Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TITLE OF THF INVENTION
HERPES SIMPLEX TYPE 1 PROTEASE MUTANTS AND VECTORS
I)ESCRIPTION OF THE INVENTION
This inventiion relates to Herpes Simplex Virus type 1
(HSV-1) viruses which ~,ontain a mutation in the protease gene, and to
vectors and host cells used in producing them.
E~CKGROUND OF T~IE INVENTION
The Herpes Simplex Type-l (HSV-1) virus is a relatively
large virus (152,260 bp). While much is known about the viral life
cycle and its general activity, it has been difficult to study the
relationship between biochemical and biophysiological properties of its
gene products and the virus life cycle since its large size makes it
difficult to create predetermined point mutations.
HSV-1 protlease is a serine protease that has both a
structural and enzymatic role in the assembly of the HSV-1 capsid. The
protease and infected ceil protein 35 (ICP-35) form a complex of
approximately 1100 molecules in a ratio of 1:10 within the nucleus of
the infected cell. Around this complex the capsid proteins assemble into
B capsids. After assembly the protease cleaves itself twice and ICP-35
once, releasing the ICP-35 and the carboxyl terminal fragment of the
protease from the capsid interior. The 247 amino acid protease remains
within the capsid. Concurrently (or subsequently) the genomic HSV-1
DNA is packaged within the capsid.
DETAILED DESCRIPTION OF THE INVENTION
This invention relates to HSV-l viruses which have a
mutated protease gene. ~Preferred mutant viruses of this invention
contain altered protease ~enes which include changes in amino acid
sequences of the resulting proteases, and which confer phenotypes which
are different from the wild-type virus. A further aspect of this
invention are the vectors and sets of vectors used to create the mutant
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viruses of this invention and host cells which are transformed with these
vectors.
The mutant viruses of this invention may also be made by
methods which are described in co-pending U.S. Application Serial
5 No. , (Attorney Docket No. 19458) filed herewith, which is hereby
incorporated by reference.
The HSV-l viruses of this invention are preferably made by
transforming a host cell with a set of vectors comprising: a first vector
comprising a HSV-l mllt~ted protease gene and overlapping DNA
10 homologous with overlapping DNA of at least one additional vector; and
additional vectors, each additional vector comprising a fragment of the
subst~nti~lly complete HSV-l genome and also comprising overlapping
DNA which is homologous with a sequential genomic fragment
contained in at least one other additional vector, so that upon co-
15 transfection of a host cell, replication of viral DNA, and recombinationof the viral DNA, a virus having a mllt~ted protease gene and which is
replicable in a wild type or host range cell line is forrned.
Preferably, the viruses of this invention may be made by a
process comprising the steps of:
(a) obtaining a set of starting vectors, each starting
vector comprising a fragment of a subst~nti~qlly
complete HSV-l genome and also comprising DNA
which is overlapping DNA with a sequential genomic
fragment contained in other starting vectors, so that
upon co-transfection of a host cell, replication of
viral DNA, and recombination of the viral DNA, a
virus is formed which is replicable in a wild type or
host range cell line;
(b) replacing a starting vector comprising a protease
gene which is to be mutated with a first replacement
vector, the first replacement vector comprising a
mutated protease gene and overlapping DNA, and at
least one additional replacement vector comprising
genomic DNA which was present in the replaced
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~ 3 ~
starting vector, but is not present in the first
replacement vector along with overlapping DNA; and
(c) co-transfecting a host cell with the replacement
vectors and the rem~ining starting vectors under
conditions allowing replication of viral DNA and
recombination of viral DNA to form a virus which is
replicable in a wild type or host range cell line.
A further a$pect of this invention is a set of vectors used to
10 make the m~lt~nt viruses of this invention. The set of vectors comprises:
a first vector which is a plasmid, comprising a HSV-l
mutated protease gene and overlapping DNA homologous with
overlapping DNA of at least one additional vector; and
additional vectors, each additional vector comprising a
fragment of the subst~n~i~lly complete HSV-1 genome and also
comprising overlapping DNA which is homologous with a sequential
genomic fragment conta~Lined in at least one other additional vector, so
that upon co-transfection of a host cell, replication of viral DNA, and
recombination of the viral DNA, a virus having a mutated protease and
which is replicable in a wild type or host range cell line is formed.
The vectors of this invention are preferably made by a
process comprising the steps of:
(a) obtaining a set of starting vectors, each starting
vector comprising a fragment of the subst~nti~lly
complete HSV-l genome and also comprising DNA
which is overlapping DNA with a sequential genomic
fragment contained in other starting vectors, so that
upon co-transfection of a host cell, replication of
viral DNA, and recombination of the viral DNA, a
virus which is replicable in a wild type or host range
cell line is formed;
(b) replaLcing a starting vector comprising a protease
gene which is to be mutated with a first replacement
vector, the first replacement vector comprising a
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mutated protease gene and overlapping DNA, and at
least one additional replacement vector comprising
genomic DNA which was present in the replaced
starting vector, but is not present in the first
replacement vector along with overlapping DNA; and
(c) co-transfecting a host cell with the replacement
vectors and the rem~ining starting vectors under
conditions allowing replication of viral DNA and
recombination of viral DNA to form a mutant virus
which is replicable in a wild type or host range cell
line.
The first replacement vector may be made by a process
comprising:
(a) creating a vector comprising a protease gene site
which is to be mllt~ted and overlapping DNA;
(b) defining a first restriction endonuclease site in a
position S' to the protease gene site which is to be
mutated;
(c) defining a second restriction endonuclease site in a
position 3' to the protease gene site which is to be
mutated to define a wild-type gene segment contained
between the first and second restriction endonuclease
sites;
(d) creating a mutant protease gene segment substantially
identical to the wild-type gene segment, except for
comprising a desired mutation; and
(e) replacing the wild-type gene segment with the mutant
protease gene segment to obtain the first replacement
vector.
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BRIEF DESCRIPTIONI OF THE FIGURES
Figure 1 is the DNA and amino acid sequences
(SEQ.ID.NO.:1&2) of the HSV-l (F) protease (Pra) BsmI fragment.
(The 82bp upstream fragment is not shown).
S Figure 2 is a diagram of HSV-l protease (Pra) cleavage
sites. Pra is a 635 amino acid serine protease which undergoes autolytic
cleavage at Ala247 and Ala610. Products of this cleavage are shown.
Figure 3 is a diagram of the plasmid/cosmid-based
mutagenesis process of this invention.
As used in the specification and claims, the following
definitions apply:
Null Mutant: an HSV- 1 mutant which lacks the ability to
grow or form plaques on Vero cells.
Overlappirg Vectors: two or more vectors, each cont~ining
a segment of a DNA which has sufficient common base pairs with the
DNA contained in a second vector so that homologous recombination
can occur when copies ~f the DNA are present in a common host.
Replacement Vector: a vector, generally a plasmid which
contains a portion of a ~ISV-l genomic fragment which was originally
present in a starting vector. Generally, a starting vector will be
replaced by two replacement vectors: the first one comprising the
mutant gene and the second one comprising the rem~ining genomic
DNA which was contained in the starting vector, but not present in the
first vector. Additionally, replacement vectors also contain sufficient
overlapping DNA so that homologous recombination can occur.
