Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 95120659 PCTIAU95100043
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TITLE
"MODIFIED PAPILLOMA VIRUS L2 PROTEIN AND
VLPs FORMED THEREFROM"
FIELD OF INVENTION
THIS INVENTION relates to a modified papilloma virus L2
' protein and VLPs formed therefrom, in particular, antigens and
vaccines containing said VLPs that may be effective in treatment of
infections caused by such viruses.
PRIOR ART
Papilloma viruses (PV) infect both humans and animals
(see for review "Papilioma Virus Infections in Animals" by J. P.
Sundberg which is described in Papilloma Viruses and Human Disease,
edited by K. Syrjanen, L. Gissmann and L. G. Koss, Springer-Verlag
1987). Human papilloma viruses are a family of small DNA viruses
which induce benign hyperproliferative lesions of the cutaneous and
mucosal epithelia. Of the 70 different virus types which have been
identified, more than 20 are associated with anogenital lesions (de
Villiers, 1989. J. Virol. 63 4898-49031.
In particular, HPV 16 is associated with pre-malignant and
malignant diseases of the genito-urinary tract, and in particular, with
carcinoma of the cervix (Durst et al., 1983. P.N.A.S. 80 3812-3815;
Gissmann et al., 1984. J. Invest. Dermatol. 83 265-285). The
detection of antibodies against HPV 16 fusion proteins (Jenison et al.,
1990. J. Virol. 65 1208-1218; Kochel et al., 1991. Int. J. Cancer 48
682-688) and synthetic HPV16L1 peptides (Dillner et al., 1990. Int.
J. Cancer 45 529-535) in the serum of patients with HPV 16 infection
confirms that there are B epitopes within the capsid proteins of HPV,
. though few patients have HPV 16L1-specific antibodies identified by
these techniques. There is no system for PV propagation in vitro, and
human genital lesions associated with HPV 16 infection contain few
PV particles and low levels of viral structural proteins. Thus further
studies on papilloma viruses have been limited.
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PV capsids comprise two virally encoded structural
proteins, designated L 1 and L2, which are assembled onto a DNA-
protein complex (Galloway et al., 1989. Adv. Virus Res. 37 125-171 ).
A single virus capsid is a T=7d icosahedron composed of 72
pentameric capsomeres, each of which contains five molecules of the
major capsid protein, L1 (Baker et al., 1991. Biophys. J. 60 1445-
1456; Finch et al., 1965. J. Mol. Biol. 13 1-12). The minor capsid
protein, L2, is present at approximately 1/10 the abundance of L1
(Doorbar et al., 1987. J. Virol. 61 2793-2799) and has an unknown
structural role. L1 protein is directed to the nucleus by a C-terminal
nuclear localization signal (Zhou et al., 1991. Virology 185 625-632);
virus assembly occurs in the nucleus (Orth et al., 1977. J. Virol. 24
108-120; Pfister et al., 1987. Papilloma viruses: particles, genome
organisation and proteins, p. 1-18 In K. Syrjanen, L. Gissmann, and L.
G. Koss (ed.), Papilloma viruses and human disease. Springer-Verlag
KG, Berlin). Recombinant L1 protein self-assembles into particles
resembling virus capsids (Zhou et al., 1993. J. Gen. Virol. 74 763-
7681, but assembly is enhanced in the presence of L2 protein, which
may be required for assembly of infectious virions (Hagensee et al.,
1993. J. Virol. 67 315-322; Zhou et al., 1991. Virology 185 251-
257).
Recombinant PV L 1 protein and the combination of
recombinant PV L1 and PV L2 proteins have formed the basis of
vaccines for the prevention and treatment of papilloma virus infections
and used as antigens for the detection of papilloma virus llnternational
Patent Application Publication No. W093/02184). The presence of
the PV L2 protein increases the immunogenicity of the vaccine (Zhou
et al., 1991. Virology 185 251-257).
Subsequent to International Publication No.
W093/02184 and the published research by Zhou and others (Zhou et
al., 1991, Virology 185 251-257), other workers have developed
expression systems for the expression of human papilloma virus VLPs.
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International Patent Application Publication No. W094/20137 is directed to
the expression of the L1 protein of human paptilloma virus and the production
of VLPs in Sf-9 insect cells using a baculovirus expression system.
As well, the formation of capsids ft>Ilowing expression of L1 and
L2 in mammalian cells and insect cells were disclosed in two research articles
(Hagensee et al.. 1993, J. Virology 67 315 and Kirnbauer et al., 1992, PNAS
89 12180). Two other International Patent Application Publications, namely
W094/00152 anti W094I05792, were directed to recombinant papilloma virus
L1 proteins and their use as vaccines and for diagnostic purposes.
One problem, however, with VLP formation which includes PV
L2 protein is the incorporation of DNA into the capsid. The inclusion of DNA
in the capsid of papilloma virus VLP is not desirable for the vaccines
comprising these VLPs. Indeed, in some countries, there are legislative
requirements limiting the amount of DNA allowed in a vaccine shot. The level
of 10 picograms of DNA per shot has been typically used as an upper limit.
This requirement may k~e due in part to fears of infection from introducing
foreign DNA into a healthy individual. The concern with vaccines comprising
native L2 protein is that amounts of DNA exceeding this level may be
included.
S~JMMARY OF THE INVENTION
Thus it is an object ~of an aspect of the present invention to
provide a virus-like particle that incorporates a substantially minimal amount
of DNA.
A turther object is to provide a vaccine which overcomes the
aforementioned problem.
Tht~ invention, therefore, in one aspect, includes a method for
production of one or more papilloma virus-like particles (VLPs) which
incorporates a substantially minimal amount of DNA including the steps of:-
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( 1 ) constructing a recombinant DNA molecule which
encodes a papilloma virus L2 protein that binds a
substantially minimal amount of DNA; and
(2) introducing said recombinant DNA molecule into a
suitable host cell so that a papilloma virus L 1
protein and said papilloma virus L2 protein is
expressed and said VLPs are formed therefrom.
The term "a substantially minimal amount of DNA"
covers the situation where essentially no DNA is bound by the PV L2
protein or where there is 10 picograms of DNA or less per vaccine
shot or other DNA limit set by legislation.
It will be appreciated that a second aspect of the
invention lies in the recombinant DNA molecule which encodes said
papilloma virus L2 protein.
Further, another aspect of the invention resides in said
papilloma virus L2 protein. The L2 protein is preferably modified so
that any one or more of the 1-12 amino acid residues adjacent the N-
terminal end of the L2 protein are different compared to the wild type
L2 protein.
Most preferably the invention includes within its scope
L2 mutants) shown hereinafter in FIG. 4.
In another aspect, the present invention resides in the
novel papilloma virus VLP formed from the said papilloma virus L1 and
L2 proteins. The L2 protein forming the VLP preferably has a minimal
number of amino acid modifications
The invention, in another aspect, includes a vaccine
containing the papilloma virus VLPs with or without a suitable
adjuvant.
In relation to step ( 1 ), the recombinant DNA molecules
are suitably constructed from a source of papilloma virus genome
whereby the L2 gene may be amplified by PCR amplification using
suitably designed primers. The recombinant DNA molecules are
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preferably amplified from a suitable plasmid containing the PV genome
or part thereof. The preferable genome is HPV16 genome.
Preferably primers include those that change one or more
of the bases 1-36 from the 5' end of the L2 gene. Changes are
preferably by deletion or substitution. A list of the most preferable
primers for amplification are listed below (see FIGS. 6, 7 and 8). The
L1 and L2 genes may be transcribed from any mammalian or viral
promoter with a mammalian or viral polyadenylation signal. Preferably
the L1 and L2 genes are transcribed from any vaccinia virus promoter
which may be an early promoter or a late promoter as considered
appropriate. A list of such promoters is given in Davidson & Moss,
1989, J. Mol. Biol. 210 749-769 and 1989, J. Mol. Biol. 210 771-
784. A suitable promoter from which to initiate transcription of the
L2 gene is the vaccinia virus late promoter 4b.
