 1999-10-19
Preparation of Competent Bacterial Cells and DNA Transformation
Single colonies of Escherichia coli (strain JM 109) grown
on M9 plates were inoculated into LB broth and grown overnight
with shaking at 37 deg. C. Overnight cultures were diluted 1:100
with fresh LB broth. Cells were grown at 37 deg.C with vigorous
agitation to an O.D. 600 of 0.5. Cells were chilled on ice 10
minutes and centrifuged at 4,000 x g for 10 minutes at 4 deg.C.
The supernatant was aspirated off and cells were resuspended in
the original volume with water followed by centrifugation as
above. The process was again repeated with one half the original
volume of H20. Cells were then resuspended in 1/50th the
original volume of 10% glycerol and spun, as described above.
Cells were resuspended in 1/500th the original volume of 10%
glycerol (greater than 5x1010 cells/ml) and either used directly
for transformation with cloned genomic DNA or frozen for further
subcloning of cloned DNA fragments. Transformation of bacterial
cells with bovine genomic DNA/vector constructs was performed by
high voltage electroporation using a Gene Pulser~apparatus (Bio
Rad). DNA (up to 500 ng in a volume of 2 ul or less was mixed with
40 ul competent cells on ice for 1 minute, added to a pre-chilled
(4 deg.C) electroporation cuvette and pulsed according to manu-
facturers settings. Immediately after pulsing, 1 ml SOC medium,
pre-warmed to 37 deg.C, was added to cells which were transferred
to 15 mi polypropylene culture tubes and shaken at 37 deg.C for
1 hour. Cells were pelleted at 4,000 x g for 10 minutes at room
temperature, resuspended in 1 ml of LB amp and grown for 1 hour
CA 02026926 1999-10-19
26
(approximately 3 cell doubling times), mixed with 0.4 ml of 100%
glycerol, frozen in a dry ice ethanol bath and stored at -70 deg.C.
Aliquots were then filtered for accurate CFU determination before
plating for colonly hybridization.
Colony Hybridization
Transformed bacteria (3,000-5,000 cfu) were suspended in
2 ml of LB amp which was plated on LB amp plates (185 mm) and
incubated at 37 deg.C until colonies were 0.5-1.5 mm in diameter.
Plates were chilled at 4 deg.C for one hour prior to being over-
lain with 182 mM Nylon Filters (Hybond N+ Amersham). Needle
pricks were used to mark alignment of plates and filters. Filters
were placed of 1.5M NaCl and 0.5M NaOH soaked filter paper for
7 minutes to denature bacterial DNA and transferred to filter
paper soaked in 1.5M NaCl, 0.5M Tri~s HC1 (pH 7.2) and 1 mM EDTA
for 3 minutes. The final step was repeated one time, after which
filters Were rinsed briefly in 2 x SSPE and dried at 80 deg.C for
1 hour in an oven.
Southern Blotting of DNA and Filter Hybridization
Southern blots of agarose gels were prepared by the capillary
method (see Maniatis et.al. 1982) to nylon membranes (Hybond N+
Amerham) using NaOH (0.4M) as the transfer buffer by the proced-
ure recommended by the manufacturer. Filters (either blotted DNA
or circles containing colony lifts) were pre-incubated in a solu-
tion containing 50% deionized formamide, 4 x SSPE, 5'x Denhards
solution. 0.5% SDS and 500 ug/ml yeast RNA for 1-4 hours at 42
CA 02026926 1999-10-19
27
deg.C. Probe was added at a concentration not exceeding 10 ng/ml
of prehybrid solution. Filters were incubated for 12-18 hours at
42 deg.C. Filters were washed once with 2 x SSPE, 0.1% SDS at
room temperature for 30 minutes, twice for 15 minutes each with
1 x SSPE, 0.1% SDS at 65 deg.C and finally once for 10 minutes
with 0.1 x SSPE, 0.1% SDS at 65 deg.C. This latter step is a high
stringency wash and was not always performed. Filters were dried
and exposed to Kodak XAR X-ray film with a "Lightening Plus
intensifying screen (Kodak) at -70 deg.C for varying amount of
time.
Preparation of Radiolabeled Probes
Radiolabeled probes were prepared as originally described
by Hodgson and Fisk (1987, NAR pg. 6295) without modification.
Probes were purified by Sephadex~G-75 chromotography (Pharmacia)
and denatured at 100 deg.C for 10 minutes prior to use in hybrid-
izations.
SubcioninQ of Recombinant DNA's
To facilitate DNA sequence analysis. recombinant clones were
thoroughly mapped with a number-of restriction endonucleases.