Starting Vector: one of a series of vectors, generally
cosmids, which together comprise the subst~nti~lly complete genome of
HSV-l along with overlapping DNA.
Subst~nti~lly Complete Genome: sufficient DNA is present
so that upon transfection of a host cell, replication of the viral DNA and
homologous recombination, a replicable HSV-l virus is formed. This
invention specifically el,lvisions: (1) an HSV-l virus cont~ining a
complete genome cont~ining desired mutations and (2) an HSV-l virus
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which does not have a complete genome, but the genes which are
missing are not essential for virus replication; (3) an HSV-l virus
missing genes which are essential for virus replication, but the missing
gene product(s) are complemented by those produced in a host range
5 cell line; and (4) an HSV-l virus according to 1), 2), or 3) and/or
comprises additional DNA, regardless of source, which does not
interfere with virus replication; or if replication is interfered with,
which can be complemented by a host range cell line.
Replicable Virus: an HSV-1 virus whose genome is neither
10 too short nor too long, so ~at functional capsid assembly and packaging
occurs.
Overlapping DNA: a segment of DNA at least about 300
base pairs in length, more preferably about 2,000 to 5,000 base pairs in
length, which is subst~nti~lly identical to a segment in another vector.
15 The vector generally contains two differing overlapping DNAs, one on
the 5' end of the vector and one on the 3' end of the vector, and each
overlapping DNA overlaps that of a different vector.
Host Range Cell line: a host cell line which has been
transformed to express a viral gene, such as HSV-l protease. Viruses
20 which do not produce a functional version of this gene are able to utilize
the protein produced by the transformed cell line.
One aspect of this invention is a convenient system which
allows researchers to study the protease gene in the context of the virus,
25 and to create any desired mutation(s) within the protease gene.
The starting point for the method according to this
invention is a set of vectors, such as cosmids. The total number of
vectors in the set is not critical, but together the set of vectors contains a
substantially complete HSV- 1 genome. In general, the total number of
30 vectors in the set should not be so large that it becomes cumbersome to
co-transfect the host cell. Preferably the number of vectors in a set
should be less than ten, and preferably, less than about eight, and most
preferably about six.
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One or more of these vectors are replaced by one or more
replacement vectors, each replacement vector cont~inin~ a smaller
HSV-l DNA insert than in ~e starting vector, but together the
replacement vectors contain the "equivalent amount" of unique, non-
5 overlapping HSV-l genomic DNA as was present in the starting vector.
("Equivalent amount" as used in this content means subst:~nti~lly the
same amount, plus or minus any DNA which was intentionally added or
deleted as mutations). If the complete protease gene which is to be
mllt~ted is contained within one starting vector, then only this single
10 vector needs to be replaced. If, however, the protease gene which is to
be mllt~ted is contained on two starting vectors (i.e., each starting vector
cont~ining only a fragment of the protease gene), then the two starting
vectors should be replaced. Replacement vectors make up one aspect of
this invention.
The first replacement vector may be a cosmid or a plasmid;
plasmids are generally preferred. The vector may be any vector which
is able to replicate in the host cell system. Any host cell may be lltili7ed,
but for general convenience, E. coli is preferred. The first replacement
vector comprises a copy of the protease gene which is to be mutated
along with a sufficient amount of overlapping DNA so that homologous
recombination can occur. While homologous recombination can occur
with a few base pairs (i.e., less than 20), it is preferred that at least
about 300 base pairs of overlapping DNA be present, and even more
preferred that at least about 2,000 to about 5,000 be present. It is
preferred that overlapping DNA be overlapping with DNA of at least
one vector, and it is preferred that it overlaps DNA of two vectors.
Additional replacement vectors of this invention contain the rem~ining
genes and/or gene fragments which were originally in the starting
vector, along with overlapping DNA.
Next, two restrictions sites should be defined in the
replacement vector containing the protease gene to be mutated. These
restriction sites, which may be naturally occurring or may be inserted as
desired using known techniques, define a protease gene fragment which
is to replaced by a newly synthesized mutated protease gene fragment.
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The first restriction site may be anywhere upstream of the position
where the mutation or mutations are to be introduced. In a preferred
embodiment, it is upstream of the initiation ATG site of the protease
gene. The second restriction site may be anywhere within the protease
5 gene, or even downstream of the gene, as long as it is downstream of the
site where desired mutation or mutations are to be made. It is also
desirable to choose a position for the second restriction site which is
close enough to the first restriction site so that with currently available
technology, the mutated gene fragment may be easily synthesized and
10 sequenced as needed. Thus, the second restriction site is generally less
than about 2,000 bp downstream of the first restriction site, and
preferably less than about 1,100 bp downstream of the first restriction
site.
The restriction sites may be the recognition sites for
15 virtually any restriction endonuclease. It is preferred, however, that
each site be unique. In order to ensure that the mutated gene fragment
is cloned into the restriction sites having the correct orientation (i.e.,
can be "force-cloned"), it is particularly preferred that the enzyme
recognizes different base pair sequences, and that the first restriction
20 site and the second restriction site be differing base pair sequences,
although recognized by the same enzyme. Numerous enzymes are
known to have this characteristic, including BsmI.
The second replacement vector according to this invention
comprises any viral DNA which was originally encoded in the first
25 starting vector, but is not present in the first replacement vector, along
with sufficient overlapping sequences so that homologous recombination
can occur.
The remAining vectors in the series of vectors according to
this invention may be any vectors, such that when the complete set of
30 vectors is co-transfected into host cells, they are able to recombine to
form a mutated virus which is replicable in a wild type or host range
cell line.
In a preferred embodiment of this invention, a set of
starting vectors to be used are the five cosmids: cos2~, cos6, cosl4,
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cos48, and cosS6, which were obtained from Dr. Andrew J. Davison.
These cosmids and/or their equivalents can be made according to the
description given in Cllnnin~h~m and Davison Virology 197:116-124
(1993), which is hereby incorporated by reference.
One of the cosmids of the Cllnningh~m and Davison system,
cosmid cos28, contains DNA encoding the protease and its substrate (the
assembly protein ICP-35) on the overlapping genes (UL26 and UL26.5).
This cosmid is replaced by two novel overlapping replacement vectors,
both of which are further aspects of this invention. This is diagrammed
in Figure 3B.
The first replacement vector should carry a copy of the
HSV-1 protease gene which has at least two restriction sites that have
been defined, according to the considerations mentioned above. One
preferrred restriction enzyme is BsmI, a degenerate restriction
endonuclease with a recognition sequence of GAATG/\CN''
(SEQ.ID.NO.:3).