The L1 and L2 genes may be encoded on separate
vectors or on the same vector. Suitable vectors include plasmids,
cosmids and recombinant viruses. Preferably the recombinant DNA
molecules are contained in one or more recombinant viruses which
may transfect those cells. Suitable viruses that may be used for this
purpose include baculovirus, vaccinia, sindbis virus, SV40, Sendai
virus adenovirus, retrovirus and poxviruses. Suitable host cells may
include host cells that are compatible with the above viruses and
these include insect cells such as Spodoptera frugiperda, CHO cells,
chicken embryo fibroblasts, BHK cells, human SW13 cells, drosophila,
mosquito cells derived from Aedes albopictus or monkey epithelial
cells. It will also be appreciated that other eukaryote cells may
comprise yeast cells or other mammalian cells.
Suitable expression systems include prokaryotic
expression systems including E. coli and any plasmid or cosmid
expression vector or eukaryotic systems including host cells described
above in combination with a recombinant virus vector or alternatively,
yeast cells and yeast plasmids.
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The VLPs may be obtained from the transfected cells by any
suitable means of purification. A preferable method of production and
purification of papilloma virus-like particles is provided in W093102184. The
VLPs may be combined with any suitable adjuvant such as ISCOMS, alum,
Freunds Incomplete or Complete Adjuvant,, Quil A and other saponins or any
other adjuvant as described for example in Vanselow, 1987, S. Vet. Bull. 57
881-896.
According to one aspect of the invention, there is provided a
protein consisting of L2 protein which does not bind DNA or which has an
impaired ability to bind DNA compared to wild-type papilloma virus L2 protein.
According to another aspect of the invention, there is provided a
papilloma virus L2 protein comprising a modified papilloma virus L2 amino acid
sequence which does not bind DNA or which as an impaired ability to bind DNA
compared to wild-type papilloma virus L2 protein, wherein said papilloma virus
L2 protein comprises an intact C-terminal region and a modified or deleted N-
terminal sequence compared to wild-type papilloma virus L2 protein.
According to a further aspect of the invention, there is provided a
papilloma virus L2 protein which does not bind DNA or which has an impaired
ability to bind DNA compared to wild-type papilloma virus L2 protein, wherein
said L2 protein is capable of binding papilloma virus L1 protein to form a
virus-
like particle.
According to another aspect of the invention, there is provided a
papilloma virus L2 protein which does not bind DNA or which has an impaired
ability to bind DNA compared to wild-type papilloma virus L2 protein, wherein
said L2 protein is not a fusion protein.
According to a further aspect of the invention, there is provided a
method of producing one or more virus-like particles comprising a papilloma
virus L1 protein and a papilloma virus L2 protein, wherein said papilloma
virus L2
protein does not bind DNA or has an impaired ability to bind DNA compared to
wild-type papilloma virus L2 protein, said method comprising the steps of:
(1 ) constructing a recombinant DNA molecule comprising a
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6a
DNA sequence encoding said papilloma virus L2 protein wherein said
sequence is operably linked to a promoter;
(2) introducing said recombinant DNA molecule into a
suitable host cell;
(3) expressing said papilloma virus L2 protein in said host
cell in the presence of said papilloma virus L1 protein to form one or more
virus-like particles; and
(4) recovering said one or more virus-like particles from said
cell.
According to another aspect of the present invention, there is
provided a papilloma virus L2 protein which does not bind DNA or which has
an impaired ability to bind DNA compared to wild-type papilloma virus L2
protein, wherein said L2 protein is capable of binding papilloma virus L1
protein to form a virus-like particle and said papilloma virus L2 protein
comprises one or more amino acid residues in the N-terminal region that are
modified or deleted as compared to wild-type papilloma virus L2 protein.
According to a further aspect of the present invention, there is
provided a papilloma virus L2 protein which does not bind DNA or which has
an impaired ability to bind DNA compared to wild-type papilloma virus L2
protein, wherein said L2 protein is not a fusion protein and said papilloma
virus L2 protein comprises one or more amino acid residues in the N-terminal
region that are modified or deleted as compared to wild-type papilloma virus
L2 protein.
According to another aspect of the present invention, there is
provided a method of producing one or more virus-like particles comprising a
papilloma virus L1 protein and a papilloma virus L2 protein, wherein said
papilloma virus L2 protein does not bind DNA or has an impaired ability to
bind DNA compared to wild-type papilloma virus L2 protein, and said
papilloma virus L2 protein comprises one or more amino acid residues in the
N-terminal region which are modified or deleted compared to wild-type
papilloma virus L2 and said method comprising the steps of:
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6b
(1 ) constructing a recombinant DNA molecule comprising a
DNA sequence encoding said papilloma virus L2 protein wherein said sequence
is operably linked to a promoter;
(2) introducing said recombinant DNA molecule into a suitable
host cell;
(3) expressing said papilloma virus L2 protein in said host cell
in the presence of said papilloma virus L1 protein to form one or more virus-
like
particles; and
(4) recovering said one or more virus-like particles from said
cell.
Reference may now be made to various preferred embodiments of
the invention as illustrated. In these preferred embodiments, it should be
noted
that the specific papilloma viruses, VLPs and specific constructs of DNA
recombinant molecules are given by way of example.
EXPERIMENTAL
1. CONSTRUCTION OF A MODIFIED PAPILLOMA VIRUS L2 PROTEIN
Materials and Methods
Plasmid construction: For expression in VV, the open reading
frame corresponding to the 474-amino-acid HPV16L2 coding region was
amplified by PCR from a plasmid containing the HPV16 genome. The 5' primer
introduced a BamHl site upstream from the L2 open reading frame ATG and the
3' primer introduced a Smal sited beyond the termination codon. The amplified
L2 fragment was recovered by elution from an agarose gel, cut with BamHl and
Smal, and ligated into RK19 (Kent, 1988. Ph.D. thesis. University of
Cambridge,
Cambridge, England), creating RK19/16L2, in which L2 expression is driven by
the VV late promoter 4b. The HPV16L2 and 4b promoter were then transferred
to the VV expression vector pSX3 (Zhou et al., 1991. Virology 185 251-257) for
rVV construction.
To create the simplified vector pUC18/4b16L2 and facilitate
transfer of mutant L2 genes between vectors used for VV
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expression, a Klenow-blunted Mlul-EcoRl fragment carrying the VV 4b
promoter and the whole HPV 16L2 open reading frame was cleaved
from RK 19116L2 and inserted into pUC 18. This plasmid was used as
the DNA template for PCR amplifications with primers designed to
create C-terminal truncations of L2. Mutants e374, e384, e394,
e404, and e414, C-terminally truncated to residues 374, 384, 394,
404, and 414 of the L2 protein, respectively, were created by using a
common 5' primer fMI3RSP) (Zhou et al., 1991. Virology 185 625-
632) and a panel of 3' primers introducing stop codons (TAA) at
codons 374, 384, 394, 404, and 414. To create N-terminal
;' truncation and point mutations, we used a panel of 5' primers. N
terminal deletions let-2, e1-3, e1-4, e1-5, e1-6, e1-7, e1-8, e1-9,
e1-10, e1-11, e1-12, e1-13, e1-14, e1-15, e1-20, e1-40, e1-60,
e1-80, and e1-100) were created by using the set of primers which
introduced ATG codons at positions corresponding to amino acids 2,
3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 20, 40, 60, 80, and 100,
respectively. For N-terminal point mutations (designated H3P; K4P;
2,4,5,N; RSP; S6N; S6P: A7P; KBP; R9P; and 8,9N), amino acids 3,
4, 5, 6, 7, 8, and 9 were changed to either proline (Pro) or asparagine
(Asn) by the mismatched-primer method (Zhou et al., 1991. Virology
185 625-632). All mutations were confirmed by direct sequencing of
the expression plasmids.