Fragments to be subcloned were isolated on DEAE cellulose paper
as described above. Vector DNA was prepared with the appropriate
restriction endonucleases and treated with CIAP as described
above. Fragments were inserted into both Bluescript + SK and KS
to facilitate sequencing from either end. Fragments whose ends
were not compatible with restriction sites in the vector poly-
CA 02026926 1999-10-19
28
linker region were converted to blunt ended fragments by treat-
ment with the Klenow fragment of E coli DNA polymerase I (see
Maniatis for method). These fragments were then cloned into
Bluescript KS digested with either Sma I or Eco RV whose restrict-
ion sites were also inside in the polylinker.
DNA Seguence Determination
Single colonies picked from M9 ampicillin plates (150 ug/ml)
were used to inoculate 2 ml of LB/ampicillin media and grown over-
night at 37 deg.C. Ten ul was then inoculated into 1 ml LB amp.
shaken for 4 hours at 37 deg.C before addition of 2 ul of helper-
phage strain M13K07 and continued shaking for one hour. The
culture was transferred to a disposable 50 ml culture tube contain-
ing 10 ml LB amp/Kanamycin (70 ug/ml) and shaken vigorously at
37 deg.C for 12-18 hours. Following centrifugation at 10.000 rpm
for 15 minutes, 8 mi of supernatant were added to 1.4 ml PEG/NaCl
and incubated for a minimum of one hour on ice. Phage were
pelleted for 20 minutes at 10,000 x g and resuspended in 400 ul
TE. DNA was isolated by standard PCI extraction and ethanol pre-
cipitation (procedure described above). DNA was dissolved in
50 ul sterile H20. Nucleotide sequence determination was per-
formed using a commercially available T7 DNA polymerase based
dideoxynucleotide system (Sequenase 2Ø United States Biochem-
icals) according to manufacturer's instructions. Nucleotide
sequence ladders were resolved by polyacrylamide gel electrophor-
esis (see Maniatis). DNA sequences were assembled using the Micro-
CA 02026926 1999-10-19
29
m
genie sequence analysis package (Beckman). Data base searches
were performed with the Genbank on line service (Intelligenetics
Inc., Stanford) using the 'Fasts' sequence analysis program.
Although the foregoing invention has been described in
some detail by way of illustration and example for purposes of
clarity and understanding, it will be obvious that many changes
and modifications may be practiced within the scope of the
appended claims.
~~a~i~~
SEQUENCE LISTING
SEQ ID N0:1; BtYt shows the double stranded DNA sequence of
BtYl; and in this illustration, C refers to deoxycytidine-5'-
phosphate; G refers to deoxyguanosine-5'-phosphate; A refers to
deoxyadenosine-5'-phosphate; and T refers to deoxythymidine-5'-
phosphate.
SEQ ID N0:2 shows the double-stranded RNA sequence correspond-
ing to the DNA sequence of SEQ ID N0:1; BtYl. In this illustra-
tion, C refers to cytidine-5'-phosphate; G refers to guanosine-5'-
phosphate; A refers to adenosine-5'-phosphate; and U refers to
uridine-5'-phosphate.
SEQ ID N0:3; BtY2 shows the double-stranded DNA sequence of
BtY2; and in this illustration C,G,A, and T are the same as in
SEQ ID N0:1; BtYl.
SEQ ID N0:4 shows the double-stranded DNA sequence correspond-
ing to the DNA sequence of SEQ ID N0:3; BtY2 where C,G,A, and U
are the same as in SEQ ID N0:2.
