Note: Descriptions are shown in the official language in which they were submitted.
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ASTHMA RELATED GENES
INTRODUCTION
Asthma is a disease of reversible bronchial obstruction, characterized by
airway inflammation, epithelial damage, airway smooth muscle hypertrophy and
bronchial hyperreactivity. Many asthma symptoms can be controlled by medical
intervention, but incidence of asthma-related death and severe illness
continue to
rise in the United States. The approximately 4,800 deaths in 1989 marked a 46
percent increase since 1980. As many as 12 million people in the United States
have asthma, up 66 percent since 1980, and annually, the disease's medical and
indirect costs are estimated at over $6 billion.
Two common subdivisions of asthma are atopic (allergic, or extrinsic) asthma
and non-atopic (intrinsic) asthma. Atopy is characterized by a predisposition
to
raise an IgE antibody response to common environmental antigens. In atopic
asthma, asthma symptoms and evidence of allergy, such as a positive skin test
to
common allergens, are both present. Non-atopic asthma may be defined as
reversible airflow limitation in the absence of allergies.
The smooth muscle surrounding the bronchi are able to rapidly alter airway
diameter in response to stimuli. When the response is excessive, it is termed
bronchial hyperreactivity, a characteristic of asthma thought to have a
heritable
component. Studies have demonstrated a genetic predisposition to asthma by
showing, for example, a greater concordance for this trait among monozygotic
twins
than among dizygotic twins. The genetics of asthma is complex, however, and
shows no simple pattern of inheritance. Environment also plays a role in
asthma
development, for example, children of smokers are more likely to develop
asthma
than are children of non-smokers.
In recent years thousands of human genes have been cloned. In many
cases, gene discovery has been based on prior knowledge about the
corresponding
protein, such as amino acid sequence, immunological reactivity, etc. This
approach
has been very successful, but is limited in some important ways. One
limitation is
that genes in these cases are identified based on knowledge of molecular level
protein properties. For a large number of important human genes, however,
there
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is little or no biochemical data concerning the encoded product. For example,
genes that predispose to human diseases, such as cystic fibrosis, Huntington's
disease, etc. are of interest because of their phenotypic effect. Biochemical
characterization of such genes may be secondary to genetic characterization.
A solution to this impasse has been found in combining classical genetic
mapping with the ability to identify genes and, if necessary, to sequence
large
regions of chromosomes. Population and family studies enable genes associated
with a trait of interest to be localized to a relatively small region of a
chromosome.
At this point, physical mapping can be used to identify candidate genes, and
various molecular biology techniques used to pick out mutated genes in
affected
individuals. This "top-down" approach to gene discovery has been termed
positional cloning, because genes are identified based on position in the
genome.
Positional cloning is now being applied to complex genetic diseases, which
affect a greater fraction of humanity than do the more simple and usually
rarer
single gene disorders. Such studies must take into account the contribution of
both
environmental and genetic factors to the development of disease, and must
allow
for contributions to the genetic component by more than one, and potentially
many,
genes. The clinical importance of asthma makes it of considerable interest to
characterize genes that underlie a genetic predisposition to this disease.
Positional
cloning provides an approach to this goal.
Relevant Literature
The symptoms and biology of asthma are reviewed in Chanez et al. (1994)
Qdy~ssev 1:24-33. A review of bronchial hyperreactivity may be found in Smith
and
McFadden (1995) Ann. Allergy. Asthma and Immunol. 74:454. Moss (1989) Annals
of All 63:566 review the allergic etiology and immunology of asthma.
The genetic dissection of complex traits is discussed in Lander and Schork
(1994) Science 265:2037-2048. Genetic mapping of candidate genes for atopy
and/or bronchial hyperreactivity is described in Postma et al. (1995)
N.EJ.M.J.M.
333:894; Marsh et al. (1994) i ce 264:1'!52; and Meyers et al. (1994) en m'
23:464.
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Lawrence ef al. (1994) Ann. Hum. Genet. 58:359 discuss an approach to the
genetic analysis of atopy and asthma. Genetic linkage between the alpha
subunit
of the T cell receptor and IgE reactions has been noted by Moffat et al.
(1994) Tit g
Lancet 343:1597. Caraballo and Hernandez (1990} Tissue Antigens 35:182 noted
an association between HLA alleles and allergic asthma. Evidence of linkage of
atopy to markers on chromosome 11 q has been seen in some British asthma
families (Cookson et al. (1989) L, ancet i:1292-1295; Young ef al. (1991 ) J.
Med.
Genet. 29:236, but not in other British families (Lympany et al. (1992) in. x
.
IA lergyr_ 22:1085-1092) or in families from Minnesota or Japan (Rich et al.
(1992)
Clin. E~ .~ Aliergiy 22:1070-1076; and Hizawa et al. (1992} Clin. E~~.
Allera_v
22:1065).
The association of a polymorphism for the FcERI-~i gene and risk of atopy is
described in Hill et al. (1995) B.M.J. 311:776; Hill and Cookson (1996) Human
Mol.
Genet. 5:959; and Shirakawa et al. (1994) Nature Genetics 7:125; an
association of
FcERI-~ with bronchial hyperreactivity is described in van Herwerden (1995)
Vie,
Lancet 346:1262.
Collections of polymorphic markers from throughout the human genome
have been tested for linkage to asthma, described in Meyers et al. (1996) Am.
J.
Hum. Genet. 59:A228 and Daniels et al. (1996} Nature 383:247-250. No linkage
to
human chromosome 11 p was detected in these studies.
SUMMARY OF THE INVENTION
Human genes associated with a genetic predisposition to asthma are
provided. The genes, herein termed ASTH1! and ASTH1J, are located close to
each other on human chromosome 11 p, have similar patterns of expression, and
common sequence motifs. The nucleic acid compositions are used to produce the
encoded proteins, which may be employed for functional studies, as a
therapeutic,
and in studying associated physiological pathways. The nucleic acid
compositions
and antibodies specific for the protein are useful as diagnostics to identify
a
hereditary predisposition to asthma.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 7: Genomic organization of the ASTH11 and ASTH1J genes. The
sizes of the exons are not to scale. Alternative exons are hatched. The
direction of
transcription is indicated below each gene.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The provided ASTH1 genes and fragments thereof, encoded protein, ASTH1
genomic regulatory regions, and anti-ASTH1 antibodies are useful in the
identification of individuals predisposed to development of asthma, and for
the
modulation of gene activity in vivo for prophylactic and therapeutic purposes.
The
encoded ASTH1 protein is useful as an immunogen to raise specific antibodies,
in
drug screening for compositions that mimic or modulate ASTH1 activity or
expression, including altered forms of ASTH1 protein, and as a therapeutic.
Asthma, as defined herein, is reversible airflow limitation in a patient over
a
period of time. The disease is characterized by increased airway
responsiveness to
a variety of stimuli, and airway inflammation. A patient diagnosed as
asthmatic will
generally have multiple indications over time, including wheezing, asthmatic
attacks, and a positive response to methacholine challenge, i.e. a PC2o on
methacholine challenge of less than about 4 mg/ml. Guidelines for diagnosis
may
be found in the National Asthma Education Progra-1rr Expert Panel. Guidelines
for
diagnosis and management of asthma. National Institutes of Health, 1991; Pub.
#91-3042. Atopy, respiratory infection and environmental predisposing factors
may
also be present, but are not necessary elements of an asthma diagnosis. Asthma
conditions strictly related to atopy are referred to as atopic asthma.
The human ASTH11 and ASTH1J gene sequences are provided, as are the
genomic sequences 5' to ASTH1J. The major sequences of interest provided in
the
sequence listing are as follows:
ASTH1J 5' Genomic Region DNA (SEQ ID N0:1)
ASTH1J alt1 cDNA (SEQ ID N0:2)
ASTH1J alt2 cDNA (SEQ ID N0:3)
ASTH1J alt3 cDNA (SEQ ID N0:4)
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ASTH1J protein protein (SEQ ID N0:5)
ASTH11 alt1 cDNA (SEQ ID N0:6}
ASTH11 alt1 protein protein (SEQ ID N0:7)
ASTH11 alt2 cDNA (SEQ ID N0:8)
ASTH11 alt2 protein protein (SEQ ID N0:9)
ASTH11 alt3 cDNA (SEQ ID N0:10)
ASTH11 alt3 protein protein (SEQ ID N0:11)
CHAT box "A" form DNA (SEQ ID N0:12)
CHAT box "G" form DNA (SEQ ID N0:13)
ASTH1J 5' promoter region DNA (SEQ ID N0:14)
Mouse asthlj cDNA (SEQ ID N0:338)
Mouse asth1j protein (SEQ ID N0:339)
Polymorphisms DNA (SEQ ID N0:16-159)
Microsatellite flanking sequences DNA (SEQ ID N0:160-281)
Microsatellite repeats DNA (SEQ ID N0:282-292)
Intron-Exon boundaries DNA (SEQ ID N0:293-335)
The ASTH1 locus has been mapped to human chromosome 11 p. The traits
for a positive response to methacholine challenge and a clinical history of
asthma
were shown to be genetically linked in a genome scan of the population of
Tristan
da Cunha, a single large extended family with a high incidence of asthma
(discussed in Zamel et al. (1996) Ams J. Respir. Crit. Care Med. 153:1902-
1906).
The linkage finding was replicated in a set of Canadian asthmatic families.
The
region of strongest linkage was the marker D115907 on the short arm of
chromosome 11. Additional markers were identified from the four megabase
region
surrounding D11S907 from public databases and by original cloning of new
polymorphic microsatellite markers. Refinement of the region of interest was
obtained by genotyping new markers in the studied populations, and applying
the
transmission disequilibrium test (TDT), which reflects the level of
association
between marker alleles and disease status. TDT curves were superimposed on the
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physical map. Molecular genetic techniques for gene identification were
applied to
the region of interest. A one megabase genomic region was sequenced to high
accuracy, and the resulting data used for the sequence-based prediction of
genes
and determination of the intron/exon structure of genes in the region.
Nucleic Acid Compositions
ASTH11 produces a 2.8 kb mRNA expressed at high levels in trachea and
prostate, and at lower levels in lung and kidney and possibly other tissues.
ASTH11
cDNA clones have also been identified in prostate, testis and lung libraries.
Sequence polymorphisms are shown in Table 3. ASTH11 has at least three
alternate forms denoted as alt1, alt2, and alt3. The alternative splicing and
start
codons give the three forms of ASTH1 i proteins different amino termini. The
ASTH11 proteins, alt1, alt2 and alt3 are 265, 255 and 164 amino acids in
length,
respectively.
A domain of the ASTH11 and ASTH1J proteins is similar in sequence to
transcription factors of the ets family. The ets family is a group of
transcription
factors that activate genes involved in a variety of immunological and other
processes. The family members most similar to ASTH11 and ASTH1J are: ETS1,
ETS2, ESX, ELF, ELK1, TEL, NET, SAP-1, NERF and FLI. The ASTH1I and
ASTH1J proteins show similarity to each other. Over the efs domain they are
66%
similar (ie. have amino acids with similar properties in the same positions)
and 46%
identical to each other. All forms of ASTH 1 I and ASTH 1 J have a helix turn
helix
motif, characteristic of some transcription factors, located near the carboxy
terminal
end of the protein.
ASTH1J produces an approximately 6 kb mRNA expressed at high levels in
the trachea, prostate and pancreas and at lower levels in colon, small
intestine, lung
and stomach. ASTH1J has at least three forms, consisting of the alt1, alt2 and
alt3
forms. The open reading frame is identical for the three forms, which differ
only in
the 5' UTR. The protein encoded byASTH1J is 300 amino acids in length.
Mouse coding region sequence of asthlj is provided in SEQ ID N0:326, and
the amino acid sequence is provided in SEQ ID N0:327. The mouse and human
proteins have 88.4% identity throughout their length. The match in the ets
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domain is 100%. The mouse cDNA was identified by hybridization of a full-
length
human cDNA to a mouse lung cDNA library (Stratagene).
The term "ASTH1 genes° is herein used generically to designate
ASTH11
and ASTH1J genes and their alternate forms. The two genes lie in opposite
orientations on a native chromosome, with the 5' regulatory sequences between
them. Part of the genomic sequence between the two coding regions is provided
as
SEQ ID N0:1. The term ~ASTH1 locus" is used herein to refer to the two genes
in
all alternate forms and the genomic sequence that lies between the two genes.
Alternate forms include splicing variants, and polymorphisms in the sequence.
Specific polymorphic sequences are provided in SEQ ID NOs:16-159. For some
purposes the previously known EST sequences described herein may be excluded
from the sequences defined as the ASTH1 locus.
The DNA sequence encoding ASTH1 may be cDNA or genomic DNA or a
fragment thereof. The term "ASTH1 gene" shall be intended to mean the open
reading frame encoding specific ASTH1 polypeptides, introns, as well as
adjacent 5'
and 3' non-coding nucleotide sequences involved in the regulation of
expression,
up to about 1 kb beyond the coding region, but possibly further in either
direction.
The gene may be introduced into an appropriate vector for extrachromosomal
maintenance or for integration into the host.
The term "cDNA" as used herein is intended to include all nucleic acids that
share the arrangement of sequence elements found in native mature mRNA
species, where sequence elements are exons and 3' and 5' non-coding regions.
Normally mRNA species have contiguous exons, with the intervening introns
removed by nuclear RNA splicing, to create a continuous open reading frame
encoding the ASTH1 protein.
The genomic ASTH1 sequence has non-contiguous open reading frames,
where introns interrupt the protein coding regions. A genomic sequence of
interest
comprises the nucleic acid present between the initiation codon and the stop
codon,
as defined in the listed sequences, including all of the introns that are
normally
present in a native chromosome. It may further include the 3' and 5'
untranslated
regions found in the mature mRNA. It may further include specific
transcriptional
and translational regulatory sequences, such as promoters, enhancers, etc.,
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including about 1 kb, but possibly more, of flanking genomic DNA at either the
5' or
3' end of the transcribed region. The genomic DNA may be isolated as a
fragment
of 100 kbp or smaller; and substantially free of flanking chromosomal
sequence.
Genomic regions of interest include the non-transcribed sequences 5' to
ASTH1J, as provided in SEQ ID N0:1. This region of DNA contains the native
promoter elements that direct expression of the linked ASTH1J gene. Usually a
promoter region will have at least about 140 nt of sequence located 5' to the
ASTH1
gene and further comprising a TATA box and CART box motif sequence (SEQ ID
N0:14, nt. 597-736). The promoter region may further comprise a consensus ets
binding motif, (C/A)GGA(ArT') (SEQ ID N0:14, nt 1-5). A region of particular
interest, containing the ets binding motif, TATA box and CHAT box motifs 5' to
the
ASTH1J gene, is provided in SEQ ID N0:14. The position of SEQ ID N0:14 within
the larger sequence is SEQ ID N0:1, nt 60359-61095. The promoter sequence
may comprise polymorphisms within the CART box region, for example those
shown in SEQ ID N0:12 and SEQ tD N0:13, which have been shown to affect the
function of the promoter. The promoter region of interest may extend 5' to SEQ
ID
N0:14 within the larger sequence, e.g. SEQ ID N0:1, nt 59000-61095; SEQ ID
N0:1, nt 5700-61095, etc.
The sequence of this 5' region, and further 5' upstream sequences and 3'
downstream sequences, may be utilized for promoter elements, including
enhancer
binding sites, that provide for expression in tissues where ASTH1J is
expressed.
The tissue specific expression is useful for determining the pattern of
expression,
and for providing promoters that mimic the native pattern of expression.
Naturally
occurring polymorphisms in the promoter region are useful for determining
natural
variations in expression, particularly those that may be associated with
disease.
See, for example, SEQ ID N0:12 and 13. Alternatively, mutations may be
introduced into the promoter region to determine the effect of altering
expression in
experimentally defined systems. Methods for the identification of specific DNA
motifs involved in the binding of transcriptional factors are known in the
art, e.g.
sequence similarity to known binding motifs, gel retardation studies, etc. For
examples, see Blackwell et al. (1995) Mol Med 1: 194-205; Mortlock et al.
(1996)
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Genome Res. 6: 327-33; and Joulin and Richard-Foy (1995) Eur J Biochem 232:
620-626.
The regulatory sequences may be used to identify cis acting sequences
required for transcriptional or translational regulation of ASTH1 expression,
especially in different tissues or stages of development, and to identify cis
acting
sequences and trans acting factors that regulate or mediate ASTH1 expression.
Such transcription or translational control regions may be operably linked to
a
ASTH1 gene in order to promote expression of wild type or altered ASTH1 or
other
proteins of interest in cultured cells, or in embryonic, fetal or adult
tissues, and for
gene therapy.
The nucleic acid compositions of the subject invention may encode all or a
part of the subject polypeptides. Fragments may be obtained of the DNA
sequence
by chemically synthesizing oligonucleotides in accordance with conventional
methods, by restriction enzyme digestion, by PCR amplification, etc. For the
most
part, DNA fragments will be of at least 15 nt, usually at least 18 nt, more
usually at
least about 50 nt. Such small DNA fragments are useful as primers for PCR,
hybridization screening, etc. Larger DNA fragments, i.e. greater than 100 nt
are
useful for production of the encoded polypeptide. For use in amplification
reactions,
such as PCR, a pair of primers will be used. The exact composition of the
primer
sequences is not critical to the invention, but for most applications the
primers will
hybridize to the subject sequence under stringent conditions, as known in the
art. It
is preferable to choose a pair of primers that will generate an amplification
product
of at least about 50 nt, preferably at least about 100 nt. Algorithms for the
selection
of primer sequences are generally known, and are available in commercial
software
packages. Amplification primers hybridize to complementary strands of DNA, and
will prime towards each other.
The ASTH9 genes are isolated and obtained in substantial purity, generally
as other than an intact mammalian chromosome. Usually, the DNA will be
obtained
substantially free of other nucleic acid sequences that do not include an
ASTH9
sequence or fragment thereof, generally being at least about 50%, usually at
least
about 90% pure and are typically "recombinant", i.e. flanked by one or more
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nucleotides with which it is not normally associated on a naturally occurring
chromosome.
The DNA sequences are used in a variety of ways. They may be used as
probes for identifying ASTH1 related genes. Mammalian homologs have
substantial sequence similarity to the subject sequences, i.e. at least 75%,
usually
at least 90%, more usually at least 95% sequence identity with the nucleotide
sequence of the subject DNA sequence. Sequence similarity is calculated based
on a reference sequence, which may be a subset of a larger sequence, such as a
conserved motif, coding region, flanking region, etc. A reference sequence
will
usually be at least about 18 nt long, more usually at least about 30 nt tong,
and may
extend to the complete sequence that is being compared. Algorithms for
sequence
analysis are known in the art, such as BLAST, described in Altschul et al.
(1990) ,~
Mol Bi~_I 215:403-10.
Nucleic acids having sequence similarity are detected by hybridization under
low stringency conditions, for example, at 50°C and 10XSSC (0.9 M
saline/0.09 M
sodium citrate) and remain bound when subjected to washing at 55°C in
1XSSC.
Sequence identity may be determined by hybridization under stringent
conditions,
for example, at 50°C or higher and 0.1XSSC (9 mM saline/0.9 mM sodium
citrate).
By using probes, particularly labeled probes of DNA sequences, one can isolate
homologous or related genes. The source of homologous genes may be any
species, e.g. primate species, particularly human; rodents, such as rats and
mice,
canines, felines, bovines, ovines, equines, yeast, Drosophila, Caenhorabditis,
etc.
The DNA may also be used to identify expression of the gene in a biological
specimen. The manner in which one probes cells for the presence of particular
nucleotide sequences, as genomic DNA or RNA, is well established in the
literature
and does not require elaboration here. mRNA is isolated from a cell sample.
mRNA may be ampl~ed by RT-PCR, using reverse transcriptase to form a
complementary DNA strand, followed by polymerase chain reaction amplification
using primers specific for the subject DNA sequences. Alternatively, mRNA
sample
is separated by gel electrophoresis, transferred to a suitable support, e.g.
nitrocellulose, nylon, etc., and then probed with a fragment of the subject
DNA as a
probe. Other techniques, such as oligonucleotide ligation assays, in situ
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hybridizations, and hybridization to DNA probes arrayed on a solid chip may
also
find use. Detection of mRNA hybridizing to the subject sequence is indicative
of
ASTH1 gene expression in the sample.
The subject nucleic acid sequences may be modified for a number of
purposes, particularly where they will be used intracellularly, for example,
by being
joined to a nucleic acid cleaving agent, e.g. a chelated metal ion, such as
iron or
chromium for cleavage of the gene; or the like.
The sequence of the ASTH1 locus, including flanking promoter regions and
coding regions, may be mutated in various ways known in the art to generate
targeted changes in promoter strength, sequence of the encoded protein, etc.
The
DNA sequence or product of such a mutation will be substantially similar to
the
sequences provided herein, i.e. will differ by at least one nucleotide or
amino acid,
respectively, and may differ by at least two but not more than about ten
nucleotides
or amino acids. The sequence changes may be substitutions, insertions or
deletions. Deletions may further include larger changes, such as deletions of
a
domain or exon. Other modifications of interest include epitope tagging, e.g.
with
the FLAG system, HA, etc. For studies of subcellular localization, fusion
proteins
with green fluorescent proteins (GFP) may be used. Such mutated genes may be
used to study structure-function relationships of ASTH1 polypeptides, or to
after
properties of the protein that affect its function or regulation. For example,
constitutively active transcription factors, or a dominant negatively active
protein
that binds to the ASTH1 DNA target site without activating transcription, may
be
created in this manner.
Techniques for in vitro mutagenesis of cloned genes are known. Examples
of protocols for scanning mutations may be found in Gustin et al.,
8iotechniques
14:22 (1993); Barany, Gene 37:111-23 (1985); Colicelli et al., MoI Gen Genet
199:537-9 (1985); and Prentki ef al., Gene 29:303-13 (1984). Methods for site
specific mutagenesis can be found in Sambrook et al., Molecular Cloning: A
Laboratory Manual, CSH Press 1989, pp. 15.3-15.108; Weiner et al., Gene 126:35-
41 (1993); Sayers et al., Biotechniques 13:592-6 (1992); Jones and
Winistorfer,
Biotechniques 12:528-30 (1992}; Barton et al., Nucleic Acids Res 18:7349-55
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(1990); Marotti and Tomich, Gene Anal Tech 6:67-70 (7989); and Zhu Anal
Biochem 177:120-4 (1989).
Synthesis of ASTH1 Proteins
The subject gene may be employed for synthesis of a complete ASTH 1
protein, or polypeptide fragments thereof, particularly fragments
corresponding to
functional domains; binding sites; etc.; and including fusions of the subject
polypeptides to other proteins or parts thereof. For expression, an expression
cassette may be employed, providing for a transcriptional and translational
initiation
region, which may be inducible or constitutive, where the coding region is
operably
linked under the transcriptional control of the transcriptional initiation
region, and a
transcriptional and translational termination region. Various transcriptional
initiation
regions may be employed that are functional in the expression host.
The polypeptides may be expressed in prokaryotes or eukaryotes in
accordance with conventional ways, depending upon the purpose for expression.
For large scale production of the protein, a unicellular organism, such as E.
coli, B.
subtilis, S. cerevisiae, or cells of a higher organism such as vertebrates,
particularly
mammals, e.g. COS 7 cells, may be used as the expression host cells. In many
situations, it may be desirable to express the ASTH1 gene in mammalian cells,
where the ASTH1 gene will benefit from native folding and post-translational
modifications. Small peptides can also be synthesized in the laboratory.
With the availability of the polypeptides in large amounts, by employing an
expression host, the polypeptides may be isolated and purifred in accordance
with
conventional ways. A lysate may be prepared of the expression host and the
lysate
purified using HPLC, exclusion chromatography, gel electrophoresis, affinity
chromatography, or other purification technique. The purified polypeptide will
generally be at least about 80% pure, preferably at least about 90% pure, and
may
be up to and including 100% pure. Pure is intended to mean free of other
proteins,
as well as cellular debris.
The polypeptide is used for the production of antibodies, where short
fragments provide for antibodies specific for the particular polypeptide, and
larger
fragments or the entire protein allow for the production of antibodies over
the
surface of the polypeptide. Antibodies may be raised to the wild-type or
variant
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forms of ASTH 1. Antibodies may be raised to isolated peptides corresponding
to
these domains, or to the native protein, e.g. by immunization with cells
expressing
ASTH 1, immunization with liposomes having ASTH 1 inserted in the membrane,
etc.
Antibodies are prepared in accordance with conventional ways, where the
expressed polypeptide or protein is used as an immunogen, by itself or
conjugated
to known immunogenic carriers, e.g. KLH, pre-S HBsAg, other viral or
eukaryotic
proteins, or the like. Various adjuvants may be employed, with a series of
injections, as appropriate. For monoclonal antibodies, after one or more
booster
injections, the spleen is isolated, the lymphocytes immortalized by cell
fusion, and
then screened for high affinity antibody binding. The immortalized cells, i.e.
hybridomas, producing the desired antibodies may then be expanded. For further
description, see Monoclonal Antibodies: A Laboratory Manual, Harlow and Lane
eds., Cold Spring Harbor Laboratories, Cold Spring Harbor, New York, 1988. If
desired, the mRNA encoding the heavy and light chains may be isolated and
mutagenized by cloning in E. coli, and the heavy and light chains mixed to
further
enhance the affinity of the antibody. Alternatives to in vivo immunization as
a
method of raising antibodies include binding to phage "display" libraries,
usually in
conjunction with in vitro affinity maturation.
Detection of ASTH 1 Associated Asthma
Diagnosis of ASTH1 associated asthma is performed by protein, DNA or
RNA sequence and/or hybridization analysis of any convenient sample from a
patient, e.g. biopsy material, blood sample, scrapings from cheek, etc. A
nucleic
acid sample from a patient having asthma that may be associated with ASTH1, is
analyzed for the presence of a predisposing polymorphism in ASTH1. A typical
patient genotype will have at least one predisposing mutation on at least one
chromosome. The presence of a polymorphic ASTH9 sequence that affects the
activity or expression of the gene product, and confers an increased
susceptibility to
asthma is considered a predisposing polymorphism. Individuals are screened by
analyzing their DNA or mRNA for the presence of a predisposing polymorphism,
as
compared to an asthma neutral sequence. Specific sequences of interest include
any polymorphism that leads to clinical bronchial hyperreactivity or is
otherwise
associated with asthma, including, but not limited to, insertions,
substitutions and
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deletions in the coding region sequence, intron sequences that affect
splicing, or
promoter or enhancer sequences that affect the activity and expression of the
protein. Examples of specific ASTH1 polymorphisms in asthma patients are
listed
in Tables 3-8.
The CART box polymorphism of SEQ ID N0:12 and 13 (which is located
within SEQ ID N0:14) is of particular interest. The "G" form, SEQ ID N0:13,
can be
associated with a propensity to develop bronchial hyperreactivity or asthma.
Other
polymorphisms in the surrounding region affect this association. It has been
found
that substitution of "G" for "A" results in decreased binding of nuclear
proteins to the
DNA motif.
The effect of an ASTH1 predisposing polymorphism may be modulated by
the patient genotype in other genes related to asthma and atopy, including,
but not
limited to, the FcE receptor, Class I and Class II HLA antigens, T cell
receptor and
immunoglobulin genes, cytokines and cytokine receptors, and the like.
Screening may also be based on the functional or antigenic characteristics of
the protein. Immunoassays designed to detect predisposing polymorphisms in
ASTH1 proteins may be used in screening. Where many diverse mutations lead to
a particular disease phenotype, functional protein assays have proven to be
effective screening tools.
Biochemical studies may be performed to determine whether a candidate
sequence polymorphism in the ASTH1 coding region or control regions is
associated with disease. For example, a change in the promoter or enhancer
sequence that affects expression of ASTH1 may result in predisposition to
asthma.
Expression levels of a candidate variant allele are compared to expression
levels of
the normal allele by various methods known in the art. Methods for determining
promoter or enhancer strength include quantitation of the expressed natural
protein;
insertion of the variant control element into a vector with a reporter gene
such as
~i-galactosidase, luciferase, chloramphenicol acetyltransferase, etc. that
provides
for convenient quantitation; and the like. The activity of the encoded ASTH1
protein
may be determined by comparison with the wild-type protein.
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A number of methods are available for analyzing nucleic acids for the
presence of a specific sequence. Where large amounts of DNA are available,
genomic DNA is used directly. Alternatively, the region of interest is cloned
into a
suitable vector and grown in sufficient quantity for analysis. Cells that
express
ASTH1 genes, such as trachea cells, may be used as a source of mRNA, which
may be assayed directly or reverse transcribed into cDNA for analysis. The
nucleic
acid may be amplified by conventional techniques, such as the polymerase chain
reaction (PCR), to provide sufficient amounts for analysis. The use of the
polymerase chain reaction is described in Saiki, ef al. (1985) ci ~g 239:487,
and
a review of current techniques may be found in Sambrook, et al. Molecular
Cloning;
A Laborator~Manual, CSH Press 1989, pp.14.2-14.33. Amplification may also be
used to determine whether a polymorphism is present, by using a primer that is
specific for the polymorphism. Alternatively, various methods are known in the
art
that utilize oligonucleotide ligation as a means of detecting polymorphisms,
for
examples see Riley et al. (1990) N.A.R. 18:2887-2890; and Delahunty et al.
(1996)
Am. J. Hum. Genet. 58:1239-1246.
A detectable label may be included in an amplification reaction. Suitable
labels include fluorochromes, e.g. fluorescein isothiocyanate (F1TC),
rhodamine,
Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM),
2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X-
rhodamine
(ROX), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 5-
carboxyfluorescein
(5-FAM) or N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive
labels,
e.g. s2P~ 355 sH; etc. The label may be a two stage system, where the
amplified
DNA is conjugated to biotin, haptens, etc. having a high affinity binding
partner, e.g.
avidin, specific antibodies, etc., where the binding partner is conjugated to
a
detectable label. The label may be conjugated to one or both of the primers.
Alternatively, the pool of nucleotides used in the amplification is labeled,
so as to
incorporate the label into the amplification product.
The sample nucleic acid, e.g. amplified or cloned fragment, is analyzed by
one of a number of methods known in the art. The nucleic acid may be sequenced
by dideoxy or other methods, and the sequence of bases compared to a neutral
ASTH1 sequence. Hybridization with the variant sequence may also be used to
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determine its presence, by Southern blots, dot blots, etc. The hybridization
pattern
of a control and variant sequence to an array of oligonucleotide probes
immobilised
on a solid support, as described in US 5,445,934, or in W095/35505, may also
be
used as a means of detecting the presence of variant sequences. Single strand
conformational polymorphism (SSCP) analysis, denaturing gradient gel
electrophoresis (DGGE), mismatch cleavage detection, and heteroduplex analysis
in gel matrices are used to detect conformational changes created by DNA
sequence variation as alterations in electrophoretic mobility. Alternatively,
where a
polymorphism creates or destroys a recognition site for a restriction
endonuclease
(restriction fragment length polymorphism, RFLP), the sample is digested with
that
endonuclease, and the products size fractionated to determine whether the
fragment was digested. Fractionation is performed by gel or capillary
electrophoresis, particularly acrylamide or agarose gels.
The hybridization pattern of a control and variant sequence to an array of
oligonucleotide probes immobilised on a solid support, as described in US
5,445,934, or in W095/35505, may be used as a means of detecting the presence
of variant sequences. In one embodiment of the invention, an array of
oligonucleotides are provided, where discrete positions on the array are
complementary to at least a portion of mRNA or genomic DNA of the ASTH1 locus.
Such an array may comprise a series of oligonucleotides, each of which can
specifically hybridize to a nucleic acid, e.g. mRNA, cDNA, genomic DNA, etc.
from
the ASTH9 locus.
An array may include all or a subset of the polymorphisms listed in Table 3
(SEQ ID NOs:16-126). One or both polymorphic forms may be present in the
array,
for example the polymorphism of SEQ ID N0:12 and 13 may be represented by
either, or both, of the listed sequences. Usually such an array will include
at least 2
different polymorphic sequences, i.e. polymorphisms located at unique
positions
within the locus, usually at least about 5, more usually at least about 10,
and may
include as many as 50 to 100 different polymorphisms. The oligonucleotide
sequence on the array will usually be at least about 12 nt in length, may be
the
length of the provided polymorphic sequences, or may extend into the flanking
regions to generate fragments of 100 to 200 nt in length. For examples of
arrays,
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see Hacia ef al. (1996) Nature Genetics 14:441-447; Lockhart-et al. (1996)
Nature
Biotechnol. 14:1675-1680; and De Risi et al. (1996) Nature Genetics 14:457-
460.
Antibodies specific for ASTH1 polymorphisms may be used in screening
immunoassays. A reduction or increase in neutral ASTH1 and/or presence of
asthma associated polymorphisms is indicative that asthma is ASTH1-associated.
A sample is taken from a patient suspected of having ASTH1-associated asthma.