Cells and virus. CV-1 cells were maintained in
Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10%
fetal or newborn bovine serum ~(CSL, Melbourne, Australia). Plaque
purified isolates of rVVs were propagated in CV-1 cells grown in
Dulbecco's modified Eagle's medium supplemented with 2.5% fetal
bovine serum (CSL, Melbourne, Australia).
rVV construction. We used previously described
methods (Zhou et al., 1991. Virology 185 251-257) for rVV
construction. Briefly, plasmids including the HPV 16L2 gene with
various mutations driven from the VV late promoter 4b, the
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Escherichia coli gpt gene (Cougar ef al., 1988. Gene 68 1-10; Falkner et al.,
1988. J.
Virol. 62 1849-1854) as a selectable marker, and flanking fragments of the W
B24R
gene (Kotwal et al., 1989. J. Virol. 63 600-606; Smith et aG, 1989. J. Gen.
Virol. 70 233-
2343) or thymidine kinase (TK) gene were transfected into W WR strain-infected
(0.05
PFU per cell) CV-1 cells by calcium phosphate precipitation. Virus plaques
were purified
twice in CV-1 cells in the presence of mycophenolic acid at a concentration of
25 pglml.
Immunoprecipitation of L1 and L2 proteins. CV-1 cells were infected with
HPV16L1 rW or HPV16L2 rW at a multiplicity of infection of about 20 PFU per
cell. At
48 h, 5 x 105 infected cells were lysed with RIPA buffer (150 mM NaCI, 1 %
Nonidet T"'' P
40, 0.5% deoxycholate, 0.1 % sodium dodecyl sulfate [SDS), 50 mM Tris [pH
8.0)). Lysed
cells were centrifuged briefly at 12,000 x g, and the supernatant was used for
immunopreciptation. Immunoprecipitation were carried out with a 1:20 dilution
of
monoclonal anti-HPV16L1 antibody (McLean et al., 1990. J. Clin. Pathol. 43 488-
492) or
a 1:2,000 dilution of rabbit anti-HPV16L2 antibody (provided by D. A.
Galloway). The
precipitated L1 or L2 protein was collected with protein A-Sepharose beads T""
and
washed four times in RIPA buffer. Proteins were removed from protein A-
Sepharose T""
beads by boiling in polyacrylamide gel electrophoresis (PAGE) sample buffer
and
separated by SDS-PAGE for analysis.
Southwestern assays. The Southwestern (DNA-protein) assays were based on
previously published procedures (McCall et al., 1991. J. Invest. Dermatol. 97
111-114;
Moreland et al., 1991. J. Virol. 65 1168-1176). Immunoprecipitated HPV16L1 and
HPV16L2 proteins were separated on a SDS-10% polyacrylamide gel and
transferred to
a nitrocellulose filter by electroblotting. Filters were blocked with blocking
buffer (10 mM
Tris [pH 7.5], 5% nonfat skim milk, 10% glycerol, 2.5% Nonidet T"" P-40, 0.1
mM
dithiothreitol [DTT], 150 mM NaCI) at 4°C for 12 h. The filters were
then washed with
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binding buffer ( 10 mM Tris (pH 7.5], 40 mM NaCI, 1 mM EDTA, 1
mM DTT, 8°i° glycerol, 0.125°r° skim milk). 32P-
labelled probes were
added to binding buffer, and incubation was continued for 4 h at 4°C.
The filters were washed with five changes of binding buffer. After
being air dried, they were wrapped and exposed to X-ray films. They
were subsequently reprobed with anti-HPV16L1 or anti-HPV16L2 anti-
serum and '251-protein A to confirm protein transfer.
Binding specificity assay. DNA sequences binding
specifically to L2 were selected from a pool of double-stranded, 76-
mer oligonucleotides (R76) containing a central stretch of 26 random
base pairs, flanked by two unique sequences of 25 by each (Sorger et
al., 1986. J. Mol. Biol. 191 639-658). The sequences of these
oligonucleotides were:-
R76
5'-CAGGTCAGATCAGCGGATCCTGTCG (N)2s
GAGGCGAATTCAGTGCATGTGCAGC-3'
Forward primer 5'-GCTGCACATGCACTGAATTCGCCTC-3'
Back primer 5'-CAGGTCAGATCAGCGGATCCTGTCG-3'
Random oligonucleotide-binding assays were performed
essentially as described previously (Treacy et al., 1991. Nature 350
577-584). Briefly, L1 and L2 proteins purified by immunoprecipitation
were electrophoresed on an SDS-PAGE gel ( 10% polyacrylamide) and
transferred to a nitrocellulose filter. Filters were incubated for 4 h at
4°C in buffer A (20 mM N-2-hydroxyethylpiperazine-N'-2-
ethanesulfonic acid (HEPES]-HCI [pH 7.9), 1 mM DTT, 10% glycerol,
0.01 % Nonidet P-40) containing 50 mM KCI and 5% skim milk. The
s2P-labelled pool of random oligonucleotides was prepared by primed
synthesis with the forward primer annealed to the random 76-mer
oligonucleotide template, added to the filter, and incubated overnight
at 4°C. Washes were performed at 4°C in buffer A adjusted to 100
mM KCl (two 10-min washes) and then in buffer A adjusted to 200
mM KCI (one 10-min wash). Filters were autoradiographed, the area
CA 02182272 2004-12-15
of the filter corresponding to bound DNA was excised, and the DNA was eluted
by
heating at 100°C in water. Eluted DNA was amplified by 30 cycles of PCR
with forvvard
and backward primers and purified on 2% agarose gels. For subsequent rounds of
binding, the 76-by product was labelled by 18 cycles of PCR in the presence of
32P-
5 labelled nucleotide, as described previously (Sorger et al., 1986. J. Mol.
Biol. 191 639-
658). DNA from the fifth round of selection which remained bound to L2 after
washing in
buffer A was eluted and amplified as described above, cloned into pUClB, and
sequenced.
Immuno-DNA binding assay. Extracts from HPV16L2 rW-infected cells were
10 prepared and immunoprecipitated as described above. Immune complexes
attached to
protein A-SepharoseTM beads were incubated with BamHl-Pstl-digested HPV16 DNA
for
2 h at 4°C in DNA-binding buffer (10 mM Tris [pH 7.4], 100 mM NaCI,1 mM
MgCl2, 1 mM
EDTA, 8% glycerol, 1 mM DTT, 5% skim milk). Following incubation, the beads
were
washed five times with the same buffer at room temperature. Protein-DNA
complexes
were eluted in 1 % SDS-5 mM EDTA at 65°C. DNA was extracted with phenol
twice and
precipitated with ethanol. Samples were run on a 1.5% agarose gel and blotted
onto
nylon membranes. The DNA bound to the membranes were detected by Southern
blotting with 32P-labelled HPV16 DNA digested with Pstl-BamHl.
DNA sequencing. Dideoxy DNA sequencing was performed with the Sequenase
TM version 2.0 kit (U.S. Biochemicals, Cleveland, Ohio). To denature double-
stranded
DNA for sequencing, we incubated 2 pg of DNA for 30 min at 37°C in 200
mM sodium
hydroxide. DNA was ethanol precipitated and sequencing carried out as
specified by the
manufacturer.
RESULTS
HPV16L2 protein binds DNA. To investigate the DNA-binding activity of PV
structural proteins, we expressed HPV16L1 or HPV16L2 in eukaryotic cells by
using rW.
L1 and L2 proteins were
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purified from cell lysates by immunoprecipitation, separated by SDS-
PAGE ( 10°io polyacrylamide), and transferred to nitrocellulose
filters.