SEG1 TD No ~ l
C~TGCAGAGCT TCAGGCAGGG TTGGAAATGC TCGCCCTGGA CAGCTGAATG AGTTCTGCCT 60
GCATTCTATA TTCTCCCATT ACCTTGGACA GCTTCACAGT ACCAGTCACA CTGGCCTGAT 120
CCr~I'TGCCTG TGCATTCTCT CAGGGGACCA GAAAACAAGG ACGTCTGGGC TCAGCTGACT 180
TGGAGAACTG CTTTCTCAGT GTGCCCCTTC TAAGTCATTC CTGGTCAAAA CTGTGTCCCT 240
ATTGCTAGCC TACCACATCA GCATTCTGAG TGAGGTCCCC TGTTCTTTCT ACCTGTGTAG 300
TTTTCTGTGT GCACCTGTCT ACCTGTGCCT CCAAGCACTA TCTCCCTTTA GCAGGAAAAG 360
ACCTGTGCCT CCAAGCACTA TCTCCCTTTA GCAGGAAAAG GCCAAAGAGA TGCCTGAGCC 420
TCCAAGGGCC CCCAGAGTCT GTGAGAGACC TGGGTGTGAT CCAATGTTGT GAAGAAGGTG 480
CCCATAGATA GAGGGTCTCT TCTGAAACAA GGCATGAAGC CCGAGACCAT AATGGTAAGG 540
TGGCATTCCT ACAGGTGGTC CCTTCTGTTT ATTCCTACCC AGACCCATGG AGTCCCCAAA 600
CAGATGATGA TCTGGGAATC CTGCCCTTTC TGGGCCCACA GCTCATGCCT CCCTTGGACA 660
GAAAGCAGCT TTTCTATCTC AAAAACACCA AGAGGGCTTG ATTCCACCCA GGCCTCATTG 720
ATTTGCTAAA TCAAATACTC TCTTTCATTG GGTTCATTAA GCCCAGGTAG GACTCCCTGG 780
AGTCAGGCAT CCCTGCTTAC CTACACAGCC CACGTGCCAA GTTAGCCAGT CCTTGGTTGG 840
CCACAGGGGC ATCCAAGACT GTCACCTGGA ATGCAGCTTC CTTCTGAGTG TCAGCTGGTG 900
CAGATCCCCT ACGACAAAAT CAGAGATTAT GCTCCAGAGA AACTGCCAAA ATCCTCCCCC 960
AGGTGCAAAC ACACACCTTT GCCCTCAGGT CCCCAAAGCC AGGGGAAAGA CCCAGAGAAA 1020
AGAAGGAATT TATATCAGGA CTTTCAGCAC AAGCCATGGG GTATCTTTGG CAGGAGCGTT 1080
ATTGCCTTTC CCCTGGACCC TGAAAACCAG CAGGCCCTAA ACTGCACCCA GGGGCTTCCC 1140
TGTCTCCCAC TCTCATGAGG TCCTTCAGAC ACGCAATAAG CCCATCATCC TTGCTTCCTC 1200
CCTGTTCCCT CCCTTATAGG CACACCTCGG CAGAAGAGCA CACACGTAAA ACACCTGCAC 1260
TTTCTACGCC TTTCTGCACT GCCAGGGAGA CTGGAAGTGC CTGGAGGCAT GCCACACTCA 1320
CATCTTGTCT CTCCTAGGAT GCCTGTGGTT TTGCACGACA GCCTACCTTA GCATGTCTCG 1380
CATTTTGTGT CACATCGTTC CAGTGTGTGA AACCCTCATG GAGAGAGGGT GCTGGCTGAT 1440
GGGCTGATCC TGGGAAGCAC TGGCCCAGGA CCTTCCCAGG TCTCCTTCTC ACATGTGTAG 1500
AGCAAGTCTC CAGTACACAA GTCAATCTGT GCCTCTTTCT CTTCGGGTCT CTGTCCTTCT 1560
CAGCAAGACC TTAGCCTCCT CACCCATCCC AGGTCCTCTG TATCCACATC CACCATTTCC 1620
GCCTGCCAGC CCATGTCCCC ACAGGCTGTG GGCTCCACAG GCGGTGGTTT TAAAGCCTCA 1680
CTCCACCTGA TTTGCCCTGG GTGAATCCAC AGACCATGCA CTCACTCTTC CTGGTCCAAA 1740
CACATACAAG AACACGGTAG AAATGGTGAG TGTGTTTTTG TATTTCATCT CATGGCAGAT 1800.