In a preferred embodiment of this invention, the first
replacement vector is plasmid pR700 (or a plasmid carrying the same
inserts as pR700). Plasmid pR700 was made from the commercially
available plasmid pGEM-4Z (Promega Corp, Madison, VVI), and
contains the UL26 protease gene in a 13.3 kb insert of HSV-l (base
pairs 44440-57747). Plasmid pR700 also contains two naturally
occurring BsmI sites, a first one 82 base pairs 5'- of the HSV-l protease
start site and one at amino acid 348 of the protease. The "N" at the 5'
BsmI site is "T" whereas at the 3' BsmI site, the "N" is "G", so that the
mutant PCR fragments may be force-cloned into the vector. PCR
mutagenesis of this 1.1 kb BsmI fragment was used to introduce various
desired mutations into the HSV-l protease gene fragment.
A second replacement vector according to this invention is
plasmid pR710 (or a plasmid carrying the same inserts as pR710) which
is derived from commercially available plasmid pNEB93 (New England
Biolabs, Beverly, MA). Plasmid pR710 contains a 24.7 kb insert of
HSV-l (base pairs 24699-49435) that does not include the HSV-l
protease.
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Thus~ a further aspect of this invention is a set of vectors
comprising at least one vector selected from the group consisting of
cos48, cos6, cosl4, cos56, and pR710 and at least one additional
plasmid. Preferably the additional plasmid carries subst~nti~lly the
5 same insert as a plasmid selected from the group consisting of: pR700,
pR711, pR712, pR713, pR714, pR715, pR716, pR717, pR718, pR725,
pR728, pR729, and pR730. Preferred plasmids are selected from the
group consisting of: pR700, pR711, pR712, pR713, pR714, pR715,
pR716, pR717, pR718, pR725, pR728, pR729 and pR730.
In this preferred embodiment, the two replacement
plasmids and four rem~ining starting vectors, which together make up a
further aspect of this invention, are introduced into HSV-l host cells.
The HSV-l host cell chosen is generally not a critical aspect of this
invention. Generally, any cell in which HSV-l can replicate is an
15 appropriate host cell. Particularly preferred host cells are Vero cells.
DNA which is replicated during the virus life cycle homologously
recombines in the host cells to create the mllt~nt HSV-l viruses of this
invention. This is illustrated in Figure 3C.
The above-described mutagenesis method allows one to
20 make the desired HSV-l protease mutations in the virus in a short
period of time, i.e., within about 2 weeks. It has the further advantage
that pure mllt~nt virus cultures are generated; there are no wild type
background viruses in the transfections of Vero cells.
In creating the mutant protease gene fragments of this
25 invention, virtually any known method of synthesizing and mutating
DNA may be used. PCR mutagenesis is a preferred method. In
performing the PCR mutagenesis of the target DNA, standard PCR
techniques may be used in general, such as those described in H. Russell,
1990, "Recombinant PCR" in PCR Protocols (Innis, et al., Eds.),
30 Academic Press, Inc. San Diego, CA, pages 177-183, which is hereby
incorporated by reference. However, since HSV-l DNA is quite GC
rich and if the region which is to be mutated is also high in GC content
(as is the case with the protease gene) it is preferred that a higher than
usual melting temperature be employed during the PCR cycle,
,
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preferably at least about 99~C to maximize product formation. A
second consideration with PCR mutagenesis in general is maintAining
fidelity. While any suitable polymerase enzyme may be employed,
VentR DNA polymerase (commercially available from New Fngl~nd
S Biolabs) is a preferred polymerase for the PCR reactions used herein
because of its proofre~clin~ ability and thermal stability at 99~C.
Virtually any mnt~tion which is desired may be introduced
into the protease gene using the PCR mutagenesis method. For instance,
in order to obtain viruses which have altered phenotypes, it is desirable
10 to change an amino acid sequence. Further type of mutations which are
preferred are those which introduce new restriction endonuclease
recognition sites.
In order to demonstrate the versatility of the mllt~tion
procedure of this invention, the following mutant viruses were made.
15 Throughout the specification and claims, the virus nomenclature is the
same as that used for the replacement plasmid cont~inin~ the mutation,
except that the virus uses the prefix "V" and the replacement plasmid
uses "pR".
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TABLE 1
Representative protease mllt~nt~
VIRUS MUTATION ADDED SITE*
V711 His61 to Val61 AatII
V730 His61 to Ala61 Pspl406I
V715 His61 to Tyr61 none
V717 Leul25 to Vall25 BsaAI
V718 Prol26 to Glyl26 BstXI
V713 Serl29 to Alal29 NheI
V714 Serl29 to Alal29 none
V712 His148 to Alal48 PstI
V716 His148 to Tyrl48 none
V725 His 148 to Argl48 MulI
V728 His148 to Glu148 Eco47III
V732** Alal 29 to Serl29 HindI~
V729 His 148 to Lys 148 StyI
5 * restriction endonuclease site
**back-mutation of V713
The active site serine of HSV- 1 protease has been
previously identified by chemical mutations methods to be Serl29.
10 Therefore, changes of amino acids at the active serine site and near the
active serine site were of particular interest.
Mutations At Serl29:
A mutation was made in HSV- 1 protease gene to change the
15 protease amino acid Serl29 to Alal29. This virus is designated V713,
and is a further aspect of this invention. Recombinant virus could only
be rescued on a host range cell line (PHS-23) which expresses protease.
When V713 was used to super-infect Vero cells, the Western analysis
showed an accumulation of the ~0 kD protease (Pra) along with several
-
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other peptides r~ngin~ in molecular wieght from 29 kD to 75 kD. A 24
kD band seen in wild-type infections was absent.
Mutation At Leu 125
S V717 contains a mutation of Leul 25 to Val 125. This virus
did not grow on Vero cells at 31~, 34~, 37~, or 39~C, and showed by
western blot analysis no protease activity at 20 hours after infection. A
light 27 kd No protease band was observed in the western analysis. This
band may reflect protease formed via recombination or carried over
from the host range cell line PHS-23 during propagation of the virus.
Mutations At Pro 126
V718 contains a mutation of Prol26 to Glyl26. This virus
did not grow on Vero cells at 31~, 34~, 37~, or 39~C, but after 20 hours,
substantial processing of the 80 kd protease (Pra) occurred. However,
even extended incubation for 7 days failed to produce plaques. The
inability of the virus to replicate may reflect a requirement for proper
structural assembly of the capsid. VVhile not wishing to be bound by
theory, this may result from the protease activity not being properly
synchronized with the replication cycle, i.e., the protease may be cutting
itself in the cytoplasm, or that the protease activity observed in this
mutant is insufficient to digest all of the assembly protein within the
capsid. If so, then the intact ICP-35 protein that is retained within the
capsid may block DNA pack~ging.
Mutations At His 14~s
Mutations which changed the histidine at position 148 were
mixed. Changing this amino acid to Ala (V712) resulted in a small
plaque phenotype and Western analysis showed a reduction in protease
30 activity. This result was unexpected because in the prior art, where the
protease gene having the same mutation,but not contained within the
virus showed no protease activity in in vitro assays. (Liu et al., 1992
Proc. Natl. Acad, Sci. USA 89:2076-20~s0 and Deckman et al., 1992 J.
Virology 66:7362-7367, both of which are incorporated by reference.)