Binding of these proteins to DNA was investigated by Southwestern
blotting with 31P-labelled PV genomic DNA and bacteriophage ~1 DNA
in a buffer containing 40 mM NaCI. HPV 16L2 protein bound both
HPV genomic DNA and ~i DNA (FIG. 1B, lanes 2 to 5). HPV16L1
protein, in contrast, failed to bind any labelled DNA (FIG. 1 A, lanes 2
to 5), although the purified L1 protein was detectable on the filters by
using an anti-HPV16L1 monoclonal antibody (FIG. 1A lanes 1 and 6).
Further experiments in buffers containing up to 150 mM NaCI showed
no binding of DNA to HPV16L1 (data not shownl. These results
suggest that HPV 16L2 protein, but not HPV 16L 1 protein, contains
DNA-binding sequences and that the recognition of DNA by HPV 16L2
may not be sequence specific.
HPV 16L2 N terminus is important for DNA binding. To
identify the protein sequence responsible for binding of L2 to DNA,
we first checked for DNA-binding motifs in the predicted amino acid
sequence of HPV16L2. HPV16L2 has two highly charged regions,
rich in lysine and arginine. The first makes up the N terminus of the
protein (MRHKRSAKRTKR) from amino acids 1 to 12; the second lies
within the C terminus (RKRRKR) from amino acids 456 to 461.
To determine whether either charged region was involved ;
in DNA binding to L2, we made a series of deletion mutants with
mutations in HPV 16L2. Constructs encoding various C-terminal or N-
terminal mutations of HPV 16L2 protein were inserted into plasmid
pSX3, which had been previously shown to efficiently direct the
synthesis of the HPV16L1 proteins in rVVs (Zhou et al., 1991.
Virology 185 625-6321. CV-1 cells were infected with rVV containing
each mutant L2 gene, and cell lysate was analyzed by
immunoprecipitation with a rabbit anti-HPV16L2 antibody. The
expected relative size of each mutated protein was confirmed by
comparing the electrophoretic mobility of truncated proteins with wild-
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type L2 protein (data not shown).
The deletion mutants were examined for binding to 3ZP-
labelled HPV 16 genomic DNA by the Southwestern procedure under
conditions associated with binding of native L2 protein. All C-terminal
deletion mutants tested, including e374, which had the longest
deletion, bound HPV DNA in proportion to the amount of
immunoreactive L2 protein present (FIG. 2A, lane e374). To delineate
the contribution of the N terminus of L2 to DNA binding, we
constructed N-terminal deletion mutants of L2. Three mutants, in
which the first 60, 80 and 100 amino acids of L2 were deleted, each
failed to bind HPV DNA (FIG. 2A, lanes e1-60 through e1-100). To
further characterize the N-terminal DNA-binding region, we tested a
series of smaller deletions between amino acids 1 and 15 for their
DNA-binding activity. Each of these, including the smallest deletion
(which was missing only the arginine residue at position 2), failed to
bind DNA (FIG. 3A, lanes e1-2 to e1-15). These results suggested
that critical DNA-binding sequences were in the N terminus of L2 and
that binding was dependent on charged amino acids including the
arginine at position 2.
L2 protein uses an arginine-rich motif for DNA binding.
The amino acid sequence of the N terminus for HPV 16L2, starting
from position 1, is MRHKRSAKRTKR (one-letter code with charged
amino acids underlined). The role of the N terminus of L2 protein in
DNA binding was further assessed by site-specific mutagenesis. Ten
substitution mutants with mutations of the first 9 amino acids were
expressed by rVV, and their DNA-binding activities were examined by
Southwestern blotting with 3zP-labelled HPV 16 genomic DNA. Similar
amounts of the various mutant L2 proteins constructed were available
for DNA binding, as determined by analysis of immunoreactive L2
protein on the blots, with the exception of K8P (FIG. 3B, lower panel).
Mutation of some charged amino acid residues (Lys-4, Lys-8, Arg-9)
to Pro or Asn (K4P and 8,9N) abolished DNA binding (FIG. 3B, lanes
SUBSTfTUTE SHEET (RULE 26)
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K4P and 8,9N1, while substitution of Arg-5 with Pro (R5P) reduced
binding activity (lane R5P). In contrast, substitution of Arg-9 with Pro
IR9P) had no effect on DNA binding (lane R9P). Mutation of the
neutral amino acids between the Lys-Arg clusters had less effect on
DNA-binding activity. Mutations termined H3P, S6P, and A7P, in
which substitution of His-3, Ser-6, and Ala-7 for Pro had been
produced, showed binding of DNA comparable to that with wild-type
L2 (lanes H3P, S6P, and A7P1; in contrast, changing Ser-6 to Asn
(S6N) abolished DNA binding (lane S6N). These results suggest that
the four charged amino acid clusters are important for DNA binding.
,' In each of these charged amino acid clusters, retention of at least one
charged amino acid appears necessary for DNA binding. A flexible
secondary structure might also be important for L2-DNA interaction
because substitution of Ser-6 with Pro and of Arg-5 and Arg-9 did not
abolish the DNA binding, whereas the substitution of Ser-6 with Asn
removed the L2 DNA-binding function. A summary of L2-DNA
interaction results is given in FIG. 4.
L2-DNA interaction has no DNA sequence specificity.
We used a library of oligonucleotides and bound these to purified
HPV16L2 protein to select for any high-affinity target DNA
sequences. The oligonucleotides were random at 26 positions and
were flanked by primer and cloning sequences. After incubation of L2
with a pool of these oligonucleotides, oligonucleotides bound to L2
protein were eluted and amplified by PCR for subsequent rounds of
selection. Selection and amplification were carried out six times.
DNA clones recovered after the selection rounds 5 and 6 were
sequenced. Probability theory predicts that among 52 random 26-mer
sequences, any trinucleotide would be found in 20 clones (95°ro
confidence interval, 15 to 26) and any specified sequence of 4
nucleotides would be found in 5 of the 26-mers (95% confidence
interval, 0 to 9). Among 52 26-mer clones were observed that the
most commonly observed trinucleotide (GGG) was present in 24
WO 95120659 PCT/AU95/00043
14
clones (twice in 5, three times in 1 ), whereas the most common
series of 4bp (GGGG) was observed in 8 clones. Thus there was no
evidence that any short nucleotide sequence was represented among
these clones more frequently than would be expected by chance
alone. Further, no more complex conserved nucleotide patterns were
observed by using standard sequence alignment programs. These
results suggested that high-affinity binding between L2 and DNA is a
DNA sequence-independent process. Two additional experiments
confirmed this observation. First, extract from cells infected with
HPV16L2 rVV was immunoprecipitated with anti-L2 antibody, and the
precipitated protein-antibody complexes, attached to Sepharose
beads, were allowed to bind to a mixture of restriction fragments from
HPV 16 genomic DNA. After unbound DNA was washed away,
bound DNA fragments were resolved on an agarose gel and detected
by Southern blotting with 32P-labelled HPV 16 DNA. An HPV 16L2-
containing extract, bound to Sepharose beads with anti-L2 antibody,
retained each of the HPV16 DNA fragments (FIG. 5A, lanes 3, 6, and
7), whereas none of these fragments were bound by an L1 rVV-
infected cell extract bound to the bead with anti-L1 antibody (data not
shown) or by wild-type VV-infected cell extract bound to the beads
with L2-specific antibodies (FIG. 5A, lane 2). Second, a fixed amount
of L2 protein was incubated with 32P-labelled HPV DNA fragments, in
the presence of a 100-fold (lane 4) or 1,000-fold (lane 5) excess of
unlabelled ~I DNA, and L2, together with any bound DNA, was
immunoprecipitated from the mixture by antibody to L2. Phage ~1
DNA was able to prevent binding of HPV DNA. We conclude that L2
does not interact with DNA in a DNA sequence-specific manner by
each of these criteria.