Samples, as used herein, include biological fluids such as tracheal lavage,
blood,
cerebrospinal fluid, tears, saliva, lymph, dialysis fluid and the like; organ
or tissue
culture derived fluids; and fluids extracted from physiological tissues. Also
included
in the term are derivatives and fractions of such fluids. Biopsy samples are
of
particular interest, e.g. trachea scrapings, etc. The number of cells in a
sample will
generally be at least about 103, usually at least 104 more usually at least
about 105.
The cells may be dissociated, in the case of solid tissues, or tissue sections
may be
analyzed. Alternatively a lysate of the cells may be prepared.
Diagnosis may be performed by a number of methods. The different
methods all determine the absence or presence or altered amounts of normal or
abnormal ASTH1 in patient cells suspected of having a predisposing
polymorphism
in ASTH1. For example, detection may utilize staining of cells or histological
sections, performed in accordance with conventional methods. The antibodies of
interest are added to the cell sample, and incubated for a period of time
sufficient to
allow binding to the epitope, usually at least about 10 minutes. The antibody
may
be labeled with radioisotopes, enzymes, fluorescers, chemiluminescers, or
other
labels for direct detection. Alternatively, a second stage antibody or reagent
is used
to amplify the signal. Such reagents are well known in the art. For example,
the
primary antibody may be conjugated to biotin, with horseradish peroxidase-
conjugated avidin added as a second stage reagent. Final detection uses a
substrate that undergoes a color change in the presence of the peroxidase. The
absence or presence of antibody binding may be determined by various methods,
including flow cytometry of dissociated cells, microscopy, radiography,
scintillation
counting, etc.
An alternative method for diagnosis depends on the in vifro detection of
binding between antibodies and ASTH1 in a lysate. Measuring the concentration
of
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ASTH1 binding in a sample or fraction thereof may be accorrtplished by a
variety of
specific assays. A conventional sandwich type assay may be used. For example,
a
sandwich assay may first attach ASTH1-specific antibodies to an insoluble
surface
or support. The particular manner of binding is not crucial so long as it is
compatible with the reagents and overall methods of the invention. They may be
bound to the plates covalentiy or non-covalently, preferably non-covalently.
The insoluble supports may be any compositions to which polypeptides can
be bound, which is readily separated from soluble material, and which is
otherwise
compatible with the overalt method. The surface of such supports may be solid
or
porous and of any convenient shape. Examples of suitable insoluble supports to
which the receptor is bound include beads, e.g. magnetic beads, membranes and
microtiter plates. These are typically made of glass, plastic (e.g.
polystyrene),
polysaccharides, nylon or nitrocellulose. Microtiter plates are especially
convenient
because a large number of assays can be carried out simultaneously, using
small
amounts of reagents and samples.
Patient sample lysates are then added to separately assayable supports (for
example, separate wells of a microtiter plate) containing antibodies.
Preferably, a
series of standards, containing known concentrations of normal and/or abnormal
ASTH1 is assayed in parallel with the samples or aliquots thereof to serve as
controls. Preferably, each sample and standard will be added to multiple wells
so
that mean values can be obtained for each. The incubation time should be
sufficient for binding, generally, from about 0.1 to 3 hr is sufficient. After
incubation,
the insoluble support is generally washed of non-bound components. Generally,
a
dilute non-ionic detergent medium at an appropriate pH, generally 7-8, is used
as a
wash medium. From one to six washes may be employed, with sufficient volume to
thoroughly wash non-specifically bound proteins present in the sample.
After washing, a solution containing a second antibody is applied. The
antibody will bind ASTH1 with sufficient specificity such that it can be
distinguished
from other components present. The second antibodies may be labeled to
facilitate
direct, or indirect quantification of binding. Examples of labels that permit
direct
measurement of second receptor binding include radiolabels, such as 3H or
'251,
fluorescers, dyes, beads, chemilumninescers, colloidal particles, and the
like.
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Examples of labels which permit indirect measurement of binding include
enzymes
where the substrate may provide for a colored or fluorescent product. In a
preferred
embodiment, the antibodies are labeled with a covalently bound enzyme capable
of
providing a detectable product signal after addition of suitable substrate.
Examples
of suitable enzymes for use in conjugates include horseradish peroxidase,
alkaline
phosphatase, malate dehydrogenase and the like. Where not commercially
available, such antibody-enzyme conjugates are readily produced by techniques
known to those skilled in the art. The incubation time should be sufficient
for the
labeled ligand to bind available molecules. Generally, from about 0.1 to 3 hr
is
sufficient, usually 1 hr sufficing.
After the second binding step, the insoluble support is again washed free of
non-specificaily bound material. The signal produced by the bound conjugate is
detected by conventional means. Where an enzyme conjugate is used, an
appropriate enzyme substrate is provided so a detectable product is formed.
Other immunoassays are known in the art and may find use as diagnostics.
Ouchterlony plates provide a simple determination of antibody binding. Western
blots may be performed on protein gels or protein spots on filters, using a
detection
system specific for ASTH1 as desired, conveniently using a labeling method as
described for the sandwich assay.
Other diagnostic assays of interest are based on the functional properties of
ASTH 1 proteins. Such assays are particularly useful where a large number of
different sequence changes lead to a common phenotype, i.e. altered protein
function leading to bronchial hyperreactivity. For example, a functional assay
may
be based on the transcriptional changes mediated by ASTH1 gene products. Other
assays may, for example, detect conformational changes, size changes resulting
from insertions, deletions or truncations, or changes in the subcellular
localization of
ASTH1 proteins.
in a protein truncation test, PCR fragments amplified from the ASTH1 gene
or its transcript are used as templates for in vivo transcription/translation
reactions
to generate protein products. Separation by gel electrophoresis is performed
to
determine whether the polymorphic gene encodes a truncated protein, where
truncations may be associated with a loss of function.
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Diagnostic screening may also be performed for polyrriorphisms that are
genetically linked to a predisposition for bronchial hyperreactivity,
particularly
through the use of microsatellite markers or single nucleotide poiymorphisms.
Frequently the microsatellite polymorphism itself is not phenotypically
expressed,
but is linked to sequences that result in a disease predisposition. However,
in some
cases the microsatellite sequence itself may affect gene expression.
Microsatellite
linkage analysis may be performed alone, or in combination with direct
detection of
polymorphisms, as described above. The use of microsatellite markers for
genotyping is well documented. For examples, see Mansfield et aL (1994)
Genomics 24:225-233; Ziegle et al. (1992) Genomics 14:1026-1031; Dib et al.,
supra.
Microsatellite loci that are useful in the subject methods have the general
formula:
U (R)~ U', where
U and U' are non-repetitive flanking sequences that uniquely identify the
particular
locus, R is a repeat motif, and n is the number of repeats. The repeat motif
is at
least 2 nucleotides in length, up to 7, usually 2-4 nucleotides in length.
Repeats
can be simple or complex. The flanking sequences U and U' uniquely identify
the
microsatellite locus within the human genome. U and U' are at least about 18
nucleotides in length, and may extend several hundred bases up to about 1 kb
on
either side of the repeat. Within U and U', sequences are selected for
amplification
primers. The exact composition of the primer sequences are not critical to the
invention, but they must hybridize to the flanking sequences U and U',
respectively,
under stringent conditions. Criteria for selection of amplification primers
are as
previously discussed. To maximize the resolution of size differences at the
locus, it
is preferable to chose a primer sequence that is close to the repeat sequence,
such
that the total amplification product is between 100-500 nucleotides in length.
The number of repeats at a specific locus, n, is polymorphic in a population,
thereby generating individual differences in the length of DNA that lies
between the
amplification primers. The number will vary from at least 1 repeat to as many
as
about 100 repeats or more.
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The primers are used to amplify the region of genomic DNA that contains the
repeats. Conveniently, a detectable label will be included in the
amplification
reaction, as previously described. Multiplex amplification may be pertormed in
which several sets of primers are combined in the same reaction tube. This is
particularly advaritageous when limited amounts of sample DNA are available
for
analysis. Conveniently, each of the sets of primers is labeled with a
different
fluorochrome.
After amplification, the products are size fractionated. Fractionation may be
performed by gel electrophoresis, particularly denaturing acrylamide or
agarose
gels. A convenient system uses denaturing polyacrylamide gels in combination
with
an automated DNA sequencer, see Hunkapillar et al. (1991) Scien,~g 254:59-74.
The automated sequencer is particularly useful with multiplex amplification or
pooled products of separate PCR reactions. Capillary electrophoresis may also
be
used for fractionation. A review of capillary electrophoresis may be found in
t_anders, et al. (1993) BioTechnicpes 14:98-111. The size of the amplification
product is proportional to the number of repeats (n) that are present at the
locus
specified by the primers. The size will be polymorphic in the population, and
is
therefore an allelic marker for that locus.
A number of markers in the region of the ASTH1 locus have been identified,
and are fisted in Table 1 in the Experimental section (SEQ ID NOs:160-273). Of
particular interest for..diagnostic purposes is the marker D1152008, in which
individuals having alleles C or F at this locus, particularly in combination
with the
CART box polymorphism and other polymorphisms, are predisposed to develop
bronchial hyperreactivity or asthma. The association of D11S2008 alleles is as
follows:
Allele Association with Numt~er of TATC repeats relative
asthma to allele C
(SEQ ID N0:15)
A no -2
B no _1
C yes equivalent
D no +1
E no +2
F yes +3
G no +4
H no +5
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A DNA sequence of interest for diagnosis comprises the D1152008 primer
sequences shown in Table 1 (SEQ ID N0:242 and 243), flanking one or three
repeats of SEQ ID N0:15.
Other microsatellite markers of interest for diagnostic purposes are CA39 2;
774F; 774J; 7740; L19PENTA1; 65P14TE1; AFM205YG5; D11S907; D11S4200;
774N; CA11-11; 774L; AFM283WH9; ASM114 and D11S1900 (primer sequences
are provided in Table 1, the repeats are provided in Table 1B).
Regulation of ASTH1 Expression
The ASTH1 genes are useful for analysis of ASTH1 expression, e.g. in
determining developmental and tissue specific patterns of expression, and for
modulating expression in vitro and in vivo. The regulatory region of SEQ ID
N0:1
may also be used to investigate analysis of ASTH1 expression. Vectors useful
for
introduction of the gene include plasmids and viral vectors. Of particular
interest
are retroviral-based vectors, e.g. Moloney murine leukemia virus and modified
human immunodeficiency virus; adenovirus vectors, etc. that are maintained
transiently or stably in mammalian cells. A wide variety of vectors can be
employed
for transfection and/or integration of the gene into the genome of the cells.
Alternatively, micro-injection may be employed, fusion, or the like for
introduction of
genes into a suitable host cell. See, for example, Dhawan et al. (1991 )
Science
254:1509-1512 and Smith et al. (1990) Molecular and Cellular Biology 3268-
3271.
Administration of vectors to the lungs is of particular interest. Frequently
such methods utilize liposomal formulations, as described in Eastman et al.
(1997)
Hum Gene Ther 8:765-773; Oudrhiri ef al. (1997) P.NA.S.A.S. 94:1651-1656;
McDonald et al. (1997) Hum Gene Ther 8:411-422.
The expression vector will have a transcriptional initiation region oriented
to
produce functional mRNA. The native transcriptional initiation region, e.g.
SEQ ID
N0:14, or an exogenous transcriptional initiation region may be employed. The
promoter may be introduced by recombinant methods in vitro, or as the result
of
homologous integration of the sequence into a chromosome. Many strong
promoters are known in the art, including the ~i-actin promoter, SV40 early
and late
promoters, human cytomegalovirus promoter, retroviral LTRs, methallothionein
responsive element (MRE), tetracycline-inducible promoter constructs, etc.
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Expression vectors generally have convenient restriction sites located near
the promoter sequence to provide for the insertion of nucleic acid sequences.
Transcription cassettes may be prepared comprising a transcription initiation
region,
the target gene or fragment thereof, and a transcriptional termination region.
The
transcription cassettes may be introduced into a variety of vectors, e.g.
plasmid;
retrovirus, e.g. lentivirus; adenovirus; and the like, where the vectors are
able to
transiently or stably be maintained in the cells, usually for a period of at
least about
one day, more usually for a period of at least about several days to several
weeks.
Antisense molecules are used to down-regulate expression of ASTH? in
cells. The anti-sense reagent may be antisense oligonucleotides (ODN),
particularly synthetic ODN having chemical modifications from native nucleic
acids,
or nucleic acid constructs that express such anti-sense molecules as RNA. The
antisense sequence is complementary to the mRNA of~the targeted gene, and
inhibits expression of the targeted gene products. Antisense molecules inhibit
gene
expression through various mechanisms, e.g. by reducing the amount of mRNA
available for translation, through activation of RNAse H, or steric hindrance.
One or
a combination of antisense molecules may be administered, where a combination
may comprise multiple different sequences.
Antisense molecules may be produced by expression of all or a part of the
target gene sequence in an appropriate vector, where the transcriptional
initiation is
oriented such that an antisense strand is produced as an RNA molecule.
Alternatively, the antisense molecule is a synthetic oligonucleotide.
Antisense
oiigonucleotides will generally be at least about 7, usually at least about
12, more
usually at least about 20 nucleotides in length, and not more than about 500,
usually not more than about 50, more usually not more than about 35
nucleotides in
length, where the length is governed by efficiency of inhibition, specificity,
including
absence of cross-reactivity, and the like. It has been found that short
oligonucleotides, of from 7 to 8 bases in length, can be strong and selective
inhibitors of gene expression (see Wagner et al. (1996) Nature Biotechnoloav
14:840-844).
A specific region or regions of the endogenous sense strand mRNA
sequence is chosen to be complemented by the antisense sequence. Selection of
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a specific sequence for the oligonucleotide may use an empirical method, where
several candidate sequences are assayed for inhibition of expression of the
target
gene in an in vitro or animal model. A combination of sequences may also be
used,
where several regions of the mRNA sequence are selected for antisense
complementation.
Antisense oligonucleotides may be chemically synthesized by methods
known in the art (see Wagner et al. (1993} supra. and Milligan et aL, supra.)
Preferred oligonucleotides are chemically modified from the native
phosphodiester
structure, in order to increase their intracellular stability and binding
affinity. A
number of such modifications have been described in the literature, which
after the
chemistry of the backbone, sugars or heterocyclic bases.
Among useful changes in the backbone chemistry are phosphorothioates;
phosphorodithioates, where both of the non-bridging oxygens are substituted
with
sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates.
Achiral
phosphate derivatives include 3'-O'-5'-S-phosphorothioate, 3'-S-5'-O-
phosphorothioate, 3'-CH2-5'-O-phosphonate and 3'-NH-5'-O-phosphoroamidate.
Peptide nucleic acids replace the entire ribose phosphodiester backbone with a
peptide linkage. Sugar modifications are also used to enhance stability and
affinity.
The a-anomer of deoxyribose may be used, where the base is inverted with
respect
to the natural ~i-anomer. The 2'-OH of the ribose sugar may be altered to form
2'-
O-methyl or 2'-O-allyl sugars, which provides resistance to degradation
without
comprising affinity. Modification of the heterocyclic bases must maintain
proper
base pairing. Some useful substitutions include deoxyuridine for
deoxythymidine;
5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine. 5-
propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have been shown to
increase affinity and biological activity when substituted for deoxythymidine
and
deoxycytidine, respectively.
As an alternative to anti-sense inhibitors, catalytic nucleic acid compounds,
e.g. ribozymes, anti-sense conjugates, etc. may be used to inhibit gene
expression.
Ribozymes may be synthesized in vitro and administered to the patient, or may
be
encoded on an expression vector, from which the ribozyme is synthesized in the
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targeted cell (for example, see International patent application WO 9523225,
and
Beigelman et al. (1995) Nucl. Acids Res 23:4434-42). Examples of
oiigonucleotides
with catalytic activity are described in WO 9506764. Conjugates of anti-sense
ODN
with a metal complex, e.g. terpyridylCu(II), capable of mediating mRNA
hydrolysis
are described in Bashkin et al. (1995) Cpl Biochem Biotechnol 54:43-56.
Ther ~eutic Use of ASTH1 PrQ~gin
A host may be treated with intact ASTH1 protein, or an active fragment
thereof to modulate or reduce bronchial hypereactivity. Desirably, the
peptides will
not induce an immune response, particularly an antibody response. Xenogeneic
analogs may be screened for their ability to provide a therapeutic effect
without
raising an immune response. The protein or peptides may also be administered
to
in vitro cell cultures.
Various methods for administration may be employed. The polypeptide
formulation may be given orally, or may be injected intravascularly,
subcutaneously,
peritoneally, etc. Methods of administration by inhalation are well-known in
the art.
The dosage of the therapeutic formulation will vary widely, depending upon the
nature of the disease, the frequency of administration, the manner of
administration,
the clearance of the agent from the host, and the like. The initial dose may
be
larger, followed by smaller maintenance doses. The dose may be administered as
infrequently as weekly or biweekly, or fractionated into smaller doses and
administered daily, semi-weekly, etc. to maintain an effective dosage level.
In many
cases, oral administration will require a higher dose than if administered
intravenously. The amide bonds, as well as the amino and carboxy termini, may
be
modified for greater stability on oral administration.
The subject peptides may be prepared as formulations at a
pharmacologically effective dose in pharmaceutically acceptable media, for
example normal saline, PBS, etc. The additives may include bactericidal
agents,
stabilizers, buffers, or the like. In order to enhance the half life of the
subject
peptide or subject peptide conjugates, the peptides may be encapsulated,
introduced into the lumen of liposomes, prepared as a colloid, or another
conventional technique may be employed that provides for an extended lifetime
of
the peptides.
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The peptides may be administered as a combination therapy with other
pharmacologically active agents. The additional drugs may be administered
separately or in conjunction with the peptide compositions, and may be
included in
the same formulation.
~tlodels for Asthr~
The subject nucleic acids can be used to generate genetically modified
non-human animals or site specific gene modifications in cell lines. The term
Ntransgenic" is intended to encompass genetically modified animals having a
deletion or other knock-out of ASTH1 gene activity, having an exogenous ASTH1
gene that is stably transmitted in the host cells, or having an exogenous
ASTH1
promoter operably linked to a reporter gene. Transgenic animals may be made
through homologous recombination, where the ASTH1 locus is altered.
Alternatively, a nucleic acid construct is randomly integrated into the
genome.
Vectors for stable integration include plasmids, retroviruses and other animal
viruses, YACs, and the like. Of interest are transgenic mammals, e.g. cows,
pigs,
goats, horses, etc., and particularly rodents, e.g. rats, mice, etc.
A "knock-out" animal is genetically manipulated to substantially reduce, or
eliminate endogenous ASTH1 function. Different approaches may be used to
achieve the "knock-out". A chromosomal deletion of all or part of the native
ASTH1
homolog may be induced. Deletions of the non-coding regions, particularly the
promoter region, 3' regulatory sequences, enhancers, or deletions of gene that
activate expression of ASTH1 genes. A functional knock-out may also be
achieved
by the introduction of an anti-sense construct that blocks expression of the
native
ASTH1 genes (for example, see Li and Cohen {1996) Cell 85:319-329).
Transgenic animals may be made having exogenous ASTH1 genes. The
exogenous gene is usually either from a different species than the animal
host, or is
otherwise altered in its coding or non-coding sequence. The introduced gene
may
be a wild-type gene, naturally occurring polymorphism, or a genetically
manipulated
sequence, for example those previously described with deletions, substitutions
or
insertions in the coding or non-coding regions. The introduced sequence may
encode an ASTH1 polypeptide, or may utilize the ASTH1 promoter operably linked
to a reporter gene. Where the introduced gene is a coding sequence, it usually
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operably linked to a promoter, which may be constitutive or inducible, and
other
regulatory sequences required for expression in the host animal.
Specific constructs of interest, but are not limited to, include anti-sense
ASTH1, which will block ASTH1 expression, expression of dominant negative
ASTH 1 mutations, and over-expression of a ASTH 1 gene. A detectable marker,
such as lac 2 may be introduced into the ASTH1 locus, where upregulation of
ASTH1 expression will result in an easily detected change in phenotype.
Constructs utilizing the ASTH1 promoter region, e.g. SEQ ID N0:1; SEQ ID
N0:14,
in combination with a reporter gene or with the coding region of ASTH1J or
ASTH1l
are also of interest.
The modified cells or animals are useful in the study of ASTH1 function and
regulation. Animals may be used in functional studies, drug screening, etc.,
e.g. to
determine the effect of a candidate drug on asthma. A series of small
deletions
and/or substitutions may be made in the ASTH1 gene to determine the role of
different exons in DNA binding, transcriptional regulation, etc. By providing
expression of ASTH1 protein in cells in which it is otherwise not normally
produced,
one can induce changes in cell behavior. These animals are also useful for
exploring models of inheritance of asthma, e.g. dominant v. recessive;
relative
effects of different alleles and synergistic effects between ASTH1l and ASTH1J
and
other asthma genes elsewhere in the genome.
DNA constructs for homologous recombination will comprise at least a
portion of the ASTH1 gene with the desired genetic modification, and will
include
regions of homology to the target locus. DNA constructs for random integration
need not include regions of homology to mediate recombination. Conveniently,
markers for positive and negative selection are included. Methods for
generating
cells having targeted gene modifications through homologous recombination are
known in the art. For various techniques for transfecting mammalian cells, see
Keown et aL (1990) Methods in Enzymology 185:527-537.
For embryonic stem (ES) cells, an ES cell fine may be employed, or
embryonic cells may be obtained freshly from a host, e.g. mouse, rat, guinea
pig,
etc. Such cells are grown on an appropriate fibroblast-feeder layer or grown
in the
presence of appropriate growth factors, such as leukemia inhibiting factor
(LIF).
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When ES cells have been transformed, they may be used to produce transgenic
animals. After transformation, the cells are plated onto a feeder layer in an
appropriate medium. Cells containing the construct may be detected by
employing
a selective medium. After sufficient time for colonies to grow, they are
picked and
analyzed for the occurrence of homologous recombination or integration of the
construct. Those colonies that are positive may then be used for embryo
manipulation and btastocyst injection. Blastocysts are obtained from 4 to 6
week
old superovulated females. The ES cells are trypsinized, and the modified
cells are
injected into the blastocoel of the blastocyst. After injection, the
blastocysts are
returned to each uterine horn of pseudopregnant females. Females are then
allowed to go to term and the resulting litters screened for mutant cells
having the
construct. By providing for a different phenotype of the blastocyst and the ES
cells,
chimeric progeny can be readily detected.
The chimeric animals are screened for the presence of the modified gene
and males and females having the modification are mated to produce homozygous
progeny. If the gene alterations cause lethality at some point in development,
tissues or organs can be maintained as allogeneic or congenic grafts or
transplants,
or in in vitro culture.
Investigation of genetic function may utilize non-mammalian models,
particularly using those organisms that are biologically and genetically
well-characterized, such as C. elegans, D. melanogaster and S. cerevisiae. For
example, transposon (Tc1 ) insertions in the nematode homolog of an ASTH1 gene
or promoter region may be made. The subject gene sequences may be used to
knock-out or to complement defined genetic lesions in order to determine the
physiological and biochemical pathways involved in ASTH1 function. A number of
human genes have been shown to complement mutations in lower eukaryotes.
Drug screening may be performed in combination with the subject animal
models. Many mammalian genes have homologs in yeast and lower animals. The
study of such homologs' physiological role and interactions with other
proteins can
facilitate understanding of biological function. In addition to model systems
based
on genetic complementation, yeast has been shown to be a powerful tool for
studying protein-protein interactions through the two hybrid system described
in
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Chien et al. (1991) P.NiA.S. 88:9578-9582. Two-hybrid system analysis is of
particular interest for exploring transcriptional activation by ASTH9
proteins.
Drug Screenings Assayrs
By providing for the production of large amounts of ASTH1 protein, one can
identify ligands or substrates that bind to, modulate or mimic the action of
ASTH1.
Areas of investigation are the development of asthma treatments. Drug
screening
identifies agents that provide a replacement or enhancement for ASTH1 function
in
affected cells. Conversely, agents that reverse or inhibit ASTH1 function may
stimulate bronchial reactivity. Of particular interest are screening assays
for agents
that have a low toxicity for human cells. A wide variety of assays may be used
for
this purpose, including labeled in vitro protein-protein binding assays,
protein-DNA
binding assays, electrophoretic mobility shift assays, immunoassays for
protein
binding, and the like. The purified protein may also be used for determination
of
three-dimensional crystal structure, which can be used for modeling
intermolecular
interactions, transcriptional regulation, etc.
The term "agent" as used herein describes any molecule, e.g. protein or
pharmaceutical, with the capability of altering or mimicking the physiological
function of ASTH1. Generally a plurality of assay mixtures are run in parallel
with
different agent concentrations to obtain a differential response to the
various
concentrations. Typically, one of these concentrations serves as a negative
control,
i.e. at zero concentration or below the level of detection.
Candidate agents encompass numerous chemical classes, though typically
they are organic molecules, preferably small organic compounds having a
molecular weight of more than 50 and less than about 2,500 daltons. Candidate
agents comprise functional groups necessary for structural interaction with
proteins,
particularly hydrogen bonding, and typically include at least an amine,
carbonyl,
hydroxyl or carboxyl group, preferably at least two of the functional chemical
groups. The candidate agents often comprise cyclical carbon or heterocyclic
structures and/or aromatic or polyaromatic structures substituted with one or
more
of the above functional groups. Candidate agents are also found among
biomolecules including, but not limited to: peptides, saccharides, fatty
acids,
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steroids, purines, pyrimidines, derivatives, structural analogs or
combinations
thereof.
Candidate agents are obtained from a wide variety of sources including
libraries of synthetic or natural compounds. For example, numerous means are
available for random and directed synthesis of a wide variety of organic
Compounds
and biomolecules, including expression of randomized oligonucleotides and
oligopeptides. Alternatively, libraries of natural compounds in the form of
bacterial,
fungal, plant and animal extracts are available or readily produced.
Additionally,
natural or synthetically produced libraries and compounds are readily modified
through conventional chemical, physical and biochemical means, and may be used
to produce combinatorial libraries. Known pharmacological agents may be
subjected to directed or random chemical modifications, such as acylation,
alkylation, esterification, amidification, etc. to produce structural analogs.
Where the screening assay is a binding assay, one or more of the molecules
may be joined to a label, where the label can directly or indirectly provide a
detectable signal. Various labels include radioisotopes, fluorescers,
chemiluminescers, enzymes, specific binding molecules, particles, e.g.
magnetic
particles, and the like. Specific binding molecules include pairs, such as
biotin and
streptavidin, digoxin and antidigoxin etc. For the specific binding members,
the
complementary member would normally be labeled with a molecule that provides
for detection, in accordance with known procedures.
A variety of other reagents may be included in the screening assay. These
include reagents like salts, neutral proteins, e.g. albumin, detergents, etc
that are
used to facilitate optimal protein-protein binding and/or reduce non-specific
or
background interactions. Reagents that improve the efficiency of the assay,
such
as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may
be used.
The mixture of components are added in any order that provides for the
requisite
binding. Incubations are performed at any suitable temperature, typically
between 4
and 40°C. Incubation periods are selected for optimum activity, but may
also be
optimized to facilitate rapid high-throughput screening. Typically between 0.1
and 1
hours will be sufficient.
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Other assays of interest detect agents that mimic ASTW1 function. For
example, candidate agents are added to a cell that lacks functional ASTH1, and
screened for the ability to reproduce ASTH1 in a functional assay.
The compounds having the desired pharmacological activity may be
administered in a physiologically acceptable carrier to a host for treatment
of
asthma attributable to a defect in ASTH1function. The compounds may also be
used to enhance ASTH1 function. The therapeutic agents may be administered in
a variety of ways, orally, topically, parenterally e.g. subcutaneously,
intraperitoneally, by viral infection, intravascularly, etc. Inhaled
treatments are of
particular interest. Depending upon the manner of introduction, the compounds
may be formulated in a variety of ways. The concentration of therapeutically
active
compound in the formulation may vary from about 0.1-100 wt.%.
The pharmaceutical compositions can be prepared in various forms, such as
granules, tablets, pills, suppositories, capsules, suspensions, salves,
lotions and the
like. Pharmaceutical grade organic or inorganic carriers andlor diluents
suitable for
oral and topical use can be used to make up compositions containing the
therapeutically-active compounds. Diluents known to the art include aqueous
media, vegetable and animal oils and fats. Stabilizing agents, wetting and
emulsifying agents, salts for varying the osmotic pressure or buffers for
securing an
adequate pH value, and skin penetration enhancers can be used as auxiliary
agents.
Pharmacogenetics
Pharmacogenetics is the linkage between an individual's genotype and that
individual's ability to metabolize or react to a therapeutic agent.
Differences in
metabolism or target sensitivity can lead to severe toxicity or therapeutic
failure by
altering the relation between bioactive dose and blood concentration of the
drug. In
the past few years, numerous studies have established good relationships
between
polymorphisms in metabolic enzymes or drug targets, and both response and
toxicity. These relationships can be used to individualize therapeutic dose
administration.
Genotyping of polymorphic alleles is used to evaluate whether an individual
will respond well to a particular therapeutic regimen. The polymorphic
sequences
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are also used in drug screening assays, to determine the dose and specificity
of a
candidate therapeutic agent. A candidate ASTH1 polymorphism is screened with a
target therapy to determine whether there is an influence on the effectiveness
in
treating asthma. Drug screening assays are performed as described above.
Typically two or more different sequence polymorphisms are tested for response
to
a therapy.
Drugs currently used to treat asthma include beta 2-agonists,
glucocorticoids, theophylline, cromones, and anticholinergic agents. For
acute,
severe asthma, the inhaled beta 2-agonists are the most effective
bronchodilators.
Short-acting forms give rapid relief; long-acting agents provide sustained
relief and
help nocturnal asthma. First-line therapy for chronic asthma is inhaled
glucocorticoids, the only currently available agents that reduce airway
inflammation.
Theophylline is a bronchodilator that is useful for severe and nocturnal
asthma, but
recent studies suggest that it may also have an immunomodulatory effect.
Cromones work best for patients who have mild asthma: they have few adverse
effects, but their activity is brief, so they must be given frequently.
Cysteinil
leukotrienes are important mediators of asthma, and inhibition of their
effects may
represent a potential breakthrough in the therapy of allergic rhinitis and
asthma.
Where a particular sequence polymorphism correlates with differential drug
effectiveness, diagnostic screening may be performed. Diagnostic methods have
been described in detail in a preceding section. The presence of a particular
polymorphism is detected, and used to develop an effective therapeutic
strategy for
the affected individual.
EXPERIMENTAL
The following examples are put forth so as to provide those of ordinary skill
in the art with a complete disclosure and description of how to make and use
the
subject invention, and are not intended to limit the scope of what is regarded
as the
invention. Efforts have been made to ensure accuracy with respect to the
numbers
used (e.g. amounts, temperature, concentrations, etc.} but some experimental
errors and deviations should be allowed for. Unless otherwise indicated, parts
are
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parts by weight, molecular weight is average molecular weight; temperature is
in
degrees centigrade; and pressure is at or near atmospheric.
MATERIALS AND METHODS
Asthma families for genetic mapping studies
Asthma phenotype measurements and blood samples were obtained from
the inhabitants of Tristan da Cunha, an isolated island in the South Atlantic,
and
from asthma families in Toronto, Canada (see Zamel et al., (1996) supra.) The
282
inhabitants of Tristan da Cunha form a single large extended family descended
from
28 original founders. Settlement of Tristan da Cunha occurred beginning in
1817
with soldiers who remained behind when a British garrison was withdrawn from
the
island, followed by the survivors of several shipwrecks. In 1827 five women
from
St. Helena, one with children, emigrated to Tristan da Cunha and married
island
men. One of these women is said to have been asthmatic, and could be the
origin
of a genetic founder effect for asthma in this population. Inbreeding has
resulted in
kinship resemblances of at least first cousin levels for all individuals.
The Tristan da Cunha family pedigrees were ascertained through review of
baptismal, marriage and medical records, as well as reliably accurate
historical
records of the early inhabitants (Zamel (1995) Can. Resl i~ r-J. 2:18). The
prevalence of asthma on Tristan da Cunha is high; 23% had a definitive
diagnosis
of asthma.
The Toronto cohort included 59 small families having at least one affected
individual. These were ascertained based on the following criteria: (i) an
affected
proband; (ii) availability of at least one sibling of the proband, either
affected or
unaffected; (iii) at least one living parent from whom DNA could be obtained.
A set
of 156 "triad" families consisting of an affected proband and his or her
parents were
also collected. Signed consent forms were obtained from each individual prior
to
commencement of phenotyping and blood sample collection. The Toronto patients
were mainly of mixed European ancestry.
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Clinical characterization -
A standardized questionnaire based on that of the American Thoracic
Society (American Lung Association recommended respiratory diseases
questionnaire for use with adults and children in epidemiology research. 1978.
American Review of es iratoryr Disease 118(2):7-53) was used to record the
presence of respiratory symptoms such as cough, sputum and wheezing; the
presence of other chest disorders including recent upper respiratory tract
infection,
allergic history; asthmatic attacks including onset, offset, confirmation by a
physician, prevalence, severity and precipitating factors; other illnesses and
smoking history; and all medications used within the previous 3 months. A
physician-confirmed asthmatic attack was the principal criterion for a
diagnosis of
asthma.