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While not wishing to be bound by theory, this surprising result may be
due to a difference in the three dimensional structure of the protein
wi~in the virus environment, or the presence of a hi~erto unknown
accessory protein which lends activity to the protease.
Viruses V716 (His148 to Tyrl48) V725 (His148 to
Argl48) V729 (His148 to Lysl48) were not viable on Vero cells, but
each exhibited a different level of protease activity. V729 showed no
protease activity by Western blot analysis; V716 had greater than 50%
protease activity, and V725 exhibited wild-type activity against Pra, but
did not process ICP-35.
Mutations At His 61
Three mutations at His61 to Val61 (V711), Tyr61 (V715),
and Ala61 (V730) all created null mutant viruses and in Western
analysis had the same extra bands as the V713 mutant.
Taken with the observations of the His148 mutations, the
results suggest ~at His61 is required for protease activity whereas
His148 is not.
The following non-limitin~ Examples are presented to
better illustrate the invention.
EXAMPLES
GENERAL METHODS
Viral Strains
Two strains of viruses were used, HSV-l strain 17
[designated HSV- 1 (17)] and HSV- 1 strain F [designated HSV- 1 (F)] .
30 Mutations to the protease have been made in HSV-l(F) (see Liu, F.
etal., l991,J. Virol. 65:5149-5156, hereby incorporated by reference)
and temperature sensitive mutants have been isolated in HSV-1(17).
(See Preston, V. et al., 1983, J. Virol. 45: 1056- 1064, hereby
incorporated by reference). Sequence analysis of the BsmI fragment
35 revealed that the two strains differ by two amino acids (Leu300/Ser300
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and Ser301/Pro301) and six silent mutations (in Prol5, Arg46, Gly84,
Gln90, Glyl99 and His341). To make an equivalent comparison of
in vitro and in vivo studies, a protease chimera (pR73 1) was made.
Plasmid pRHS2, cont~ining the HSV- l (F) protease was digested with
5 BsmI and the l.lkb fragment was cloned into pR700 cont~ining HSV-
1(17) protease. Both viruses were equivalent in virus titer and plaque
morphology on Vero cells.
PCR mutagenesis
Four oligonucleotides and a DNA template were amplified
in two rounds of PCR to create a variety of mutated DNA fragments
which were subsequently cloned into plasmid pR700 and used to create
the mutant viruses. The first round of PCR mutagenesis was carried out
in two separate reactions. In one reaction, a positive strand
15 oligonucleotide homologous to the DNA 5' to the first BsmI site, was
paired with the negative strand oligonucleotide specified below. In the
other reaction, a negative strand oligonucleotide homologous to the
DNA 3' to the second BsmI site was paired with the positive strand
oligonucleotide specified. The two specified oligonucleotides are
20 complementary to each other, mutate the same amino acid residue, and
most, but not all, concurrently introduce a new endonuclease restriction
site. The specified DNA template (from pR700, pRHS2, or V713,
below) was added to both reaction mixtures and PCR amplification
initi~ted. In the second round of the procedure, the DNA fragments
25 generated by the first round PCR reactions were gel purified and mixed
together with oligonucleotides flanking the BsmI sites (SEQ.ID.NOS:4
and 5, below), and subjected to PCR amplification.
PCR mutagenesis was performed with VentR DNA
polymerase (New England Biolabs) in a DNA thermal cycler from
30 Perkin Elmer Cetus. The cycle was melt for 1 minute at 99~C; anneal at
40~C for two minlltes; extend at 71~C for 3 minutes; for 30 cycles. The
product of the second round PCR reaction and extended BsmI fragment,
was digested with BsmI, gel purified and ligated into the BsmI sites of
pR700.
-
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Oli~onucleotides used for mutagenesis: Unless otherwise indicated, all
oligos were from Midland Certified Reagent Co., Midland, TX. (In
each pair, the plus stand oligo is listed first):
5 5' and 3' oligonucleotides flanking the two BsmI cloning sites:
5'-GTACTCAAAAGGTCATAC-3' (SEQ.ID.NO.:4) (This oligo is 5' to
the first BsmI site and was used for the generation of all mutations in
the protease from amino acid 1 to 348).
10 5'-GGGAAACCAAACGCGGAATG-3' (SEQ.ID.NO.:5) (This oligo is
3' to the second BsmI site and was used in generation of mutations in the
protease from amino acids 1 to 34~.)
Oligonucleotides for the temperature sensitive protease mllt~nt pR701:
15 5'-GATACGGTGCGGGCAGTACTGCCTCCGGAT-3 '
(SEQ.ID.NO.:6)
5'-ATCCGGAGGCAGTACTGCCCGCACCGTATC-3' (SEQ.~.NO.:7)
These oligos add a SacI site to the Ala48 to Val48 mutation.
20 Oligonucleotides for the temperature sensitive protease mutant pR701:
5'- l~ l GGCGCTCTTCGACAGCGGGGAC-3' (SEQ.ID.NO.:8)
5'-GTCCCCGCTGTCGAAGAGCGCCAAAAA-3' (SEQ.ID.NO.:9)
These oligos add a SapI site at the Thr30 to Phe30 mutation.
25 Linker oligonucleotides (BspHI-PacI-HindIII) for pR710:
S'-CATGATTAATTA-3' (SEQ.ID.NO.:10)
5'-AGCTTAATTAAT-3' (SEQ.ID.NO.: 11)
Oligonucleotides used for the His61 to Val61 mutation for pR711:
30 5'-CCCACTCCCGATTAACGTGGACGTCCGCGCTGGCTGCGAGG-
TG-3' (SEQ.ID.NO.:12)
5 '-CCTCGCAGCCAGCGCGGACGTCCACGTTAATCGGGAGT-
GGG-3' (SEQ.ID.NO.: 13)
This also adds an AatII restriction site.
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W 096/38~46 PCT~US96~77
Oligonucleotides used for the His148 to Alal48 mutation for pR712:
S ' -CCCCGATCGCACGCTGTTCGCTGCAGTCGCGCTGTGCGCGA-
TCGGGCGG-3 ' (SEQ.ID.NO.: 14)
S ' -GATCGCGCACAGCGCGACrGCAGCGAACAGCGTGCGATC-
5 GGGG (SEQ.ID.NO.:15)
This also adds a PstI restriction site.
Oligonucleotides used for the Serl29 to Alal29 mutation for pR713:
5 '-CACCAACTACCTGCCCTCGGTCGCGCTAGCCACAAAACGCC-
10 TGGGGGG-3' (SEQ.ID.NO.: 16)
5'-CAGGCG l l~GTGGCTAGCGCGACCGAGGGCAGGTAG-
TTG-3'(SEQ.ID.NO.: 17)
This also adds a NheI restriction site.