2. PRODUCTION OF VIRUS-LIKE PARTICLES WITH MODIFIED
PAPILLOMA VIRUS L2 PROTEIN
Virus-like particles formed with the modified papilloma
virus L2 protein can be produced by the procedures outlined in
SUBSTITUTE SHEET (RULE 2~1
WO 95/20659 PCTlAU95/00043
15 21822?2
W093/02184. A suitable method for the production of virus-like
particles is given below by way of example.
CV-1 cells were grown under standard cell culture
conditions at 37 ° C in an atmosphere of 5 % COZ to a 80% confluency
in Dulbecco's modified Eagle's medium (Gibco or CSL) supplemented
wth 10% foetal calf serum (CSL) in a tissue culture flask. Cells were
then infected with 1 to 2 pfu/cell of recombinant vaccinia virus 16L 1
(pSXl6L,) and recombinant vaccinia virus 16L2 07 (Rkgptl9 n1-7) in
Dulbecco's modified Eagle's medium supplemented with 2.5% foetal
calf serum. Mycophenolic acid was added to the medium at a final
concentration of 25 Ng/ml. The cell culture was incubated for a
further 48-60 hours. Cells were then scraped from the tissue culture
flask and pelleted by centrifugation 1,500 x g at 4°C for 10 mins.
The pellet was dissolved and the cells were resuspended in 20 ml of
phosphate buffered saline (pH 7.4) containing 2 mM PMSF (protease
inhibitor). The cell suspension was stored at 4°C or frozen at -
20°C
when the purification procedure was interrupted.
The cell suspension was then placed in a Wheaton glass
Dounce homogeniser and kept on ice for 10 mins. Cells were
disrupted by 50 strokes of the Dounce homogeniser. A small sample
of the resuspension was checked by microscopy to determine
whether the cells were disrupted. Homogenization was continued
until all the cells were disrupted.
The lysate was centrifuged at 1500 x g for 10 mins at
4°C. The cloudy supernatant was discarded and the pellet was
resuspended by aspiration in 20 ml of phosphate buffered saline (pH
7.4) containing 2 mM PMSF. The resuspended material was then
sonicated (Vibra Celi Sonicator from Sonics Materials Inc. USA,
setting 80) for 30 sec on ice in order to release viral particles from the
nuclei. The sonicate was diluted to 60 m1 with phosphate buffered
(pH 7.4) saline containing 2 mM PMSF.
In each of four 38 ml ultra clear centrifuge tubes, 15 ml
CA 02182272 2004-12-15
16
of resuspended ice cold sonicate was layered on top of 23 ml of ice cold 20%
w/v
sucrose in phosphate buffered saline (pH 7.4). The sonicate was centrifuged at
95000 x
g (rotor midpoint) for 2 hrs at 4°C in a SW-28 rotor. The supernatant
and sucrose were
discarded and the pellet was washed with phosphate buffered saline (pH 7.4).
The pellet
was resuspended in 10 ml of phosphate buffered saline (pH 7.4) containing 2 mM
PMSF
by sonication (Vibra T"" Cell Sonicator from Sonics Materials Inc USA, setting
80) for 60
sec on ice. The resuspension was diluted to 20 ml with phosphate buffered
saline (pH
7.4) containing 2 mM PMSF. Cesium chloride was added at 0.481 g/ml to a final
volume
of 23 ml. The resuspension was centrifuged at 220000 x g (mid-tube) for 18
hours at
21 °C in a SW-41 rotor. After centrifugation, two bands were observed.
An upper band
was observed approximately 1 cm below the meniscus whereas a lower band was
noted
approximately 1 cm below the upper band. Both bands contained virus-like
particles.
Both bands were removed by aspiration. The virus-like particles in the upper
band were
often not as well formed as the virus-like particles from the lower band. The
bands were
dialysed against 5 litres of phosphate buffered saline (pH 7.4) for 2 hrs at
room
temperate or up to 24 hrs at 4°C. The virus-like particle preparations
were stored at -
20°C.
(a) Detection of L~ and L2 proteins
Samples of cesium chloride purified preparations of virus like particles were
diluted (1:10) in 5x reducing buffer (0.05 M (final concentration) Tris-CI (pH
6.8), 10%
glycerol, 10% sodium dodecyl sulphate (SDS), 10% 2-(3-mercaptoethanol (0.05%)
and
made up with water to 100%). The diluted samples were loaded on to SDS-PAGE
(10%
polyacrylamide) gel and electrophoresed. Standard procedures and conditions
were
followed (Towbin et aL, 1979, Virology 175 1-9).
Molecular weight determination of proteins: Following electrophoresis,
the gel was stained for 1-24 hours with coomassie blue stain (coomassie
brilliant blue (1
g/1), 40% methanol, 10% acetic acid and 50% water). The gel was destained in
methanol-
CA 02182272 2004-12-15
17
acetic acid solution (40% methanol, 10% acetic acid and 50% water) for 1-24
hours and
dried.
Identification of L1 and L2 proteins: On a separate but identifical SDS-
PAGE gel the protein species contained therein were analsysed by western
blotting to
determine whether the L2 protein was present on the virus like particles.
Standard
western blotting techniques were employed (Harlow and Lane, 1988,
Immunoblotting
(Chapter 12) In: Antibody - A laboratory manual, Harlow and Lane (Eds.), Cold
Spring
Harbour Laboratory Press).
Proteins from the SDS-PAGE gel were transferred to a nitrocellulose filter
paper. The nitrocellulose filter paper was cut into strips and blocked with
phosphate
buffered saline (pH 7.4) containing 5% skim milk powder at 4°C
overnight. The primary
antibody was incubated with the nitrocellulose paper strips in phosphate
buffered saline
(pH 7.4) containing 5% milk powder for 1.5 hours at room temperature with
agitation. The
primary antibody included monoclonal mouse anti-HPV16L1 antibody and
polyclonal
rabbit anti-HPV16L1 antibody for the detection of HPV16L1 protein and
polyclonal rabbit
anti-HPV16L2 antibody for the detection of HPV16L2 protein. Following
incubation with
the primary antibody the nitrocellulose paper strips were washed three times
(10 mins per
wash) in phosphate buffered saline (pH 7.4) containing 0.5% TweenT"" 20. A
second
antibody was incubated with the nitrocellulose paper strips in phosphate
buffered saline
2.0 (pH 7.4) containing 5% skim milk powder for 1 hr at room temperature with
agitation. The
second antibody was either horse radish peroxidase anti-rabbit or horse radish
peroxidase anti-mouse immunoglobulin. Three washes as described above were
repeated. The nitrocellulose paper strips were rinsed for 30 sec to 1 min with
phosphate
buffer (pH 7.4). The nitrocellulose paper strips were developed after placing
them in a
solution
WO 95/20659 PCT/AU95/00043
218~21~
containing 18 ml DAB (Di aminobenadine), 27 ml of phosphate buffer
(pH 7.6), 3 ml 0.3°'o cobalt chloride and 30 NI of 30% hydrogen
peroxide for 10-90 sec or as soon as the band appears. The
nitrocellulose paper strips were rinsed with water and dried.
Total protein determination: Three 5 NI samples from
each cesium chloride gradient purified preparation was analysed for
the total amount of protein present by BCA protein assay reagent as
described by the manufacturer /Pierce).
(b) Detection of virus-like particle formation
A sample (approximately 0.05 ml) of a virus-like particle
preparation that had been purified by centrifugation on a cesium
chloride gradient and dialysed against 0.1 M to 0.5 M Tris-HCI for at
least 2 hrs but preferably 24 hrs at 4°C to remove the cesium
chloride from the preparation was placed onto a formvar coated EM
grid and negatively stained with either 1 % or 2% ammonium
molybdate (pH 6.5). The grids containing the samples were examined
using a Hitachi H-800 transmission electron microscope.