Skin atopy was determined by skin prick tests to common allergens:
A. fumigatus, Cladosporium, Altemaria, egg, milk, wheat, tree, dog, grass,
horse,
house dust, cat, feathers, house dust mite D. farinae, and house dust mite
D. pferonyssinus. Atopy testing of Toronto subjects omitted D. pteronyssinus
and
added cockroach and ragweed allergens. Saline and histamine controls were also
performed (Bencard Laboratories, Mississauga, Ontario). Antihistamines were
withdrawn for at least 48 hours prior to testing. Wheat diameters were
corrected by
subtraction of the saline control wheat diameter, and a corrected wheat size
of >3
mm recorded 10 min after application was considered a positive response.
Airway responsiveness was assessed by a methacholine challenge test in
those subjects with a baseline FEV1 (forced exhalation volume in one second) >
70% of predicted (Crapo et al: (1981) Am. Rev. Res ir. Dis. 123:659).
Methacholine challenge response was determined using the tidal breathing
method
(Cockcroft et al. (1977) Clin. Allergyr 7:235). Doubling doses of methacholine
from
0.03 to 16 mg/ml were administered using a Wright nebulizer at 4-min intervals
to
measure the provocative concentration of methacholine producing a 20% fall in
FEV1 (PC20). If FEV1 was <70% of predicted, a bronchodilator response to 400
mg salbutamol aerosol was used to determine airway responsiveness. Both
methacholine challenges and bronchodilator responses were measured using a
computerized bronchial challenge system (S&M Instrument Co. Inc., Doyleston,
PA)
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consisting of a software package and interface board installed in a Toshiba
T1850C
laptop computer and connected to a flow sensor (RS232FS). The power source for
instruments used on Tristan da Cunha has been described (Zamel et al. (1996)
supra.) Increased airway responsiveness was defined as a PC20 < 4.0 mg/ml or a
> 15% improvement in FEV1 15 min postbronchodilator. Participants were asked
to
withhold bronchodilators at least 8 h before testing; inhaled or systemic
steroids
were maintained at the usual dosage. Subjects with a history of an upper
respiratory tract infection within a month of testing were rechallenged at a
later date.
Genotyping
PCB primer pairs were synthesized using Applied Biosystems 394
automated oligo synthesizer. The forward primer of each pair was labeled with
either FAM, HEX, or TET phosphoramidites (Applied Biosystems). No oligo
purification step was performed.
~ Genomic DNA was extracted from whole blood. PCB was performed using
PTC100 thermocyclers (MJ Research). Reactions contained 10 mM Tris-HCI, pH
8.3; 1.5-3.0 mM MgCl2; 50 mM KCI; 0.01 % gelatin; 250 ~M each dGTP, dATP,
dTTP, dCTP; 20 p,M each PCB primer; 20 ng genomic DNA; and 0.75 U Taq
Polymerase (Perkin Elmer Cetus) in a final volume of 20 ~.I. Reactions were
performed in 96 well polypropylene microtiter plates (Bobbins Scientific) with
an
initial 94°C, 3 min. denaturation followed by 35 cycles of 30 sec. at
94°C, 30 sec. at
the annealing temp., and 30 sec. at 72°C, with a final 2 min. extension
at 72°C
following the last cycle. Dye label, annealing temperature, and final
magnesium
concentration were specific to the individual marker.
Dye label intensity and quantity of PCB product (as assessed on agarose
gels) were used to determine the amount to be pooled for each marker locus.
The
pooled products were precipitated and the product pellets mixed with 0.4 ~.I
Genescan 500 Tamra size standard, 2 ~I formamide, and 1 p,l ABI loading dye.
Plates of PCB product pools were heated to 80°C for 5 minutes and
immediately
placed on ice prior to gel loading.
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PCR products were electrophoresed on denaturing 6%-polyacrylamide gels
at a constant 1000 volts using ABI 373a instruments. Peak detection, sizing,
and
stutter band filtering were achieved using Genescan 1.2 and Genotyper 1.1
software (Applied Biosystems). Genotype data were subsequently submitted to
quality control and consistency checks (Hall et al. (1996) Genome Res. 6:781).
Genotyping of 'saturation' markers in the ASTH1 region was done by the
method described above with several exceptions. In most cases, the unlabeled
primer of each pair was modified with the sequence GTTTCTT at the 5' end
(Smith
et al. 1995 Genome Res. 5:312). Amplitaq Gold (Perkin Elmer Cetus) and buffer
D
(2.5 mM MgCl2, 33.5 mM Tris-HCI pH 8.0, 8.3 mM (NH4)2S04, 25 mM KCI, 85 Ng/ml
BSA) were used in the PCR. A 'touchdown' amplification profile was employed in
which the annealing temperature began at 66°C and decreased one degree
per
cycle to a final 20 cycles at 56°C. Products were run on 4.25%
polyacrylamide gels
using ABI 377 instruments. The data was processed with Genescan 2.1 and
Genotyper 1.1 software.
The Genome Scan
A genome scan was performed in the population of Tristan da Cunha using
274 polymorphic microsatellite markers chosen from among those developed at
Oxford (Reed et al. (1994) Nature Genetics 7:390), Genethon (Dib et al. (1996)
Nature 380:152) and the Cooperative Human Linkage Center (CHLC, Murray et at.
(1994) ' nc 265:2049). Markers with heterozygosity values of 0.75 or greater
were selected to cover all the human chromosomes, as well as for ease of
genotyping and size of PCR product for multiplexing of markers on gels.
Fifteen
multiplexed sets were used to provide a ladder of PCR products in each of
three
dyes when separated by size. Published distances were used initially to
estimate
map resolution. More accurate genetic distances were calculated using the
study
population as the data was generated. The 274 markers gave an average 14 cM
interval for the genome scan.
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Linkage analysis -
Parametric linkage analyses of marker data were conducted using the
methods of Haseman and Elston (1972} Sehav. Genet. 2:3, and FASTLINK
(Schaffer et al. (1996) Hum. Hered. 46:226), assuming a dominant mode of
transmission with incomplete penetrance. Linkage to three primary phenotypes
including asthma diagnosis (history), airway responsiveness (PC20 < 4 mg/ml
for
methacholine challenge) and atopy (one or more skin-prick test which yielded a
wheat diameter > 3 mm) and combinations of these, were tested.
Small scale yeast artificial chromosome (YAC) DNA preparation
Small scale isolation of YAC DNA for STS mapping was done by a procedure
which uses glass beads and physical shearing to damage the yeast cell wall
(Scherer and Tsui (1991) Cloning and analyrsis of largie DNA molecules, In
Advanced Techniques in Chromosome Research. (K.W. Adolph, ed.) pp. 33-72.
Marcel Dekker, Inc. New York, Basel, Hong Kong.)
YAC block prep and pulsed field gel electrophoresis (PFGE)
A 50 ml culture of each YAC was grown in 2 x AHC at 30°C. The
cells were
pelleted by centrifugation and washed twice in sterile water. After
resuspension of
the cells in 4 ml of SCEM (1 M sorbitol, 0.1 M sodium citrate (pH 5.8), 10 mM
EDTA, 30 mM ~i-mercaptoethanol), 5 ml of 1.2% low melting temperature agarose
in SCEM was added, mixed, pipetted into 100 ml plug molds and allowed to
solidify.
Plugs were incubated overnight in 50 ml of SCEM containing 30 U/ml lyticase
(Sigma). Plugs were rinsed 3 times in TE (10 mM Tris pH 8.0, 1 mM EDTA) and
incubated twice for 12 hours each at 50°C in lysis solution (0.5 M
EDTA, pH 8.0;
1 % wlv sodium lauryl sarcosine; 0.5 mglml proteinase K}. They were washed 5
times with TE and stored in 0.5 M EDTA (pH 8.0) at 4°C.
YACs and yeast chromosomes were separated on pulsed field gels using a
CHEF Mapper (BIO-RAD) and according to methods supplied by the manufacturer,
then transferred to nitrocellulose. YACs which comigrated with yeast
chromosomes
were visualized by hybridization of the blot with radiolabelled YAC vector
sequences (Scherer and Tsui (1991) supra.)
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Hybridization of YAC DIVA to bacterial artificial chromosome (BAC) and cosmid
grids
Size-purified YAC DNA was prepared by pulsed field gel electrophoresis on a
low melting temperature Seaplaque GTG agarose (FMC) gel, purified by
GeneClean (810101) and radiolabeled for 30 mins with 32P-dCTP using the Prime-
It
II kit (Stratagene). 50 ~I of water was added and unincorporated nucleotide
was
removed by Quick Spin Column (Boehringer Mannheim). 23 p,l of 11.2 mg/ml
human placenta( DNA (Sigma) and 36 p,l of 0.5 M Na2HP04, pH 6.0 were added to
the approximately 150 ~I of eluant. The probe was boiled for 5 mins and
incubated
at 65°C for exactly 3 hours, then added to the prehybridized gridded
BAC (Shizuya
et al. (1992) Proc. Natl. Acad. Sci. 89:8794; purchased from Research
Genetics) or
chromosome 11 cosmid [Resource Center! Primary Database of the German
Human Genome Project, Berlin; Lehrach et al. (7990), In Davies, K.E. and
Tilghman, S.M. (eds.), Genome Analyrsis Volume 1: Genetic and Physical
Mapping.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 39-81 ] filters
in
dextran sulfate hybridization mix (10% dextran sulfate, 1 % SDS, 1 M NaCI).
Hybridizations were at 65°C for 12 - 48 hours, followed by 2 washes
at room
temperature in 2x SSC for 10 mins each, and 3 washes at 65°C in 0.2X
SSC, 0.2%
SDS for 20 mins each.
Metaphase fluorescence in situ hybridization (FISH) and direct visual in situ
hybridisation (DIRVISH)
Metaphase FISH was carried out by standard methods (Heng and Tsui
(1994) FISH detection on DAPI banded chromosomes. In Methods of Molecular
Biolog~r: In Situ HKbridisation Protocols (K.H.A. Choo, ed.) pp. 35-49. Human
Press,
Clifton, N.J.). High resolution FISH, or DIRVISH, was used to map the relative
positions of two or more clones on genomic DNA. The protocol used was as
described by Parra and Windle (1993) Nature Genet. 5:17. Briefly, slides
containing stretched DNA were prepared by adding 2 ~I of a suspension of
normal
human lymphoblast cells at one end of a glass slide and allowing to dry. 8 ~,I
lysis
buffer (0.5% SDS, 50 mM EDTA, 200 mM Tris-HCL, pH 7.4) was added and the
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slide incubated at room temperature for 5 minutes. The slide was tilted so
that the
DNA ran down the slide, then dried. The DNA was fixed by adding 400 ~I 3:1
methanol/acetic acid. Probes were labeled either with biotin or with
digoxygenin by
standard nick translation (Rigby et al. (1977) J. Mol. Biol. 113:237).
Hybridization
and detections were carried out using standard fluorescence in situ
hybridization
techniques (Heng and Tsui (1994) supra.). Results were visualised using a
Mikrophot SA microscope (Nikon) equipped with a CCD camera (Photometrics).
Images were recorded using Smartcapture software (Vysis).
Gap filling
Clones flanking gaps in the map were end cloned by digestion with enzymes
that do not cut the respective vector sequences (Nsil for BAC clones and Xbal
for
PAC clones), followed by religation and transformation into competent DHSa.
Clones which produced two end fragments and plasmid vector upon digestion with
Notl and Nsil or Xbal were sequenced. Gaps in the tiling path were filled by
screening a gridded BAC library with the end clone probes or by screening DNA
pools of a human genomic PAC library (loannou et al. (1994) Nature Genetics
6:84;
licensed from Health Research, Inc.) by PCR using primers designed from end
clone sequences.
Direct cDNA selection
Direct cDNA selection (Lovett et al., (1991) Proc. Natl. Acad. Sci. 88:9628)
was carried out using cDNA derived from both adult whole lung tissue and fetal
whole lung tissue (Clontech). 5 ~,g of Poly(A)+ RNA was converted to double
stranded cDNA using the Superscript Choice System for cDNA synthesis and the
supplied protocol (Gibco BRL). First strand priming was achieved by both
oligo(dT)
and random hexamers. The resulting cDNA was split into 2 equal aliquots and
digested with either Mbol or Taql prior to the addition of specific linker
primers.
Linker primers for Mbol-digested DNA were as described by Morgan et al. (1992)
Nucleic Acid Res. 20:5173. Linker primers for Taql-digested DNA were a
modification of these:
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(SEQ ID N0:336 ) Taq1a: 5'-CGAGAATTCACTCGAGCATCAGG;
(SEQ ID N0:337 ) Taq1b: 5'-CCTGATGCTCGAGTGAATTCT. The modified cDNA
was ethanol precipitated and resuspended in 200 ~I of H20. 1 ~.I of cDNA was
amplified with the linker primer Mbo1 b in a 100 ~.I PCR reaction. The
resulting
cDNA products, approximately 1 fig, were blocked with 1 ~g of COT1 DNA (Gibco
BRL) for 4 hours at 60°C in 120 mM NaP04 buffer, pH 7Ø
Approximately 1 ~g of the appropriate genomic clones was biotinylated using
the BioNick Labeling System (Gibco BRL). Unincorporated biotin was removed by
spin column chromatography. Approximately 100 ng of biotinylated genomic DNA
was denatured and allowed to hybridize to 1 ~g of blocked cDNA in a total
volume
of 20 ~,I in 120 mM NaP04 for 60 hours at 60°C under mineral oil. After
hybridization, the biotinylated DNA was captured on streptavidin-coated
magnetic
beads (Dynal) in 100 ~.i of binding buffer (1 M NaCI, 10 mM Tris, pH 7.4, 1 mM
EDTA) for 20 minutes at room temperature with constant rotation. Two 15 minute
washes at room temperature with 500 ~I of 1 X SSC/0.1 % SDS were followed by
four washes for 20 minutes at 65°C with 500 ul of 0.1X SSC/0.1 % SDS
with
constant rotation. After each wash, the beads were collected on the side of
the tube
using magnet separation and the supernatant was removed with a pipette.
Following the last wash, the beads were briefly rinsed once with wash solution
prior
to eluting the bound cDNA with 50 ~,I of 0.1 M NaOH for 10 minutes at room
temperature. The supernatant was removed and neutralized with 50 ~I 1 M Tris
pH
7.4. The primary selected cDNA was desalted using a Sephadex G-50 column
(Boehringer Mannheim). PCR was performed on 1, 2, 5, and 10 ~,I of eluate with
Mbo1b primers. Amplified products were analyzed on a 1.4% agarose gel. The
reaction with the cleanest bands and least background was scaled up to produce
approximately 1 ~,g of primary selected cDNA. This amplified primary selected
cDNA was blocked with 1 pg of COT1 at 60°C for 1 hour followed by a
second
round of hybridization to 100 ng of the appropriate genomic DNA under the same
conditions as the first round of selection. Washing of the bound cDNA,
elution, and
PCR of the selected cDNA was identical to the first round. 1 ~I of PCR
amplified
secondary selected cDNA was cloned using the TA cloning system according to
the
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manufacturers protocol (Invitrogen). Colonies were picked into 96-well
microtiter
plates and grown overnight prior to sequencing.
Exon Trapping
Exon trapping was performed by the method of Buckler et al. (1991, Proc.
Natl. Acad. Sci. USA 88:4005) with modifications described in Church et al.,
(1994)
Nature Genetics 6:98. Each BAC clone of the minimal set of clones required to
the
cover the ASTH1 region (i.e. the tiling path) was subject to exon trapping
separately. Briefly, restriction fragments (Pstl or BamHI/Bglll) of each
cosmid were
shotgun subcloned into Pstl- or BamHl-digested and phosphatase-treated pSPL3B
which had been modified as in Burns et al. (1995) Gene 161:183 (GIBCO BRL).
Ligations were electroporated into ElectroMax HB101 cells (Gibco BRL) and
plated
on 20 cm diameter LB ampicillin plates. DNA was prepared from plates with >
2000
colonies by collection of the bacteria in LB ampicillin liquid and plasmid DNA
purification by a standard alkaline lysis protocol (Sambrook et al. (1989)
supra.) 5
~g of DNA from each plasmid pool preparation were electroporated into Cos 7
cells
(ATCC) and RNA harvested using TRIZOL (Gibco BRL) after 48 hours of growth.
RT-PCR products were digested with BstXl prior to a second PCR amplification.
Products were cloned into pAMP10 (Gibco BRL) and transformed into DH5 cells
(Gibco BRL). 96 colonies per BAC were picked and analyzed for insert size by
PCR.
Northern blot hybridisation
Northern hybridisation was performed using Multiple Tissue Northern (MTN)
blots (Clontech). DNA probes were radioactively labeled by random priming
[Feinberg and Vogelstein (1984) Anal. Biochem. 137:266] using the Prime-It II
kit
(Stratagene). Hybridizations were performed in ExpressHyb hybridisation
solution
(Clontech) according to the manufacturer's recommendations. Filters were
exposed
to autoradiographic film overnight or for 3 days.
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cDNA library screening
Phage cDNA libraries were plated and screened with radiolabeled probes
(exon trapping or cDNA selection products amplified by PCR from plasmids
containing these sequences) by standard methods (Sambrook et al. (1989)
supra.)
Rapid amplification of cDNA ends (RACE)
RACE libraries were constructed using polyA+ RNA and the Marathon cDNA
amplification kit (Clontech). Nested RACE primer sets were designed for each
cDNA or potential gene fragment (trapped exon, predicted exon, conserved
fragment, efc). The RACE libraries were tested by PCR using one primer pair
for
each potential gene fragment; the two strongly positive libraries were chosen
for
RACE experiments.
Genomic sequencing
DNA from cosmid, PAC, and BAC clones was prepared using Qiagen DNA
prep kits and further purified by CsCt gradient. DNA was sonicated and DNA
fragments were repaired using nuclease BAL-31 and T4 DNA polymerase. DNA
fragments of 0.8-2.2 kb were size-fractionated by agarose gel electrophoresis
and
ligated into pUC9 vector. Inserts of the plasmid clones were amplified by PCR
and
sequenced using standard ABI dye-primer chemistry.
ABI sample file data was reanalyzed using Phred (Phil Green, University of
Washington) for base calling and quality analysis. Sequence assembly of
reanalyzed sequence data was accomplished using Phrap (Phil Green, University
of Washington). Physical gaps between assembled contigs and unjoined but
overlapping contigs were identified by inspection of the assembled data using
GFP
(licensed from Baylor College of Medicine) and Consed (Phil Green, University
of
Washington). Material for sequence data generation across gaps was obtained by
PCR amplification. Low coverage regions were resequenced using dye-primer and
dye-terminator chemistries (ABI). Final base-perfect editing (to > 99%
accuracy)
was accomplished using Consed.
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Single stranded conformational polymorphism (SSCP) analysis -
PCR primers flanking each exon of the ASTH11 and ASTH1J genes, or more
than one primer pair for large exons, were designed from genomic sequence
generated using Primer (publicly available from the Whitehead institute for
Biomedical Research) or Oligo 4.0 (licensed from National Biosciences).
Radioactive SSCP was performed by the method of Orita et al. (1989, Proc.
Natl.
Acad. Sci. 86:2766). Briefly, radioactively labeled PCR products between 150
and
300 by and spanning exons of the ASTH 1 I and ASTH 1 J genes were generated
from a set of asthma patient and control genomic template DNAs, by
incorporating
a-32P dCTP in the PCR. PCR reactions (20 ~I) included 1x reaction buffer, 100
~M
dNTPs, 1 pM each forward and reverse primer, and 1 unit Taq DNA polymerase
(Perkin-Elmer) and 1 ~,Ci a 3zP dCTP. A brief denaturation at 94°C was
followed by
30-32 cycles of: 94°C for 30 sec, 30 sec at the annealling temperature,
and 72°C for
30 sec; followed by 5 mins at 72°. Radiolabeled PCR products were
diluted 1:20 in
water, mixed with an equal volume of denaturing loading dye (95% formamide,
0.25% bromophenol blue), and denatured for 10 minutes at 80°C
immediately prior
to electrophoresis. 0.5x MDE (FMC} gels with and without 8% glycerol in 1 x
TBE
were run at 8-12 Watts for 16-20 hours at room temperature. Dried gels were
exposed to autoradiographic film (Kodak XAR) for 1-2 days at -80°C. PCR
products
from individuals carrying SSCP variants were subcloned into the PCR2.1 or
pZeroBlunt plasmid vector (Invitrogen). Inserts of the plasmid clones were
amplified
by PCR and sequenced using standard ABI dye-primer chemistry to determine the
nature of the sequence variant responsible for the conformational changes
detected
by SSCP.
Fluorescent SSCP was carried out according to the recommended ABI
protocol (ABI User Bulletin entitled 'Multi Color Fluorescent SSCP').
Unlabeled
PCR primers were used to amplify genomic DNA segments containing different
exons of the ASTH11 or ASTH1J genes, in patient or control DNA. Nested
fluorescently labeled (TET, FAM or HEX) primers were then used to amplify
smaller
products, 150 to 300 by containing the exon or region of interest.
Amplification was
done using a 'touchdown' PCR protocol, in which the annealing temperature
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decreased from 57°C to 42°C, and Amplitaq Gold polymerase
fPerkin Elmer,
Cetus}. In most cases the fluorescently labeled primers were identical in
sequence
to those used for conventional radioactive SSCP. The fluorescent PCR products
were diluted and mixed with denaturing agents, GeneScan size standard
(Genescan 500 labelled with Tamra) and Blue dextran dye. Samples were heated
at 90°C and quick chilled on ice prior to loading on 6.5% standard or
0.5 X MDE
(manufacturer) polyacrylamide gels containing 2.5% glycerol and run using
externally temperature controlled modified ABI 377 instruments. Gels were run
at
1240V and 20°C for 7-9 hrs and analyzed using GeneScan software (ABI}.
Comparative (heterozygote detection) sequencing
Unlabeled PCR primers were used to amplify genomic DNA segments
containing different exons of the ASTH11 orASTH1J genes, from patient or
control
DNAs. A set of nested PCR primers was then used to reamplify the fragment.
Unincorporated primers were removed from the PCR product by Centricon-100
column (Amicon), or by Centricon-30 column for products less than 130 bp. The
nested primers and dye terminator sequencing chemistry (ABI PRISM dye
terminator cycle sequencing ready reaction kit) were then used to cycle
sequence
the exon and flanking region. Volumes were scaled down to 5 p.l and 10% DMSO
added to increase peak height uniformity. Sequences were compared between
samples and heterozygous positions detected by visual inspection of
chromatograms and using Sequence Navigator (licensed from ABI).
For some exons, PCR products were also compared by subcloning and
sequencing, and comparison of sequences for ten or more clones.
RESULTS
Genome scanning and linkage analysis
A genome scan was performed using polymorphic microsatellite markers
from throughout the human genome, and DNA isolated from blood samples drawn
from the inhabitants of Tristan da Cunha. Linkage analysis, an established
statistical method used to map the locations of genes and markers relative to
other
markers, was applied to verify the marker orders and relative distances
between
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markers on all human chromosomes, in the Tristan da Cunha population. Linkage
analysis can detect cosegregation of a marker with disease, and was used as a
means to detect genes influencing the development of asthma in this
population.
The most highly significant linkage in the genome scan (p = 0.0001 for history
of
asthma and p = 0.0009 for methacholine challenge) was obtained at D11S907, a
marker on the short arm of chromosome 11. This significant linkage result
indicated
that a gene influencing predisposition to asthma in the Tristan da Cunha
population
was located near D115907.
Replication of this finding was obtained in a collection of asthma families
from Toronto, in which D115907 and several nearby markers were tested for
linkage. The significant linkage seen (p = 0.001 for history of asthma and p =
0.05
for methacholine challenge) supported the mapping of an asthma gene near
D115907 and indicated that the gene was likely to be relevant in the more
diverse
outbred Toronto group as well as in the inbred population of Tristan da Cunha.
The approximate genetic location of the ASTH1 gene in the Tristan da
Cunha population was confirmed by genotyping and analyzing data from several
markers near D11S907, spaced at intervals no greater than 5 cM across a
possible
linked region of about 30 cM. Sib-pair and affected pedigree member linkage
analyses of these markers yielded confirmatory evidence for linkage and
refined the
genetic interval.
Physical mapping at ASTH?: YAC contig construction
Yeast artificial chromosome (YAC) clones were derived from the CEPH
megaYAC library (Cohen et al. 1993 Nature 366:698). Individual YAC addresses
were obtained from a public physical map of CEPH megaYAC STS (sequence
tagged site; Olson ef al. (1989) i a 245:1434) mapping data maintained by the
Whitehead Institute and accessible through the world wide web (Cohen et al.
1993.
supra.; http://www-genome.wi.mit.edulcgi-bin/contig/phys map). YAC clones
spanning or overlapping other YACs containing D11S907 were chosen for map
construction; STSs mapping to these YACs were used for map and clone
verification. Some YACs annotated in the public database as being chimeric
were
excluded from the analyses. Multiple colonies of each YAC, obtained from a
freshly
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streaked plate inoculated from the CEPH megaYAC library masterplate, were
scored using STS markers from the ASTH1 region. These markers included
polymorphic microsatellite repeats, expressed sequence tags (ESTs) and STSs.
Comparison of STS mapping data for each clone with the public map allowed
choice of the individual clone which retained the greatest number of ASTH1
region
STSs, and was therefore least likely to be deleted. YAC addresses for which
clones differed in STS content were interpreted to be prone to deletion; those
for
which a subset of clones contained no ASTH1 region STSs were presumed to be
contaminated with yeast cells containing a YAC from another region of the
genome.
Chimerism of the chosen clones was assessed by metaphase fluorescent in situ
hybridization (FISH). Their sizes were determined by pulsed field gel
electrophoresis (PFGE), Southern blotting and hybridization with a YAC vector
probe. The PFGE analyses also showed that no YAC clone chosen contained
more than one yeast artificial chromosome.
An STS map based on assuming the least number of deletions in the YAC
clones was generated. The STS marker order was in agreement with that of the
Whitehead map. The STS retention pattern of individual YACs, however, was
slightly different from that of the public data. In general, the chosen clones
were
positive for a greater number ASTH1 region markers, showing that the data set
was
likely to have fewer false negatives than the public map. Non-chimeric YAC
clones
spanning the region of greatest interest were chosen for use as hybridization
probes for the identification of smaller BAC, PAC, P1 or cosmid clones from
the
region.
Conversion to a plasmid based clone map
The YAC map at ASTH1 provided continuous coverage of a 4 Mb region, the
central 1 Mb of which was of greatest interest. YAC clones comprising a
minimal
tiling path of this region were chosen, and the size purified artificial
chromosomes
were used as hybridization probes to identify BAC and cosmid clones. Gridded
filters of a 3x human genomic BAC library and of a human chromosome 11-
specific
cosmid library were hybridized with radiolabeled purified YAC. Clones
corresponding to the grid coordinates of the positives were streaked to colony
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purity, and filters gridded with four clones of each BAC or cosmid. These
secondary filters were hybridized with size-purified YAC DNAs. A proportion of
both
the BACs and cosmids were found to be non-clonal by these analyses. A
positively
hybridizing clone of each was chosen for further analysis.
The BAC and cosmid clones were STS mapped to establish overlaps
between the clones. The BACs were further localized by DIRVISH. BACs which
did not contain an STS marker were mapped in pairwise fashion by simultaneous
two-color DIRVISH with another BAC. The map produced had three gaps which
were subsequently filled by end cloning and hybridization of the end clones to
a
human genomic PAC library. Genetic refinement of the ASTH1 region had
occurred concurrently with mapping, rendering it unnecessary to extend the BAC-
contigged region. Mapping data was recorded in ACeDB (Eeckman and Durbin
(1995) Methods Cell Biol. 48:583).
Genomic sequencing and gene prediction
A minimal tiling path of BAC and cosmid clones was chosen for genomic
sequencing. Over 1 Mb of genomic sequence was generated at ASTH1. On
average, sequencing was done to 12x coverage (12 times redundancy in
sequences). Marker order was verified relative to the STS map.
BLAST searches (Altschul et al. (1990) supra.) were performed to identify
sequences in public databases that were related to those in the ASTH1 region.
Sequence-based gene prediction was done with the GRAIL [Roberts (1991)
~jence 254:805] and Geneparser [Snyder and Stormo (1993) Nucleic Acids Res.
21: 607] programs. Genomic sequence and feature data was stored in ACeBD.
Development of new microsatellite markers for genetic refinement of the ASTN1
region
Additional informative polymorphic markers were important for the genetic
refinement of the ASTH1 region. 'Saturation' cloning of every microsatellite
in the
1 Mb region surrounding D11S907 was performed. Plasmid libraries were
constructed from PFGE purified DNA from each YAC, prescreened with a primer
from each known microsatellite marker, then screened with radiolabeled (CA)15
or
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a pool of trinucleotide and tetranucleotide repeat oligonucleotides. The
plasmid
inserts were sequenced, the set of sequences compared with those of the known
microsatellite markers in the region, using Power assembler (ABI) or
Sequencher
(Alsbyte). Primer pairs flanking each novel microsatellite repeat were
designed,
and the heterozygosity of each new marker was tested by Batched Analysis of
Genotypes (BAGs; LeDuc ef al., 1995, PCR Methods and ARplications 4:331 ).
Additional microsatellites were found by analysis of the genomic sequence in
AceDB. Table 1 lists all the microsatellite markers used for genotyping in the
ASTH1 region and their repeat type, source and primers. Table 1B lists some
repeat sequences.