15 Oligonucleotides used for the Serl29 to Alal29 mllt~hon for pR714:
5 '-CCAACTACCTGCCCTCGGTCGCCCTGGCCACAAAACGCCTG-
GGG-3' (SEQ.ID.NO.: 18)
5'-GCCAGGGCGACCGAGGG-3' (SEQ.ID.NO.:l9) Oligonucleotides
used for the His61 to Tyr61 mutation for pR715:
20 5'-CCCACTCCCGATTAACGTGGACTACCGCGCTGGCTGCGAGG-
TG-3' (SEQ.ID.NO.:20)
5 ' -CGCGGTAGTCCACGTTA-3 ' (SEQ.~D.NO. :21)
Oligonucleotides used for the His148 to Tyrl48 mutation for pR716:
25 5'-CCCCGATCGCACGCTGTTCGCGTACGTCGCGCTGTGCGCGA-
TCGG-3' (SEQ.ID.NO.:22)
5'-GCGACGTACGCGAACAGC-3' (SEQ.ID.NO.:23)
Oligonucleotides used for the Leul25 to Vall25 mutation for pR717:
30 5 '-CACCAACTACGTGCCCTCGGTCTCCCTG-3 ' (SEQ.ID.NO. :24)
5'-CCGAGGGCACGTAGTTGGTGATCAGG-3' (SEQ.ID.NO.:25)
This also adds a BsaAI restriction site.
CA 02222877 1997-11-28
W O 96/38546 PCTrUS96/07795
- 18-
Oligonucleotides used for the Prol26 to Glyl26 mutation for pR718:
5'-CAACTACCTGGGCTCGGTCTCCCTGGCC-3' (SEQ.ID.NO.:26)
5 '-GAGACCGAGCCCAGGTAGTTGGTGATCAG-3 '
(SEQ.ID.NO.:27)
5 This also adds a BstXI restriction site
Oligonucleotides used for the His148 to Argl48 mutation for pR725:
5 ' -CGCTGTTCGCACGCGTCGCGCTGTGCGCGATCG-3 '
(SEQ.ID.NO.:28)
10 5 ' -CAGCGCGACGCGTGCGAACAGCGTGCGATCGGG-3 '
(SEQ.ID.NO.:29)
This also adds a MulI restriction site.
Oligonucleotides used for the His148 to Glu148 mutation for pR728:
15 5 ' -CTGTTCGCGGAAGTAGCGCTGTGCGCGATCGG-3 '
(SEQ.ID.NO.:30)
S ' -CGCACAGCGCTA( ~TTCCGCGAACAGCGTGCGATCGGG-3 '
(SEQ.ID.NO.:31)
This also adds a Eco47III restriction site.
Oligonucleotides used for the His148 to Lysl48 mutation for pR729:
5 ' -CGCTGTTCGCCAAGGTCGCGCTGTGCGCGATCG-3 '
(SEQ.ID.NO.:32)
5 ' -CACAGCGCGACCTTGGCGAACAGCGTGCGATCGGG-3 '
25 (SEQ.ID.NO.:33)
This also adds a StyI restriction site.
Oligonucleotides used for the His61 to Ala61 mutation for pR730:
5 ' -CCGATTAACGTTGACGCCCGCGCTGGCTGCGAGGTGGG-3 '
30 (SEQ.ID.NO.:34)
5 ' -CAGCCAGCGCGGGCGTCAACGTTAATCGGGAGTGGG-3 '
(SEQ.ID.NO.:35)
This also adds a Pspl406I restriction site.
CA 02222877 1997-11-28
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- 19 -
Oligonucleotides used for the Alal29 to Serl29 back mutation for
pR732:
J 5'-CCTGCCCTCGGTAAGCTTGGCCACAAAACGCCTGG-3'
(SEQ.ID.NO.:36)
5'-GGCGlYmGTGGCCAAGCTTACCGAGGGCAGGTAG-3'
(SEQ.ID.NO.:37)
This also adds a HindIII restriction site.
Constructs:
Plasmids derived from HSV-l (F):
pRHSl: This plasmid contains HSV-l(F) DNA base pairs 44590-54473,
starting within the UL22 gene and ending within UL28. This was made
by digesting HSV-l (F) DNA with XbaI and ScaI. The 9884 base pair
fragment was gel purified and subcloned into pGEM-7Zf(-) (Promega)
at the XbaI and SmaI sites.
pRHS2: This plasmid contains HSV-l(F) DNA base pairs 49126-53272,
starting within UL25 and ending within UL27. To prepare this plasmid,
pRHS 1 was digested with NotI and NheI, and the 414~ base pair
fragment was subcloned into the pGEM-7Zf(-) vector at the Bspl20I
and XbaI sites. This clone was used for the creation of the host range
cell line PHS23, and plasmids pR711, pR712, pR713, pR714, pR715,
pR716, pR725, pR728, pR729 and pR730.
pR731: pRHS-2 was digested with BsmI, and the 1.1 kb fragment was
then subcloned into the BsmI sites of pR700. This created a F strain
protease in the 17 strain virus.
pR732: V713 virus DNA was digested with NotI and the 6.5 kb
fragment cont~ining the HSV-l protease was gel purified. This
fragment was used as a template for PCR to back-mutate the Serl29 to
Alal29 back to Ser. The back mutation also created a new HindIII site.
pR732 exhibited a wild-type phenotype. The back mutation was
performed to demonstrate that the mutant phenotypes observed for the
various mutants of this invention were due to the mutagenesis process,
and were not artifacts of the transfection procedure.
CA 02222877 1997-11-28
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- 20 -
Plasmids derived from HSV-l (17):
pR700: This plasmid contains HSV-1(17) DNA base pairs 44440-
57747, starting within UL22 and ending within UL28 to prepare HSV-l
cos-28 was digested with StuI and NdeI, the 13,308 base pair fragment
was gel purified and ligated into pGEM-4Z (Promega) at the NdeI and
SmaI sites. This plasmid was use for both generation and sub-cloning of
m~lt~nts pR701, pR717 and pR718 into the BsmI sites.
pR710: This plasmid contains HSV-1(17) DNA base pairs 24699-
49435, starting between UL10/ULl l and ending within UL25. Cos 28
was digested with PacI and BspHI and the resulting 24,736 bp fragment
was subcloned with the two linker oligos (SEQ.ID.NOS. 10 & 11)
cont~ining BspHI-PacI-HindIII into the PacI/HindIII sites of New
England Biolabs vector pNEB93.
pR701: HSV-l temperature sensitive mutant was created from pR700
by PCR mutagenesis. It has a Thr30 to Phe30 mutation which contains a
SapI site and an Ala48 to Val48 mutation a cont~ining a new ScaI site.
Sequencing
Sequencing reactions were done using a Sequenase(~) Quick
Denature Plasmid Sequencing kit (United States Biochemical) according
to the manufacturer's instructions. S-35 dATP was obtained from
Amersham.
Host Range Cell Line PHS-23 (Expressing Protease).
pRHS2 was co-transfected with pSVNeo (Southern et al.,
1982. J. Mol. Appl. Gen. 1:327-341) into Vero cells and cultured in
800 ,ug/ml of G418 sulphate (GIBCO). Drug resistant cell lines were
screened for the ability to complement the temperature sensitive
protease virus, V701, at 39~C.