PCT/AU95/00043
WO 95/20659
19 2182272
FIGURE LEGENDS
FIG. 1 A
Characterization of HPV16L1 DNA-binding activity. HPV16L1 protein,
from rVV-infected CV-1 cells, were immunoprecipitated by L1-
specific antibodies, separated by SDS-PAGE, and transferred to
nitrocellulose. Proteins were renatured in blocking buffer containing
DTT and incubated with 32P-labelled DNA from HPV16 (lane 2),
HPV6b (lane 31, HPV11 (lane 4), or phage ~I (lane 5). Unbound DNA
was removed, and DNA-binding proteins were detected by
autoradiography. The position of the L 1 protein, determined by
immunoblotting, is shown (lanes 1 and 6).
FIG. 1 B
Characterization of HPV 16L2 DNA-binding activity. HPV 161 L2
protein, from rVV-infected CV-1 cells, were immunoprecipitated by
L2- specific antibodies, separated by SDS-PAGE, and transferred to
nitrocellulose. Proteins were renatured in blocking buffer containing
DTT and incubated with 32P-labelled DNA from HPV16 (lane 2),
HPV6b (lane 3), HPV11 (lane 4), or phage ~t (lane 5). Unbound DNA
was removed, and DNA-binding proteins were detected by
autoradiography. The position of the L2 protein, determined by
immunoblotting, is shown (lanes 1 and 61.
FIG. 2A
Definition of the HPV 16L2 DNA-binding region. L2 proteins were
separated by SDS-PAGE, transferred to a nitrocellulose filter, and
probed with 32P-labelled HPV 16 DNA. C-terminal amino acid deletions
are designated as follows: amino acids 374 to 474 as e374, 384 to
474 as e384, 394 to 474 as e394, 404 to 474 as e404, and 414 to
. 474 as e414. Removal of amino acids up to 100 from the C-terminal
end had no effect on DNA binding. N-terminal amino acid deletions
- 30 were designated as follows: amino acids 1 to 60 as e1-60, 1 to 80
as e1-80, and 1 to 100 as e1-100. Removal of any N-terminal
sequence diminished DNA binding markedly.
SUBSTITUTE SHEET {RULE 26)
WO 95/20659 PCT/AU95I00043
21822'2
FIG. 2B
Definition of the HPV16L2 DNA-binding region. L2 proteins were
separated by SDS-PAGE, transferred to a nitrocellulose filter, and
probed with L2-specific antiserum. C-terminal amino acid deletions
are designated as follows: amino acids 374 to 474 as n374, 384 to
474 as e384, 394 to 474 as e394, 404 to 474 as e404, and 414 to
474 as n414. N-terminal amino acid deletions were designated as
follows: amino acids 1 to 60 as n1-60, 1 to 80 as e1-80, and 1 to
100 as o1-100. The L2 bands are indicated by arrows.
FIG. 3A
HPV DNA L2 protein interactions defined by using mutants with L2 N-
terminal mutations. N terminus-truncated L2 proteins, with deletions
indicated above each lane, were separated by SDS-PAGE, transferred
to nitrocellulose membranes, and incubated with 32P-labelled HPV 16
DNA lupper panel). The 32P-labelled DNA was removed, and the filter
was reprobed with a rabbit anti-HPV16L2 antibody for quantitation of
the L2 protein (lower panel). L2 bands are indicated by arrows, and
molecular mass markers are indicated on the left. Substitutions are
coded as follows: WT, wild-type VV; H3P, His-3 to Pro; K4P, Lys-4
to Pro; 2,4,5N, Arg-2 to Asn, Lys-4 to Asn, Arg-5 to Asn; RSP, Arg-5
to Pro; S6N, Ser-6 to Asn; S6P, Ser-6 to Pro; A7P, Ala-7 to Pro; KBP,
Lys-8 to Pro; R9P, Arg-9 to Pro; 8,9N, Lys-8 to Asn, Arg-9 to Asn.
FIG. 3B
HPV DNA L2 protein interactions defined by using mutants with L2 N
terminal mutations. Substitution mutants of L2 proteins, as indicated
above each lane, were incubated with 32P-labelled HPV 16 genomic
DNA (upper panel) or with rabbit anti-HPV16L2 antiserum (lower
panel). L2 bands are indicated by arrows, and the molecular mass
markers are shown on the left. Substitutions are coded as follows:
WT, wild-type VV; H3P, His-3 to Pro; K4P, Lys-4 to Pro; 2,4,5N, Arg-
2 to Asn, Lys-4 to Asn, Arg-5 to Asn; RSP, Arg-5 to Pro; S6N, Ser-6
to Asn; S6P, Ser-6 to Pro; A7P, Ala-7 to Pro; KBP, Lys-8 to Pro; R9P,
SUBSTITUTE SHEET (RULE 26)
WO 95/20659 PC'T/AU95100043
21 218222
Arg-9 to Pro; 8,9N, Lys-8 to Asn, Arg-9 to Asn.
FIG. 4
Binding of mutant L2 proteins to HPV DNA. For each mutant, the
sequence of the protein is given (single-letter code, with conserved
amino acids shown as dashes) and the binding of DNA to the protein
by Southwestern blot analysis is indicated by ( + or -).
FIG. 5A
DNA-binding assay for HPV16L2 proteins from rVVs. L2 protein was
immunoprecipitated with anti-L2 antibody. Equal amounts of L2
protein were incubated with Pstl-BamHl-cleaved HPV 16 genomic
DNA. The bound DNA fragments were eluted with 1 % SDS and
subjected to Southern blotting with 32P-labelled HPV16 DNA (lanes 3,
6, and 7). In some experiments, a 100-fold (lane 4) or 1,000-fold
(lane 5) molar excess of phage ~1 DNA was added to the initial
incubation of HPV DNA with L2 protein. Mock assay of a control
precipitate from wild-type VV-infected cells is also shown (lane 2).
On the left the input DNA fragments are labelled from A to G.
FIG. 5B
A linearized map of the HPV16 DNA is shown below. Restriction
sites are Pstl (p) and BamHl (b), and the corresponding fragments are
labelled A to G according to size.
F1G. 6
Amino acid sequence of wild type HPV 16L2 protein
FIG. 7
Deoxyribonucleic acid sequence of wild type HPV 16L2 gene.
FIG. 8
Nucleotide sequence of the PCR primers used to construct HPV 16L2
mutants.
C-terminal deletions
The PCR amplified 4b promoter/L2 fragments were cut with Smal and
cloned into pSX3 (Zhou et al., 1990. J. Gen.~ Virol. 77 2185-2190)
to create vaccinia expression plasmids.
SUBSTITUTE SHEET (RULE 26)
WO 95/20659 PCT/AU95100043
21a 2~ ~2~~~
N-terminal mutations
The restriction enzyme BamHl and Smal sites are underlined and start
codons ATG and stop codons TAA are in bold. The amplified PCR
products were digested with BamHl and Smal and cloned into the
RK19 BamHl/Smal sites (Kent, 1988). The vaccinia 4b promoter and
L2 mutant ORF was cloned into pSX3 (Zhou et al., 1990) to produce
vaccinia expressing plasmid containing various L2 mutant ORF.