TTCTGAAGCC AAGGTCCTGA GTTATCAGTG GCCATCCTTT CCTCATTCCC ATCCTGGACA 1860
GGGTCACTGC AG 1872
~EI~IU ~~l4;at 32 ~~~~(~~~
CUGCAGAGCU UCAGGCAGGG UUGGAAAUGC UCGCCCUGGA CAGCUGAAUG AGUUCUGCCU 60
GCP.~)UCUAUA UUCUCCCAUU ACCUUGGACA GCUUCACAGU ACCAGUCACA CUGGCCUGAU 120
CCAUUGCCUG UGCAUUCUCU CAGGGGACCA GAAAACAAGG ACGUCUGGGC UCAGCUGACU 180
UGGAGAACUG CUUUCUCAGU GUGCCCCUUC UAAGUCAUUC CUGGUCAAAA CUGUGUCCCU 240
AUUGCUAGCC UACCACAUCA GCAUUCUGAG UGAGGUCCCC UGUUCUUUCU ACCUGUGUAG 300
UUUUCUGUGU GCACCUGUCU ACCUGUGCCU CCAAGCACUA UCUCCCUUUA GCAGGAAAAG 360
ACCUGUGCCU CCAAGCACUA UCUCCCUUUA GCAGGAAAAG GCCAAAGAGA UGCCUGAGCC 420
UCCAAGGGCC CCCAGAGUCU GUGAGAGACC UGGGUGUGAU CCAAUGUUGU GAAGAAGGUG 480
CCCAUAGAUA GAGGGUCUCU UCUGAAACAA GGCAUGAAGC CCGAGACCAU AAUGGUAAGG 540
UGGCAUUCCU ACAGGUGGUC CCUUCUGUUU AUUCCUACCC AGACCCAUGG AGUCCCCAAA 600
CAGAUGAUGA UCUGGGAAUC CUGCCCUUUC UGGGCCCACA GCUCAUGCCU CCCUUGGACA 660
GAAAGCAGCU UUUCUAUCUC AAAAACACCA AGAGGGCUUG AUUCCACCCA GGCCUCAUUG 720
AUUUGCUAAA UCAAAUACUC UCUUUCAUUG GGUUCAUUAA GCCCAGGUAG GACUCCCUGG 780
AGUCAGGCAU CCCUGCUUAC CUACACAGCC CACGUGCCAA GUUAGCCAGU CCUUGGUUGG 840
CCACAGGGGC AUCCAAGACU GUCACCUGGA AUGCAGCUUC CUUCUGAGUG UCAGCUGGUG 900,
CAGAUCCCCU ACGACAAAAU CAGAGAUUAU GCUCCAGAGA AACUGCCAAA AUCCUCCCCC 960
AGGUGCAAAC ACACACCUUU GCCCUCAGGU CCCCAAAGCC AGGGGAAAGA CCCAGAGAAA 1020
AGAAGGAAUU UAUAUCAGGA CUUUCAGCAC AAGCCAUGGG GUAUCUUUGG CAGGAGCGUU 1080
AUUGCCUUUC CCCUGGACCC UGAAAACCAG CAGGCCCUAA ACUGCACCCA GGGGCUUCCC 1140
UGUCUCCCAC UCUCAUGAGG UCCUUCAGAC ACGCAAUAAG CCCAUCAUCC UUGCUUCCUC 1200
CCUGUUCCCU CCCUUAUAGG CACACCUCGG CAGAAGAGCA CACACGUAAA ACACCUGCAC 1260
UUUCUACGCC UUUCUGCACU GCCAGGGAGA CUGGAAGUGC CUGGAGGCAU GCCACACUCA 1320
CAUCUUGUCU CUCCUAGGAU GCCUGUGGUU UUGCACGACA GCCUACCUUA GCAUGUCUCG 1380
CAUUUUGUGU CACAUCGUUC CAGUGUGUGA AACCCUCAUG GAGAGAGGGU GCUGGCUGAU 1440
GGGCUGAUCC UGGGAAGCAC UGGCCCAGGA CCUUCCCAGG UCUCCUUCUC ACAUGUGUAG 1500
AGCAAGUCUC CAGUACACAA GUCAAUCUGU GCCUCUUUCU CUUCGGGUCU CUGUCCUUCU 1560
CAGCAAGACC UUAGCCUCCU CACCCAUCCC AGGUCCUCUG UAUCCACAUC CACCAUUUCC 1620
GCCUGCCAGC CCAUGUCCCC ACAGGCUGUG GGCUCCACAG GCGGUGGUUU UAAAGCCUCA 1680
CUCCACCUGA UUUGCCCUGG GUGAAUCCAC AGACCAUGCA CUCACUCUUC CUGGUCCAAA 1740
CACAUACAAG AACACGGUAG AAAUGGUGAG UGUGUUUUUG UAUUUCAUCU CAUGGCAGAU 1800