TABLE 1
Polymorphic microsatellite markers in the ASTH1 region
SEQ ID MARKER PRIMER 1
160. 11005GT1 CTGCTGTGGACGAATAGG
161. TCAATATAATCTTGCTTAACTTGG
162. 139C7GT1 GACCTGTTTGGGTTGATTTCAG
163. GTTTCTTACAGTGTCTTGCTATCACATCACC
164. 171L24AT1 GAGGACTGGCAGTACCAAGTAAAC
165. GTTTCTTTGGTTCATTCTAAGATGGCTGG
166. 253E6GT1 GCTGAGGCAGGAGAAAAGACAAG
167. GTTTCTTCATGCAAAGGTCAGGAGGTAGG
168. 253E6TE1 GTTGCTTCCAGACGAGGTACATG
169. GTTTCTTCAATGGCTCCACAAACATCTCTG
170. 253E6TR1 AGGTTTAGGGGACAGGGTTTGG
171. GTTTCTTTCCTGGCTAACACGGTGAAATC
172. 65P14 GTTTCTTATTGCCTCCTCCCAAAATTC
173. AGAGGCCACTGGAAGACGAA
174. 65P14GT1 AACTGGAGTCAGGCAAAACGTG
175. GTTTCTTTGGCTGGTAAGGAAAGAAACCAC
3O 176. 65P14TE1 GGCTAGGTTCATAAACTCTGTGCTG
177. GTTTCTTGATTGTTTGAGATCCTTGACCCAG
178. 65P14TE2 GCCGAAATCACAACACTGCATC
179. GTTTCTTGATTCTGCTCTTACTCTTGCCCC
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180. 65P14TR1 GTAATAGAACCAAAGGGCTGAGAC
181. GTTTCTTCGGAGTCAGACCTTACATTGTTGAG
182. 774F ATCTCCCTGCTACCCACCTT
183. GTTTCTTGTTTTCAGTGAGTTTCTGTTGGG
184. 774J GTGTGCCAAACAACATTTGC
185. GTTTCTTCAAGCCATCAAGCTAGAGTGG
186. 774L GGGCTTTTAAACCCTTATTTAACC
187. GTTTCTTAGGTGATCTCAGAGCCACTCA
188. 774N AGGGCAGGTGGGAACTTACT
O 189. GTTTCTTTGGAGTCAGTTGAGCTTTCTACC
190. 7740 TGAACTTGCCTACCTCCCAG
191. GTTTCTTAGCATATATCCTTACACAAGCACA
192. 774T CATGGTTCCAAAGGCAAGTT
193. GTTTCTTTTGAGGCTGAATGAGCTGTG
194. 86J5AT2 ACAGGTGGGAAGACTGAATGTC
195. GTTTCTTGCAGTACACATCACATGACCTTG
196. 86J5CA1 GAAATAGGCGGAAACTGGTTC
197 GTTTCTTCGTTGTGGTTGTTCAGAAAGG
198. 86J5GT1 GGTCAAGTGTTCAGAACGCATC
199. GTTTCTTGCAGGGATTATGCTAGGTCTGTAG
200. 86J5GT2 AGCACTTCTGAGGAAGGGACAC
201. GTTTCTTAGGGCAGGCAGACATACAAAC
202. 86J5TE1 GCCAATGTGTTCCTAGAGCGAC
203. GTTTCTTTTAAAGGGGGTAGGGTGTCACC
25 204. 8E.PENTA1 GGAAGGGAAAAGGACAAGGTTTTG
205. GTTTCTTAGCAAGAGCACTGGTGTAGGAGTC
206. 8EP04D05 GCTTTTCAAGCACTTGTCTC
207. TGGGATTGTGACTTACCATG
208. 8016GT1 ACTTGGTGTCTTATAGAAAGGTG
3O 209. GTTTCTTAGCTGTGTTTGCTGCATC
210. 8016GT2 AGATGTGTGATGAGATGCAG
211. GTTTCTTCAAATAGTGCAACAAACCC
212. AFM198YB10(G) TGTCATTCTGAAAGTGCTTCC
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213. GTTTCTTCTGTAACTAACGATCTGTAGTGGTG
214. AFM205YG5(G) TATCAAGGTAATATAGTAGCCACGG
215. AGGTCTTTCATGCAGAGTGG
216. AFM206XB2(G) ATTGCCAAAACTTGGAAGC
217. AGGTGACATATCAAGACCCTG
218. AFM283WH9(G) TTGTCAACGAAGCCCAC
219. GTTTCTTGCAAGATTGTGTGTATGGATG
220. AFM324YH5(G) GCTCTCTATGTGTTTGGGTG
221. AAGAGTACGCTAGTGGATGG
'I0 222. AFMA154ZD1(G) TCCATTAGACCCAGAAAGG
223. GTTTCTTCACCAGGCTGAGATGTTACT
224. ASMI14 AATCGTTCCTTATCAGGTAATTTGG
225. GTTTCTTCAAAGAAAGCAATTCCATCATAACA
226. ASMI14T GCATTTGTTGAAGCAAGCGG
227. CTTTGTTCCTTGGCTGATGG
228. CAll 11 AATAGTACCAGACACACGTG
229. CAATGGTTCACAGCCCTTTT
230. CA39 2 AGCCTGGGAGACAGAGTGAG
231. GTTTCTTGCACTTTTTGGGGAAGGTG
232. CD59(L) GTTCCTCCCTTCCCTCTCC
233. GTTTCTTTCAGGGACTGGATTGTAG
234. D11S1301(U) GTGTTCTTTATGTGTAGTTC
235. GTTTCTTGGCAACAGAGTGAGACTCA
236. D11S1751(G) GTGACATCCAGTGTTGGGAG
Z5 237. GTTTCTTCCTAAGCAAGCAAGCAATCA
238. D11S1776(G) AAAGGCAATTGGTGGACA
239. GTTTCTTTTCAATCCTTGATGCAAAGT
240. D11S1900(U) GGTGACAGAGCAAGATTTCG
241. GTTTCTTGTAGAGTTGAGGGAGCAGC
242. D11S2008/D11S1392 CATCCATCTCATCCCATCAT
(C)
243. GTTTCTTTTCACCCTACTGCCAACTTC
244. D11S2014(C) CCGCCATTTTAGAGAGCATA
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245. GTTTCTTTTCTGGGACAATTGGTAGGA
246. D11S4200(G) TTTGTGTTATTATTTCAGGTGC
247. GTTTCTTGTTTTTTGTTTCA GTTTAGGAAC
248. D11S907(G) CATACCCAAATCGTTCTCTTCCTC
S 249. GTTTCTTGGAAAAGCAAAG GCATCGTAGAG
250. D11S935(G) TACTAACCAAAAGAGTTGGGG
251. CTATCATTCAGAAAATGTTGGC
252. GATA-P18492(C) GTATGGCAGTAGAGGGCATG
253. AAGGTTACATTTCAAGAAATAAAGT
1O 254. GATA-P6915(C) CTGTTCAGGCCTCAATATATACC
255. AAGAGGATAGGTGGGGTTTG
256. L19CA3 CCTCCCACCTAGACACAAT
257. ATATGATCTTTGCATCCCTG
258. L19PENTA1 AAGAAAGACCTGGAAGGAAT
15 259. AAACAGCAAAACCTCATCTC
260. L19TETRA5 CCACCACTTATTACCTGCAT
261. TGAATGAATGAATGAACGAA
262. LMP2 AACTGTGATTGTGCCACTGCACTC
263. GTTTCTTCACCGCCTTTATCCCTCAAATG
20 264. LMP3 GATGGGTGGAGGGCAGTTAAAG
265. GTCAAGCAACTTGTCCAAGGCTAC
266. LMP4 CAGGCTATCAGTTTCCTTTGGAG
267. GGCAGGTAATACTGGAGAATTAGG
268. LMP7 GACGGATCTCAGAGCCACTC
25 269. GTTTCTTAAAAGATAAGGGCTTTTAAACC
270. T18-5 AGTTTCACAGCTTGTTATGG
271. GGTTGATGAAGTGAGACTTT
272. T29 9 ATGGTGGATGCATCCTGTG
273. GTTTCTTGTATTGACTCCTCCTCTGC
30 274. 774L CAGTAAACAT
275. TGTTGAGTGG
276. 774N TCTCCTCAATGTGCATGT
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277. ATTCTACATA
278. ASMI14 GTGTTTGCAT
279. ACAAGTTGGC
280. CAll TAGTACCAGA
11
281. TACATCCAAGAAAA
The source
of marker
was Sequana
Therapeutics,
Inc.
unless
a
letter in parenthesis is indicated after the name, where G -
Genethon;
L = Nothen
and Dewald
(1995)
Clin.
Genet,
47:165;
U =
the Utah center, see: The Utah Marker Development Group
genome
(1995) Am. J. Hum. Genet. 57:619; c= the cooperative Human
Lineage Center.
Table 1B
SEQ Marker Repeat and flanking sequence
282. CA39 GAGACTCTGA(CA)nAATATATATA
2
283. 774F TGTTGATCGC(CA)nAACCAAAATC
284. 774J AATGCATGTA(TG)2TATA(TG)nGTGTGGTATG(TG)3TACATATG
CG
285. 7740 CCTCCCAGAA(CA)n ATCATGATAA
286. L19PENT AGACAGTCTCAAAAAAT(ATTTT)nAAAGAAAAAGCTGGATAAAT
A1
287. 65P14TE AACTAGCTTTAAGAAAATAAGAAGAAA.AAGAAAGAAG(AAAG)2TAA
1 G ( AAAG ) nAGAAAGAAAAG ( AAAG ) r~PrAAAG ( AAAG
) nAGGAATGAT
TGAC
288. 65P14 CGCGCACATA(CA)nCCCTTTCTCT
289. 774L CAGTAAACAT(CA)n TGTTGAGTGG
290. 774N TCTCCTCAATGTGCATGT (GTGC)2 ATGA {GTGC)2 (AC)n
ATTCTACATA
291. ASMI14 GTGTTTGCAT (GT)n T (GT)3 ACAAGTTGGC
2 9 2 CAl 1-11TAGTACCAGA ( CA ) 2 CG ( TG ) 2 ( CA ) 2 GGCAAGCG
. ( CA ) n C
(CA}3 TACATCCAAGAAAA
Genetic refinement of the ASTH1 region
The microsatellite markers isolated from YACs from the ASTH1 region were
genotyped in both the Tristan da Cunha and Toronto cohorts. Genetic refinement
of the ASTH1 region was accomplished by applying the
transmission/disequilibrium
test (TDT; Spielman et al. (1993} Am. J. Hum. Genet. 52:506) to genetic data
from
the Tristan and Toronto populations, at markers throughout the ASTH1 region.
The
TDT statistic reflects the level of association between a marker allele and
disease
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status. A multipoint version of the TDT test controls for variability in
heterozygosities between foci, and results in a smoother regional TDT curve
than
would a plot of single locus TDT data. Significance of a TDT value is
determined by
means of the x2 test; A x2 value of 3.84 or greater is considered
statistically
significant at a probability level of 0.05. Figure 1 shows graphs of xz values
for key
ASTH1 region markers for both history of asthma with positive methacholine
challenge, for the Toronto triad families. x2 is plotted vs. genomic location
of the
marker on the physical map.
The Toronto TDT peak is located at marker D1152008 (x2= 11.6, p < .0001 ).
The marker allele in disequilibrium is fairly rare (freq = 6%), representing
the fourth
most common allele at this marker. The relative risk of affection vs. normal
for this
allele is 5.25. This is also the peak marker for linkage and linkage
disequilibrium in
Tristan da Cunha, indicating that the ASTH1 gene is very close to this marker.
The
markers defining the limits of linkage disequilibrium were D11S907 and
65P14TE1.
The physical size of the refined region is approximately 100 kb.
A significant TDT test reflects the tendency of alleles of markers located
near
a disease locus (also said to be in "linkage disequilibrium" with the disease)
to
segregate with the disease locus, while alleles of markers located further
from the
disease locus segregate independently of affection status. An expectation that
derives from this is that a population for which a disease gene (ie a disease
predisposing polymorphism) was recently introduced would show statistically
significant TDT over a larger region surrounding the gene than would a
population
in which the mutant gene had been segregating for a greater length of time. In
the
latter case, time would have allowed more opportunity for markers in the
vicinity of
the disease gene to recombine with it. This expectation is fulfilled in our
populations. The Tristan da Cunha population, founded only 10 generations ago,
shows a broader TDT curve than does the set of Toronto families, which are
mixed
European in derivation and thus represent an older and more diverse, less
recently
established population.
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Gene isolation and characterization
The tiling path of BACs, cosmids and PAC clones was subjected to exon
trapping and cDNA selection to isolate sequences derived from ASTH1 region
genes. Exon trap clones were isolated on the basis of size and ability to
cross-
hybridize. Approximately 300 putatively non-identical clones were sequenced.
cDNA selection was performed with adult and fetal lung RNA using pools of
tiling
path clones. The cDNA selection clones were sequenced and the sequences
assembled with those of the exon trap clones. Representative exon trapping
clones spanning each assembly were chosen, and arranged as "masterplates" (96-
well microtitre dishes) of clones. Exon trap masterplate clones and cDNA
selection
clones were subjected to expression studies.
Human multi-tissue Northern blots were probed with PCR products of
masterplate clones. In some cases, exon trapping clones did not detect RNA
species, either because they did not represent expressed sequences, or
represented genes with very restricted patterns of expression, or due to small
size
of the exon probe.
Masterplate clones detecting discrete RNA species on Northern blots were
used to screen lambda phage based cDNA libraries chosen on the basis of the
expression pattern of the clone. The sequences of the cDNAs were determined by
end sequencing and sequence walking. cDNAs were also isolated, or extended, by
5' and 3' rapid amplification of cDNA ends (RACE). In most cases, 5' RACE was
necessary to obtain the 5' end of the cDNA.
ASTH 1 I and ASTH 1 J were detected by exon trapping. ASTH 1 I exons
detected a 2.8 kb mRNA expressed at high levels in trachea and prostate, and
at
lower levels in lung and kidney. ASTH11 exons were used as probes to screen
prostate, lung and testis cDNA libraries; positive clones were obtained from
each of
these libraries. Isolation of a ASTH11 cDNA clone from testis demonstrates
that this
gene is expressed in this tissue, and possibly others, at a level not
detectable by
Northern blot analysis.
ASTH1J exons detected a 6.0 kb mRNA expressed at high levels in the
trachea, prostate and pancreas and at lower levels in colon, small intestine,
lung
and stomach. Pancreas and prostate libraries were screened with exon clones
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from ASTH1J. cDNA clone end sequences were assembled using Sequencher
(Alsbyte) with the sequences of the exon trapped clones, producing sequence
contigs used to design sequence walking and RACE primers. The additional
sequences produced by these methods were assembled with the original
sequences to produce longer contigs of cDNA sequences. It was evident from the
sequence assemblies that both ASTH11 and ASTH1J are alternatively spliced
and/or have alternative transcription start sites at their 5' ends, since not
all clones
of either gene contained the same 5' sequence.
ASTH1J has three splice forms consisting of the alt1 form, found in prostate
and lung cDNA clones, and in which the exons (illustrated in Figure 1 ) are
found in
the order: 5' a, b, c, d, e, f, g, h, i 3'. A second form, alt2, in which the
exon order is:
5' a2, b, c, d, e, f, g, h, i 3' was seen in a pancreas cDNA clone. A third
form, alt3,
contains an alternate exon, a3, between exons a2 and b. The start codon is
within
exon b, so that the open reading frame is identical for the three forms, which
differ
only in the 5' UTR. The ASTH1J cDNAs shown as SEQ ID N0:2 (form alt1); SEQ
ID N0:3 (form alt2); SEQ ID N0:4 (form alt3) are 5427, 5510 and 5667 by in
length,
respectively. The sequence of the entire protein coding region and alternate
5'
UTRs are provided. The 3' terminus, where the polyA tail is added, varies by 7
by
between clones: The provided sequences are the longest of these variants. The
encoded protein product is provided as SEQ ID N0:5.
ASTH11 was seen in three isoforms denoted as alt1, alt2, and alt3. The
exons of ASTH11 and ASTH1J were given letter designations before the
directionality of the cDNA was known, the order is different for the two
genes. In
the alt1 form of ASTH11, exons are in the following order: 5' i, f, e, d, c,
b, a 3'. In
the alt2 form of ASTH11, an alternative 5' exon, j, substitutes for exon i,
with the
following exon arrangement: 5' j, f, e, d, c, b, a 3'. The alt3 form of the
gene has
the exon order: 5' f, k, h, g, e, d, c, b, a 3'. The alternative splicing and
start
codons in each of exons i, f and a give the three forms of ASTH1 I protein
different
amino termini. The common stop codon is located in exon a, which also contains
a
long 3' UTR. Two polyadenylation signals are present in the 3' UTR; some cDNA
clones end with a polyA tract just after the first polyA signal and for others
the polyA
tract is at the end of the sequence shown. Since the sequences shown for the
alt1,
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alt2, and alt3 forms of ASTH11 (2428 bp; 2280 by and 2498 bp; respectively)
are
close to the estimated Northern blot transcript size of 2.8 kb, these
sequences are
essentially full length.
EST matches
The nucleotide sequences of the alt1, alt2 and alt3 forms of ASTH1J and the
alt1, alt2 and alt3 forms of ASTH11 were used in BLAST searches against dbEST
in
order to identify EST sequences representing these genes. Perfect or near
perfect
matches were taken to represent sequence identity rather than relatedness.
Accession numbers T65960, T64537, AA055924 and AA055327 represent the
forward and reverse sequences of two clones which together span the last 546
by
(excluding the polyA tail) of the 3' UTR of ASTH1 I. No ESTs spanned any part
of
the coding region of this gene. One colon cDNA clone (accession number
AA149006) spanned 402 by including the fast 21 by of the ASTH1J coding region
and part of the 3' UTR.
Intronlexon structure determination
The genomic organization of genes in the ASTH1 region was determined by
comparison by BLAST of cDNA sequences to the genomic sequence of the region.
The genomic sequence of the ASHT1 region 5' to and overlapping ASTH1J, is
provided in SEQ ID N0:1. Genomic structure of the ASTH11 and ASTH1J genes is
shown in Figure 1; the intron/exon junction sequences are in Table 2.
TABLE 2: Genomic organization of the ASTH 1 I and ASTH I J genes.
*Exonic sequences are upper case, flanking sequences lower case.
SEQ NO Exon Size of Sequences at the ends of and
exon flanking the exons of ASTH1I and
(bp) ASTH1J*
ASTH1I
293. i >214 ggaggctgagCAGGGGTGCC...
294. ...ACTCCCACAGgtacctgcag
295. j >66 ...CTGCCCTCACgtaagcgcct
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296. f 125 gctgttgcagGGTAATGTTG...
297. ...CATCAGACAGgtgcgtaca
298. k 226 ggctggtgagGAGGGGCTGA...
299. ...CGCTCTGTGGgtgagcttca
300. h 93 tgtggaatagCCCAATTACA...
301. ...AGGGTGCTGAgtgagtagta
302. g 79 ttcttttcagGCCCTCGTGT...
303. ...TGCTGACCCGgtatggtggt
304. a 232 tttggtgcagCCTGTGACTC...
305. ...CGCACACAAGgtcagtgttc
306. d 51 tctttcccagGTTACTCCTT...
307. ...ATCAAAGACTgtaagtaacc
308. c 69 tctatttcagATGCTGATTC...
309. ...AGTAGAACAAgtaagtgcag
310. b 196 ttttcaaaagGCCTCCAAAG...
311. ...GAGCCCTGAGgtaagttaat
312. a 1522 gctttttcagATACTACTAT...
313. ...TAACATGTTCaactgtctgt
314. a 146 tgttatatgcATTTATCTTC...
315. . ...GGTAAATGAGgtaagtcctg
316. a2 229 tcttgttaagATCGCTCTCT...
317. ...CCTTGCCCAGgttctcttaa
318. a3 157 gcaatcgcacCTGCACACCC...
319. ...ACTGCCCATTtctggtaaag
320. b 100 cccctaacagATCATGATTC...
321. ...ACGTGCAATGgtaagagggc
322. c 246 tgttttgcagTTTCCAGTGG...
323. ...AAGTGGAACGgtgactctct
324. d 63 tccttcacagGCCAGTGCAG...
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325. ...GAACAAACTGgtg agtagta
326. a 69 ttttttgtagAGCCTTCCAT...
327. ...AGCACAGTAGgtaactaact
328. f 69 atggccacagATTTGTTGGA...
329. ...CTTCCTGTTGgtaagctgtc
330. g 63 ttctccttagCAGAGTCACC...
331. ...AAAAAGCACAgtaagttggc
332. h 196 ttttcatcagACCCGAGAGG...
333. ...GAGCTATGAGgtgaggagtt
334. i 4457 tttgttacagATATTACTAC...
335. ...AGCCTGGAAAtgcgtgtttc
The deduced ASTH1l and ASTH1J proteins
The protein encoded by ASTH1J (SEQ ID N0:5) is 300 amino acids in
length. A BLASTP search of the protein sequence against the public
nonredundant
sequence database (NCBI) revealed similarity to one protein domain of
transcription
factors of the ets family. The ets family, named for the E26 oncoprotein which
originally defined this type of transcription factor, is a group of
transcription factors
which activate genes involved in a variety of immunological and other
processes, or
implicated in cancer. The family members most similar to ASTH11 and ASTH1J
are:
ETS1, ESX, ETS2, ELF, ELK1, TEL, NET, SAP-1, NERF and FLI. Secondary
structure analysis and comparison of the protein sequence to the crystal
structure of
the human ETS1-DNA complex (Wemer et al. (1995) II 83:761 ) confirmed that it
has a winged helix turn helix motif characteristic of some DNA binding
proteins
which are transcription factors.
Multiple sequence alignment of ASTH11, ASTH1J, and other ETS-domain
proteins detected a second, N-terminal domain shared by ASTH1 I, ASTH1J and
some, but not all, ETS-domain proteins. Conservation of this motif have been
observed (Tei et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6856-6860), and its
involvement in protein self association have been documented for TEL, an ETS-
domain protein, upon its fusion with platelet-derived growth factor ~i
receptor (Carrot
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et al. (1996} Proc. Natl. Acad. Sci. USA 93:14845-14850). Alignment of the N-
terminal conserved domain in the ETS proteins was converted into a generalized
sequence profile to scan the protein databases using the Smith-Waterman
algorithm. This search revealed that the N-terminal domain in ASTH11, ASTH1J
and
other ETS-domain proteins belongs to the SAM-domain family (Schultz et al.
(1997)
Protein Science 6:249-253). SAM domains are found in diverse developmental
proteins where they are thought to mediate protein-protein interactions. Thus,
both
ASTH11 and ASTH1J are predicted to contain two conserved modules, the N-
terminal protein interaction domain (SAM-domain) and the C-terminal DNA-
binding
domain (ETS-domain}. The sequence segments between these two domains is
predicted to have elongated, non-globular structure and may be hinges between
the
two functional domains in ASTH 1 I and ASTH 1 J.
The ASTH11 alt1 (SEQ ID N0:7), alt2 (SEQ ID N0:9) and alt3 (SEQ ID
N0:11 ) forms are 265, 255 and 164 amino acids in length, respectively, and
differ at
their 5' ends. The ASTH11 and ASTH1J proteins show similarity to each other in
the ets domain and between ASTH1J exon c and ASTH11 exon e. They are more
related to each other than to other proteins. Over the ets domain they are 66%
similar (ie. have amino acids with similar properties in the same positions)
and 46%
identical to each other. All three forms of ASTH1 I have the helix turn helix
motif
located near the carboxy terminal end of the protein.
The alternate forms of the ASTH 1 I protein may differ in function in critical
ways. The activity of ets transcription factors can be affected by the
presence of
independently folding protein structural motifs which interact with the ets
protein
binding domain (helix loop helix). The differing 5' ends of the ASTH1 I
proteins may
help modulate activity of the proteins in a tissue-specific manner.
Polymorphism analysis ofASTHI1 and ASTH1J
Affected and unaffected individuals from the Toronto cohort were used to
determine sequence variants, as were approximately 25 controls derived from
populations not selected for asthma. Affected and unaffected individuals from
the
Tristan da Cunha population were also chosen; the set to be assayed was also
selected to represent all the major haplotypes for the ASTH1 region in that
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population. This ensured that all chromosome types for Tristan were included
in the
analysis.
Polymorphism analysis was accomplished by three techniques: comparative
(heterozygote detection) sequencing, radioactive SSCP and fluorescent SSCP.
Polymorphisms found by SSCP were sequenced to determine the exact sequence
change involved.
PCR and sequencing primers were designed from genomic sequence
flanking each exon of the coding region and 5' UTRs of ASTH11 and ASTH1J. For
fluorescent SSCP, the forward and reverse PCR primers were labeled with
different
dyes to allow visualization of both strands of the PCR product. In general, a
variant
seen in one strand of the product was also apparent in the other strand. For
comparative sequencing, heterozygotes were also detected in sequences from
both
DNA strands.
Polymorphisms associated with the ASTH11 locus are listed in Table 3. The
sequence flanking each variant is shown. Polymorphisms were also deduced from
comparison of sequences from multiple independent cDNA clones spanning the
same region of the transcripts, and comparison with genomic DNA sequence. The
polymorphisms in the long 3' UTR regions of these genes were found by this
method. One polymorphism in each gene is associated with an amino acid change
in the protein sequence. An alanine/valine difference in exon c of ASTH1J is a
conservative amino acid change. A serine/cysteine variant in exon g of ASTH1 I
is
not a conservative change, but would be found only in the alt3 form of the
protein.
The polymorphisms in the ASTH11 and J transcribed regions were genotyped
in the whole Tristan da Cunha and Toronto populations, as well as in a larger
sample of non-asthma selected controls, by high throughput methods such as OLA
(oligonucleotide ligation assay; Tobe et al. (1996) Nucl. Acids Res. 24:3728)
or
Taqman (Holland et al. (1992) Clin. Chem. 38: 462), or by PCR and restriction
enzyme digestion. The population-wide data were used in a statistical analysis
for
significant differences in the frequencies of ASTH 1 I or ASTH1 J alleles
between
asthmatics and non-asthmatics.
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TABLE 3: POLYMORPHISMS IN THE ASTH1I AND ASTH1J GENES.
Polymorphism Sequence
Location
SEQ ASTH1I Transcribed region
16. EXON B (+)170 ACAGAATGAC$TATGAAAAGT
17. INTRON D (+)15 GTAACCAAGC$CAAGCCACCC
18. INTRON F (+)24 AAGGAGCCCA~'CTGAGTGCAG
19. EXON G (+)62 ser-~cys CGTTCCATCT$TGCTCTGTGC
20. EXON H (+)77 AGCGCCTCGGYTGGCTGAGGG
21. EXON A 3' UTR (+)1176 TGTATTCAAG~GCTATAACAC
22. EXON I (+)76 CACTGAGAAGCC~ACAGGCCTGT
2 3 EXON I ( + ) 8 6 CCCACAGGCC~jGTCCCTCCAA
.
24. INTRON J (+)93 CGTCCATCTC~AGCTCCAGGG
ASTH1J Transcribed region
25. EXON A 5' UTR (+}38 GACTTGATAAYGCCCGTGGTG
26. EXON A 5' UTR (+)39 ACTTGATAAC$CCCGTGGTGC
27. EXON A 5' UTR (+)99 CTCCCCTCCAjjGAGCCACAGC
28. INTRON A (+) 224/225 ATTTCCTGCAT~GTCTGGACTT
29. INTRON A (+)48 ATCCAAACAC~TGAGTGGAAA
30. EXON A3 (+)28 AGTTTCCTCA$TGCGGGAGCT
31. EXON C (+)158 GCGAGCACCT~TGCAGCATGA
32. EXON C (+)190 ala->val TTCACCCGGG~GGCAGGGACG
33. INTRON D (-)36/37 CTGGGGAAAA(GA),/TGATCGCTGAC
34. INTRON F (-)22 GTCAATTAAA~GGCTCTCATT
35. INTRON G (-}27 TAGATCATTC$TAACCTGCCT
36. EXON I (3' UTR) (+)22 AAAGAGAAATunCTGGAGCGTG
37. EXON I (3' UTR) (+)220 ATGAGGGGAA~AAGAAACTAC
38. EXON I (3' UTR) (+)475 TTTTGTATGTKACATGATTTA
39. EXON I (3' UTR) (+)871 AGCTTGGTTC~TTTTTGCTCC
40. EXON I (3' UTR) (+)1084 TTGACACCAG$AACCCCCCAG
5' to
ASTH1J
41. CAAT box AAATGAGCCA$TGTTTGTAAT
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42. 5PW1J PO1+399 ATCCATTTTG~ATTCCTCATT
43. 5PW1J PO1+1604 CTGGAGCTCA$ACCAGACAGC
44. 5PW1J _P02+1382 GCCAGTGCAG$CATCATTACC
45. 5PW1J -P03+128 AGTTCAAATC$TAATTTTTAT
46. 5PW1J P03+556 TCATCAGAAT~TAAATCTCCC
47. 5PW1J P03+712 GGAGATTCAG~TGAAGCAAGA
48. 5PW1J P03+781 TTTTTCCACAyCCAGCCTGGC
49. 5PW1J P03+791 CCCAGCCTGG~GAACCCTGGC
50. 5PW1J P03+820 CTCTTCATCAyGGTCAAATAC
51. 5PW1J -P03+1530 CAACTTGCTGyCAAAGTGCTG
52. 5PW1J P03+1605 TACTATGTGC~AGATACTAAG
53. 5PW1J P04+542/543 ATGCCACTTT$$ACAACTTGAG
54. 5PW1J -P04+973 CGCATGCCTG$AAAGAAGAGA
55. 5PW1J P04+1079 GGATAAGCAC~AGTGAGCCTG
56. 5PW1J P04+1153 AAAGCCAGAC$GCAACTTGTG
57. 5PW1J_ P04+1430 TCTCAAAAAG$GTGATAGGAG
58. 5PW1J P05+334 TCTGAATCCT$TCTCCTCCTT
59. 5PW1J P05+749 TAGAACCAGGjdTGTGGGACCA
60. 5PW1J P05+915 TTCTTGTGTC$GGCGCAAAAC
61. 5PW1J P06+529 AACCAACATG$AGAAACCCCA
62. 5PW1J P06+1290 AATAAACTAT$GTTCACCTAG
63. 5PW1J P06+1573 ACATATTTGT$TCTCATATGA
64. 5PW1J P06+1661 CAAAGCAGTTyCTAATAATCC
65. 5PW1J P07+335 AGATCCTAACyGGGGCCTCCT
66. 5PW1J P07+731 CTCTTTCTCT~TGCTTCCTCC
67. 5PW1J P07+1024 TTAGGAATCCunCAAATATGTA
68. 5PW1J P07+1610 GTCTGACTCC$CCTCCCTCAT
69. 5PW1J P08+398 GAATCACATC$TGAGAAATGT
70. 5PW1J P08+439 AATTCAATCC~TCACAGACTT
71. 5PW1J P08+580 GTGTAGCCAG$GTTGCTAATT
72. 5PW1J P08+762 CCTAGAAATA~CCAAGGGCAC
73. 5PW1J_ P08+952 AAATTCTCAT$CCTCACCCTC
74. 5PW1J_ P08+1172 TCCCACCCCT$TCACCTTCAT
75. 5PW1J P08+1393 CCTCATTCTC$GAAGCCAACA
76. 5PW1J P08+1433 GAAGAGCCGT~CAGTCCCTTT
77. 5PWIJ P08+1670 TCCATAGGCT~TTTATTTGGC
78. SPWIJ_ P08+1730 TCGTTTAGTA~ACAGGCTTTG
79. 5PW1J P09+59 GCCTCAGTTGYCCCAGCTATA
80. 5PW1J P09+145 AGCAAAATGC~nCTATGCACTG
81. 5PW1J P09+892 GTGTCCTGAC ('r'~'GC_A-CTCCAC)
/-
ACACTGCCTG
82. 5PW1J P10+1070 ATCAGATAAC$CCTACACTTA
83. 5PW1J P10+1511 TCTCTCTTCT$CCTGCCCTGT
84. 5PW1J P09+1132 TGGACACAGGKAGGGGAATAT
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85. 5PW1J P09+1688 TGTCACTTGC$CATACAAGGC
86. 5PW1J P09+1900 ATCATCAGAT~AGCCCAGAAT
87. 5PW1J W1R1-1060 TCAACAGAGA$AGTTAATGGT
88. 5PW1J W1R1-1831 AGCAATAATG~TTCCCTTTTC
89. 5PW1J W1R1-2355 TCTAGCTTTTyTGTGTTTTTT
90. 5PW1J W1R1-3160 GATTCCTTAA~GCTTGATACT
91. 5PW1J W1R1-3787 CCTCCTCCAG~ACCAAAGTGG
92. W1J CD+24 ATGGCCACAG$TCAAATCCTG
93. W1J CA+564 ACTGAGTGTT~ATGCCAATTT
5' to ASTHlI
94. WI CL+94 GACAAGCCCT$TCTGACACAC
95. WI-CN+134 TGAAAAGCCT~CTTGCTGCCT
96. WI CQ-28 TCCTGGAGTTyCTTTGCTCCC
97. WI CQ+39 GATTCCAAATLnIAACTAAAGAT
98. P14-16+191662 GACCTCAAGTC$TCCACCCGCC
99. P14-16+192592 AACAAATACT~CCCCGCAACCC
100. P14-16+192762 ATTTTTTTTTT/-AAGGAAAATA
101. P14-16+195066 AAATTTCCCC~AAACAAGCAG
102. P14-16+196590 GAGAAAGGGT$TGTGTGTGTG
103. P14-16+196617 GTGTGTGTGTGT-/GTGTATGTGCGCGTG
104. P14-16+196902 ATCGGGAACCyCATACCCCAA
105. P14-16+198040 TTTGTTTCGCbATGAGGTACG
106. P14-16+198240 TGAGGGTGTT$TGGGCTGGAC
107. P14-16+198840 TCTTCATTGG~ATCTGAATGT
108. P14-16+200120 GCGAGCACCT~TGCAGCATGA
109. P14-16+200617 AACCCCCCCC~CACACACACA
110. J5-16+4454 TCAGTGCTCT$TAATCAGTCA
111.. J5-16+4825 TCTTTGTGAAA-/(~AATTAGTCTG*
112. J5-16+5426 GCTGCCCTGA~AGCTGGGCCA
113. J5-16+5623 CCTTCTGATC~TTGTTTGCTG
114. J5-16+7386 GGAACACTGA~TCTTGATTAG
115. J5-16+7904 TAGGCTTCTC~TGATAATTGA
116. J5-16+8055 TCTTAAAATA~TTGGCTTGTA
117. J5-16+10595 TAGATCATTA$TAACCTGCCT
118. J5-16+11140 ATGAGGGGAA~AAGAAACTAC
119. J5-16+12004 TTGACACCAG$AACCCCCCAG
120. J5-16+12219 TGTTTTAAAT$TTAGGGACAA
121. J5-16+12303 GTAAGCATAG~AATGTAGCAG
122. J5-16+13504 GGCTCTTTCTK,CAACCTTTCC
123. J5-16+14120 GACCCAGGTT$TGAGTTTTCC
124. ASTH1I, exon B +169 GACAGAATGAyATATGAAAAG
125. ASTH1I, exon I +69 TGTGTGACAC~GAGAAGCCCA
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126. ASTH1J, AGTACTGGAC~AAGTACCAGG
exon
C
+56
127. 5' CCTGGGAGCA$GTATTGCATT
ASTH1J,
WI
Cg
-9
ASTH1J
Intron
A
128. WIJ _Ia01 +39 AGATTTGAGG~CTCAGGTCCC
129. WIJ -Ia01 +140 TGTCAATGTC$CATGATAAGC
130. WIJ _Ia01 +678 TTGCCCCAGTgTTCTCCGGGC
131. WIJ Ia01 +855 TATGAGCAGC$TAGGGAGTGG
132. WIJ Ia01 +929 AGTTGACTGA( TAAGAC
133. WIJ _Ia +362 ATTCAAATAG$CTCTAGAAAC
03
134. WIJ Ia +918 CCCAGAATTT~ATATCCATTC
03
135. WIJ Ia +943 TGACCCAACA$AAACTCACTG
03
136. WIJ Ia +1569 CCAGAATATA~CATCAGCCCT
03
137. WIJ Ia +1580 CATCAGCCCTnuCTGAGGAGAT
03
138. WIJ _Ia +435 CCAGAACAGA~TTTATTCTGT
02
139. WIJ Ia +583 TTCAGCCATC~TTCCAGTTGT
02
140. WIJ Ia +643 TCACTAACTCgAAAACGACAT
02
141. WIJ Ia +648 AACTCAAAAA~GACATCCTCC
02
142. WIJ Ia +1048 GAACTGCACA$GTTGCACACT
02
143. WIJ _Ia +1061 TTGTTCCATG~CTACCTCCT
02
144. WIJ Ia +1142 ACAGCAGGCA~'TCAACAAATT
02
145. WIJ Ia +410 TTATTTTTGG~TTTGTTTTAA
04
146. WIJ Ia +1056 TAGGCTGTTCyCTGCCATCAC
04
147. WIJ Ia +1484 GTGCTCTGGGr(CACACAGCTC
05
148. WIJ_ Ia +1103 AGACCCGATA$GAGCTCCTTC
05
149. WIJ Ia +1823 CATCTTGCGC$GTCATGTAAG
05
150. WIJ Ia +1852 CAGCACAGCT$TTCCCTCAAA
05
151. WIJ Ia +1906 TTTGGAAACA,yGGTGAAGTAT
05
152. WIJ Ia +19,13 ACACGGTGAA$TATTGTCTCC
05
153. WIJ_ Ia +794 AAAAGTGGAT~CTCTGCAAAC
06
154. WIJ Ia +814 CTTCAAATGC$GCTATTAAAG
06
155. WIJ Ia +1197 CCTGGGAGCA~GGTAAATCAG
06
156. WIJ Ia +1231 TGAAAATGTC$CTTTCTCACCT
06
157. WIJ Ia +1256 CCTGATATTT$CCAACAAGAA
06
158. WIJ_ Ia +1535 AAAGGGTTAGyTTGTCCCCTT
06
159. WI 63 TGAAAATAAAA$ACAATTTTTT
Caa
+1
The
sequences
are
listed
with
the
variant
residues
represented
by
the
appropriate
single
letter
designation, or
i.e. G
A is
shown
by
NR".