SUBSTITUTE SHEET (RULE 26)
CA 02182272 2005-06-27
1
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: The University of Queensland
(B) STREET: ------------------
(C) CITY: St. Lucia
(D) STATE: Queensland
(E) COUNTRY: Australia
(F) POSTAL CODE (ZIP): 4072
(A) NAME: CSL Limited
(B) STREET: 45 Poplar Road
(C) CITY: Parkville
(D) STATE: Victoria
(E) COUNTRY: Australia
(F) POSTAL CODE (ZIP): 3052
(ii) TITLE OF INVENTION: Modified Papilloma Virus L2 Protein and VLPs
formed therefrom
(iii) NUMBER OF SEQUENCES: 62
(iv) 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 #1.25 (EPO)
(v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: CA 2182272
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/AU95/00043
(B) FILING DATE: 31-JAN-1995
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: AU PM 3588
(B) FILING DATE: 31-JAN-1994
(vii)PATENT AGENT INFORMATION
(NAME: John H. Woodley
(REFERENCE NUMBER: 8996-2 JHW
(2) INFORMATION FOR SEQ ID NO: l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
CAGGTCAGAT CAGCGGATCC TGTCG 25
(2) INFORMATION FOR SEQ ID N0: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
GAGGCGAATT CAGTGCATGT GCAGC 25
(2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 3:
GCTGCACATG CACTGAATTC GCCTC 25
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4:
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CAGGTCAGAT CAGCGGATCC TGTCG 25
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:
Met Arg His Lys Arg Ser Ala Lys Arg Thr Lys Arg
1 5 10
(2) INFORMATION FOR SEQ ID N0: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Arg Lys Arg Arg Lys Arg
1 5
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Met Arg His Lys Arg Ser Ala Lys Arg Thr Lys Arg
1 5 10
CA 02182272 2005-06-27
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(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 8:
Met Arg His Lys Arg Ser Ala Lys Arg Thr Lys Arg Ala Ser Ala Thr
1 5 10 15
Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro
20 25 30
(2) INFORMATION FOR SEQ ID N0: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Met His Lys Arg Ser Ala Lys Arg Thr Lys Arg Ala Ser Ala Thr Gln Leu
1 5 10 15
Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro
20 25
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
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Met Thr Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro
1 5 10 15
(2) INFORMATION FOR SEQ ID N0: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Met Pro His Lys Arg Ser Ala Lys Arg Thr Lys Arg Ala Ser Ala Thr
1 5 10 15
Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro
20 25 30
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 12:
Met Arg Pro Lys Arg Ser Ala Lys Arg Thr Lys Arg Ala Ser Ala Thr
1 5 10 15
Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro
20 25 30
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
CA 02182272 2005-06-27
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Met Arg His Pro Arg Ser Ala Lys Arg Thr Lys Arg Ala Ser Ala Thr
1 5 10 15
Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro
20 25 30
(2) INFORMATION FOR SEQ ID N0: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Met Asn His Asn Asn Ser Ala Lys Arg Thr Lys Arg Ala Ser Ala Thr
1 5 10 15
Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro
20 25 30
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Met Arg His Lys Pro Ser Ala Lys Arg Thr Lys Arg Ala Ser Ala Thr
1 5 10 15
Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro
20 25 30
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Met Arg His Lys Arg Asn Ala Lys Arg Thr Lys Arg Ala Ser Ala Thr
1 5 10 15
Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro
20 25 30
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 17:
Met Arg His Lys Arg Pro Ala Lys Arg Thr Lys Arg Ala Ser Ala Thr
1 5 10 15
Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro
20 25 30
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Met Arg His Lys Arg Ser Pro Lys Arg Thr Lys Arg Ala Ser Ala Thr
1 5 10 15
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Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro
20 25 30
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 19:
Met Arg His Lys Arg Ser Ala Pro Arg Thr Lys Arg Ala Ser Ala Thr
1 5 10 15
Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro
20 25 30
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Met Arg His Lys Arg Ser Ala Lys Pro Thr Lys Arg Ala Ser Ala Thr
1 5 10 15
Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro
20 25 30
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 21:
Met Arg His Lys Arg Ser Ala Asn Asn Thr Lys Arg Ala Ser Ala Thr
1 5 10 15
Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro
20 25 30
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 22:
Met Arg His Lys Arg Asn Ala Lys Arg Asn Lys Arg Ala Ser Ala Thr
1 5 10 15
Gln Leu Tyr Lys Thr Cys Z,ys Gln Ala Gly Thr Cys Pro Pro
20 25 30
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
Met Arg His Lys Arg Ser Ala Lys Arg Thr Asn Asn Ala Ser Ala Thr
1 5 10 15
Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro
20 25 30
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
CA 02182272 2005-06-27
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
Met Arg His Lys Arg Asn Ala Lys Arg Asn Lys Arg Ala Asn Ala Thr
1 5 10 15
Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro
25 30
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 473 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Met Arg His Lys Arg Ser Ala Lys Arg Thr Lys Arg Ala Ser Ala Thr
1 5 10 15
Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile
20 25 30
Ile Pro Lys Val Glu Gly Lys Thr Ile Ala Glu Gln Ile Leu Gln Tyr
35 40 45
Gly Ser Met Gly Val Phe Phe Gly Gly Leu Gly Ile Gly Thr Gly Ser
50 55 60
Gly Thr Gly Gly Arg Thr Gly Tyr Ile Pro Leu Gly Thr Arg Pro Pro
65 70 75 80
Thr Ala Thr Asp Thr Leu Ala Pro Val Arg Pro Pro Leu Thr Val Asp
85 90 95
Pro Val Gly Pro Ser Asp Pro Ser Ile Val Ser Leu Val Glu Glu Thr
100 105 110
Ser Phe Ile Asp Ala Gly Ala Pro Thr Ser Val Pro Ser Ile Pro Pro
115 120 125
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Asp Val Ser Gly Phe Ser Ile Thr Thr Ser Thr Asp Thr Thr Pro Ala
130 135 140
Ile Leu Asp Ile Asn Asn Thr Val Thr Thr Val Thr Thr His Asn Asn
145 150 155 160
Pro Thr Phe Thr Asp Pro Ser Val Leu Gln Pro Pro Thr Pro Ala Glu
165 170 175
Thr Gly Gly His Phe Thr Leu Ser Ser Ser Thr Ile Ser Thr His Asn
180 185 190
Tyr Glu Glu Ile Pro Met Asp Thr Phe Ile Val Ser Thr Asn Pro Asn
195 200 205
Thr Val Thr Ser Ser Thr Pro Ile Pro Gly Ser Arg Pro Val Ala Arg
210 215 220
Leu Gly Leu Tyr Ser Arg Thr Thr Gln Gln Val Lys Val Val Asp Pro
225 230 235 240
Ala Phe Val Thr Thr Pro Thr Lys Leu Ile Thr Tyr Asp Asn Pro Ala
245 250 255
Tyr Glu Gly Ile Asp Val Asp Asn Thr Leu Tyr Phe Ser Ser Asn Asp
260 265 270
Asn Ser Ile Asn Ile Ala Pro Asp Pro Asp Phe Leu Asp Ile Val Ala
275 280 285
Leu His Arg Pro Ala Leu Thr Ser Arg Arg Thr Gly Ile Arg Tyr Ser