UUCUGAAGCC AAGGUCCUGA GUUAUCAGUG GCCAUCCUUU CCUCAUUCCC AUCCUGGACA 1860
GGGUCACUGC AG 1872
,spa zo nra:.~ ; ~* r~,
GAGCTCGCTC TGTGTCTCTT TATCTCTGCT TCTGGCATAG CACTGTTTTG GGCTATCCTT 60
CTfTGTGTGT ACCAGGGCTG GTGTCTATGT AGTTCCATCT CTTTAAGTGA TGCTGTTACC 120
CTTTGCCACT GTCTGGACAC CAGCACTCAT ACGAGAAGCT TATCCTTGGC ATGAAGGCAA 180
GCCCTTCTCC TCCTGAGTGA GTTTCACTAA CGGGAGATCA GACTCTTTTT TAATTTAAAT 240
TTATTTATTT TAAGTAGAGG CTAATTTACA ATATTATATT GGTTTTGCCA TACATCAACA 300
TGAGTCCACC ACGGGTGTAC AAAATCAGAC ATTTTTAGTC CACACTTTCA GACAGTACTT 360
TCTCAAACTT GAAAGCCAAC AGTGGGCGGT CACTGCTGAA CCTCAGAAGG GGCCGGTTTA 420
TCCTCCCTCC CTCACTGGAC AGATGTGAAC ACTGCACAGG TCTGCAGCGT CTTGGCCACA 480
CTTTGCACAG AGGGAGAAAT TGGGGCATGC TCTGCTGGCA TGAGGAAACT CCCTGAACCT 540
TGTTCAAATG CCTACCAGTG AGATGCTAAG GACAACTCCC TGTTAAGTTC CAGGACTTCC 600
TGGTGCCCGA GACATGCACG TCTGCCCATT TACCCTACCA AGGTCCTTTC AAAATGGTTC 660
TGTTCTCCAT GTAAGAACAC GTACCAGCCT GCCCAATAGG CCAATCCTGT GGGGCCAGGA 720
GCAGCAAGAG GATCAAGCTA ATCCATCCAT CCTATAGTCC TTACTCCCGA TATATGCCTT 780
CTTCAAAGAG TACAAAGAGT ATTTTTCAAA CTGGAAGACT ATGCAGGTAG CAGGTGCTTA 840
TGCTGCAGTG CTGGTCCGTT TCTGAAACCT CAAAAAAAGG CAAGGGGTGC TCATGCTCAT 900
GGCAAGGACA GGGAAAGAAA GCCCAGTGTT CTAGAAGGAT AGAACNCCCT GGTAGCTGCA 960
TGTCCAAGGG GCTGTGGGGC CACCCACTAT GACCTCTGTG TATTGGAATT GCAGCCTGTT 1020
CTGTGTCTCG GATCCTCTGC ACTCTCATTG GTCACCCCCA GGCACTTTCT TCCTGCCTCT 1080
CCTTTCTGCC AGGCATCCTG GGTGCACCAC TGTGATTCCA CTTAGAAGTT GCTCACTCAG 1140
TGAGATAACA GGGATTGGCA ACCCGGCTCC AGTGCTTCTG ATGGCCAGAG CATGTACCTT 1200
TCCTATGGCT TTTATGTTTT CCCTATTCAA CTTCTATCAC CTGGTTAGGT CAGTTTCTAC 1260
ACCTCATACT CACAAGCATA CTATCAGGCG CTTTTCATGC ATATATGCAC ACACATGCGT 1320
GTGTACACAT TAACATCCTG AGAGGAAACT TGCACATATA CAGACATACA AACTTTCTTC 1380
TCCAGGAACA TCAATTTTGG TAAGCACCTG ACTTTCTTTG TCCTGATTAT TTTCTTTCAC 1440
TTTCTCATCG ATCCTGTCAG GTTACACTTC TAACCCTTTG ACTTAGCCTC AAAGGTCACA 1500
AAATTTTGGC ATTTGCTCCT GACAAGGACC GATCTGCAGA GCTTCAGGCA GGGTTGGAAA 1560
TGCTCGCCCT GGACAGCTGA ATGAGTTCTG CCTGCATTCT ATATTCTCCC ATTACCTTGG 1620
ACAGCTTCAC AGTACCAGTC ACACTGGCCT GATCCATTGC CTGTGCATTC TCTCAGGGGA 1680
CCAGAAAACA AGGACGTCTG GGCTCAGCTG ACTTGGAGAA CTGCTTTCTC AGTGTGCCCC 1740
TTCTAAGTCA TTCCTGGTCA AAACTGTGTC CCTATTGCTA GCCTACCACA TCAGCATTCT 1800