Digests
Prior to transfection, cosmid DNA and pR710 were
digested with PacI. Plasmids pR700, pR701, pR711, pR712, pR713.
pR714, pR715, pR716, pR717, pR71~, pR725, pR728, pR729, pR730,
CA 02222877 1997-11-28
W 096J38546 PCT~US961077
and pR73 1 were digested with Hinclm and NdeI, while pR732 was
digested with XbaI. The digested DNA was precipitated in 2M final
NH40Ac pH 7.5, and 2 volumes of isopropanol, centrifuged l0 minlltes
then washed in 70% ethanol and dried. The DNA was re-suspended in
10 mM Tris, 1 mM EDTA pH 7.g. Restriction endonucleases were
purchased from New Fngl~nd Biolabs and Promega (Madison, WI).
Western Blots
12% SDS-PAGE gels were transferred to Immobilon-P
(Millipore, Bedford, MA) and blocked in phosphate buffered saline, 2%
bovine fetal calf serum (FCS) (Hyclone Laboratories, Logan, UT), 2%
nonfat dry miLk, and 0.1% Tween-20. A peptide made to correspond to
the N-terminus of the protease, DAPGDRMEEPLPDRAC-NH2
(SEQ.ID.NO.:38), was conjugated to keyhole limpet hemocyanin, and
was used to generate a polyclonal rabbit antibody (Multiple Peptide
Systems, San Diego, CA). The second antibody was Goat Anti-Rabbit
IgG (H+L) ~lk~line phosphatase conjugate (Bio-Rad, Hercules, CA).
Western blots were developed with an ~lk~line phosphatase conjugate
substrate kit from Bio Rad or with a ECL kit from Amersham.
Southern Blots
Viral DNA was digested with the restriction endonuclease
corresponding to those sites which were added at the site of mutation.
Agarose gels were transferred to Zeta Probe (Millipore) in 0.4M NaOH,
and hybridized at room temperature with P-32 kinased oligonucleotides
(below) in 5 X SSC, 20 mM Na2HPO4 pH 7.2, 7% SDS, 1 X Denhardts
and 100 ,ug/ml herring sperm DNA, for two hours, then washed with
5X SSC at 50~C for four changes at 15 minutes.
Oligonucleotides used to probe Southerns:
SH-2 5'-CAGCGCTGGGAl~ lCG-3' (SEQ.ID.NO.:39)
SH-10 5'-GTTAACAACATGATGCTG-3' (SEQ.ID.NO.:40)
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- 22 -
Transfections
Vero or PHS-23 cells were plated at 3 x 105 cells per well
in six well clusters the day before transfection. The following day the
cells were washed in Delbeco's Modi~1ed Eagles Medium (DMEM)
S (from GIBCO, Gaithersburg, MD) without FCS and then 1 ml of
transfection cocktail was added. Transfection cocktail was made as
follows. To 100 ,~Ll of DMEM media, 0.5 ,ug of digested DNA was
added, followed by 14 ,ul of LiptofectAM~ETM. (GIBCO) This
transfection mixture was incubated for 30 minlltes at room temperature,
10 then 900 ,ul of DMEM was added. The cells (90% confluent) were
washed twice with DMEM without FCS and then the one ml of
transfection mixture was added. The transfection was incubated for 18
hours at 37~C, 5% CO2. Transfected cells were then washed and fresh
media, DMEM, 4% FCS, 100 units/ml penicillin and 100 ~lg/ml
15 streptomycin, was added. At day six or seven the recombinant virus
were harvested and the virus was plaque purified.
Plaque Purification.
After transfection with LipofectAMINETM Reagent
20 (GIBCO/BRL) the cells were scraped off the plates and were either
frozen and thawed three times, or sonicated. Serial dilutions 1:10,
1:100, 1:1000, 1:10,000, 1:100,000, and 1:1,000,000 were done in
DMEM. Cells in six well clusters were incubated with 0.5 ml of each
dilution and were rocked every 15 minutes for 2 hours. The cells were
25 then over-layed with 0.5% agarose, DMEM without phenol red, and
10% FCS and incubated at 37~C in 5% CO2 for three to five days.
Plaques were picked with a cotton-plugged sterile Pasteur pipette by
piercing the agarose and lifting a plug cont~ining the recombinant virus.
The plug was placed in a sterile eppendorf tube cont:~ining 0.5 ml of
30 DMEM and 20% FCS. The plaque was sonicated and then repuified a.s
described.
CA 02222877 1997-11-28
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- 23 -
Recombinant virus expansion
After plaque purification the virus was expanded on Vero
cells, or if the the mllt~nt was a null mllt~nt, it was expanded on the host
range cell line PHS-23.
s
Virus titers
Expanded virus stocks were titered on Vero and PHS-23
cells. Serial dilutions 1:10, 1:100, 1:1000, 1:10,000, 1:100,000, and
1:1,000,000 were done in DMEM. Six well clusters were then infected
10 with 0.5 ml of each dilution, rocked every 15 minutes, and adsorbed for
2 hours at 37~C. The cells were then fed with DMEM, 4% FCS and
0.16% hllm~n IgG (Armour, ~nk~kee, LL). Two to six days later the
cells were fixed in 1 ml of methanol for 7 minlltes and then air dryed.
Fixed cells were stained with Gemsa stain for 45 minlltes, washed with
15 water and dryed. The plaques were then counted under a microscope.
Virus DNA Mini Preps
Mini preps of virus DNA were made as follows: a T-225
flask of vero or PHS-23 cells was infected at a MOI of S and harvested
20 at 18 hours post infection. Cells were pelleted and then washed in PBS
three times. The cells were re-suspended in 400 ,ul 10mM Tris pH 8.0,
SmM NaCl, SmM EDTA and incubated on ice for 10 minutes. NP-40
was added to a final concentration of 1% and incubated for ten minutes
on ice. The nuclei were pelleted at 10,000 x g for 15 minutes. The
25 resulting supern~t~nt solution was incubated with proteinase K
(Boehringer ~nnheim, Indianopolis, IN) 100 ,ug/ml at 37~C overnight.
DNA was extracted twice with phenol and once with chloroform and
precipitated
EXAMPLE 2
In order to test the mutagenesis process of this invention,
the temperature sensitive mutation described by Preston et al.. 1983
J. Virol. 45:1056-1064, which is hereby incorporated by reference, was
35 made. This required the introduction of two amino acid changes. Four
CA 02222877 1997-11-28
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- 24 -
separate transfections were done. Vero or host range cell line PHS-23
were plated at approximately 3.0 x 105 cells per well in six well
clusters. This resulted in a cell density of 80-90% the following day.
The cells were washed in DMEM without FCS just prior to transfection.
S The transfection mix was then added to the cells and incubated for 1~
hours at 37~C. The cells were washed lX and 3 ml of DMEM with 4%
FCS was added. At day six or seven, plaques were observed and
recombinant virus was harvested. Each transfection gave rise to 50 or
more plaques five to six days post transfection. Recombinant virus was
10 titered on both Vero and PHS-23 cell lines. A minimllm of four plaques
were picked per transfection, all of the isolates grew and plaqued
similarly. Table 2 shows the titer of both wild type HSV-l (17) from
one of the temperature sensitive (ts) mutant isolates on Vero and PHS-23
cells at 31~C and 39~C.