290 295 300
Arg Ile Gly Asn Lys Gln Thr Leu Arg Thr Arg Ser Gly Lys Ser Ile
305 310 315 320
Gly Ala Lys Val His Tyr Tyr Tyr Asp Leu Ser Thr Ile Asp Pro Ala
325 330 335
Glu Glu Ile Glu Leu Gln Thr Ile Thr Pro Ser Thr Tyr Thr Thr Thr
340 345 350
Ser His Ala Ala Ser Pro Thr Ser Ile Asn Asn Gly Leu Tyr Asp Ile
355 360 365
Tyr Ala Asp Asp Phe Ile Thr Asp Thr Ser Thr Thr Pro Val Pro Ser
370 375 380
Val Pro Ser Thr Ser Leu Ser Gly Tyr Ile Pro Ala Asn Thr Thr Ile
385 390 395 400
Pro Phe Gly Gly Ala Tyr Asn Ile Pro Leu Val Ser Gly Pro Asp Ile
405 410 415
Pro Ile Asn Ile Thr Asp Gln Ala Pro Ser Leu Ile Pro Ile Val Pro
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420 425 430
Gly Ser Pro Gln Tyr Thr Ile Ile Ala Asp Ala Gly Asp Phe Tyr Leu
435 440 445
His Pro Ser Tyr Tyr Met Leu Arg Lys Arg Arg Lys Arg Leu Pro Tyr
450 455 460
Phe Phe Ser Asp Val Ser Leu Ala Ala
465 470
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1422 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
NO: 26:
ATGCGACACAAACGTTCTGCAAAACGCACAAAACGTGCATCGGCTACCCAACTTTATAAA 60
ACATGCAAACAGGCAGGTACATGTCCACCTGACATTATACCTAAGGTTGAAGGCAAAACT 120
ATTGCTGAACAAATATTACAATATGGAACTATGGGTGTATTTTTTGGTGGGTTAGGAATT 180
GGAACAGGGTCGGGTACAGGCGGACGCACTGGGTATATTCCATTGGGAACAAGGCCTCCC 240
ACAGCTACAGATACACTTGCTCCTGTAAGACCCCCTTTAACAGTAGATCCTGTGGGCCCT 300
TCTGATCCTTCTATAGTTTCTTTAGTGGAAGAAACTAGTTTTATTGATGCTGGTGCACCA 360
ACATCTGTACCTTCCATTCCCCCAGATGTATCAGGATTTAGTATTACTACTTCAACTGAT 420
ACCACACCTGCTATATTAGATATTAATAATACTGTTACTACTGTTACTACACATAATAAT 480
CCCACTTTCACTGACCCATCTGTATTGCAGCCTCCAACACCTGCAGAAACTGGAGGGCAT 540
TTTACACTTTCATCATCCACTATTAGTACACATAATTATGAAGAAATTCCTATGGATACA 600
TTTATTGTTAGCACAAACCCTAACACAGTAACTAGTAGCACACCCATACCAGGGTCTCGC 660
CCAGTGGCACGCCTAGGATTATATAGTCGCACAACACAACAGGTTAAAGTTGTAGACCCT 720
GCTTTTGTAACCACTCCCACTAAACTTATTACATATGATAATCCTGCATATGAAGGTATA 780
GATGTGGATAATACATTATATTTTTCTAGTAATGATAATAGTATTAATATAGCTCCAGAT 840
CCTGACTTTTTGGATATAGTTGCTTTACATAGGCCAGCATTAACCTCTAGGCGTACTGGC 900
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ATTAGGTACAGTAGAATTGG TAATAAACAA ACACTACGTACTCGTAGTGG AAAATCTATA960
GGTGCTAAGGTACATTATTA TTATGATTTA AGTACTATTGATCCTGCAGA AGAAATAGAA1020
TTACAAACTATAACACCTTC TACATATACT ACCACTTCACATGCAGCCTC ACCTACTTCT1080
ATTAATAATGGATTATATGA TATTTATGCA GATGACTTTATTACAGATAC TTCTACAACC1140
CCGGTACCATCTGTACCCTC TACATCTTTA TCAGGTTATATTCCTGCAAA TACAACAATT1200
CCTTTTGGTGGTGCATACAA TATTCCTTTA GTATCAGGTCCTGATATACC CATTAATATA1260
ACTGACCAAGCTCCTTCATT AATTCCTATA GTTCCAGGGTCTCCACAATA TACAATTATT1320
GCTGATGCAGGTGACTTTTA TTTACATCCT AGTTATTACATGTTACGAAA ACGACGTAAA1380
CGTTTACCATATTTTTTTTC AGATGTCTCT TTGGCTGCCTAG 1422
(2) INFORMATION
FOR
SEQ
ID NO:
27:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)
MOLECULE
TYPE:
DNA
(genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 27:
CAGGAAACAG CTATGAC 17
(2) INFORMATION FOR SEQ ID N0: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
CGCCCGGGTT AAAAGTCATC TGCATAAATA TCATATAATC C 41
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
CGCCCGGGTT ATGGTACCGG GGTTGTAGAA GTATCTGTAA TAAAG 45
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
CGCCCGGGTT AATAACCTGA TAAAGATGTA GAGGGTACAG ATGG 44
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
CGCCCGGGTT AACCAAAAGG AATTGTTGTA TTTGCAGGAA TATAACC 47
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
CGCCCGGGTT AACCTGATAC TAAAGGAATA TTGTATGCAC C 41
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 33:
GCGGATCCAT GCACAAACGT TCTGCAAAAC GC 32
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 34:
GCGGATCCAT GAAACGTTCT GCAAAACGCA CAAAACG 37
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
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GCGGATCCAT GCGTTCTGCA AAACGCACAA AACGTGC 37
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
GCGGATCCAT GTCTGCAAAA CGCACAAAAC GTGCATCGG 39
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
GCGGATCCAT GGCAAAACGC ACAAAACGTG CATCGGC 37
(2) INFORMATION FOR SEQ ID N0: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
GCGGATCCAT GAAACGCACA AAACGTGCAT CGGCTACC 38
(2) INFORMATION FOR SEQ ID NO: 39:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 39:
GCGGATCCAT GCGCACAAAA CGTGCATCGG CTACCC 36
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH. 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
GCGGATCCAT GACAAAACGT GCATCGGCTA CCCAAC 36
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
GCGGATCCAT GAAACGTGCA TCGGCTACCC AACTTTATAA AAC 43
(2) INFORMATION FOR SEQ ID NO: 42.
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
(B) TYPE. nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
GCGGATCCAT GCGTGCATCG GCTACCCAAC TTTATAAAAC 40
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
GCGGATCCAT GGCATCGGCT ACCCAACTTT ATAAAAC 37
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
GCGGATCCAT GTCGGCTACC CAACTTTATA AAACATG 37
(2) INFORMATION FOR SEQ ID N0: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
GCGGATCCAT GGCTACCCAA CTTTATAAAA CATGC 35
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
GCGGATCCAT GACCCAACTT TATAAAACAT GCAAACAGG 39
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
GCGGATCCAT GACATGCAAA CAGGCAGGTA CATGTCC 37
(2) INFORMATION FOR SEQ ID N0: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
GCGGATCCAT GATTGCTGAA CAAATATTAC AATATGG 37
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(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
GCGGATCCAT GGGAACAGGG TCGGGTACAG GCGGACG 37
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
GCGGATCCAT GACAGCTACA GATACACTTG CTCCTG 36
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS:-single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
GCGGATCCAT GTCTGATCCT TCTATAGTTT CTTTAG 36
(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 52:
GCGGATCCAT GCGACCCAAA CGTTCTGCAA AACG 34
(2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 53:
GCGGATCCAT GCGACACCCA CGTTCTGCAA AACG 34
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
GCGGATCCAT GAATCACAAT AATTCTGCAA AACGCACAAA ACG 43
(2) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
GCGGATCCAT GCGACACAAA CCTTCTGCAA AACG 34
(2) INFORMATION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
GCGGATCCAT GCGACACAAA CGTAATGCAA AACGCACAAA ACG 43
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
GCGGATCCAT GCGACACAAA CGTCCTGCAA AACG 34
(2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
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GCGGATCCAT GCGACACAAA CGTTCTCCAA AACGCAC 37
(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
GCGGATCCAT GCGACACAAA CGTTCTGCAC CACGCACAAA ACG 43
(2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
GCGGATCCAT GCGACACAAA CGTTCTGCAA AACCCACAAA ACG 43
(2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
CGGGATCCAT GCGACACAAA CGTTCTGCAA ATAACACAAA ACGTGC 46
(2) INFORMATION FOR SEQ ID NO: 62:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:
CGCCCGGGCT AGGCAGCCAA AGAGACATCT GAAAAAAAAT ATGG 44