The
variant
residues
are
underlined.
Where
the
polymorphism deletion,the underlined
is residues are
a underlined,
and the alternative
form shown
as a
=".
aWhere n 3' to exon
intron 'a', etc.
'a'
is
the
intro
bPosition correspond the intron or exon, with nucleotide
numbers to +1 being the
the
position
within
5'-most the intron.
base Alternatively,
of negative numbers
the denote the
exon number of bases
or
from '
the end
3 of
an
intron.
Position
in
cDNA
= position
# for
the
exon
a form
of
ASTH1J
or
the
exon
i form
of
ASTH1
I.
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dExonic sequences are uppercase, intronic sequences lower case.
UTR = untranslated region. NIA = not applicable.
Cross-species sequence conservation
Cross-species sequence conservation can reveal the presence of
functionally important areas of sequence within a larger region. Approximately
90
kb of sequence lie between ASTH11 and ASTH1J, which are transcribed in
opposite
directions (Figure 1). The transcriptional orientation of these genes may
allow
coordinate regulation of their expression. The expression patterns of these
genes
are similar but not identical. Sequences found 5' to genes are critical for
expression. To search for regulatory or other important regions, the genomic
sequence between ASTH11 and ASTH1J, was examined and plasmid clones
derived from genomic sequencing experiments chosen for cross-species
hybridization experiments. The criterion for probe choice was a lack of repeat
elements such as Alu or LINEs. Inserts from these clones were used as probes
on
Southern blots of EcoRl-digested human, mouse and pig or cow genomic DNA.
Probes that produced discrete bands in more than one species were considered
conserved.
Conserved probes clustered in four locations. One region was located 5' to
ASTH11 and spanned exon j of this gene. A second conserved region was located
5' to ASTH11J, spanning approximately 10 kb and beginning 6 kb 5' to ASTH1J
exon a (and is within SEQ ID N0:1 ). Two other clusters of conserved probes
were
noted in the region between ASTH11 and J. They are approximately 10 and 6 kb
in
length.
Promoters, enhancers and other important control regions are generally
found near the 5' ends of genes or within introns. Methods of identifying and
characterizing such regions include: luciferase assays, chloramphenicol acetyl
transferase (CAT) assays, gel shift assays, DNAseI protection assays
(footprinting),
methylation interference assays, DNAseI hypersensitivity assays to detect
functionally relevant chromatin-ree regions, other types of chemical
protection
assays, transgenic mice with putative promoter regions linked to a reporter
gene
such as ~i-galactosidase, etc. Such studies define the promoters and other
critical
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control regions of ASTH11 and ASTH1J and establish the functional significance
of
the evolutionarily conserved sequences between these genes.
Discussion
The ASTH1 locus is associated with asthma and bronchial hyperreactivity.
ASTH11 and ASTH1J are transcription factors expressed in trachea, lung and
several other tissues. The main site of their effect upon asthma may therefore
be in
trachea and lung tissues. Since ets family genes are transcription factors, a
function for ASTH11 and ASTH1J is activation of transcription of particular
sets of
genes within cells of the trachea and lung. Cytokines are extracellular
signalling
proteins important in inflammation, a common feature of asthma. Several ets
family
transcription factors activate expression of cytokines or cytokine receptors
in
response to their own activation by upstream signals. ELF, for example,
activates
IL-2, IL-3, IL-2 receptor a and GM-CSF, factors involved in signaling between
cell
types important in asthma. NET activates transcription of the IL-1 receptor
antagonist gene. ETS1 activates the T cell receptor a gene, which has been
linked
to atopic asthma in some families (Moffatt et al. (1994) supra.)
Activation of genes involved in inflammation by other members of the ets
family suggest that the effect of these ASTH1 genes on development of asthma
is
exerted through influencing cytokine or receptor expression in trachea and/or
lung.
Cytokines are produced by structural cells within the airway, including
epithelial
cells, endothelial cells and fibroblasts, bringing about recruitment of
inflammatory
cells into the airway.
A model for the role of ASTH11 and ASTH1J in asthma that is consistent with
the phenotype linked to ASTH1, the expression pattern of these genes, the
nature
of the ASTH1 IIJ genes, and the known function of similar genes is that
aberrant
function of ASTH11 and/or ASTH1J in trachea or lung leads to altered
expression of
factors involved in the inflammatory process, leading to chronic inflammation
and
asthma.
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Functional analyrsis of a ASTH1J promoter seclyPnce variant and location of
the
SA TH1J rp omot~r
Primer extension analyses performed using total RNA isolated from both
bronchial and prostate epithelial cells have revealed one major and five minor
transcription start sites for ASTH 1 J. The major site accounts for more than
90% of
ASTH1J gene transcriptional initiation. None of these sites are found when the
primer extension analysis is performed using mRNA isolated from human lung
fibroblasts that do not express ASTH1J.
Identification of the ASTH1J transcriptional start site has allowed the
localization of a putative TATA box (TTTAAAA) between positions -24 and -30
(24
to 30 by 5' to the transcription start site). Although the sequence is not
that of a
typical TATA box, it conforms to the consensus sequence (TATAAAA) for TATA box
protein binding as compared with 389 TATA elements (Transfac database:
http:/Itransfac.gbf braunschweig.del, ID: V$TATA 01).
Analysis of the CART box "G'~~ymo hism byrgel shift assay
Binding of nuclear proteins to a polymorphism in the GCCAAT motif
(GCCAAT or GCCAGT) found at position -140 (140 by 5' to the transcription
start of
ASTH1J as defined by primer extension experiments, previously referred to as
"-165 by"), has been assessed using electrophoretic mobility shift assays.
These
experiments clearly showed a remarkable difference when binding of nuclear
proteins to radioactively-labelled double stranded oligonucleotides containing
the
normal "A" vs the mutant "G" nucleotide was examined. A specific set of
nuclear
proteins was able to bind to the normal oligonucleotide, but did not bind to
the "G"
oligonucleotide. The specificity of the DNA binding complexes was further
addressed by competition with either normal or mutant unlabeled
oligonucleotides.
Addition of increasing amounts of normal unlabeled oligonucleotide effectively
competed binding of nuclear proteins to the labeled normal oligonucleotide,
while
the addition of increasing amounts of unlabelled "G" oligonucleotide did not.
The GCCAAT cis-element is found in many promoters at various locations
relative to genes, as well as in distal enhancer elements. There is no known
correlation between location of these elements and activity. Both positive and
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negative regulatory trans-acting factors are known to bind this class of cis
element.
These factors can be grouped into the NF-1 and C/EBP families.
The nuclear factor-1 (NF-1) family of transcription factors comprises a large
group of eukaryotic DNA binding proteins. Diversity within this gene family is
contributed by multiple genes (including: NF-1A, NF-1B, NF-1C and NF-1X),
differential splicing and heterodimerization.
Transcription factor C/EBP (CCAAT-enhancer binding protein) is a heat
stable, sequence-specific DNA binding protein first purified from rat liver
nuclei.
C/EBP binds DNA through a bipartite structural motif and appears to function
exclusively in terminally differentiated, growth arrested cells. C/EBPa was
originally
described as NF-IL-6; it is induced by IL-6 in liver, where it is the major
C/EBP
binding component. Three more recently described members of this gene family,
designated CRP 1, C/EBP a and C/EBP 8, exhibit similar DNA binding
specificities
and affinities to C/EBP a. Furthermore, C/EBP (i and C/EBP 8 readily form
heterodimers with each other as well as with C/EBP a.
Members of the C/EBP family of transcription factors, but not members of the
NF-1 family, bind to the ASTH1J promoter region, as determined by the use of
commercially available antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA)
that recognize all NF-1 and C/EBP family members known to date, in
electrophoretic mobility shift assays.
Fabricating a DNA arrayr of l~lrmon~hic sea,~uences
DNA array: is made by spotting DNA fragments onto glass microscope slides
which are pretreated with poly-L-lysine. Spotting onto the array is
accomplished by
a robotic arrayer. The DNA is cross-linked to the glass by ultraviolet
irradiation, and
the free poly-L-lysine groups are blocked by treatment with 0.05% succinic
anhydride, 50% 1-methyl-2-pyrrolidinone and 50% borate buffer.
The spots on the array are oligonucleotides synthesized on an ABI
automated synthesizer. Each spot is one of the alternative polymorphic
sequences
indicated in Tables 3 to 8. For each pair of polymorphisms, both forms are
included. Subsets include (1 ) the ASTH?J polymorphisms of Table 3, (2) the
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ASTH1l polymorphisms of Table 3; and (3) the polymorphisms of Table 4. Some
internal standards and negative control spots including non-polymorphic coding
region sequences and bacterial controls are included.
Genomic DNA from patient samples is isolated, amplified and subsequently
labeled with fluorescent nucleotides as follows: isolated DNA is added to a
standard
PCR reaction containing primers (100 pmoles each), 250uM nucleotides, and
5 Units of Taq polymerase (Perkin Elmer). In addition, fluorescent nucleotides
(Cy3-dUTP (green fluorescence) or Cy5-dUTP (red fluorescence), sold by
Amersham) are added to a final concentration of 60 uM. The reaction is carried
out
in a Perkin Elmer thermocycler (PE9600) for 30 cycles using the following
cycle
profile: 92°C for 30 seconds, 58°C for 30 seconds, and
72°C for 2 minutes.
Unincorporated fluorescent nucleotides are removed by size exclusion
chromatography (Microcon-30 concentration devices, sold by Amicon).
Buffer replacement, removal of small nucleotides and primers and sample
concentration is accomplished by ultrafiltration over an Amicon
microconcentrator-
30 (mwco = 30,000 Da) with three changes of 0.45 ml TE. The sample is reduced
to 5 NI and supplemented with 1.4 girl 20X SSC and 5 Ng yeast tRNA. Particles
are
removed from this mixture by filtration through a pre-wetted 0.45N microspin
filter
(Ultrafree-MC, Millipore, Bedford, Ma.). SDS is added to a 0.28% final
concentration. The fluorescently-labeled cDNA mixture is then heated to
98°C for 2
min., quickly cooled and applied to the DNA array on a microscope slide.
Hybridization proceeds under a coverslip, and the slide assembly is kept in a
humid~ed chamber at 65°C for 15 hours.
The slide is washed briefly in 1X SSC and 0.03% SDS, followed by a wash in
0.06% SSC. The slide is kept in a humidified chamber until fluorescence
scanning
was done.
Fluorescence scanning and data acquisition. Fluorescence scanning is set
for 20 microns/pixel and two readings are taken per pixel. Data for channel 1
is set
to collect fluorescence from Cy3 with excitation at 520 nm and emission at 550-
600 nm. Channel 2 collects signals excited at 647 nm and emitted at 660-705
nm,
appropriate for CyS. No neutral density filters are applied to the signal from
either
channel, and the photomultiplier tube gain is set to 5. Fine adjustments are
then
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made to the photomultiplier gain so that signals collected from the two spots
are
equivalent.
Construction of an asfh9J Transgenic Mouse
Isolation of mouse asth1-J genomic fragment:
Phage MW1-J was isolated by screening a mouse 129Sv genomic phage
library (Stratagene) with the 443bp BamHl-Smal fragment from the 5' region of
the
human asth1-J cDNA clone PA1001A as probe. The 23kb insert in MW1-J was
sequenced.
Assembly of asth 1-Jexb targeting construct:
A 2.65kb Sacl fragment (bp7115-bp9765) from MW1-J was isolated, cloned
into the Sacl site of pUC19, isolated from the resultant plasmid as an EcoRl-
Xbal
fragment, inserted into the EcoRl-Xbal sites of pBluescriptll KS+
(Stratagene), and
the 2.5kb Xhol-Mlul fragment isolated. A 5.4kb Hindlll fragment (bp11515-
bp16909)
was isolated from MW1-J, inserted into the Hindlll site of pBluescriptll KS+,
reisolated as a Xhol-Notl fragment, inserted into the Xhol-Notl sites of pPNT,
and
the 9.5kb Xhol-Mlul fragment isolated. The two Xhol-Mlul fragments were
ligated
together to produce the final targeting construct plasmid, asth1exb. Asth1exb
was
linearized by digestion with Nott and purified by CsCI banding.
Identification of targeted ES clones:
Approximately 10 million RW4 ES cells (Genome Systems) were
electroporated with 20 Ng of linearized asth1exb and grown on mitomycin C
inactivated MEFs (Mouse Embryo Fibroblasts) in ES cell medium (DMEM + 15%
fetal bovine serum+1000U/ml LIF (Life Technologies)) and 400 Ng/ml 6418. After
24-48hrs, the cells were refed with ES cell medium. After 7-10 days in
selection
culture approximately 200 colonies were picked, trypsinized, grown in 96 well
microtiter plates, and expanded in duplicate 24 well microtiter plates. Cells
from
one set of plates were trypsinized, resuspended in freezing medium (Joyner,
A.,
ed., Gene Targeting, A Practical Approach. 1993. Oxford University Press), and
stored at -85C. Genomic DNA was isolated from the other set of plates by
standard
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methods (Joyner, supra.) Approximately 10 Ng of genomic DNA per clone were
digested with Ndel and screened by southern blotting using a 100 by fragment
(bp61fi4-bp6260) as probe. A banding pattern consistent with targeted
replacement by homologous recombination at the asth1-J locus was detected in
10
of 113 clones screened.
Production of asth1-J knockout mice:
Two of the targeted clones, cl#117 and cl#58, were expanded and injected
into C57BU6 blastocysts according to standard methods (Joyner, supra). High
percentage male chimeric founder mice (as ascertained by extent of agouti coat
color contribution) were bred to A/J and C57BU6 female mice. Germline
transmission was ascertained by chinchilla or albino coat color offspring from
A/J
outcrosses and by agouti coat color offsprint from C57BU6 outcrosses. The Ndel
southern blot assay employed for ES cell screening was used to identify
germline
offspring carrying the targeted allele of Asth1-J. Germline offspring from
both A/J
and C57BU6 outcrosses were identified and bred with A/J or C57BU6 mates
respectively.
Mice heterozygous for the Asth1-J targeted allele are interbred to obtain
mice homozygous for the asth1-J targeted allele. Homozygotes are identified by
Ndel Southern blot screening described above. The germline offspring of the
chimeric founders are 50% A/J or C57BL6 and 50% 129SvJ in genetic background.
Subsequent generations of backcrossing with wild type A/J or C57BU6 mates will
result in halving of the 129SvJ contribution to the background. The percentage
A/J
or C57BU6 background is calculated for each homozygous mouse from its
breeding history.
Molecular and cellular analysis of homozygous mice:
Various tissues of homozygotes, heterozygotes and wild type littermates at
various stages of development from embryonic stages to mature adults are
isolated
and processed to obtain RNA and protein. Northern and western expression
analyses as well as in situ hybridizations and immunohistochemical analyses
are
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performed using cDNA probes and polyclonal andlor monoclonal antibodies
specific
for asth1-J protein.
Phenotypic analysis of homozygous mice:
A/J, C57BU6, wild type, heterozygous and homozygous mice in both A/J and
C57BU6 backgrounds at varying stages of development are assessed for gross
pathology and overt behavioral phenotypic differences such as weight, breeding
performance, alertness and activity level, etc.
Metacholine challenge tests are performed according to published protocols
(De Sanctis et al. (1995). Quantitative Locus Analysis of Airway
Hyperresponsiveness in A/J and C57BU6J mice. Nat. Genet. 11:150-154.).
Targeting at asthl-J exon C:
Assembly of axon C targeting construct:
A 3.2kb Hindlll-Xbal fragment (bp11515-bp14752) from MW1-J was isolated,
cloned into the Hindlll-Xbal site of pUC19, isolated from the resultant
plasmid as a
Kpnl-Xbal fragment,. inserted into the Kpnl-Xbal sites of pBluescriptll KS+
(Stratagene), and the 4.5kb Rsrll-Mlul fragment isolated. A 3.4kb Hindlll
fragment
(bp17217-bp20622) was isolated from MW1-J, inserted into the Hindlll site of
pBluescriptll KS+, reisolated as a Xhol-Notl fragment, inserted into the Xhol-
Notl
sites of pPNT, and the 9.5kb Rsrll-Mlul fragment isolated. The two Rsrll-Mlul
fragments were ligated together to produce the final targeting construct
plasmid,
Asth1exc. Asth1exc was linearized by digestion with Notl and purified by CsCI
banding.
Identification of targeted ES clones:
Approximately 10 million RW4 ES cells (Genome Systems) were
electroporated with 20pg of linearized asth1exc and grown on mitomycin C
inactivated MEFs (Mouse Embryo Fibroblasts) in ES cell medium (DMEM + 15%
fetal bovine serum+1000U/ml LIF (Life Technologies)) and 400 Ng/ml 6418. After
24-48hrs, the cells were refed with ES cell medium. After 7-10 days in
selection
culture approximately 200 colonies were picked, trypsinized, grown in 96 well
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microtiter plates, and expanded in duplicate 24 well microtiter plates. Cells
from one
set of plates were trypsinized, resuspended in freezing medium (Joyner,
supra),
and stored at -85C. Genomic DNA was isolated from the other set of plates by
standard methods (Joyner, supra). Approximately 10 Ng of genomic DNA per clone
were digested with Ncol and screened by southern blotting using a 518bp
fragment
(bp8043-bp8560) as probe. A banding pattern consistent with targeted
replacement
by homologous recombination at the Asth1-J locus was detected in 3 of 46
clones
screened.
Targeted clones are injected into blastocysts and high percentage chimeras
bred to A/J and C57BU6 mates analogously to that done for asth1-Jexb knockout
mice. Heterozygote, homozygote and wild type littermates are obtained and
analyzed analogously to that done for asth1-Jexb knockout mice.
The data presented above demonstrate that ASTH 1 I and ASTH 1 J are novel
human genes linked to a history of clinical asthma and bronchial
hyperreactivity in
two asthma cohorts, the population of Tristan da Cunha and a set of Canadian
asthma families. A TDT curve in the ASTH1 region indicates that ASTH11 and
ASTH1J are located in the region most highly associated with disease. The
genes
have been characterized and their genetic structure determined. Full length
cDNA
sequence for three isoforms of ASTH11 and three isoforms of ASTH1J are
reported.
The genes are novel members of the ets family of transcription factors, which
have