Table 2. HSV-l Temperature Sensitive Protease Mutants V701 and
HSV-l titer on Host Range And Vero Cells
HostCell Line Virus Temp~C Titer (pfu)
Vero V701 31 6.2 x 105
Vero V701 39 1.2 x 102
Vero HSV-1(17) 31 1.0 x 107
Vero HSV-1(17) 39 2.1 x 107
PHS-23 V701 31 1.3 x 106
PHS-23 V701 39 1.0 x 106
PHS-23 HSV-1(17) 31 2.5 x 107
PHS-23 HSV-1(17) 39 1.0 x 107
CA 02222877 l997-ll-28
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: MERCK & CO., INC.
Register, Robert B.
Shafer, Jules A.
(ii) TITLE OF INVENTION: HERPES SIMPLEX TYPE 1 PROTEASE MUTANTS
AND VECTORS
(iii) NUMBER OF SEQUENCES: 40
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Ms. Joanne M. Giesser
(B) STREET: 126 East Lincoln Avenue, P.O. Box 2000-0907
(C) CITY: Rahway
(D) STATE: New Jersey
(E) COUNTRY: US
(F) ZIP: 07065-0907
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Giesser, Joanne M.
(B) REGISTRATION NUMBER: 32,838
(C) REFERENCE/DOCRET NUMBER: 19457 PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (908) 594-3046
(B) TELEFAX: (908) 594-4720
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1050 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
.
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
CA 02222877 l997-ll-28
WO 96138546 PCT/US96/07795
- 26 -
( ix ) FEATURE:
( A ) NAME /KEY: CDS
(B) LOCATION: 1. .1050
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
ATG GCA GCC GAT GCC CCG GGA GAC CGG ATG GAG GAG CCC CTG CCA GAC 48
Met Ala Ala Asp Ala Pro Gly Asp Arg Met Glu Glu Pro Leu Pro Asp
AGG GCC GTG CCC ATT TAC GTG GCT GGG TTT TTG GCC CTG TAT GAC AGC 96
Arg Ala Val Pro Ile Tyr Val Ala Gly Phe Leu Ala Leu Tyr Asp Ser
GGG GAC TCG GGC GAG TTG GCA TTG GAT CCG GAT ACG GTG CGT GCG GCC 144
Gly Asp Ser Gly Glu Leu Ala Leu Asp Pro Asp Thr Val Arg Ala Ala
35 40 45
CTG CCT CCG GAT AAC CCA CTC CCG ATT AAC GTG GAC CAC CGC GCT GGC 192
Leu Pro Pro Asp Asn Pro Leu Pro Ile Asn Val Asp His Arg Ala Gly
50 55 60
TGC GAG GTG GGG CGG GTG CTG GCC GTG GTC GAC GAC CCC CGC GGG CCG 240
Cys Glu Val Gly Arg Val Leu Ala Val Val Asp Asp Pro Arg Gly Pro
65 70 75 80
TTT TTT GTG GGA CTG ATC GCC TGC GTG CAA CTG GAG CGC GTC CTC GAG 2 88
Phe Phe Val Gly Leu Ile Ala Cys Val Gln Leu Glu Arg Val Leu Glu
85 90 95
ACG GCC GCC AGC GCT GCG ATT TTC GAG CGC CGC GGG CCG CCG CTC TCC 336
Thr Ala A1A Ser Ala Ala Ile Phe Glu Arg Arg Gly Pro Pro Leu Ser
100 105 110
CGG GAG GAG CGC CTG TTG TAC CTG ATC ACC AAC TAC CTG CCC TCG GTC 384
Arg Glu Glu Arg Leu Leu Tyr Leu Ile Thr Asn Tyr Leu Pro Ser Val
115 120 125
TCC CTG GCC ACA AAA CGC CTG GGG GGC GAG GCG CAC CCC GAT CGC ACG 43 2
Ser Leu Ala Thr Lys Arg Leu Gly Gly Glu Ala His Pro Asp Arg Thr
130 135 140
CTG TTC GCG CAC GTC GCG CTG TGC GCG ATC GGG CGG CGC CTC GGC ACT 4 80
Leu Phe Ala His Val Ala Leu Cys Ala Ile Gly Arg Arg Leu Gly Thr
145 150 155 160
ATC GTC ACC TAC GAC ACC GGT CTC GAC GCC GCC ATC GCG CCC TTT CGC ~ 528
Ile Val Thr Tyr Asp Thr Gly Leu Asp Ala Ala Ile Ala Pro Phe Arg
165 170 175
CAC CTG TCG CCG GCG TCT CGC GAG GGG GCG ~GG CGA CTG GCC GCC GAG 576
His Leu Ser Pro Ala Ser Arg Glu Gly Ala Arg Arg Leu Ala Ala Glu
180 185 190
CA 02222877 l997-ll-28
WO 96138546 PCTtUS96107795
- 27 -
GCC GAG CTC GCG CTG TCC GGA CGC ACC TGG GCG CCC GGC GTG GAG GCG 624
Ala Glu Leu Ala Leu Ser Gly Arg Thr Trp Ala Pro Gly Val Glu Ala
195 200 205
CTG ACC CAC ACG CTG CTT TCC ACC GCC GTT AAC AAC ATG ATG CTG CGG 672
Leu Thr His Thr Leu Leu Ser Thr Ala Val Asn Asn Met Met Leu Arg
210 215 220
GAC CGC TGG AGC CTG GTG GCC GAG CGG CGG CGG CAG GCC GGG ATC GCC 720
Asp Arg Trp Ser Leu Val Ala Glu Arg Arg Arg Gln Ala Gly Ile Ala
225 230 235 240
GGA CAC ACC TAC CTC CAG GCG AGC GAA AAA TTC AAA ATG TGG GGG GCG 768
Gly His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Met Trp Gly Ala
245 250 255
GAG CCT GTT TCC GCG CCG GCG CGC GGG TAT AAG AAC GGG GCC CCG GAG 816
Glu Pro Val Ser Ala Pro Ala Arg Gly Tyr Lys Asn Gly Ala Pro Glu
260 265 270
TCC ACG GAC ATA CCG CCC GGC TCG ATC GCT GCC GCG CCG CAG GGT GAC 864
Ser Thr Asp Ile Pro Pro Gly Ser Ile Ala Ala Ala Pro Gln Gly Asp
275 280 285
CGG TGC CCA ATC GTC CGT CAG CGC GGG GTC GCC TCG CCC CCG GTA CTG 912
Arg Cys Pro Ile Val Arg Gln Arg Gly Val Ala Ser Pro Pro Val Leu
290 295 300
CCC CCC ATG AAC CCC GTT CCG ACA TCG GGC ACC CCG GCC CCC GCG CCG 960
Pro Pro Met Asn Pro Val Pro Thr Ser Gly Thr Pro Ala Pro Ala Pro
305 310 315 320
CCC GGC GAC GGG AGC TAC CTG TGG ATC CCG GCC TCC CAT TAC AAC CAG 1008
Pro Gly Asp Gly Ser Tyr Leu Trp Ile Pro Ala Ser His Tyr Asn Gln
325 330 335
CTC GTC GCC GGC CAC GCC GCG CCC CAA CCC CAG CCG CAT TCC 1050
Leu Val Ala Gly His Ala Ala Pro Gln Pro Gln Pro His Ser
340 345 350
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 350 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ala Ala Asp Ala Pro Gly Asp Arg Met Glu Glu Pro Leu Pro Asp
1 5 10 15
CA 02222877 l997-ll-28
W O 96/38546 PCTrUS96/07795
~rg Ala Val Pro Ile Tyr Val Ala Gly Phe Leu Ala Leu Tyr Asp Ser
~ly Asp Ser Gly Glu Leu Ala Leu Asp Pro Asp Thr Val Arg Ala Ala
.