GAGTGAGGTC CCCTGTTCTT TCTACCTGTG TAGTTTTCTG TGTGCACCTG TCTACCTGTG 1860
CCTCCAAGCA CTATCTCCCT TTAGCAGGAA AAGACCTGTG CCTCCAAGCA CTATCTCCCT 1920
SEQ ID N0:3; 8tY2 34
TTAGCAGGAA AAGGCCAAAG AGATGCCTGA GCCTCCAAGG GCCCCCAGAG TCTGTGAGAG 1980
AC. :GGTGT GATCCAATGT TGTGAAGAAG GTGCCCATAG ATAGAGGGTC TCTTCTGAAA 2040
CAAGGCATGA AGCCCGAGAC CATAATGGTA AGGTGGCATT CCTACAGGTG GTCCCTTCTG 2100
TTTATTCCTA CCCAGACCCA TGGAGTCCCC AAACAGATGA TGATCTGGGA ATCCTGCCCT 2160
TTCTGGGCCC ACAGCTCATG CCTCCCTTGG ACAGAAAGCA GCTTTTCTAT CTCAAAAACA 2220
CCAAGAGGGC TTGATTCCAC CCAGGCCTCA TTGATTTGCT AAATCAAATA CTCTCTTTCA 2280
TTGGGTTCAT TAAGCCCAGG TAGGACTCCC TGGAGTCAGG CATCCCTGCT TACCTACACA 2340
GCCCACGTGC CAAGTTAGCC AGTCCTTGGT TGGCCACAGG GGCATCCAAG ACTGTCACCT 2400
GGAATGCAGC TTCCTTCTGA GTGTCAGCTG GTGCAGATCC CCTACGACAA AATCAGAGAT 2460
TATGCTCCAG AGAAACTGCC AAAATCCTCC CCCAGGTGCA AACACACACC TTTGCCCTCA 2520
GGTCCCCAAA GCCAGGGGAA AGACCCAGAG AAAAGAAGGA ATTTATATCA GGACTTTCAG 2580
i
CACAAGCCAT GGGGTATCTT TGGCAGGAGC GTTATTGCCT TTCCCCTGGA CCCTGAAAAC 2640
CAGCAGGCCC TAAACTGCAC CCAGGGGCTT CCCTGTCTCC CACTCTCATG AGGTCCTTCA 2700
GACACGCAAT AAGCCCATCA TCCTTGCTTC CTCCCTGTTC CCTCCCTTAT AGGCACACCT 2760
CGGCAGAAGA GCACACACGT AAAACACCTG CACTTTCTAC GCCTTTCTGC ACTGCCAGGG 2820
AGACTGGAAG TGCCTGGAGG CATGCCACAC TCACATCTTG TCTCTCCTAG GATGCCTGTG 2880
GTTTTGCACG ACAGCCTACC TTAGCATGTC TCGCATTTTG TGTCACATCG TTCCAGTGTG 2940
TGAAACCCTC ATGGAGAGAG GGTGCTGGCT GATGGGCTGA TCCTGGGAAG CACTGGCCCA 3000
GGACCTTCCC AGGTCTCCTT CTCACATGTG TAGAGCAAGT CTCCAGTACA CAAGTCAATC 3060
TGTGCCTCTT TCTCTTCGGG TCTCTGTCCT TCTCAGCAAG ACCTTAGCCT CCTCACCCAT 312PJ
CCCAGGTCCT CTGTATCCAC ATCCACCATT TCCGCCTGCC AGCCCATGTC CCCACAGG~T 318f~J'
GTGGGCTCCA CAGGCGGTGG TTTTAAAGCC TCACTCCACC TGATTTGCCC TGGGTGAATC
3240;°
CACAGACCAT GCACTCACTC TTCCTGGTCC AAACACATAC AAGAACACGG TAGAAATGGT 3300
GAGTGTGTTT TTGTATTTCA TCTCATGGCA GATTTCTGAA GCCAAGGTCC TGAGTTATCA 3360
GTGGCCATCC TTTCCTCATT CCCATCCTGG ACAGGGTCAC TGCAGAGATA GGGCGACCAA 3420
CCACCCTCAA ACTGGGGGTG CCTTGTGTCT CGTCTTCTGA TTGCTTGGCA TTTCCTCTCC 3480
TGTAGCCTTT TTCTCTGATA TTTCCCTGGG CCACACACAC ACACACACAC ACACACACAC 3540
ACACACGCAC GCAAACACAG GTGACACAAG CACACACGGT ATACACACAC AGGCTCGATA 3600
CAGGGACACA AACACAAACA GGGAACACAG GTGTTTCAGG AGCTGAAGAC GCCCATGTGT 3660