been implicated in the activation of a variety of genes including the TCRa
gene and
cytokine genes known to be important in the aetiology of asthma. Polymorphisms
in the ASTH11 and ASTH1J genes are described. These polymorphisms are useful
in the presymptomatic diagnosis of asthma susceptibility, and in the
confirmation of
diagnosis of asthma and of asthma subtypes.
All publications and patent applications cited in this specification are
herein
incorporated by reference as if each individual publication or patent
application
were specifically and individually indicated to be incorporated by reference.
The
citation of any publication is for its disclosure prior to the filing date and
should not
be construed as an admission that the present invention is not entitled to
antedate
such publication by virtue of prior invention.
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Although the foregoing invention has been described in some detaii by way
of illustration and example for purposes of clarity of understanding, it will
be readily
apparent to those of ordinary skill in the art in light of the teachings of
this invention
that certain changes and modifications may be made thereto without departing
from
the spirit or scope of the appended claims.
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SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: AxyS Pharmaceuticals, Inc.
(ii) TITLE OF THE INVENTION: Asthma Related Genes
(iii) NUMBER OF SEQUENCES: 339
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Bozicevic & Reed, LLP
(B) STREET: 285 Hamilton Ave, Suite 200
(C) CITY: Palo Alto
(D) STATE: CA
(E) COUNTRY: USA
(F) ZIP: 94301
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 21-JAN-1998
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sherwood, Pamela J
(B) REGISTRATION NUMBER: 36,677
(C) REFERENCE/DOCKET NUMBER: SEQ-4P
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 650-327-3231
{B) TELEFAX: 650-327-3231
( C ) TELEX
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 72928 base pairs
(B) TYPE: nucleic acid
{C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GCACTTTTTG GGGAAGGTGG AAGAATAAAA GTAAGGGAGG TGTGCTGAGA CTTCAATTTT 60
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AATATCTTATTTCTTAGGTTGAGTGTTACA TAATCATATATACTTTTGTA120
CAGGCATTTG
CACTTGAAATATATATATTTGTGTGTGTGTGTGTGTGTGTGTCAGAGTCTCACTCTGTCT180
CCCAGGCTGGAGTGCAGTGGTGTGATCTTGGCTCATTGCAACCTCCACCTCCCAGGTTCA240
AGAGCTTTTTGTGCCTCCATCTCCTGAGTAGCTGAGACTACAGGCAAGCACCACCACACC300
GGCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTGCCCAGGCTGGTCTCA360
AATTCCTGGCCTCAAGTGATCCAGTCACCTTGGCCTCCCAAAGTGCTGGAATTACAGGCG420
TGAGCCACCATGCCCGGTCTGAAATATTTCAAAATGTAAAAAAGCTAAACCCAAATCCAG480
ATGTCTACTTTCAAGGTGCTCACAGGTCAGATCTAGGATTATTGCTACTAACTGATATTT540
ATTATCCCAGCACCAGCATGTTTGGCTGTGTGTCATGGGTAAGTTACTCACCTTCTCTGC600
GACAGTGTCATCATTGTAAAATAGGGATAAAAGAGTTTAGACCTTGCAGAGTCCTTCAGA660
TTAAAGGAGATAATCAGTACGTGGCACTGAGTACCTGCAATATATTAAGTGGTGTGTGCT720
CAGAGATATGATCACATACAGTATCTTGGATCTGCCCAGCAACTCTATGAAGATGAGGAA780
ACAGACTCAGGCAGGTCAGAGCCAGAACATAATGTTTCTGGAATTTGAACGTAAACGTTC840
CCCTTTCTCTTATCCAGGCTGAGTGCTAAAGGAATTGTAAAAATGGAATTTGCCTGTTGC900
CTGCATCTCCCTCTCTTTTTCTTCCTCTGTGTCCTCTGAATATCTAGCACCAGTGGGACT960
TTACAGTGTTGGCCTCAATGCTGTAGGGTGCTGTGTGCACACTTGTCTTCAGCTCCCTGA1020
GTTAGCAGAGCATTGCCCCAACTCTGCCCTCTGGCCAGCTCATGTGCCTTACAACTTTCT1080
GTTGCCAGAAGAGAGCCCTGCTCATTCTCTAGACTCAACCAACAAAAGCTGCCTACCATT1140
TTCAGAATGCCAGTGGGCAGTGAGAAGTGCAGAGCTTGTGTCCTGAGCTTGGCAGCCATC1200
TTGCTTGGTGTTAACAAAGAGTAATTAAGTGATCTCATAAAACTCAGTGGTGGAGGTTGT1260
GGTTCAGAGCAAGCTGGGTCAATGCCAAGGCTACTTTGGCTTCATCTGGTCCATAGCCCC1320
ACATTTCTCTTCTGATGGTTCAGTTCCGGGAATGAGAACCAGTCTGAGTGTAAGAAGACT1380
TGGGTTTGAATCTGTCTCCTCCAATCACTAGCTGACCTTAGAAAAGTGACTTAACCTCCC1440
GAGCTGCTATTTCCTCATCTTAAATGGTGATAGTAATCTTTCCTTACCTTAAGGTTGTTG1500
AGCAGCTTAAATAATATAATGAGTTGAAAGCTTTTTGTATGATCTGTTATTAGGAGTCCA1560
GATAGTGTTTTATAAACAAGAGGATAAAAAFI~AAAAAAAAAAAAAAAAACAGGATTCTGAA1620
GGCTGGACTCATTGCATTCCTTGCAAACTACCCACTGAGCCCCAACTCTTCCGTCAGCTC1680
AAAGTCACTTCTCAGAGCAAACCAGATTGTCCTGAACCCAGCACTTGCCAACATCTCCTC1740
CTCTTCCCTGATGAAAACTCTGGGCTGGAGTTGTGGTGGGTGAGGGGAAGGCAGGATAAA1800
TCAAAAATTGATGTTTTAAGAAAACTATGGTATTCTTGGATGCAAAGGCATGAGAATGAT1860
ACCTTAGACTTTGGGGCTTGGGGAAAAGGGTGGGGGGTGGCGAGGGATAAAAGACTACAC1920
ATTGGGTTTAGTGGACACTGCTCGGGTTATGGGTGCACCAAAATCTCAGAAATCACCACT1980
AAAGAACTGATTCAGGTAACCAAACACCACCTGTTCCCCAAAAACCTATTGAAATAAAAA2040
CAGAAAATTAAF~AAAAAGAAAACCTATGGTATTCTTGGAAGAAGCACAGTGGTGAAGTGG2100
AGTAGACACAGATGTGGAAGTGATGTGAACTTTGGTAAGTTGCTGAGCCTCTGAGGATGA2160
TTTCCCTCATCTGTCAATCAGGGAACAAAATCCCTTACTTGTACAATGAGTATTATAAAG2220
ATCAATTCAGATGACGCATGTAAAGATGCAATGTGGGACTGGTAGGTAGTAAGCATCCCA2280
TAAATGGCAGCTATTAATAAGTAATAATCACCGAGTGGTGGGCTGCCTTTCATGAAAACA2340
TTCCCAGCAAGCTGCTCTTCTGTCGGCTCAAAGTCACTTCTCAGAGTAAATGAGATTGGC2400
CAGTTCTTTCTTTCCAAGGCTTTTCTGGATATTCATTTGTCCCAGATTTCTCCTGTATAC2460
AAAGCTCAGGAGTGAGGACCCCCACAGTGGGGCTTGCACAAGGATAGCCTTGGGGGGCTT2520
TTTCTAAGAGCTATGACTTTGAATGCTCTCTTCATCGATGCTGACAGATGAGGGCTGATG2580
GAAGTGGTCATGTTTTAAAATGTCTGATGTCCAGAAACACAGAGATGTGTACGCAAAACA2640
TTCATTCATTCAAGATGGAATTAGTGCCCCAGACACAGAGGCAGGGGATAAATAGCAAAC2700
AAGGCTTGATTCCTGCCTTCATAGAGCTTACTGTCTTGTAGGGGAAACATGAGTAAATTC2760
AGCAGAGTAAGGGCTCTAATTGGGTAAATGGGGGCTAGGCTGCCTGTGTCCTTGGGGTGG2820
TGGGAAGGCTGCTGATCTGGGGTGCCAGAAGACCTGAGTTTTGATGCAGGCTCTGTGACT2880
TTGAGCAGGTCGTTTCCAACTTCTGAGCTTCCATTTCCCTAGCTGAAAATGGGGGCTTGC2940
CATACTCGATGCTGTACTCTATGAGTCTTTGCAGCTCTGTCATCTTTTTTTCTTTTGGTC3000
ACTCAGAGACTCCAGGATTGGGAGAACAACCTGCATTCTGATTTAAAGTGTGAATCTAAT3060
AATTTCAAAAAGAAAGGGACTAAAAGGGACAAACTTGTTTCTGTTTATTTTCCATCCTTC3120
TTTGGGGAAGTGTAACATTTGAAATCAAATTCTCATTGGCTTAGCCAATGTGTAGACTTC3180
GAGGGGAAATTCTCACTGCCCAGAGAAGTGACTAAAAATGACCATTACAGCCAAAAAGAG3240
AAGTTTTTTTTTTTTTAAAATCTGTGCTCTACAGATGGATGAAGTGCTGCTGCACATGGA3300
CAGAGTGGATCTGGACATTCTGCATGAGCCCAGGGATCCTGAGAATGGATTGGCTGAGCA3360
TAGACAGGGTGACCTATCGATGTTCACTGTGGTCCTGATCTATGTGGCCTCTTCCTAAGG3420
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GAAGATTTTTCTTAAGGTTGTTTCCTTTCTCAGCAGATATTTGTGAAGAA 3480
~rCTGTATCTG
TAGTCTCATTTTGTCCTTATAATGACCCTGATGGATGGGAGGTAGAGGGATGATGATCAG3540
TAAGAGCTGGGAAAGCACCAGGAACTAGCAAGAGCAGGACACCTTTTCCACCACTAGGTA3600
AATGGACCTAGTGACTGCTGGCACCGTGGGTGAGGGGACTGCCTGGCAGGAGCTGTGGCC3660
GTAGCTAGGGGATTACAGCTACGGCCACAACTCTGGCCCTGTACGGAGGGAGTGGGGGAA3720
ATAAAGAGTTCATATCACTCCCCTCTTTCCCTGGAGTCTCCTGCTGGTACCTTGCATTGG3780
CTGAGTCTAACTGGAAGCCAGAGGGCAAAGGAGGTACCCTTTCCAGCTCTGCAATTCTCT3840
TCAGACAGGGCTGGGATTTCTGGAGAGAATTTGCAGAATCAGAAAGCAGAGCTTTCCAAT3900
CAATGCCAAGCAAGAGACTCTGCAGACTCTCATAGCCTTGGGACCTGAGAAACCAGGTAT3960
CCAGTGAGCAGTCACTTAAGCCTGTTCACCTGGCCCTCTCTTACTTTCTCTCCTATAGCA4020
GCAGCAAAGGAGCGATGGGCCGAAGrGGACTTGCTGGGTAGAAGTGGACCCACATTCTAAA4080
AAGGAATGGAAGAGAAACCTGATTTCTTTGACTCGCCCTGTCCCTGAAGATGAGGGGCAG4140
GCACAGACCAGCCCTCTCCAGAAAGACAAATATATTCTTCCATTCATGGGAGGGGTAGTA4200
GAGACTAACATTTGTTAAGTATCTATTACATGGGGGGTATGGAGGTAGGCCCTTTGTGTG4260
TGTTGCCTCTTTTAATCCTTTGGTGATCAACTCATGAAAATAAACAGCTCCAGAGCCAGC4320
TGTCTTTGGAGGGTGTAGGCAGGCCCGGCTCTGGGAAACCTGGTGACACTGACCTAGTTT4380
GACTTCCAAATCTTCTCTCTTCTTCGATTCTGGTGAGCCCCACTCTAGCCCCATAGTATG4440
TATGGCCAAGCACCCAGATACTGCTTCCATCAGGAGGAAATAACATACCTGATGAATTTC4500
TTCACTCAAGGTGTTAGGAGCTTAATGTGTTTCCCCCGCCCCCCGCACCAAGAGAATTTG4560
TGTTTTCCAAGACAGTCAGAGAGTGGGTGGTGCTGAACTCAAAGGAGTGAATCACTAATA4620
GTGGAATCCCAGGCATTCAGGGAGGTCCTATTTCTGGGGTGGGTTCCTTCCTGACACTTC4680
ATTTTCTACAAAGGTGGCAGCCACCTATTGTCTCCAGAAAGGAGGCTGTCCCTGTGGGTG4740
TGGTGACGGTGGGAAAGGAGAGGCACCTGCAGGCTGAAGCCAAGATCACCTGATTTTCAA4800
AACCAAATCTGTCCCTACAAAGGAGAAGTGGCTTAAAAATCCACACAGCCTCCCGAGTGG4860
AGGGAAGAATTCCCTCTCCTCTCTGGAACAGGGTTCCCTTCACCCAGAACACGGTGCTGT4920
TGTTATGCAATGTCCCTGTTGGCAAAGATATTTGAGCCCCTTGTTTTCAGGTCTGTGTCA4980
TTTCCAAGAAAGAGCTGTGGCCTTTGAGTAGGACTGGGCTCCTGAATAGGGTCCCTGGTG5040
CCAAATGAGGGAGCCAAGAAAAGGCAGAGAAGAGGAAAGTCCTGACTTTTACATGAAGAT5100
GAGACAGCCAGCCCTGTGGCAGCCAGATGGCAGTCCTGTTGCTCTGTAGTGGCCTTGGGG5160
TCAGACTAGGGGCAGAGCTGGGCTGAAGGCAGGAAGGCCAGGACAAGACAGGTGAGAAGGS220
GCAAAGTCTCCTGTAACCTGGTGAGAAAATGTGGGCTAAGCCATTCTCATCTGGAGCTGA5280
AGGCTTGGTGGAGAATGGCCCTCAACATTCAAGTTCACACCCATGGATTTATAAAAGGCA5340
GGGCTGGGGGGAAAGGTTTTTCCCATTATACTTAATAACATTATCAACAACAATAATCAC5400
TACTATCATTTATTGAGCATTGACTCAAAAGACAGTCCTTTTATGAAAATTATTTACTTA5460
AATCCTTACAAAGCTTCTATTCATTCACCCAACACATATTTATTGAGTTCCTACTATGAG5520
CCAGGCATTATTCTAGGTGCTTAATTTAGATCAAGGGACAAGACAGACAAAATCCCTGTT5580
CTGGTGGCAGGGCTACTACATGCAATTAACAGCACACAACTCTAGGGGGAGCCACATACA5640
TGGGCCACCTTATGAATGGTGTGCCCTGAGGTTAAGCATCCTGGCAGCCCCTTTCTGTGA5700
CATTTGCATTCTAGTGAAGGGAGTCTAATACCAATGAAGTAGATGTCATTATCCCCTGAC5760
TACAGTTTAGGAAACAGAGACACATAGGAATTAAGTAACTTGCTGAGTTTTTCAGCCAAA5820
AATGACTGACCCATGATTTATACTGAAGTCAGTCCTTGCAATTCACCTGTGCCACGTACT5880
TGCCTTTCTCTCCCTGGTGGGCACAGGGAAGAGGGAGTAGCCAGGCTGGCCAGATGAGTG5940
CTGGGCTGGCTGGCCCAGTAGAGGCACCATGTCCTGACTGGGTGGACAAAGACTGGGTAG6000
GAGGTAACAGAGAATCCCTTGGTGAGTCTAACTTAGCTATAAGAAGGCTTGCTGAGAGCA6060
GCTGCCTCCATGCAGAGGGTGGGGTGACCGGCCTTTAATCCTTCCCAGCTGAGGATTTAG6120
TCAAAGAAGCTTGTCTCTGGGGATAGCCTATGGTCTTGAAGGGCCTGAGTTAGCTATTAG6180
TTCACCCATTTATTTAACATTCATTCATTATTTTTAAAAAATTTCCTAGCTATGTTTGGG6240
GGCAGAGAAGTGGGTCCAGAGACCTAGAGGTTTGCAAGGGTAGCTTCTAAACTCCTTTGG6300
TTCAGAACAGAATAGAAAGTGTCCTCGGGTGACCTTGGGTCTGCTTCCCAAGCAAATTGA6360
GCATACGCAGCCAGAACAAAGACTGCACTCTACTCTAGTGAGCTCAGCCTGCTAGGCTTG6420
GATCTAGATTTTATAGCAATAAGCTTGGAGTCTCACCTTTGGGTCAGACAGAGTACTACC6480
CCAGACATGAGGTAGGGAGAGCCTAGTCTATATTCCTCTGCCTTTGTCCAAGCCTGCTTT6540
GTCCTTCCTCTTGACGAGGAATAAAGATGGCTTCTGGGTGTGCATCCCCTTCCTTCTTCC6600
ACCTGCAGATGTACCTGTTTGTGTGCAGTGGGCTTCTGAGTCCTGGGCAGGGATGCCAGA6660
GACCGCAAGCCAGATGCTTGGGATGCCAATCCTTGGGACTTTGAGGAGAAAGAGAGGTTC6720
TGAGGGGCATCTGTCTATGGCACAGAGTCAAATGGAACACATGGAAGTCCCTTAGAAGGC6780
_77_
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
TGGTATCTAA GTGTTGGCCA CACAATGTCC GTTCTTCCTC CATTATTTGA ATTTCTCCTT 6840
CTCTATCCTT CTATCTTTCT TGGCACCTTG AGCCAGGTCT GGGGTGAGAG AAGGGATGGT 6900
GTAGGTGAAT TAGTGGTAGT TATTGGAGGA AGGCAATAAA CCCAGAAAAA GTGTCACGTG 6960
ACTTCTTTCT TGGGCCCAGT GTGACGCTTC TAGTTAGGCT AACGTGGGTC TTGGGACTGT 7020
TCCTGAGATT TTGTGGAAAA CTCTTTGTAT TTGTGCTGGT AACAGAAGGA AACCAGAGTT 7080
AGGGCTGGTG GGATGAAGCA GTGGGAACAC TGATTTCTCC TTTTTTTCAG ATTCAGGGAT 7140
TTCTGTCAGA GACATCCGTG GGGGAGGGAT GGGATTGGGA GTGAGGAGAA TCCCTTTCCT 7200
CTCCTCTCAC CATCTGGTGG TCCCCGTGCC CACGCACCAG CTCGTTGGAT GGACATTTTG 7260
ATTCCCTTAA GATGTACATT CTTCAAATCA TTGTTTGTCA TTAGCTCCCT GGAGAAAATG 7320
GAGGGGCTGA GATATTAGTG AGAAAACATA AAGTTAATTG GGTGATGGAG ACTGGGAGAA 7380
GGGGAATGTT AGAAGAAAGT GAGCGAGGTC TGCTAAAAGT GAACTTTATC TTCTTCTCAA 7440
TTTTGCCTAA GACTCGTGTT GCCTGGGCAG TCTCTTTTTG GAAGAGAAAT TTTCATGACA 7500
GTTTGGGCCA GAGATGGCAA ATAAATGCCT GACATGGTTG CTGCCAGCCC CTGTCTCCCG 7560
ACACGTTCAC AAGGGTGCAC ACCACTTCTC CTCTCTGTGA CCATAGACTC AGACCCATTG 7620
CAATCCAGCA TCCTGCATGG CCCCATTGGT CAGAGTTGAC ATTTGCAATG AAGCTGCTTC 7680
CCTATGCCTG GTTAGGCCTT TTGCTATGAA TTCTCTGGAG TTAACTATTT CCAAGGGGCT 7740
CCAACTTATT CTTGTGATT"F CCACGGGATT TGGAGCCCCA GAAGACAATC CCATGTGGAT 7800
TCACAAAATG CCCTCTAAAT TTGATGGCTG TCAGTGCATA CTAAGTATGA CTGACTCACT 7860
GGTATCTGTT TCCTCCGCTG ACACAGCTGG TTCTTAGGCT CGGCAGGAGT TTGGGCTGAG 7920
ACCTCTCATT GCTCTATATT CCCTCTGTTA CTAATGAGGT GTTGTTCCTT AATTACTAGG 7980
TGCTGGATAC TAGAATTGCT TTTCTTTGTT TCAGGGGATT TAGCAAAGGG CTTATAAATA 8040
TTTCTTGTGT CTGGCATGAA CTACCTGATT TTTTTATTCT TCAGGTCACT GAGCTGGCAA 8100
TAAAGGCAAC TCAAAGTTAG CTGGGAATCA GAATGAAGGG GGACTAGGAA AAGTGATGCC 8160
TAGAACACCA ACAGGTGTGG GATCATCTTC ATTGTACCTT TCAGAGCCTA AGATATAAGT 8220
CCTCTGGATA CTCTCTGCTT GTTTATTTAA AGGAAAAAAT AATCAGAATG TGGGAGAAAT 8280
GGGTGCTTTG GGTAATTTCA TATTCTAATT GATGAACGTG TATGAAATTA TAATATTAAA 8340
CCACTACTAG CCCTTGCCGT AAA74AACTAT TCCAAAATAG CTGAGTCTAA GTTTCCTGCC 8400
TCAGTGTGTC CCACCTCTTG CGCTTGAGTC CTTAATGATC CAGAGTTTCA AGTCCCCAGT 8460
GCCCTAATCT TGAAAAGCAG AAACTTTAGA AGTTTGCTGA AGTTTATTAG TTGGCTATAC 8520
GATCCATCAA GAAATTGACT TTTTTGGATT AAATTCAAGA TAGTTTTTAA AAAATCAGAA 8580
GTTTCTTTAT CATGAAAGCT F~74AAAAATAA TTGAAGGTAG AGGCTAGTTG GAATCCCAGT 8640
TAATAGATGG ATTTCTTCCT TCTTGAAGAA ACTTGTGTCC AAGGGCAAAC TGAATCCTGG 8700
TGGTCTATGC TGGCCACATT CAGCAAAAAA TGGCCCGAGG TTTTGATGGT TATCATTCTC 8760
AAAACTGTTC CTGCCAACAC ACTCTGATCC CAGGAGGTTA CCTGACCTTT ATAAGGCTCA 8820
GTTTCCTCCC CTGTAAAATG GGCAGGGTAA TCAAGCTAGG CAAAATATTT AACCTAAGTG 8880
AGGAAATTGT GCTATTAGTG CCCTGAAAAA CATGTAGAAA GACATTAGAC ATTATTTTAT 8940
TTAATATCAT GTTGAACTTA GTTTTTAAAA AGAAGACCTA TTGGATTTTC CAAGAACAAC 9000
TAAACTGATT CCTTGTAGAC AGTTTAGAGA ATACAGAAAA TTAGAAATAG GAAAAAAGCA 9060
AAACAAAACA AAAACCATCA AACAAAGTCT ACGCAAATAC AGTTTCTCTT AACTTTTGGT 9120
TTATTTCCTT CTAGTCATTT TTTAGGTGCA TTTTTAAATT GTGGTAAAAT ATATGTAATG 9180
TAGAATTTAC CATTGTAGCC ATTTTTAAGT GTAGAGTTCA GTGGCATTAA GTACATTTAT 9240
ATTGCCGTGC AACCATCACC ACCATCTATC TCCAGATTTT ATAACCCCAG ACTGAAACTC 9300
CATATCCATT AAATGATAAC TCCCCATTCC CCTCTCCCTA CCCTGGTGAC CACCATTTTA 9360
CTTTCTGTTT TTATGAATTT GACTTTCTTG GCGCCTCTTA TAAGTGGGAT CATTTTTAGT 9420
TGTTTTTATA ATCGGTTTCC TTCCTTTAAA AATATGAATG GAGCCTAATG AATATTGAAT 9480
TTAGTGTACT GGTTTCTTTG AACATTTCAG CATCATAAAC ATGTTTTTGT ATTCTACATT 9540
CTTCTTGTAT TGCTATATTC TCTATAGGAA TTTTTTTTTT TTTTTTGACA GAGTCTCACT 9600
CTGTTGCCCA GGCTGGAGTG CAGTGGCACA ATTTCAGCTC ACTGCAACCT CCGCCTACTG 9660
GGTTCAAATG ATTCTCCTGC CTCAGCCTCC CAAGTAGCTG GGACCAGAGG TGCATGCCAC 9720
CATGCCTGGC TAATTTTTGC ATTTTTAGTA GAGATGGGGT TTCATCATGT TGGCCAGGCT 9780
GGTCTTGAAC TCCTGACCTC AGGTGATCCG CCCACCTTGG CCTCCCAAAG TGCTGGAATT 9840
ACAGGTGTGA GCCATTGGCC CCAGCCTTGA ACATCATTTT TAATGGCTGA AGATTATAGA 9900
ATCCAGTGGG TGTGCCATCC ATTATTAGTA TTCTGTTGTT TCCAAATATT TGCTGTTTTA 9960
AACAGTGTTG TGAAAACATA TTTTTGTGTT GAACTTTTAT CATATTGAGA GGCACTTCCT 10020
CTGTGCAGAA TCAAGAAATT AATTACCGGT TTATAAGGAA TGTGAACCTT TCAGGCTCAT 10080
AATCTGTATT ACCAAATGGT TAGGAAAAAA ATGTTCAGAA GGTGCCATTC ACAGATGGAG 10140
_7$_
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
TGGGCTTCCA CCAGGGGCTG TGAAGCTCTA ATCTCAAAGG ATGTTGACTA CTGGTAGGGC 10200
TGATTCAAGT ATTAGATATC TAGGAAGGGT GGGAAGGGCA GAGAAGCTTC CAAAATTCCT 10260
ATGTAGGAGA GGCATAGGGG TGCTGATCTC TTCATAAGGG GTGACGGGAA TTTTCCTTGA 10320
AACAGCATGT GCAGATCAAG CACTGTTCTT TCCTTTAGAG TGTGTGTTTA TTTGGGGCGA 10380
CTTGGAGGGT TGCTAATTGA GATTATGGGG AATCTAAAGC CACACCCCAA ACCGCCCCTT 10440
GGTTCCCCTA CCTGGGGGAG AGTTGACACT AGTCAAACCT CTCCCATCTC TGAGATTTTG 10500
TGAATCTAGG ACTCTTGCCA CTGCACAGAC TCCAGCTGGA CCCAGGGACT CCAGCTTCTC 10560
ACATCACCCT GGCTCATCCA TAACTCTCTT TTGTTTCATC TCAAACATCA CTGAGAGATG 10620
GCTGCCTCTT CTCCCTTCCT AGGAAAGCCC ATGTCACAAT AAGCGCGCCT GTGCTTCTCA 10680
TCAGTGCTTT CCTGGTAGCA CCACCTGACA AACACTGCTC GCGGCTGCCT TCAGCTGCTC 10740
TCCAAGAAGA CGTCATAACC ACAAGAGATC TGAATCAGCC CATTTTTTCC CCTGTGGCAC 10800
TGTGTGCTTT GGCTGCCTGG CCAGAAAGCT GGGACTGTAT TTACCTATCA TTTTGATACT 10860
ATCTTGGGGT GTAATTGGAA TTGAGCTCTT AGTGTGGAAA TTCTTACTCA GAACACAAAG 10920
GATTGAAGAG TGCTTGGAGG CTGAACTCTG GAAGGACTCT TCCCTGAGGC CTCTTGGCAT 10980
CTGGCTCTTG TTTCTTGGAG CGGTGGTATG GCCCACAGGT GGGTGTTTCC TTTGGGAGCA 11040
ATTTCTTGCT TTTTCAGTAG CTCTGGGCTG TCATCGAGCC CACTGTTCCT TGTCTTCTCT 11100
GCACTGTTTA GTGATGATGT AGGTGAATTG CTCCACAGTT TAATTCCAGT GGTAGAGCAG 11160
TCACCATTTG TTGGTTTCTT TTTCTTATGG GAACTCTGGT CTGCATCTCA CTGTGTTTCC 11220
CTTGAACGTG TCTGGGGTCC TCCAAACAGC TTCGTGTCCC TCTGAGTGCG GACACTCAGA 11280
TTCTAACTCA GATTCTAAGT CAATGGTCTC AGCCTTTAGA ACCGCAGGAG GCCAGGCGCG 11340
GTGACTCACG CCTGTAATCC CAGGACTTTG GGAGGCCTAC GCGGGTGGAT CACCTAAGGT 11400
CAGGAGTTCG AGACCAGCCT GGCCAACACA GTGAAACCCC ATCTCTACTA AAAATACAAA 11460
AATTAGCCAG ATGTGGTGGC ATGTGCCTGT AATCCCGGCT ACTCAGGAGG CTGAGGCAGA 11520
GGCAGGAGAA TCGCTTGAAC ACGGGAGGTG GAGGTTGCAG TGAGCCGAGA TTGTGAGATT 11580
GTGCCATTGC ACTCTAGCCT GGGCAACAGA GTGAGACTCC ATCTCAAAAA 1?~~iAAAAAAAA 11640
AAAAAAAAAA AGAACCACAG GAGGGAGAGA TCATATATGA CCCCGTATGT GTGAAAAGTC 11700
CTATCATTGC TACCCACACC AACAATATTA GTGGAAAAAT GTCTTCAAAG GACATTCGAT 11760
TCAATGATAC ATGAGATTTG CTTCCTTCCT TAATTTTTCC CTGTACAGCT ATATAATGAT 11820
TTTTTCAATC AGATCCTCTT TTCCCCCTAT TAATTGTATT TATAGGATGA GATTGATTCT 11880
AACACAATAG CAAATGATGT ATGCACATTT AACACATTTC GTGAAGGCAG GAAAGGGCAC 11940
ACTATAAATT CTGTGAAATC CACATTAGAT CATGCCTCTC CTTTCTCAGT TGGGAGGTGG 12000
GCTCTGACAG TGCTCAAGAG AAAAAAAAAT CAAGTTGTGA CAGTTTAAAA AATATTTTAA 12060
ATATTAAACT ATTTATTATG GAACTTAAAA CATACACAGA AGTTGGCAGA ATAACATCAT 12120
GTACCCTAAA TATCTATCTC CAAACCTCAA CAGTGATCAA CCTGTGGTCA GTTCTGCCTC 12180
TTCTGGTTCC CATCTGCTCT CTGACTTCAG TTTATTTTGA AGCATGTCTC AGACATCTTG 12240
TGACTTCAGT ATTGCACGAT GTATGTCCTA AACGTAAGCA TTCCCTTTAA AACATGTATC 12300
TACTTTTTAA ATGAAGAACA ATTAGGTGCA TTTTCATAAG GGTTTTAGAA AGGGAAGAAA 12360
CTGTATTTCT TTAATTTAAA AATGTATCAG ACAACTAATC CATGTTTACT GTTTCTAACA 12420
CGGATACCAT AATAATAGGA TCATTCTATT ATACATAGAC TAGTGAGATC AATTTGTCAG 12480
ATAAACTTAG AAGGGCCATT AAGAAAGTTA TGTCATAATT TTTGTCACTT GCTGAAACCA 12540
AGACTTTAAT TCTGCAGAAC ATCATACCAG GATTCACAAT TGTATACACT GATTGTGTTT 12600
GTCCAGAGGT AATCTCAGAT CCACTGTATA TAATTTTCCA TTTGCCTAGC TATGGGGTTG 12660
GACACGTCAG TTTTTTCCAG ACCAAGGGTC TCCTAGCTTT TTTTTTATTT TTATTTTTAT 12720
TTTTTGAGAC AGAGTCTCTG TTGCCTGTGC TGGAGTACAC TGGTGCGATC TCGGCTCACT 12780
GCACCCTCCA CCTCTCAGAT TCAAGTGATT CTTGTGTCTC AGCCTCCTGA GTTGTAGGTG 12840
GGACTACAGG CACCTGCCAC CATGCCTGGA TTTTTTTTTT GTTTTTTTGT ATCTTTAGTA 12900
GAGATGGAGT TTTGCCATGT TGGCCAGGCT GGTCTTGACC TCTTGATCTT AGAAGATCTG 12960
CCCACCTTGG CCTCCCAAAG CTGGGATTAC AGGCATGAGC CACTGTGCCC AGCCTCCTAG 13020
CTGTTTTGGC TGCACACTTC TATCCGTAGA TAATTAAGCA TGTACCCTTA CTATTTTCCG 13080
CAATATAAAT TATTTACTTA TAAATTACAT TATGTACTCT ATCACACTGG TAAATTAAGT 13140
ATATTATAAA ACAGAAACTA AAAGTATGAA GTGAGAATTA AAAATGAATA GCAATTCTAA 13200
TATCTTCATC TTCCCCTCAG TGGATCCTCC TGTACATACT CCAATTTGCA GACCACTGGA 13260
GGAGGCTGTA GGAGGCAATA TTATATCCCA GTGAGGTGTG TGGGTTGTAA AGCCGAACAG 13320
CCTGAGTCCA CATCCCAGCT CCACCACTCC TTAGTTCTGT GACTTGGAAA CATCACTTAA 13380
CCTCTCTGAA TCTATCTTCT CACCTGTAAT ATGAGGGCAT TAACCCCTTA CAGGTTATTG 13440
TAAGGTTTCT TACACTGTGC CTGTGGTAAG CATCAATACA TTTTAGCCAA TAATAACAGT 13500
-79-
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
AATGATAATA ACACATTCCT AGAGGGCTGG GATGGATCTA GATTTTTCTT CCCCTTTTAG 13560
TGGAAGACCR CAGCATGATG CATGAATTTA CATTTCCTCA GACATTCTGG TGCTGATGAA 13620
GGTAAAGATG GTGAGGCTGC GATGATGGTT TCAGGGATGG GTGTGTTGGG CGTGATGAAT 13680
AGCATGATGC ATATTGTCAC TCATTTAGTT TATCTGCACT GATGATGATG CTGATTATAT 13740
GATGACTGTT ACAGGGATGG TCACATTGTG GGTGATGAAT ATGACCAGAA AGGGAAGACT 13800
TTCACAGTTC CTACCCGAAC TACAACATCG ATATTTTCAT TTGTCTTTCC TAGGAACTCT 13860
TACCTTAATC ACCTGACCAA TATGCTGACG ACTAACATGT TGCGCCCTGC CTTTCTTCCG 13920
GGCCTCTCTG CCTTGCTGAT CTGTTTTGCT GGTGTGCCCT CCACTGTGCT CTTGGGTCTT 13980
TGTCTCTCGG TAAAGCCTAG TACTGTGGTT GCTGTACACA AAACCTGTAG ATGATTAAGA 14040
TCTCTGTTCA CTGCAGGGCC ATTCATCTCC CAGCAACTAT TTTATCCTTA AGTCAAGAGA 14100
CTTGCCTCTC AGCCCCTGGG GACCATGGAA AGAGTGCTAG AAACCTACAG AGTATGACCC 14160
TTTGTAGCCT TATGCAAGAA GTGACCTGTG TCTTTCCTGT CATGAGAGAG GACAGACATT 14220
GCAGGAATCA AACGCATAAC ACTAGTGCAA AACTGGGGAT AATGCCCAAA CCTGGTTAGG 14280
CAGGGGCGCC TGGAACATGC TTGTCCAGGA AATCTTCCAC TCAGTTCTGC TGCCTCCATG 14340
TCCCAGATGA TCACAGAAGC CTCCTGAGAA GGGTTGAATC CCCCGTCGCC TGGGGATCCC 14400
AAGAAAGCTG CAGAGGAAAG ACTTTCTCTT CCAAGATCAG AACAAAGGAC GGTTAGCATT 14460
GTGCCCAGTA GTGCCAAAAG GTAAGGTTGG GTTAAAATAA GAATTTGCCT TAAGCTCTTT 14520
TCCCGGGGGC TTGTTTTTTT CATTAACCTT GTTGGCTGGA CTTTAGGGAA GTATGCACCA 14580
TCTTCTCCAG AAGTGCTTCA GATTTTATAT TTTTAAGAAA TTCAAGAGTC TGAGTTAGGC 14640
ACTTTAATGT AACCTCCCCA AAGCTTTTGT TCCAGGAATT GACTTGGGGA TTAATCTGTT 14700
TAGCAAATTC TGACACAGAG GCATCTCATA ACCTTTTATT TTTTCTACAG ACCACATTGT 14760
ATCTACCTGG GATGTTTTGA AAATGAACAG TGACACCTAA GAATGTATAC TTATCTCTTC 14820
ATGCCAATTC TCCAAACTGG ATGTTGCCCA TGTCTCAAAA TTACTTGCCT CCAATTTTAG 14880