Leu Pro Pro Asp Asn Pro Leu Pro Ile Asn Val Asp His Arg Ala Gly
Cys Glu Val Gly Arg Val Leu Ala Val Val Asp Asp Pro Arg Gly Pro
~he Phe Val Gly Leu Ile Ala Cys Val Gln Leu Glu Arg Val Leu Glu
~hr Ala Ala Ser Ala Ala Ile Phe Glu Arg Arg Gly Pro Pro Leu Ser
100 105 110
Arg Glu Glu Arg Leu Leu Tyr Leu Ile Thr Asn Tyr Leu Pro Ser Val
115 120 125
Ser Leu Ala Thr Lys Arg Leu Gly Gly Glu Ala His Pro Asp Arg Thr
130 135 140
Leu Phe Ala His Val Ala Leu Cys Ala Ile Gly Arg Arg Leu Gly Thr
145 150 155 160
~le Val Thr Tyr Asp Thr Gly Leu Asp Ala Ala Ile Ala Pro Phe Arg
165 170 175
~is Leu Ser Pro Ala Ser Arg Glu Gly Ala Arg Arg Leu Ala Ala Glu
180 185 190
Ala Glu Leu Ala Leu Ser Gly Arg Thr Trp Ala Pro Gly Val Glu Ala
195 200 205
Leu Thr His Thr Leu Leu Ser Thr Ala Val Asn Asn Met Met Leu Arg
210 215 220
Asp Arg Trp Ser Leu Val Ala Glu Arg Arg Arg Gln Ala Gly Ile Ala
225 230 235 240
~ly His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Met Trp Gly Ala
245 250 255
~lu Pro Val Ser Ala Pro Ala Arg Gly Tyr Lys Asn Gly Ala Pro Glu
260 265 270
Ser Thr Asp Ile Pro Pro Gly Ser Ile Ala Ala Ala Pro Gln Gly Asp
275 280 285
Arg Cys Pro Ile Val Arg Gln Arg Gly Val Ala Ser Pro Pro Val Leu
290 295 .300
Pro Pro Met Asn Pro Val Pro Thr Ser Gly Thr Pro Ala Pro Ala Pro
305 310 315 320
CA 02222877 1997-11-28
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- 29 -
Pro Gly Asp Gly Ser Tyr Leu Trp Ile Pro Ala Ser His Tyr Asn Gln
325 330 335
Leu Val Ala Gly His Ala Ala Pro Gln Pro Gln Pro His Ser
340 345 350
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GAATGCN 7
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GTACTCAAAA GGTCATAC 18
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
CA 02222877 1997-11-28
W 096/38546 PCT~US96/07795
- 30 -
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GGGAAACCAA ACGCGGAATG 20
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GATACGGTGC GGGCAGTACT GCCTCCGGAT 30
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
ATCCGGAGGC AGTACTGCCC GCACCGTATC 30
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02222877 l997-ll-28
W 096J38546 PCTrUS96/07795
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
TTTTTGGCGC TCTTCGACAG CGGGGAC 27
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GTCCCCGCTG TCGAAGAGCG CCAAAAA 27
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
CATGATTAAT TA 12
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
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(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
AGCTTAATTA AT 12
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CCCACTCCCG ATTAACGTGG ACGTCCGCGC TGGCTGCGAG GTG 43
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
CCTCGCAGCC AGCGCGGACG TCCACGTTAA TCGGGAGTGG G 41
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
CCCCGATCGC ACGCTGTTCG CTGCAGTCGC GCTGTGCGCG ATCGGGCGG 49
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) AWTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GATCGCGCAC AGCGCGACTG CAGCGAACAG CGTGCGATCG GGG 43
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
CACCAACTAC CTGCCCTCGG TCGCGCTAGC CACAAAACGC CTGGGGGG 48
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(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
CAGGCGTTTT GTGGCTAGCG CGACCGAGGG CAGGTAGTTG 40
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
CCAACTACCT GCCCTCGGTC GCCCTGGCCA CAAAACGCCT GGGG 44
(2) INFORMATION FOR SEQ ID NO:l9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:lg:
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GCCAGGGCGA CCGAGGG 17
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
CCCACTCCCG ATTAACGTGG ACTACCGCGC TGGCTGCGAG GTG 43
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
CGCGGTAGTC CACGTTA 17
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
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- 36 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
CCCCGATCGC ACGCTGTTCG CGTACGTCGC GCTGTGCGCG ATCGG 45
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single - -
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GCGACGTACG CGAACAGC 18
(2) INFORMATION FOR SEQ ID No:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
CACCAACTAC GTGCCCTCGG TCTCCCTG . 28
(2) INFORMATION FOR SEQ ID No:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
CCGAGGGCAC GTA~ll~lG ATCAGG 26
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B~ TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
CAACTACCTG GGCTCGGTCT CCCTGGCC 28
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 5 ingle
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
GAGACCGAGC CCAGGTAGTT GGTGATCAG 29
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
~ (D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
CGCTGTTCGC ACGCGTCGCG CTGTGCGCGA TCG 33
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS: ~ :
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
CAGCGCGACG CGTGCGAACA GCGTGCGATC GGG 33
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
CTGTTCGCGG AAGTAGCGCT GTGCGCGATC GG -- 32
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 5 ingle
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
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- 39 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
CGCACAGCGC TACTTCCGCG AACAGCGTGC GATCGGG 37
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
CGCTGTTCGC CAAGGTCGCG CTGTGCGCGA TCG 33
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
CACAGCGCGA CCTTGGCGAA CAGCGTGCGA TCGGG 35
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
~ (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
CCGATTAACG TTGACGCCCG CGCTGGCTGC GAGGTGGG 38
(2) INFORMATION FOR SEQ ID No:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
CAGCCAGCGC GGGCGTCAAC GTTAATCGGG AGTGGG 36
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
CCTGCCCTCG GTAAGCTTGG CCACAAAACG CCTGG 35
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
GGC~llll~l GGCCAAGCTT ACCGAGGGCA GGTAG 35
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
Asp A1A Pro Gly Asp Arg Met Glu Glu Pro Leu Pro Asp Arg Ala Cys
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNES5: single
(D) TOPOLOGY: linear
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
CGTATCCGGA TCCAATC 17
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(iii) HYPOTHETICAL: NO
(iv) AWTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
GTTAACAACA TGATGCTG = 18