CCAGCAGTAT CAGAGATGGC ATCAGTAGGC ACCACGTCCT GCACTTGGAG CTC 3713
S~G2 r D ~Yo= ~
GAGCUCGCUC UGUGUCUCUU UAUCUCUGCU UCUGGCAUAG CACUGUUUUG GGCUAUCCUU 60
CUGUGUGUGU ACCAGGGCUG GUGUCUAUGU AGUUCCAUCU CUUUAAGUGA UGCUGUUACC 120
CVuUGCCACU GUCUGGACAC CAGCACUCAU ACGAGAAGCU UAUCCUUGGC AUGAAGGCAA 180
GCCCUUCUCC UCCUGAGUGA GUUUCACUAA CGGGAGAUCA GACUCUUUUU UAAUUUAAAU 240
UUAUUUAUUU UAAGUAGAGG CUAAUUUACA AUAUUAUAUU GGUUUUGCCA UACAUCAACA 300
UGAGUCCACC ACGGGUGUAC AAAAUCAGAC AUUUUUAGUC CACACUUUCA GACAGUACUU 360
UCUCAAACUU GAAAGCCAAC AGUGGGCGGU CACUGCUGAA CCUCAGAAGG GGCCGGUUUA 420
UCCUCCCUCC CUCACUGGAC AGAUGUGAAC ACUGCACAGG UCUGCAGCGU CUUGGCCACA 480
CUUUGCACAG AGGGAGAAAU UGGGGCAUGC UCUGCUGGCA UGAGGAAACU CCCUGAACCU 540
UGUUCAAAUG CCUACCAGUG AGAUGCUAAG GACAACUCCC UGUUAAGUUC CAGGACUUCC 600
UGGUGCCCGA GACAUGCACG UCUGCCCAUU UACCCUACCA AGGUCCUUUC AAAAUGGUUC 660
UGUUCUCCAU GUAAGAACAC GUACCAGCCU GCCCAAUAGG CCAAUCCUGU GGGGCCAGGA 720
GCAGCAAGAG GAUCAAGCUA AUCCAUCCAU CCUAUAGUCC UUACUCCCGA UAUAUGCCUU 780
CUUCAAAGAG UACAAAGAGU AUUUUUCAAA CUGGAAGACU AUGCAGGUAG CAGGUGCUUA 840
UGCUGCAGUG CUGGUCCGUU UCUGAAACCU CAAAAAAAGG CAAGGGGUGC UCAUGCUCAU 900
GGCAAGGACA GGGAAAGAAA GCCCAGUGUU CUAGAAGGAU AGAACNCCCU GGUAGCUGCA 960
UGUCCAAGGG GCUGUGGGGC CACCCACUAU GACCUCUGUG UAUUGGAAUU GCAGCCUGUU 1020
CUGUGUCUCG GAUCCUCUGC ACUCUCAUUG GUCACCCCCA GGCACUUUCU UCCUGCCUCU 1080
CCUUUCUGCC AGGCAUCCUG GGUGCACCAC UGUGAUUCCA CUUAGAAGUU GCUCACUCAG 1140
UGAGAUAACA GGGAUUGGCA ACCCGGCUCC AGUGCUUCUG AUGGCCAGAG CAUGUACCUU 1200
UCCUAUGGCU UUUAUGUUUU CCCUAUUCAA CUUCUAUCAC CUGGUUAGGU CAGUUUCUAC 1260
ACCUCAUACU CACAAGCAUA CUAUCAGGCG CUUUUCAUGC AUAUAUGCAC ACACAUGCGU 1320
GUGUACACAU UAACAUCCUG AGAGGAAACU UGCACAUAUA CAGACAUACA AACUUUCUUC 1380
UCCAGGAACA UCAAUUUUGG UAAGCACCUG ACUUUCUUUG UCCUGAUUAU UUUCUUUCAC 1440
UUUCUCAUCG AUCCUGUCAG GUUACACUUC UAACCCUUUG ACUUAGCCUC AAAGGUCACA 1500
AAAUUUUGGC AUUUGCUCCU GACAAGGACC GAUCUGCAGA GCUUCAGGCA GGGUUGGAAA 1560
UGCUCGCCCU GGACAGCUGA AUGAGUUCUG CCUGCAUUCU AUAUUCUCCC AUUACCUUGG 1620
ACAGCUUCAC AGUACCAGUC ACACUGGCCU GAUCCAUUGC CUGUGCAUUC UCUCAGGGGA 1680
CCAGAAAACA AGGACGUCUG GGCUCAGCUG ACUUGGAGAA CUGCUUUCUC AGUGUGCCCC 1740
UUCUAAGUCA UUCCUGGUCA AAACUGUGUC CCUAUUGCUA GCCUACCACA UCAGCAUUCU 1800
GAGUGAGGUC CCCUGUUCUU UCUACCUGUG UAGUUUUCUG UGUGCACCUG UCUACCUGUG 1860