GGCATAAAGT GTGAGATTCT GTAGCATGAG ATCATATGCT CTTAAAATAC TAAGTATATA 14940
TAAATTATCC CTTAGCATCT TTAACATGCA TTTTTTTTTT GTAGAGACAG TATCTCTACA 15000
AAAAAATCTC TCTGTATTGC TCAGGCTGGT CTTGAAATCC TGGGCTCAAG AGATCTTCCC 15060
ATCTCGGCTT CCCAAAATGC TAGAATTACA GGCATGAGTC TCCACACCTG GCCTAACATG 15120
AAATATTCTT TAACAGTATT CTTTAGGATA ATATATTATT CTATAGATTT GAAATAATTT 15180
ATCAGTTCTA TACTTAATTA TAAATACTCT TGGGAATAAA ACATACTTAT CTAATAAGCA 15240
AACAGTCGTG CTATTCCAAA CAATTTGGGA TTGCCTTTCC AAGCATTTTT TGGGGGTTTC 15300
TTCAACTGAT TGAGAGACCC CCGGCCGGGG AAGAGAAAGA GAATTTGATT TGTGACACTG 15360
ATGGAATGGA CTACAACCTT TTGGTGGTGA CTCTACTGGG GACTTGTCAC AGAGCTTATT 15420
TTCTAAACAG ATGTGAAAAA TGAAAGTCAG GCTGCTGTCT GGTTGGTAAG ATAAAGCTTT 15480
CATTAATACT TGGCAGCATT ATTTTAGCTA AAGTGTCAGA TCAAACGCCC ACATTATCAC 15540
CTCCCCTTCC TGATTCCAAC CGCCCATGAT AGAAAAGAAA TAAAAGACTA GGAATAGGTC 15600
CATCAACTGG TGAATGGCTA AACAAAATGA GGTATATACA TACAATAGAT GGTTATTGAA 15660
TCACAGTAGG GAATGAAGTA CTGATACATG CTACAATATA GATGATCGTC ATAAACATCA 15720
TGCTACGTGA AAGAGGCCAG ATGCAAAAAT GTCACATATT ATATGATTCT ACTTATTTGA 15780
AAAACTCAAA GTAGGCAAAT CCATAGAGAC AGAAAGCAGA CTGGTAGTTT CCCAGTGCTG 15840
GGGAGAAGGC AGACAGGGAA GTGACTGCTT AATGAGTATG AAGTTTCCTT TTGGGATGAT 15900
GAAAATGTCT TGGGACTTAG ATAGAAGTGA TGGTTGCACA ACACTGTGAA TGTAGTAATT 15960
GCCATGGAGA TGTACACCTC AAAATGGCTA AAATGAATTC TATGTTATGT GAATTTTACC 16020
TGAATTTAAA GAAGAGTAGA AACAAACACC AAGAAAAAGG GAGGAAAGGA GGCATTATTG 16080
AACAAGACAT TTCAACAAGT TTTGGAATAT GGAAAATATA CGGAGAAGTG GCAACTGACT 16140
TACCAGAGTG GCAGAAGAAA TAGTCTATGT GAGTGTGGGG AATGGGGTGG ATGTGGAACC 16200
AGTGAGAAAT AAGCCGCTTT ACTGGGAAGA ACTACAGAAA GACTGAGGCT TGGACGCAGC 16260
TTGTGCTACT ACAGGTAGCA GTAAACAGGG GGATTTGTTG AACTTCAGAA TATAGAGAAT 16320
TTTGATGTAA GAGGTTTTTT TTTTCTCGTC TCAAACCAGG AGACTTTTTT TGTTCTCTAG 16380
GTGAGGGAGA TCTAGAGACA GCCAAGTACA GGGTGCAGTA TCATCTAGAA AATAAAGAAG 16440
AGGTTTGAGT CTGCAGGTGA GACTCCTGCT~CTCTTCCTGG AATGCTGGCA GCCAGGCTTA 16500
GATCAGCCTC TCTGCCCTGC TCCAGGCAGA AGATGGAAAG ATCCCTTTCT GGAGAAACTG 16560
ACTCATCCAA GAGATAACAG CTCATATTCT TACTTTTTAG AGCTCTCCAG TAAAATGCAG 16620
CTCAACACTT GATCAGTTTC CAGCGATGAC CCCTGATCAG GCCCTCACTA CGAACCTCTG 16680
GGTTTTAATT GGTTATTTAG TATCTCAATT TTAAAGATCA AAGACAGGAT CGCTTTTGAG 16740
GAAACTTCCA ACTTTAATGA AAGAATTTAA AAAAAAAAAG GAAAAAAAAC CTGATAGTGT 16800
AAAGAGCAGA GAAATGGCAG GGAAATGAAA ATTAAGTTAA AAAACAGAAA CTTTTATATA 16860
-$0-
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
ATTCTAATCC TTTGCAGAGA TAAAAAAATA CATTGCATAC CTAAAACAAG TACAAGTTGC 16920
CATGGAAACA GATTCATTAG TGAAGAGGAA AGAGATCTTG GAAATTAAAG ACATAAAAGA 16980
CAAAATAAAA ATTAAAAAAA TTAAACAGAA TTTAGAACAT AATGTTGAAA TGAGAGAACT 17040
TTAGATCTCA AAAACAACAG AGAATCAACC CAGGAGATTG TGTGTGACTA AAGAAGTCTC 17100
AGAAAGAGAA TAGAGGAAAG GAAGGAATAT TATAAGAAAA GTTTCAAGAA TAAAAGGTCA 17160
TGGGCCTCCA GACTGATAAA AATCCATCTT GTACCCAGAA AAAATTGACT TTTCAAGAAC 17220
TGAATCAGAA CCTATCCTGT GAAATGTTAG GACAAGTAGA TCCTAAAATC TTCCAGAGGG 17280
AATCCATTCA AAGGCCTTGA ATGGCATTAG ACTTCTCCAT ATCAATACTG GATGGTGAAA 17340
GAAAAAGAGC AATACCTTAA ACTTGCTAAA AGAAAATGAT TTTTAACTAG AATTCAATTT 17400
CCATCTCAAT TAAAAAACCC ACTGTAAAGA AAAAATTCAA ATCTTCTCAG GCATATAATA 17460
ACTCTAAAAT TCTACCTCCT GTGCACCTAA TTTTGGCAAG TATCTCAGGA AGATACACTT 17520
TGCTAGAACA AGGACATAGT TTAAGAAAGT GGAAGAAATC AGATCTGGGA ATCAGGGGAT 17580
CACATGATAC AGAGGCACAG CCAGAGGGAT CCCAGGGAGA GCATGTCCAG TGTGACAAGG 17640
AGTGGACAGC TTCAGAAGGG ACAGCACCAG GGGAAAAAAC AAAATGAATA TCTGATTGGC 17700
ATAAACATTT GGAAAGTAGT ATTAAAAATG TGTGTAACAG GTGTGTTGTT ACATTTGCCA 17760
AAAAAGAGCA AAAGGGAAAA AAAACCCCAA GCAGATGAAA AGTAAAGAAG GCAATGGTTA 17820
ACTACTGGAA AAACAAAAAA CAATATTCAA GAAAGGAAAC GAAATCATGG TATACTTCTT 17880
GACTAATGGG TGAAAAATGA AGATGTACAT AGTTATTAAA ATGCAAACAT TGATTATTGA 17940
GTTAACCCAA AGTTGTGACA TTTGGAAGCA CGGGTAGGCA CAGTGGGGTG TAAGAGACCT 18000
AAATCCTCAC TTACCGTAAT GTTTAAAAAA TTGCCATGTC AAAGAATAGC AGCATATCAT 18060
ATTATTTAGA AATATGGATG CAAATGCCAG AAGAAAAATT AAAGGAAGTG AAAAATGTTT 18120
TCCTCTAGGA ATAGGACAGG GGACGTAATA GGGAACAGAT ATTCTGCATT ATCTCAATTA 18180
ATTCTCACAA CTGTGACTGA AGCTCTTTTG CTCTCCTTGT TTTGCAGATG AGCAAACTCA 18240
CAGAGGGATG CAACTTGCCT AGGATCGTAT AGCCAGCAGC TCATGAGTGT GGAATGGGGA 18300
TTCAAATAAG GTCTAGGAGA CTCCAAAATC CATGTGCTTA ACCATGAAGT TTTACTACCC 18360
CTTCTCTGCT TCTTCATTAA GTATTTTTAG TGCCTAATTG CCCATGCTCT CTGCCAGGTG 18420
CAGTAAAGGA GGATTACACA GGTGCAATAT GAGCCATGAC TCTTGTTGAA ATCAGCACGT 18480
CAAAAATAAG GCTAATGAGC ACGTGAAAAG ATGCTCAACA TCACTAATCA TTAGGGAAAT 18540
GCAAAACTGC ATTAAAATAT CACCTCATAT ACATTAGGAT GGCTACTATG AAAAAAACCA 18600
GAAAATAACA AATATTGGCA AGGATGTGGA ATAACTGGAA CACTCATGCA CTGTTGGTGG 18660
GAATGTAAAA TGGTGCAGCT GCTGTGGAAA ACAGTATGAT GGCTCTTAAA AAAATTTTAA 18720
AAAAATAGAT TTCTCATATA ATTCTGCAAT TCCATTCCTG GATATATACC CCAAAGAATG 18780
GAGAAAACAG GATCCTGGAG AGATGTTTGT ATACCCATGT TCATAGCAGC ATTATTCACA 18840
ATAGCTAACA TCTGGCAGAA CCCAATGAAT GAGTGGATAA ACAAAATGTA GTATATACAC 18900
ACAATGGGAT ATTAGTCTTA AAAAGGAAGG AAATTCTGAC ACATGCCACA ACATGGAGGT 18960
GCCTTGAGGA CATTATGCTA AGTGAAATAA AGCCAGTCAC AAAAGGACAA ATATTATATG 19020
ATTCCATTTA TATAAGCTAC TTAGAGTGGT CAAATTCATA GAGACAGAAA GTAGAATGGT 19080
GGTTGCCGGG GATGTAAAGG TGGGCATTTC TCAAAAAACT GAGAAATACA GAAAAATAAA 19140
AATCACTCAC TGTTTGCCAC ACTTCTACCC TGGTTCTTTT TAAATCTATT TTTCTTACTC 19200
AAAGAAATAC ATGTTTATAG TTTAAACATT CAAATAGTAC TACAGGTTCG TAATAAACAA 19260
GAGCGGTCCA ACTCCCCTCC TCCTAGCCCT GTGCTCCAGT CCTTTCAGAT GTTGTTTCTG 19320
GTCTTTGTAT TTCTCAATAA CATGCCTAAA TGTATTTTCT GGCTCCTTGT ATTGTTTATT 19380
TATTATTTGT TGAGTTTATT GCTATGAAAA ATAGAGATTA GATCACTTAC AGGGTCTTCC 19440
TGACACCGTG CTCACCTTCC CCACCTATAT GTACAATTCA CCTTCCCTGT CCTCATGGAA 19500
ATAATATTAC TCTTTTAGTT AAGTCACAGG TCAGTATTTA TGTTATGATT ATGTAAATAT 19560
TGTTTATGTA ATGTGCTAGG GCTACTTTTT TTTTCTTTAA TTCCTTATCC TCCTTCACCC 19620
TCACCACCCA ACCCCAATCT CATCCTGGAG TTCACAGTTA TCTCATTTTT CCTTTGCTTG 19680
GTTTTCTAAA ATCTATCTCC TGGCTCTTTC TCCAACTCTT CTCTCAGTAA GATAGTTTCT 19740
CAGCTCTACC TTTTCCCCTT GTTGACATTG CTCCAGAGCC CTTCAACCTG CTCAGGTGGC 19800
TATTCTGCTT GGTCACTCAC TTGTCCTCCT AGGTTTTCTT ATCTCCATCA TCTTGGGGAT 19860
TCTGGTCTCC AATTTCCTGT GTTAGACCAA CTGTGTCCTG GATCCCATAT CTTTCTGTCT 19920
CTTAGTTTAT TTCTTTGCTT TGATTGAACA TACTACCTAT GACATTTCTG AGAAACAATG 19980
AAAGAGAAAT GATTTTTTGA GTTGTGGGAT GAATATTAAA GTCACTACCC GGGAAGGATC 20040
ATTGTGCCTC TATCTGTATG AGGGATTCCC CTTGCACTTC TCAACCATAG ACAGCTCTGT 20100
TCTGTCTCTT GAGCTCTTGG TGAACCCATC CCCCAGGACA ACATTTCTAT GTGTCTTGGT 20160
CTGGCACAAG GTGACTACCT ATTCCCAGCA AATGCCAATC AACACCTGTC TTAATAATAC 20220
-81 _
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
CTTAGCTTCA ACACCCAAGG TTTAAGTTGC ATTAATCACT TAATAAAGAA ACCTTCACAA 20280
ATGCTAATTA CTAACCTAGT CCTTAAACCA TACTCATTTA AAGAGGTGGC ATCTTAGAAG 20340
TTACAGTGTT TATAGTCATT CAACAAACAT TTATTGTCAG CCATATAGAA GACACCATGC 20400
AAGGGCTTTA CATGGGTTAT CCAATGTAGT CCTCATGAAG GTCCTGTGAA GTGGGAATTA 20460
TTGCCATTTG TGAATGAGTT TCAGAGAGAT AAAACTTCTC CAGCCATTCA TTCAACACAT 20520
TTACTGAGTA TCTACTATGT GCTAGAAAAT GAGGATACCG CAGGGGGCAG AGGCACATGT 20580
CCCTGACCTC TTGGAGTTTC TAGTCTAGCC TAGTCTGTTT CCAAGGGTAA CAGATATTAA 20640
ATAAATAATT TCACAAATAG TCTATTAAAT ACATTTGAGA CAAGTGTCAT GAAAAAGAAG 20700
TACAAGATGC TATGGGAATG TATAAAGGCC ATAAGCTGTC CTAGTCTGGG GCTCAGAGGT 20760
GGTTTTTCTG AAGCAGTGCA TTAAGTCTGC AGGATAAGGA AGAGTCAGCC AGATGAAATG 20820
AAGTCTAAGG TTGGAGAGAG GGAGGGAACA GCATGAGCAA AGGCTCAGGG GCAGGAAGGG 20880
GCTTTGCATA TACGAAGAAC TGAAAGGCCA ATGCGGCTGG AACAAAGAAT GGAATGGTGT 20940
GGCATAAAGT GCAGCAGGGA CCGGGTCAGG GAGAAGACCA TAAAGCATTT GTGCACGCTG 21000
TTAAAGAATC TGTATGCAAC CTTGGTGGAC GTGGGAGACA TGACTGCTGA ACTTGAAGCG 21060
CATCCCTGGA GATGGGGATA AATGGAGGGA TGCGGGATGT GTGAAGCAAG AGGCTTGTTC 21120
ATGGTCAGAA CCGGCATCTG AACCCAGCTC TCATGACAAG TCTGCTGCTC TTTTTGGTAC 21180
ACAAAACCCG TTTCTTTCTC TGTGTGAGAA TGAACAAGGT GCCTGCACAT TTTTCTGTCC 21240
CAGTGCAGTG TTTGAGGATG CTAAGTTACA CCCCAACAGC TGTGCAAAAT CTGTTTCTCT 21300
CTTGTGTAGT GATGGAGGCT ATACATTGTG TTGTGAAAGG TGTCACTCAT TTGGGAAATT 21360
AGAACAAAAC ATAGTCATTG CCTTTAACAG CACACAGCCT AATAGAGGCA ATAGGAATGT 21420
AAACAGGGTC CCAAGCCAAA ACTTAACATG AGCAAGTTAT AGAATCATAT ACAATTCTTA 21480
GGGTCATAAT TCTAGGGCTA CATGTTTTGA CTGTTTGACC ACACTATATG CAGCAGTATC 21540
GTTAATGGTC CTGGATCTAG GCAGCATTTT CCGAAGTAGA CTTAAAATAA CATCACTCTT 21600
AGACTGGTCT GATTCTCTGT TTTGGCTAGA AATTGTGTTC CTCAAGAATA ATAACACATT 21660
TAAAATCATC CTTATTTTTT AAGTTCAGAT ATTCTGCTAA ATCATTGATC TCCATGAATT 21720
CATTGGTCAA TGTTTTAAAA CTTTCTCACA AACGGGCTTA TTGGAAATGG AGGCAGAAAA 21780
TAAGGTGTTC AATAATATGA CCACATGGTC TAAATTTCCT ACAATACGCT TAGTTTACAT 21840
GTGCAACACC TTTGTCAGAC ATATACCCAA TTTTGGTTTG AAAATAGCAT TTACTTCCCA 21900
GGAGTGGTGT GTAGGAACTT AAGGGTCCTA GTATGTATGT CTCTAGTGGA AACTTTGGGG 21960
TTCAGTTTGA AAAGGCAGTG TATCTCATGT GGATCCCTGT GATTCTCAGG GATTCTATAC 22020
TAGGCAGTCC CTTGTGGATG CCTGGGGAAG TCGGGCTGTG ATCCTTACAG ACCTTCTCTG 22080
AGCTGCCATA CAGATGGGGC AGAGGGTGAA TGATGGAAAA AGAACAAATG TTGCTGATGG 22140
TCCATGATTC GTCCGCAAAT ATTGTAAAAC CCTGTACTAC CTGGCTGATG CTTTAACAAA 22200
ATAGCTTCAG GGACATTAAA AAAGTAGTGT TTCCTGGTGT GCTGGTAAAT ATTTATTGAT 22260
ACAAAGATTG TGTAATCACA ATTTAAAATA TACAGTACTC TTGATTGTAA ATTCCTTATA 22320
ACCAATTGAT CCCCACAGAA TGCTCTTGTT GACTTTTGTT TGAGGCTCTT GTATCTATAG 22380
TGTATCCAAT CTATTATTGC AATTGATGGA CAAGTGCCAT TCTGATAAGA ATGTGGGCTG 22440
AGATTTCCCT TTATGTTAAT GAGTAAGAAG AAAGGGAAAC AGCAGAGCTA GACACTGGGC 22500
CTTCAATCGT TT'GTTAACAA CACGAGCAAC CTTTTTGTTG AACTGGATAA TAGTTTTTGA 22560
ATACTGGAAG AATATTTCCT CAGTCTTTTT CTGTTATTCA CCATGCATTG GCTACAGTCA 22620
CATTTTAGAA TTTAACCTGC ATTATTAGCA TTTCTCCATC ACTTTTTATA AGTCTAGACT 22680
GGGGATTATT AAACTGTGGT CTAGGGGCCA TATCTGGTCC CCTGACCTGT TTTCGTACAT 22740
AAAGTTTTCT GGAACACAAC CATGTCCACT AGTTTTATAT ATTGTATATG GCTGCTTTTG 22800
TATTACAATA GCAGAAGCAG AGTCGAGTAG GTTGGACAGA GATTTAATGG ACGCAAAGTC 22860
AAAATTATTT AATATCTGGC CTTTTGCAAA ATAAGATTTA CCAAGCCTTG GTCTGGGTGG 22920
TCAACAAAAC AATAAATCAA GCCTTGATCT GTAGTGTCTG CCAATTTCCA TGGTGTAAAT 22980
ACTCCCATCA TGGCCAATTT CTATCTACCA ACATGACACA GCAAAACATA GAGTTGGGAA 23040
GAGATGTGTA AAGTACACCG TTATAGAGTA TTCTCACTCT ATAGCTACAG TGGCTATAAA 23100
TAACTTCCAG AGCATAGACA ATAGTAAAAT GTAGTCATAA TTAAGAACTG GTAAGTTTTG 23160
AGTGTTTATT ACCTTTGTTT CTAAATACAA TTTATTTAAT TTTAAGTTTA TATTTTAATT 23220
TCGAATAATG GCTGGGTTTA ACAAGTGGTT TGCAAAATCT CTGAGAACTT AACAATCAGT 23280
TATCATGAGT TGGCACTATT GCTTTCCTTT GGTGCCCAGC TGTCTTCTTT TTTCAGCCAT 23340
TTCCCTGTCT CCAGGAGATA ATCCTTTTTT TTCTTCTCAG CCTGTCTGCT TCCCAAAGTA 23400
TCCTTTGTTC TTTTCATGGC CCTCTGGCTA CGCAGGGACC CCACTTTTTG CCAAACTAAT 23460
CTTTTAAAAC ATATGTCCCA CAGAGTACCA TTCCCTTTCA TCTGCTTCCC ATCAATACTC 23520
TTATTTCTAC AATAGGGTTG ATACCAAATG GCCAGCAACA ATTTGTAATA AGCTGTAAAT 23580
-82-
CA 02314677 2000-06-02
WO 99/37809 PC'T/US98/01260
GATTAATGGC CTGGAAACAC TTGCATTTTA AAAAAAGGAG TCTTGTTGAC CCAAAGGTTA 23640
TAGGGTTTGA ATGTCTGGCA ACATTGCAGG TGTGAGGAAC GTCTTTGGAA TTCCTAGTTC 23700
CCCCCAAAAG GTTACTGTCT TCTTCAGTGA CAAACAACCA ACCCAAGCGT GTACCCTGAT 23760
GCTCCTCATT ACCCCTCAAA ACTTTTTCCT TTTCAATCTT TTTAGTTTTA GCTCTTTATT 23820
TCCCCTCCAC TTTCATTCCT TATTTAAACC TCTCAATTGT AACTGAAGCA GATGTTATAT 23880
GGACTTGGGG AAAGGGATCA AGAAATCATT CAGTTGTTTG TGCTTATCTA GAACTGTCAG 23940
CCCCTGAATT GTGTGGTCTT GGCTGGCATC TGAGCACACC TGGTGCATCA GCAGAATCAG 24000
TGTTCTCTCA GTTCCTGGTT GGCTCTACTG TCTGGCACCA TTCGGCTGTT TGTACTTATC 24060
TGGAACTGCC AATGGGAAGA TCACATGGTC ATTGAGAAAC CGCACCCTGA AGAGATGGCT 24120
AAAAGCCTGG AGGGCATGCC CATCACAGCC TTGCCGGGAG TGTGAAAGGT GGTGTGAAGA 24180
CCCTGGGGCT CACAGGACTC CCTCACCATG GGGCACAGTG TAAGAAGGTC CACGGTGAAA 24240
ATGCAGTAGG AGGCAGTTAC ATCAGGCTCT GGATCGATGA TATCAAGGAA CAACCCAGGC 24300
TGAAGGAAAA GGCGTTTGTG TTTCAGGAAA GATGTATTGA GCCTCATCCA TGCTCCAGAC 24360
TTTGTTTAGG CCCTGGGTTA CAGCATGGAA TGGAATGAAA CCCCTGTTCT TTAGTTTCTT 24420
ACATGTTGAG TGGGTGAGAC AGAAAGCAGC AATATGGTAA AGAGGGGGGA ACAGGGGAAG 24480
AATGGTAGGA GATCAAGTTA GAGAGGGGAA TGGGCTAGAT CATGGAGCAA CCGGGGCAAG 24540
ATGTCAAGCC CTTGGAAGGT TTTGAGCAAG AGAGTGTTAT GTTCTGACTT ACGTCTTGAA 24600
ACACTCTAGT TGCTGTACAA GGAGACCAGG TCAGAGGCTA TTGCAGTTGT CCAGGTGAAG 24660
GTGGCCAGGT AGCGATGGAG GATGAGAAGT AGAAAATTCT GTGAAGGCAG AGCTGACAGG 24720
ATTTACAGAT GGATTGGCTC ATGAGAGGAA AAGAGGGACT CACGGATGAT GCCAAAGTTT 24780
TTGACCTGAG AAACTGGAAG AATGGAATTT CCACTTACTA TGATGGGAGA GGTTGTGAAA 24840
GGATGACTTA GGGGTTGGAG AAAACCAGGA GTTTGGATAT GGGCCTTAGA TATTGCCATG 24900
CAGATGTTGA GTAGACAGCT GCACATATGA GTTGGGAGTG CAGAGGGAGA GGCTGGGGTT 24960
CTGGGTATCA GTATATGAAT CATCTGTGTC CACATGGCAT TTAAAGGCAT GAGACCAGGT 25020
GACCCCCCTT ATAGAAAGAT TAGATCCAAA AGAGTAGTGG TCTGAGGACT GGGCTTTAGG 25080
CCCTGATGCT CAGAGGTGAG GACCCAGGAA AGGAGACACA GAGAATCCTC TTTGTCAGAG 25140
CATTACAAAA GGGCTATTTG GAAATAGTTC AGGTGGTGAC TGGGTGAAAA GCCCTTCGAA 25200
CAGCCTCAAG GACCCAGGCT GGTGGACTGC TGGCTGAGTC CTGTTGTGCC TCAGAGGATA 25260
TTGTAATATT TGGAAAAATT TCTCCAAGTC AAATTTAAAT TAACATGAAT GTCATATGGC 25320
TTTTTGGTAC GTCCTACAGT CAAGCAAATA ACAATTGGAT AGGGTAGCTG CAGGAAGACT 25380
GGGTGTCTCT ACAGTGGTCA AGTTGGAAGA ACAAAGAATG AGTGATTGAT CTTTTGCTAC 25440
TCCCCAAGGG GAGAAGCCAC TGATAGCTTC CTTGGAAGCA CTTTGTACCT CACCTGCCCC 25500
AGAGTAGATT AAATATTAAG TTTCCTCCCT TCTTTCAAGT CCTAGTGCTG CCATTGATAG 25560
TGCTGTGACT TCAGGAAAGT TGCTTAACTT TTCCAAACCT CTATTTCCTC ATTACTAATG 25620
AGTAATAATT CCCACCATAG GGTGTTTATA AAGATTAAAT AATTTTAAAT ATGTTGAAGC 25680
ATGTAGTGAA CTGCAAAGCA ATATGCAAAT ATAAGAGGTG GAAATGACTA TGCCTATAAT 25740
TACGTGGCTC AATTTACACA ATAATAGATT TTCACACTTT GCATAAATAA TGAGGGTTTT 25800
TATACTCAAG TCACTGAACT TACTATCTTC AGGATCCAAA ATCCCCAAAC AGAAGGCATC 25860
CCCTACTGTT AGCTCAAATA GCTCTTGCTG GTTTAGAGAG TTAATGCAAG CCCCACTGCC 25920
TCCTGAGCTG GAAACATGAA ACAGAAGTTT CAGTTCCCTA ATCAATCCAT TCTTTCTTCC 25980
TCTGGCTTCT GATAGGCCTC CTCCTTATCT TTGTAAACCC TGTAGCTGGT TGCTAGTTGA 26040
AAGTGCCTCT GATCTCCCTC TTCTGCCTCC CATGATGTTG ATAAAAAGCA CGAGGGCACA 26100
TGCAGGATGA AAACGATCGT GGTCCTGCCA GCCTGAATTA TTAAAGCATT TCAGTCCTAA 26160
GTATGAGGTG TGTATATGTT GGGGTGTGGA GTGAGTTGTG GAGATGAGAG ACAGCTGAAT 26220
TACATAAAGT TGAGAAGATC TGAGTTCTAG TCTTGAAATT CACAAGCCAT CTCTATACAA 26280
TAGTTCCGTT ACTCAGTAAA GTAAAAGCAT TGGATCTAAG CTTTAAGGAC CCTTCTAGTT 26340
CTTTCTGATT GGAATTCTGT GACTTCATCT TTTGTGGGTT AGAAACTCAT CACTCTGTCC 26400
AGTTATTTCT ATATTATGCC ACCAGATGGC AATGTTTCCT TAACCCCAAA GAAAGTTTTC 26460
ATTCTGGTAA AAAGTCAAGT TTTGTTGCCA ACTTTTCCCC CTCTGAACGT GCAAAAGAAT 26520
'GATTTTCCGA AGCTGTGGAG GAAAGAAAGA ACTCTCCTTC TGAACATCTC AGGTGGTTTA 26580
TGCTGGAAAC AGACAGGACC CTGTTTAGAG AAGATCTCTC TTTTCTTCGT GGACTGGGAA 26640
CTCCAGTTGG AATGATGTCT CCTGTGATTG CGTATGGTGG GAGGTGGGAG ATGTTGGAAT 26700
TGGCGTGTCC TCAGGAGGCT TGGGGGTGGG GGAGATGTGC CCTAGCTGGT GGGCCTGCAT 26760
GAGCCCTGCA AAACTCTGAC TTATAGAGGG GCATCAGATG CCAAGTTTTA CCAGACCATG 26820
CAGAACTAGG AATTGCCAGA TGCACTCATA GGGCAGCTAA AATGGTCCTG GCAGAATCAG 26880
ACTCTTTCGC TCATAAAGGT CAGAGACGCA AGAAAGTGAC ATAAAGTCCA GCCCTTTTCT 26940
-83-
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
TGTGCAGATG GGGAAATTGA GGCCTAGAGC AGGTCAGCTG TCCTGATTCT ATCTCCTTGC 27000
CAAGTTACTT TGTATTTAAA CATTTCAAGT AGACTTTTCA ATCATCTCAT CTTGCTGTGT 27060
TCAGCTAGCG CACCTTGTTA AGCCTGTTGG CCTCCGGGCC TGCCAAGCCC CTGCATCTAT 27120
ACACACCAGG GCATGCTGCA TGCGCTCAGT GAGACTTCAA CAGCTGACTG ATTCGTTCAA 27180
ACCTATCAAA CAGCAGACTT AGCTAGTTGG GGAGAAAAGT CATTTAAAGT AATTGCTTAT 27240
TAATCTGCAA AACAAGTCTC ATAGCAGGTT TTTATTTTAT TTTATTTTAT TTTTTTGCTT 27300
TTAACAACGA TATAATAACA ACAAACATTT GTTTAGTGTT TCCTGTGGAC CAGGCTCTGT 27360
GTTAAGCACT TAACATCACT ATATCATGCA CTTTTGCTAA TAAAGCTGTG AAATAGTTAT 27420
TACTATTTCT GCTTTACAGC TGCAACAGAG ACTCAGAGAG GTTAGGTAAC TTGCCCCAGG 27480
TCACAGAGCT GGAAGGAGCA GAGCCAATAT TCACACCCTG ATTTGCGTAA TTCCAGATTT 27540
GATCTTCTAG CTTCTATGCT GTGCTGCCTC TTCATGACAG TTTTTCTCAT GTACAGGATC 27600
TGATGCAGAA ACTTATCGGA GTTTCTTACC GGAGCACCAG TCACCTCTCA TCATTTTCCT 27660
GTTTTGACGT GAAGGCTCAG TGATAGTGAG CAGGCTCAGG GTCTACAGAG TTGGTGATAT 27720
CAGCATCACA CAGGACATTC AGAATGTTGA CTCCAGGGAT GTTGAGAGAT ACTCCTGCAC 27780
AAAGCTGCCA GCACCCGTGT CCAAGAAACA CTCAGAATCT AGGTCTCCTT GTATATTTTC 27840
CCCACTACCT GCAAAGGTAA AGAGGAACAG GCAGTGCTGG GACCGAGGGA GCGACAGTCC 27900
TAATGGAAGC TAGTGTGTTG AGAGTCTCCT CTGTGTCATG CTCTGAGCGA CATGTTTTAT 27960
ATGCACGATC TCATTTAGAC CTTGTGACAG CATGTTGTAG CAAGGACCCC ATCATCACAG 28020
GGGGCAAATG TCTGCAGTGC AGAAAGTCGT CCTGAAGAAA TGGATGTCAG ATAAAAACAG 28080
TCTTCATAAA TCAATGATCC TGTTTTACCT CAAAAGTGCA TGAAATGGAA ATGGAAATAT 28140
CTTGTGAAGA TGTAGACAAA TGACGGTCAT TGCCCAGAGC AGTAGTTACT GTCAGAAAAA 28200
GAGATAAGGA TTTCCAGTCT GACAGACTGG ATTCCTGGCT CAACACCACC CCCTTCTAAC 28260
CATGTGACCT TGGGCAAATT ACCTAACCTT TTCTGAGTCT CAATTTCCTC ATCTTCCAAA 28320
AGGGGATAAT ATCATATATG TTCCAAGATT GCTGTGAGTA TTAAATGAGA TGATGTATGT 28380
AAAGTACCTG GCCAGCAGTT TCTGGCACAT AGTAAGTATT CAATAAAGAC TAATGGTGGA 28440
GATGAGTATA GGGGCTACTA ATGCCCATCC TTACTCCAGA GACTTCTTTC TGACCATCAT 28500
GAGGCACTTT TGAATATCTA AACCCATTTA AAGCCCACTT TTCTCTATGG CTGGCCATTT 28560
CTGCCTATTG ACAGCTAATT TGCCTCATCC TACAGGACAC CTTCCATGTT TCCCCAGACT 28620
CCAGAAATCA GGTATTAAAT TATCAGGGCT TCAGGAGCCA TGGTCTATGA TGAGTTTACT 28680
ACCTGTGCCC AATAAATGTT TAAGAAATAA ATAAGAGCCA ATATAACTAT AAAGACCAAG 28740
AGCCAAAATA AGTCTCTTTG CTTGCGCTTT AGATCTTAAG AGTCCTTTAT ATTCAAGCTG 28800
CTCAGAGTCA AACGTGTGCC TAATAAACAT TCTACAAAGG TCCTGGCGTG GTGTGACCAA 28860
AGGAAGAGAG AGGGCTCCAG TGTCTGTCAC TGGGAGACCA GATGGACAGC CACGTGGGGC 28920
AGGGCCACTG GTGCCACATG TCCAGGTCTG TTAAGCCCTA TGAAAGACAC TTGAGTCAAA 28980
ATGTATTTCT ATCTAAGAAA GAAGACTATA AATGGAAAAG GGAGAGGGGA GAAGACCTCT 29040
CAAGGGCATC TCCCTCTAGA AGTAGAGATT GTGAATCTGC AGCAGAAAGG TTTTAAACAA 29100
GGGATAGCAG AATGCCTGGA TGGTGTTCTA GTGCCTGAAT GGAAAAAGGC CACAATGACC 29160
AACAAATCCC ACCTACATCC GCCTTCCTCG CTGCCTGAAA TCCCACCATT AGGATTTTTT 29220
TCCTTTTGGG TTAGCAACCA AGAAAGAGTA AAGTCTGGAA GACTCTTATT CCACATCTTC 29280
ACTTTGCAGC GCCTCTTTTT TTTTTTTTTT TTGAGATAGA GTCTTGCTCT GTCACCCAGG 29340
CTGGAGTGCA GTGATGCGAT CTCAGCTCAC TGCAAGCTCT GCTTCCTGGG TTCACATCAT 29400
TCTCGTGCTT CAGCCTCCCG AGTAGCTGGG ACTACAGGCA CCTGCCACCA CTCCCAGCTA 29460
TTTTTTTTTG TATTTTTAGT GGAGACAGGG TTTCACCGTG TTAGCCAGGA TGGTCTTGAC 29520
CTCATGATCC GTCCGCCTTG GCCTCCCAAA GTGCTGGGAT TACCACCTCT TCTTAATTAC 29580
AAACATAAAC AAAAACTAAC AACTTTCTAG TTTTTTCTTT TTCTTTTTTT TTAAATTACA 29640
AAAGAGATCC ATATTCGTCA GAGAATAATT GGAAAAAAGA GATAAGCAAA ATCAGAAAAA 29700
TAAATTCAGC CTGTAATCAC CCAGAGATAA CAATTATTAA AATTTAGGTA TTCACTTTGT 29760
TATTTCCTTT TATAACAAAA CTTTTTTTTC TTGTGAAATT TAATAGAATA CAATTGAACT 29820
ATTTTTTCCT TTATGGTTAA TGATTCTTGT TTCTTATTTA GGAAATCATT TCCTGAGTCA 29880
TAAAGAATTC TCTCATATTT TCTTCTAAAG CTTTATACAG TTTTGCCCTT CAAATAAGGT 29940
TAATAACCCA CCTAGAATTG ATTTCTGTGT ATGGCATGCA GTAGAGACAA GTTCTACTTT 30000
TTTCTCTCAA ATGAATATTC AGTTGGACCA AGGCTGTCCT TTCTCCACTA CTTTGCAGTT 30060
TCACTTTTTG TTGAAAAATC AATTGTTCAT ACATGGGTAG ATCTCTTTCT GGGCACTCTT 30120
GGAGTCTATT GGTCTGTCTA TAAGTTGAAC AGGATCAGAC AGGCTGTGCT TTGTTTCAGG 30180
TAACAAAGAA CCCCAACATC TCACTGATGA ATACACTAAA GTCATTTTTG TTTTCCATTG 30240
GCAGTTCACT TCTGATGCAG GAGATGCATC AGGGCAATCG CCCTTTGCAA GGTGAAGTGT 30300
-84-
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
CTGCACCATT GGAAGTACTC TCCATCCAGG GGAGAGAGAC TGGAAATGGT CCATGAGGTT 30360
TTCATCGACC CAGAATGAAA GCATCACCCA TCATTCCCTT CTTGTTACAG CCTATTTGTC 30420
AGAACCAGTC AGAGTTCCAC CCACCTGCAA AAGGTTGTGA CGTGCTGTTT GCGATTTGCC 30480
TGGAAGGAGG GAATACCCAG ATACAGGAAA ATGCTAGTGA CGTGCACTTC CATCTAACTA 30540
TCTTTGAATG AAAATGACAG TCTTAATTAC TGCAGTAAGA TAAGCAGACT CTATACCTGG 30600
TAGAGCAAGT CCTCTTACCC CATTTCTTCT TCAAGAAGGT CTTGGCTAGT TTGGAACCTT 30660
GGCAATCCCA TATAAACTTT AGAAAATGCT AGTTAAGTTC TTTP.AAAATC CTGCTGAGAC 30720
TTTTATTGAA TCCATAGCTT CATTTAGAGA GAGCTGACAT TTAAATTAGG GAGTGCTCCA 30780