CCUCCAAGCA CUAUCUCCCU UUAGCAGGAA AAGACCUGUG CCUCCAAGCA CUAUCUCCCU 1920
s~ r~ No: ~ ~
36 ~;~~ J
UUAGCAGGAA AAGGCCAAAG AGAUGCCUGA GCCUCCAAGG GCCCCCAGAG UCUGUGAGAG 1980
ACCUGGGUGU GAUCCAAUGU UGUGAAGAAG GUGCCCAUAG AUAGAGGGUC UCUUCUGAAA 2040
CI~Ir~GGCAUGA AGCCCGAGAC CAUAAUGGUA AGGUGGCAUU CCUACAGGUG GUCCCUUCUG 2100
UUUAUUCCUA CCCAGACCCA UGGAGUCCCC AAACAGAUGA UGAUCUGGGA AUCCUGCCCU 2160
UUCUGGGCCC ACAGCUCAUG CCUCCCUUGG ACAGAAAGCA GCUUUUCUAU CUCAAAAACA 2220
CCAAGAGGGC UUGAUUCCAC CCAGGCCUCA UUGAUUUGCU AAAUCAAAUA CUCUCUUUCA 2280
UUGGGUUCAU UAAGCCCAGG UAGGACUCCC UGGAGUCAGG CAUCCCUGCU UACCUACACA 2340
GCCCACGUGC CAAGUUAGCC AGUCCUUGGU UGGCCACAGG GGCAUCCAAG ACUGUCACCU 2400
GGAAUGCAGC UUCCUUCUGA GUGUCAGCUG GUGCAGAUCC CCUACGACAA AAUCAGAGAU 2460
UAUGCUCCAG AGAAACUGCC AAAAUCCUCC CCCAGGUGCA AACACACACC UUUGCCCUCA 2520
GGUCCCCAAA GCCAGGGGAA AGACCCAGAG AAAAGAAGGA AUUUAUAUCA GGACUUUCAG 2580
CACAAGCCAU GGGGUAUCUU UGGCAGGAGC GUUAUUGCCU UUCCCCUGGA CCCUGAAAAC 2640
CAGCAGGCCC UAAACUGCAC CCAGGGGCUU CCCUGUCUCC CACUCUCAUG AGGUCCUUCA 2700
GACACGCAAU AAGCCCAUCA UCCUUGCUUC CUCCCUGUUC CCUCCCUUAU AGGCACACCU 2760
CGGCAGAAGA GCACACACGU AAAACACCUG CACUUUCUAC GCCUUUCUGC ACUGCCAGGG 2820
AGACUGGAAG UGCCUGGAGG CAUGCCACAC UCACAUCUUG UCUCUCCUAG GAUGCCUGUG 2880
GUUUUGCACG ACAGCCUACC UUAGCAUGUC UCGCAUUUUG UGUCACAUCG UUCCAGUGUG 2940
UGAAACCCUC AUGGAGAGAG GGUGCUGGCU GAUGGGCUGA UCCUGGGAAG CACUGGCCCA 3000
GGACCUUCCC AGGUCUCCUU CUCACAUGUG UAGAGCAAGU CUCCAGUACA CAAGUCAAUC 3060
UGUGCCUCUU UCUCUUCGGG UCUCUGUCCU UCUCAGCAAG ACCUUAGCCU CCUCACCCAU 3120
CCCAGGUCCU CUGUAUCCAC AUCCACCAUU UCCGCCUGCC AGCCCAUGUC CCCACAGGCU 3180
GUGGGCUCCA CAGGCGGUGG UUUUAAAGCC UCACUCCACC UGAUUUGCCC UGGGUGAAUC 3240
CACAGACCAU GCACUCACUC UUCCUGGUCC AAACACAUAC AAGAACACGG UAGAAAUGGU 3300
GAGUGUGUUU UUGUAUUUCA UCUCAUGGCA GAUUUCUGAA GCCAAGGUCC UGAGUUAUCA 3360
GUGGCCAUCC UUUCCUCAUU CCCAUCCUGG ACAGGGUCAC UGCAGAGAUA GGGCGACCAA 3420
CCACCCUCAA ACUGGGGGUG CCUUGUGUCU CGUCUUCUGA UUGCUUGGCA UUUCCUCUCC 3480
UGUAGCCUUU UUCUCUGAUA UUUCCCUGGG CCACACACAC ACACACACAC ACACACACAC 3540
ACACACGCAC GCAAACACAG GUGACACAAG CACACACGGU AUACACACAC AGGCUCGAUA 3600
CAGGGACACA AACACAAACA GGGAACACAG GUGUUUCAGG AGCUGAAGAC GCCCAUGUGU 3660
CCAGCAGUAU CAGAGAUGGC AUCAGUAGGC ACCACGUCCU GCACUUGGAG CUC 3713