AGCCACTAAC ATAGAATTTC TCTCTTTTAC TCCAGGTCTT CTTTAATTTC TCTCGAGTGT 30840
TTTGTAATGT TTTGCGCAAA GTTCTTGCAC ATCTTTTGAT AGATTTCCCC CTAGGTTTTG 30900
GATATTTTTA AGATGCTAGT GTAAATGTTA TTGCTATATA TTTTTCATTT TACAAATATA 30960
TGTGTTTAGT ATATAGAAAT TTAATTCATG TTTCTGTATT GACTTTATTG AGTAACCTTA 31020
TGAAACTTTC TTAAATTCTA AAAATTATCC ACAGCTTCCC ATAGATTTTC TATGTAGGTA 31080
ATAACATAAT CCACAAAAAT GACACTTCAA TTTTTTCCTT TCTGTTTCTT ATGTCTTTAT 31140
TTCTCTTTCT TGCATTTCCC ATGTGGGGTC CCTAGACACT GTTGAATAGA TGTCGTGATA 31200
GTGAGCATCC CTGTTCTGTA CACAGCCTCG AAAGGAAAAT TTTCAGAGTT TTGTTTTAAA 31260
CAATCTGGTT GTTATAGGTT TTATTGTAGC AGCTCTTCAC CAGATTACCT GCATGTTTTC 31320
TTTTTTCTAG TTTCTAAGAC TTTTAATCCA TTAATGAGTG GATGTTGAAT TTTAACAAAT 31380
GCTTGTCTCT GCATGTATTG AAATGACTAT ATGACTTTTC CCCAATTGAT CTGTTAAGTT 31440
GGTAAATTAC ACTGATATTC CAAAGTTAAA GCAATTTTTA CACTGGCACC CTCAAGTAAG 31500
CCAAATTTGG ACATGATGTA TTTTTAAATA TATATTGCTG GTGTTGGCCT GTTAATATTT 31560
TATTTAGAAT TGTTGAGCCT ATGTTCAAGA ATAAAATTGG CTTGTGATTT TCCTTCACAT 31620
ACTGTTCATA TTGGGTTTTG GTATCAAGAT TACTCAAGCC TCACAAAATA ACATAGGGAG 31680
TCTCATTTTT TCTATTTTCT GGAAGAGTTT GCATAAGTGT GGCATTATAT CTTCTTTATC 31740
TCATAAAATT TGCTTGAGCC ATCAAATCTT AACATTTTAT GACAGGTTGA TTTTTTATTA 31800
AATCAATGAT TTTAATAGTT ATAGGATTAT TAGGATTTTT TATTTCTTCT TTTGTTAATT 31860
TTAGTAAGTA GTGTTTTCCT AGGAATTTGT CTATTTTATC AAAATTTATA AATTAATTCA 31920
CAGAGTTGTT TATAATATCT TCTAATTATC TTTCTAATGT CTGCAACACA TGTAATAATG 31980
TTATTTTTGC TTATAAATTG ACAATTTATA ATTGCGTATA CTTATGGGGC ACAAAACAAT 32040
GTTATGATTT ATGAAAGCAA TGTGGAATAA TTAAATCTAG CAAATTAATA TATCCATCAC 32100
CTTAAATACT CATCATTTTT TGTGGTGAAA ACATTTGAAA TTCACTTTTT TTCACAATTT 32160
AAAAATGCAC AGTACACTAT TATTATCTAC AGGTGGTTCC TGACTTCTTA TGATGATTTG 32220
AATTATCACT TTTCAACTTT ACAATAATGT GAAAGGAATA TGCATTCAGT ATGCTCTATG 32280
ACTTATGTTG GGATTATGTC TGGATAAACC CATAGTAAGT TGAAAATATC AATGGGCTCA 32340
TCCAGATATA ACTCCATCAT AATTTGAGAA GCAGCTGTAT ATTTATCATG GTGTGCAATA 32400
AATCTCAAAA AAAGACTTAT TCCTCCCGTC TGAGATTTTG TACCCTTTGG CCATCACTCC 32460
TTCATTCCCC TCACCCACAG CCCCTGTAAC TACCATTCTA CTCTCTGCTT CTATGGATTT 32520
GATTGCTTGA GATTCCACAT GTAAGTGAGA ACATGTGGTG TTTGTCTTTC TGTGTCTGGC 32580
TTATTTTACT TAGCATGATG TTCTCCAGTT TCAGTGATGT TGTTGCAAAT GATAGAATTT 32640
CCTTCTGTTT AAAGGCTGAA TTATCCCATT GCATGTATAT ACTACATTTT ATTTATCCAT 32700
TCATCCATTG ATAGACACTT AGGTTGATTC CATAACTTGG CTAGTGTAAA TAGTGCTGCA 32760
GTGAACATGG GAGTAAGGAC ATGTCTTAGA CAATCTGATT TCAATATTTG GATAAACACC 32820
CAGAAGTGGA GTTACTTGGT CATATGATAA TCTAGTTTTA GTTTTTAAAG TAACTTTCAA 32880
ATAGTTTTTC ATGATGGCAG TACTAACATA CACTCCCAAC AGTGTACAAG GGTTCTCCTT 32940
TCTCCACAGA TGTTCTCTTT TTCATTACTG ACATGAGTTA TCTGTGCCTT TCCCATTTTT 33000
TGTCTTCATC TGTCTCAGCA GAGGTTTATC AATTTTATCA TTTAAAAGGT AAAAATTGTT 33060
ACCTTTTAAA TCTTGTCTAT TGTATTTTTT TGTTTCATTA ATTTTTGCTC TGATTTTTGT 33120
ACTTCCTTTT TTCCATATTT TTAGGAGATG ACTTTGCTGT TCTTCTAACT TCTCTTTCTA 33180
GGACTCCTAG AAATATGTTA AGTCTGCTCA TTGTATTTTT CTCACCTTTA TATTTTCCAT 33240
TGTTTTATCT CTTTCTTATT CATTCTGGGT AGTTTCTTCT AATCTACCTT CCAGTTCATT 33300
AATTATCTCT TTACCTGTGT TGAATTTGCT ATTAAACCTA TCTGAATGAC TTTTTCATTT 33360
TTTATTGGGT TTTTAAATGT TAAAATTCTC ATTCCTATTT GGTTCTTCCT CAAATTTGCA 33420
ATGATTTTGT TTCAGCTGAT TGCCAAAACG TTTTTAGTTC AAGTTCATCT CTTTGAGCAT 33480
AGTGAGCACT GTTGTTTTAC AGTCTTTATG TAAATACCTT CTCTTTTATT AATCTTTCCA 33540
CGTTTCTGGT GGAGGGACTG GCTATGAGAG ACAAAAACTT TCTTTCAGGT GCTTTTAGGA 33600
CTTACCCATA TTTCTTTCAT GGTGTCTATT ATTTTATTAT CTCATTATTT AGATACTTTT 33660
-g5-
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
CTCCTCTACT AAACTAATGG TTCAAGGCTT ATCAAAGATA AATCCTCTGT CTTGTTCATC 33720
TCTGTGTCTC TCATGGTATC TAGCAGACTT CCACCCAAGA TATAAAGACA CTATGACTAA 33780
GTGAATGATT TTAGTCTTAC CTACCTGCCT GTTAACTTAC CTACTTGCAT CTCACTTATA 33840
CTTCAACTTT TGGCTTCTTC CTCAACCTCA ACTACCCCAT TCTTCCCATG GCTCACTGTG 33900
CTCACTGGCC TCCATACTGT CCCTTAAATA AGGAAAGCTG CCCTAGCCTC AGGGCCTTTG 33960
CACCTGCTCT GCCTGCTGTT TGGAATGCTC TTCTTCCCAT ATACCCATCT GTTTTAATCC 34020
CTCATCTTTT ATTCCCTCAT CCCATCTCTT CAAATGTGAT TTCTACAGAG GGTTCTCTGA 34080
CCACCTTATC CAATAACCAG CATTCCGTCT CCCCTCTGCC ATTCTCCATC ATCTCACCAT 34140
GCTTTATATC ACATATCACT AAGTGACAGT ATACTATAAA CGTACCCATT TGTTTACTGT 34200
CTGCCTCCCT AACTAATGTA TAAGCTCTCT GAGGGCAGGG ACTCTGTTTT ATTTGTACAC 34260
CACAATTATC TCCAGTGCCT TGAATAGTGT CTGGCATGTA GAAGGAATTC AAGAAATACT 34320
TGTCAAGCTA GGTGCTGTGA TAACTACTTT ATATGAAATT AAGTATTTCT CCTCCAGCAG 34380
CTCTAAAAGT TTAGTATGTT ATTATTGTCT CTGTTTTACT GATGAGTGAA CTGAGGTTCA 34440
GAGAGGTTAT TTAGCATACG TATGAAGACA GAATTAGTGA GTGATTGACC TGAGATTTGA 34500
ACTCAACCTG TGCTGTCTAA AGCTAGCCAG GCAGCCTCAC ATACATGGCA AATGCCTACT 34560
GAGACATGAA CATGCAGGTT GGGATCCCAA ACTGTTGGGA AGCATAAAAG AAAAACACTA 34620
AAGATGTGGG GAGTGTAGGA CTTTTTTTTT TAATAGGCCA GTGGCCCTCT CTGCAACCCT 34680
TTGAATGATC AGCTTGATCA GAGAATCCCC TACCCCTACC CCTGCCTCAG CCAGTTTCTA 34740
TCTGGCTGTG TCATCAGCTG GCTGATCCAA ACAGCAATGT CAACAAAAGA ATGGTGATCA 34800
GGCACGTAAA GCAATGTGTC AGAAAGAAAG AAAAGGCAGC TCAGATGATG CAAGATCATC 34860
CAGATGTCAA GCACTGTGTG GTGGCACACT TGCCCGTTCA TGTTGTTGAT TTTTTAAACA 34920
TTTGTGATAA GAACAAAAAC TTAGTTGCTT CCCTCAGGTC CTCCCTGTAT GGATTAGTGC 34980
AGACATCTGC CGCTTCAGGC TTTCTGATTG GTTCCCACTG GTTTGGGGCA AAACCGGAAA 35040
CTTCTGAGCC AAGTGCAGGG GCAGAAGAGC TCCCAAGAGC TCCTGGGAAA ACTAGGAAGG 35100
ACAATCAAGA AACCACCGGC AGCTCCATTT GCAGGATCTC ATCCCATCAG GGGCTGTCTC 35160
AGGAGGGGGA ATTGGAATAC CATTCACCTG TCCCCTTTGC AGATACACCA ATGTCTCGTT 35220
CAAGAACAAG CAGAAAGGAA ACACCAGATT GCCCAGAGCA CAGGATTAGG ACACACCACA 35280
CAGAGCCAAC TCAGCGTATC ATTGTTTGCA TTGATCATCT GGGGATGAAG CAGGCTCCGT 35340
TCTGGAAGGG GCAACCTGAA TAGAGAAGAG TCTGACATTG GAGTCAAGCA GAACTTGGTT 35400
GGAATTTGGC TCATTGCTGG GTGATCCAGA GACAGTTATT TAATCTGAGA ATCAGATATC 35460
TTGTCTGTTA AATGGAAATT ATAGTAGCCA CTTCACAGGA TTGCTGTAAA GAGTACATAA 35520
AACCAGGTAC CTGCAATGTA TAGTGCTAAG CCTGACACGT AGCAGGGTGT TAGTAAGTGG 35580
TACCTCTGAC TGGGGATGGA AGCCAGAGGA GCTGGACCTT TATTTGACTG GCCAGAAGCC 35640
AGCTCTCTAG TCACCTTCCT GATCCTTCCT TCTTCTGTGT GTACACGGAC AATGTTTTTC 35700
TACATAATGG AACAGTGGCC CTCAAAACTT GTTTTCATAA GAATTATCCA GGTTGCTAGT 35760
TATTAATACT AGTTATCCAG GTTGCTAGTT ATTAATACTA GTTATCTGTG TTGCTAGCTA 35820
AAAATACACT CAGTTCCCAT CCCCAGATTT TTCTATTTCA GTAGGTGGTA GTGGGTTCAG 35880
GAAATCTGTG TTTTTACCAA AGTATCCCCT ACTATAGAAT TAATTTTTGT GTTCCCCCCT 35940
CATTCATATG TTGACATTTA AACCTCCACT GTGATGATAC CAGGTGGCTT TGGGAGGTGA 36000
TTAGGTGATA ACGATGAAGC CCTCATAAAT GTGATTACTG ACCTAATAAA AGAGACCCCA 36060
GAATGCCCCC TTGTCCCTTC TGCCATGTGA GGTCACGGTG AGAAGATGGC ATCTATGAAC 36120
TAGGAAGTGG GCCCTCACCA GACGCTGAAT CTGCTGGTGC CTTGCTCTTG GACTTCCCAG 36180
CCTCTAGAAT TCTGAATAAT AAATTTCCGT TGCTTGTAGC CTAGTCTATG ACATTCTTTT 36240
GTGGCAGCGT AAATGGACTA AGATGTGCAC CCTCATGCCC TTTAGGGAAT TGTGACTTTG 36300
AGAAATGCTG CCCTAGGATT TACAGAATGC TGACAAAGCT TTGTTGACTC AAATGCAAAA 36360
TATTCTTATA AAGACCAAAA TAGAAATGAA TACTCCCTTG AACTCCTTTG GATGTGCACT 36420
TTGCGTAGTT ATAGCACCTT TTCATCATGT GCAAATGAGA CGCAAATGAA TCCTTAGTTT 36480
GACCCAGAAA GAATGTCTTT GCTGGTAGGG ACTACGGGAG AGAGAGAAGA GCCAGAATAC 36540
TGTAGGAAAA TTAACACCGG CCACGAGACA ACTGGTTGCT AGCTCGGTAG CTGTGCAACA 36600
TTGGCATGTT ACTTGAACTT CTAGAAATCT GTTCTTTCTT CTGTAAAATG AATATGGTCT 36660
GGAAAGTAAA GACCAGTCAC CTCCTCTATC AGTTGGAGTC TAATCAGGAA GAAACCTAAG 36720
TGTCTTCAAC AGAGGGAATT TAATGCAGGG AATGGGTCAC ACCAGTGTTA GAAAAGCTGC 36780
AATGCCAAAG AGGGGATAAA GAGATAGCTC AAAGGTTAAT AAGAGCAGAA AGTCACTAGT 36840
ATTCATAGGC TGAAAAGAGA AAGGGAGGAG ATAGTGTTCC CGGAATCCCT GATGGGCTTG 36900
TCTGGAGGGC GCTGGGGCCA TGGAGGAAAT GTAGTAGCTG CTGGAGGCAT GCTCAGGGCA 36960
GAGAGGGAGC AGAGAAATAC CCTGGCTTCT CATTTTCTTT CTCCAGTCCT TGCAGGCACC 37020
-86-
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
TCACTGGCTG AACTCAGGGG AGCATTTCTC CTCTACAGAA CAGAGTCTCC TTGCATACAA 37080
CAAGAGGGTC AAACAGAGGA TGGCTTAATT TTTCCTTCCA TTTCTCACTT CTATGATTCT 37140
CTCCCTTCAG GTTAAGTAAG TGAGGGTAAG TAAGCTGCCC AGTAAGTGAA CAGTTTTCCA 37200
AACAAGCCCA CAGCACCACC TCTATATACA GCAACTCTCT GTTTATCAGC ACTGCATTAA 37260
CCAGGACTCT CTATTAACTG GGACTTCCAG TTCCTTAAAT TTCTTCATGG TTCCTGTGTA 37320
CTCCCAAAGC ATCTTCATCA AACAAACATT AAGTTACGCT TAGAGACCAT TTCTCAATTG 37380
AATATAGATA AAAGATTCTA AGGCCTTGAA AAAAATTAAT ACATGCATAT TAGATATAGC 37440
TATAAAAGCC AGACTATCTG ATTAATTATG TGACTGGTGT TAAACTGTTT GGACAAAGGT 37500
TGGCTAAATT CCCTATGAAT ACTTACTTCC CTACTTCTGT GGACAAGGAA AAATAGACCA 37560
AAGGTTCAGA TAAAAGCTTG ATTCAATGTC ATCTCTTTTC TCACGAATCT TGGTCATGTG 37620
TGGGAAGTGA CCCAGATCTA GAACCTTAGC CTTTGGGACT TAAAAAAAAA ACAAAAAACT 37680
GTTGAGTTGA ATCATTAAGT GTTACTGAGG GACAGGAGAG AGGAGGGTAG CTTTCTTAGT 37740
TCCAAGACAA ATTTTGTTAA CAAAGATCTG TGGGTAGACT TGTGTCTGGG CAAAAGATCA 37800
GAAGATGTGC TGTTCTAGGC CTC2TTGCCC TCAGACCCAT TCCCTATCCT TTCCCCTTCA 37860
CTGTACCCCC TTATCTCCTC TTCTGCTGTC TTCCTCTGGG CCTGATGCTT GAGGATCCAG 37920
AAGTTTCTCA GGCTCCCATG TTCCAGCAAT CCAGGCCTCC TTCCCAGTAA GGGATGAGTA 37980
CAGGGGCCAC ACATAGCCCT GCAAGTTTTG TAATCCAACT TGAAATCCAA TGGCAGAATG 38040
AATGGTTATA TATGGTGTGA CCCAGGACCA CATGCAGTTG TATCACATGC ACTTACAAAA 38100
GAGCCCCATT TCTTGGACTC ATTCCCAGAC TCAATCTCTC TGAGGGTAGG ACCAGGAATT 38160
CGGCCCTTTT CACAATCTTC CCAGGTGATT CTCTACATAG TATAATAACA CAAACTCATG 38220
GAAATATATT TAATGAAAAA TGAATAAAAG AATAAATGAA ATAACAAATG GTGATGGCTG 38280
GCACAATGTG TGTATCCATT CTCCTACTGA GGTGCACTTA CTTTGCTTCC AAATGTTCAT 38340
TTGACAAGTA GTGATGCATT GAATATCCTT GTACATGTGA GCATGCAGTA AAGTTTCCAT 38400
GGGCTTATAT TTGCTGGATT ATGGGCACGT GCATCTTCCT CTTTTCTAGA TATTAACAAA 38460
TCACTCTCCA AAGTATTTAT AACAATCAAC ACTCCTGAAC AAGCAGTGGG TTGGAATTCC 38520
TTCCTCATCA CATCCTGGCC AACAATTATT ATCATCAGAT TTTTTAATTT TGCCAATTTG 38580
AAGGAAATGC AGTGGCTTCT CATGTGTTAG TGTTTCTGAT GATCAGTGAG GTTGAGTGTC 38640
ATTTTTTTTT TTTTTTTTTT TTTTTTTTGA GATGGAGTTT TGCTCTTGTT GCCCAGGCTG 38700
GAGTGCAATG GTGCTATCTT GGCTCACTGC AACCTCCGCC TCCCGGGTTC AAGTGATTCT 38760
CCTGCTTCAG CCTCCCAAGT AGATGGGATT ACAGGCATGC ACCACCATGC CTGGCTAAGT 38820
TTTATATTTT TAGTAGAGAC AGGGTTTCAC CATGTTGGTC AGGCTGGTCT CAAACTCCTG 38880
ACCTCAAGTG ATCTGCCTGC CTCGGCCTCC CAAAGTGCTG GGATTACAGG CACGAGCCAC 38940
TGCACCTGGC CGATTGAGCA TCTTTTTATG TGTTTAATGA TGCTCATTTT TTATTGACTT 39000
CCTTCTGTGC TTTCTTTTTT TTAGCAGTGA ATTTGAGTTG TAAGAATATG TATTTCTTTC 39060
ACTCTGGGAT TCACCTACAT AAAGTAATTT TCACTTGAAT GAAAAAGAAA TCAGTTGTAT 39120
AAACATCTGT TTTTTCTGAA TTTTACTGGT GTAAAAATGG CCACTCAGCC CTGGAAGAAA 39180
CAAAGGCACT TTGCCAACTG AAGTTGCAGA TGGGAAATTT TTAGAAAGGT CCTGTTCAAC 39240
CTCTGGAAGG GGAAGATCAT ATCTGAAAGT CAGGGTAATC CACCCAACCC.AAATGTTTCT 39300
TCTACTATGG GTTCTGAGGA TTCGTCCATG TGCTTCTTCT GCATTGCTGC CATCTGATTT 39360
CCTTTGCTAG GCTCCTCTTG CAACTTGGGC TACAAAGAGG TGCTTCATAG TCCACAGTCT 39420
TTGCCTCACC TTCAGTCTTG AGGTGGTCCC CTAGGAGTTA TTGGTAGTTG CCGCTGGAAG 39480
CCATTCTAAC AAACCTGGCG AAGGCACAAA AGGATAGAAA GCCTTTAGCC AATATGGTGC 39540
CATCAAAAAC AAACAGAGCA CGCTGCCCAG TCCTCTTCTG GTTGCCTTTA CTAATGCATC 39600
AGTCATACTT CTTCTGCACT CGATCTTAGC CAAGAGGTCG AGAAGCCATA GTCATAATTC 39660
TTCTGAAATT AATCTCTTCC TGCCCCACCT CCCCATCATC TGTCTTTGAA TTCCCAGGGC 39720
TAGTACTCAT AAGATTATCT CTTTCTTCTC CTTTATGAGG AGACCCATTC TTTTTCACAA 39780
ACCAGCCACA AAAGCAAGTG TCATTACCCC CTACCGGAAA TACCAGACAG AGAGTTCATC 39840
TGGGGTTAGT TTCTAATCAA GCCTCCTGCC CGGGTTTTTC CTGCTCCTGT CTTGAAGCGA 39900
CCACAGGGGG AGAGCAGTTT CCAAATATGA TCCCTCCTTT CCACTGTCAC TTGTCCAACC 39960
CCGACCACTA TCATTCTTTT ATTTGCTTCT CCCCTGAGCC AGCCAAGAGC CTAGGTCAGT 40020
GACAGGGCAG GCAGAAGAGA GAGGGGCTTC CAGGAAGGAG AGGGAGCAAC CCACAGAAGA 40080
GGCAGCAAGA CAGGAAGGCG GGCAGGGGCT GAAAATCCAA TACATATCTA AGTACATTTT 40140
TCTAGGATGG GCTTCTACAC TCAGCCAAAA CATATATTGC ATATTGTTTG TATTTTTTAG 40200
AGGTTTACAG GTCTCCCTGA AAGTCCCTCT GTGGAATTAT AAACCTCTAA TAAAAAATCC 40260
CAGGGTTAAA GAAAGGAAAA GATGAAGGAG AGGCCCACAC TCTGAAAGGA AAGGGTTCAG 40320
CGACTCCTGG AAGGTTCTGG ATGGTGCTTC CTTGACCAAG TCAGCTGCTT CTTCTACCTG 40380
_$7_
CA 02314677 2000-06-02
WO 99/37809 PG"f/US98/OI260
GTCTCCTTTG TGGTTCAGCT GGGGTGGGGC TTACTAGAAA AAGCTGTGGG AGGTGGTTGC 40440
TCCAACGTAT GGGGGCTGTC TGTAAGTGTA GGTGTTATCT GATGAAAGCT GCCCCGGGTG 40500
AGGGTTTGTA CAGAAAGCTC CTGGTGGTGG GGAGATAATG TCAAGCTTCT CTCTCTCTCC 40560
CAGATCCTGG TTGTATCCTC TGTCCCTCTC CACCCCCACC CACTCACCCA CAGACTTCCA 40620
AGGAACCGGC GCCTGCAGAC ATGCCTCTCT GATGCCCTCC CAGTAACCCC TGGCAGGCAG 40680
CACAGCGCCA AACCTCTTGG CCTTACCCCA CTGGGCCCAT GACCCAGTGG CTGTGCCTCT 40740
GGGTCCTCCC TGTCCTGCAA AGAGAACTGG GCCCTCAGTC AGGTTCTTCT GCTCCAACCC 40800
AGTGGCCACC TGTGCTCTTG GGGAGCTCGG GGGAGGCTGG GAAACTTTCA AAGAGCAGTT 40860
AATCACTAAC TAGCTGGAGA TAAGAGAGAG AGAATGAAAC AATTGAGAAA ATGCCCAACC 40920
CAGAGGTTAG TGCTTCCCTG CCTGCACACG CCAGAACCTG GCCCGCCCAG AGAAACTGGC 40980
GATCAAACTG AGTTTGTTCA CTGGAGAGAG CTGACATACA GTCTCTAAGG GGCTGCAGTA 41040
TCCCAGGCTG AGGTCCAGTG GCAGCCGCTG CCCCTTTCCT CCTAGGGCCC TTTCCTTCAG 41100
CCATGCCTCA GCCCTGAAGA CAAACAGGAG CAGTTTTCAA GGAGCCCTTC CCTTATCTCT 41160
AAGGTCTGGG CCTGGAATTC AGCTTGGCCC ATTTACTATG CCAGCTCTGT GCAGGGTGCA 41220
GAGATCCAAG ATAAATCAGA CAGGGTCTCT GCTGTCAGTG TGCTCAAGGA AAGAGGCTTT 41280
TAGGGGAAAC AAATCTAAAC GACTGCCAGC TGGAACTTCA ACTCTGTAAA GCAGCACCCT 41340
GCCACATCTG CCTGCTGGAA CATTTTCATC TGCTGGGCTC ACGTAGCTGT GCAACAGCTG 41400
GGGCTGGGGT CACATTCTGG GCTAATCTGA TGATTATTTT GGCTAGAGTG AGCTCATCCT 41460
TTTTGTTTTC AGGAGCTGTT CAAGGGTGGT CTGATGGTTT GGATCAAGAC TAGCTGTATC 41520
CCGGAGAAGA ATACGTTGAC TTTTCTGGGG TGGGGTCTGG GGCAGAAAGC AAGAAGGCTG 41580
CCTTACTTCA AGGAAGGCTC TCCTTCCACC TTCTGCCCTC TGAGTGCCTT GTATGCGCAA 41640
GTGACACTAG ACAAAGTGCT TAACACTTAT TACCTGACTT GAATCTCCCA ATGGCCCTGT 41700
AAAGCAGGTA CTCCATTATC ATCACCACCC TTCTTTTTAC AGGCAAGAAA ACCAAGGCAC 41760
AGTCAGTTTA AATAACTGGC TCAAGGCTGC ACGGCCGATA AGTAGCAAAT TTGGACTTCG 41820
AATCTGGGCG CTCTGGCTTC AAAGTGTGCT GTCCATTGTT CAGGTTCTGG TCTGGTACTG 41880
GCAATGTCAG CCACACCTGG AAGCTTGCTA GGACTATAGA ATCCCCAGCT GACCCCAAAC 41940
TCCCCAAATT AGCACCATGA TTTTAACAAG ATCTCAGGTG ATTGGTGTAC ACATTACAGT 42000
TAGAGAAACA CTGCCCTTTT CACATTATAT GGCTCTGTGC TCAGTACAGA TTTAATTTTC 42060
TTTTTTTTTT TTTTATTATA CTTTGAGTTC TGGGGTACAT GCGCAGAACA TGCAGGTTTG 42120
TTACATAGGT ATATATGTGC CATGGTGGCT TGCTGCACCC ATCAACAGGC CCCGGTGTGT 42180
GATATTCCCC TCCCTGTGTC CATGTGTTCT CATTGTTCAA CTCCCACTTA TGAGTGAGAA 42240
TGCGGTGTTT GGTTTTCTGT TCTTGTGTTA GTTTCACAAT CATTCTCAGA TTTAGCTTTC 42300
AAACTATTCA TTCCACCTGC CAACAATTAG CGAGCTCCAG ACATTGTGCC AGGTGAATGA 42360
TGGAGGTGAA GAGACAAATT TCCTTATAGA ACTTGGCCAT GCCCTTCATG CAGGCAGTGT 42420
GTGGAGTGCA AGTCAGGACA CTTGGATCTA AATCCAGTGC TACCACCTGC CGGCTGCGAG 42480
ACTGTGGCTG AGTCATTTCA CCTTCTTGGG TCCCAGGTTC CTAATCGGTA AAACCGGGAG 42540
GCAAGCCAGA GATGTCCGGC CCCAGCAGCA TATTCTATGT GAACAGGATG AGGTGCCCAG 42600
CAGGCAATCA GTGGGGATCT GCTGAATGAG GGAACCAGTA AATGAGTGAG TGAACCGATC 42660
ATCCACCACA AGGAAAGAGC CCTCCATTTC CAAATGAAGA AAAGAAGTAT GCTAGTGGAG 42720
GGGAGACGGG ATTATCTGCT GTGTGTCAGG GAAGAGTAGG GCCTTCCCAA GCTCCCTTAA 42780
TACTAACATT ACACAGGGGT CCTCGCTTGC CCTTCTCAAT GGTCCACTCA GATGATTTCT 42840
CTTGGCGAAT GTCTGCCCCA CATCTGTGTG TCACTCAGCA ACTTTGGCCA CCTATCCAGT 42900
GTGAGATCTC TAGATCACAA GGTGGGGAAA GGGGTGAGGA ATGACCTAGA ATCCTGGCCT 42960
CTGGCCTTAG AGCCTCACTT GTTAAAGGGA AAGGGGCAAA TAAGATCTGA ACATCAAAAA 43020
TTATTTCAGC TTGCCTTCCC TCTCACTTTT CTCTGTCCCC TTCTCCTCTT GTCTTCCCTG 43080
CAAACCACTT TGAGTCTCCT TTGGTTACCA AGATAAAACC AATCCACATT AACTATGGCT 43140
GGTATTTTTT TCGCTTTTAC TCCAAGCCAG TGCATAGTGC ATTTTGCTCA CATTAGATTA 43200
TGGAATCCTT CAAACAACCT GATGATGAGT GGGTGCCATT GATACCCCCA TTTTATAGCT 43260
GGGACAACTG AGGCACAGGG TTGTTAAGCA GCTAACCTGA GGCCACTCGG TCACTTCCTT 43320
GTGGTGGACC CAGGATTTGA ATCCAGGTTT GCTCAACTCC AAAGCCTGTG TACTAAACGA 43380
CACTTCCTGC CTTGATAAGA TAATTGTGGT TGTTACTTGG CCAAATAAAA AGCCTATGGA 43440
GAAGTTGTTT CCAATGAAGC ATATCAGCTT CTAAATCTGG CTGAACATTG GACTCTCCAA 43500
AGGGGCACAA AATACAGCTT TCCGGGCACC ATCTTGAAAT GACTGATTCA GCAAATTGGT 43560
CGTAGGCAGC GAGGCACCTG TAGTTTGGTA AAGCTCCCAG GTGATTCTGA TAATGAGCTT 43620
GTGCAGAACC CATTTACCTA AGGAGAACGC GGGTTCAAAG GGACTGGACG GCTCTTCCTT 43680
ATTTAGAGTA GGAGGCTGTT GGCTTCTGAG AATGAGGGCT AATTAACTTT GGGGAGCTTC 43740
_gg_
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
CTGCAGTGAC CTTTGCCTTC GGGGAAAGTG TGGGGATTGA GATAAGAGAG AGAAATCCTT 43800
GGCGGCTAGG AGGAAGGGTA GGGTGTTTGC TGTCAGGCTC CAGGCTTAGC CCTCGTGGTG 43860
TCCCTCCTGG AGATGGTGTG CACTGAGTGC AGTGGCTGCT GGAGAGTGGG TGGAGAGATG 43920
AAGGTGATAG GGGTGGGATT AATTAAAATA TCAGGCAGTG TGGCTGGGCG CAGTGGTTCA 43980
CACCTGTAAT CCCAGCGCTT TAGGAGGCCA AGGCAGGTGG ATCACCTGAG ATTGGGAATT 44040
CGAGTTTAGC GTGGCCAACA TGACGAAACC CTGTCTCTAC TAAAAAAATG TAAAAATTAG 44100
CTGGGTGTGG TGGTGCACTG CAATCCCAGC TACTCGGGAG GGTGAGGTAT GAGAATTTCT 44160
CAAACCCAGG AGGCAGAGGC TGCAAGTGAG CGGAGATCAC ACCACTGCAC TCCGGCTGGG 44220
ACAACAGAGA GAGACTCTGT CTCAAAGAAA AGAAGCAGTG AACCTTTAGA TTATCCCACT 44280
CTAAAAGTGA GGCAACCTTA GTTTTTCTGG GTCTTTAGAA GCAGAAGTGC CCTTGGGTAT 44340
TTCTAGGCTG AGGGCCCCAC CTAGTTCAAG CCTTCTAAAC ATCCAGTGTT TTGCTATATT 44400
CATTTACCAC TTGTCCTATT AGACTCTTAG GTCTTTTTTT TTAATGACTC ACTTATTAAA 44460
GAATGTGCAT TTATTTACAA GGCAATAATA TCACTACCTT TAATGGAAAA TTAGCAACCC 44520
TGGCTACACC TAGAAGGTAA CTGTTAATAA ATAGGATGAA ACCCAAGGCT GGAATTAACT 44580
TCTCATTGGA TCCTGCAGCC TATGCTCCTT TCACTGAAGG GTGATATCAG CCAACTGAGA 44640
CCTCCTCTAA AGTCTGTGAA GGATTGAATT AAGAGAATTG GAAAGGGCAC ACATTTCTCA 44700
TGATGTGATT CAATATTGAT TAATTCCAGG TTCACCTATT ATCTAAAACC ATGTTACTGA 44760
AAGTGGCTTA TAAATACCGC AGCACCAGAA TGTAAACTCC ACAAGGGCAG AGTTTTTGGT 44820
TTTGTTTTGT CTTTTAAAAA TCTGTTCATT GCTTTATTCC TAGACTCTGG AACAGTACTT 44880
GGAATATAGT AGGTGTTCAC ATATTTATTG ACTACGTGGA CTCTTTTTAG ACTGAGAAGC 44940
GGAATATAAA GTCAGAGGGT CCGACTGGTG ATCGAATGCC TTCGTTCTGT ACTCAAGCCC 45000
ACTCACCCAC TTAGTTTTGA GAACTCTGGT GACCCAACCT ACAGCCTGTC CCACCTTCAA 45060
CTTATTCCCA TTCCTTGGGT GCACGTGTTG CTGTGAGGAT CAGATGAGGT CATGGATGGG 45120
CAGGACTCTG AACTGCGTGC CCTCTGCACA GGGAAACAGC TGGGCCGATT ATAAATTGCA 45180
AAGGGGATGC CTGATGGTGG CCCCATGACT TTTCATATGC TTTGGGCTGT TGTGAGAGAG 45240
AGTGCCCAAA GCCTGATTCT GGAACATTTT CTTTGCTGTC TTCTAAATGA GAACCTGCTT 45300
GCTTCAATTC TCCCACTGAG CAATCATGCT GACATGAGGG AGGCGGAGTC AGACCTTACA 45360
TTGTTGAGAC CAGATTCTGT GTTCTACGAG TATTGGGAAG GGTGATGCAG GCAGGCACCC 45420
ACCATGTTCC CTGTGAGTGC TTATTTTTAA TAAAAACCTT GGTATACTGC TATTAATGAA 45480
AATAATAATA ATAATAATAA TTACTCCTGC TAATAATATA AGGAAACACC CACTGGTCTG 45540
TGACTGAGCC AGCCTTGCCT GAAGGCAGGG GAATGAATTC AATGACCTCT TGACACTGGT 45600
CTCAGCCCTT TGGTTCTATT ACCACCTTGT AAACCTGAGG TTGTTCTGTT TTTATCCCTA 45660
GGGAGTTGTG GTTAGAACCT GCCAGAAATT TCTCACTATG AATCAATCTT CCATTGGTCA 45720
CTGCCCTTTT CAACATGCCT GTCATTCAAG ACTTACGATT TCCTAGGCAT TGACAGAGAG 45780
AAACTGGCCA TGTGGACCAA GGCAGTGGGA TTTACGTGAC ACCCGCCAAG CCGGTGGGGC 45840
TAAGTTCCAT TGCTGAAGTC TGATACCTGT CATCTGCTGT GGGGTGACAT CCACACCATG 45900
TCATTCTCCA TTCGTTCAAT ACATATTTGT GGATTCCTAA AATGCCCCTG CTGCTGTGAT 45960
AGTCCAGCTC AAGAGAGAGG AAGTACATGA GATGTTACCA CACAGTGTGG TATGTGCTGG 46020
AGAGGTGAAG ACTCTGGAGC AGAGAGGCAA CAACTCAGGT GGGGACTGAA TGGTGGCGGG 46080
GTGAGCTCAT CAGGAAAGGC CCCCCCAGGG AAGCTGTGTT TGGGCTGGGG TCTAAGGATG 46140
AGCAGCAGTT AGCCAGGGAA GACAAGGAGT AAATGTACCT AGGCATGTGG GGCAGTCTAT 46200
GCAATAATGT GGGGAGGAAG CAAAGAGAAA GAGAATGGGA GAATGGCCTG CCTGTTTGGG 46260
GAAATGAAAG GAGCCAGTAT GTAAAAATCA GGTGAGAGAC AGCTGGAGAT GAGGCTGCAG 46320
AAATAGGTAG GTGCCAGGTC ACAGAGGGCC TTGTGAATAG TATCATGGAC GCTGGACTTT 46380
ACTCCAAAGG GCATGGGAGC CATCAAAGGG TGTTGAACAA GGAGATGCAC ATTATAGAAA 46440
GGCCAGGAAG GCCTCTGGGA TCTCCTCTTC TCCAAACTGT GGCTCTGGGG ACAGCTCCCT 46500
ATAGTGGTCT TGGGCAGCAC CAAACTGGTG TTTAGGCTCA GCTCACATGC AGCTCACAGC 46560
AAGATGGTGA CAAATGACTC ATCCTCAAAC AACAGAGCAG GCATAGGAAG GAGGCCCCAG 46620
TTAGGATCTT GCTTACCTGG TTTGCTGGTG GCCTATGCAT TTAATTGTAG AACAGAATGC 46680
CAAGCCACTT TTTAACCTTT CTTCTACACC ATGCCCTGCA CCTCCCCTTC TCTCTCTGCT 46740
CTTCTCCCTT CCACCCTCAA ATTTCTAAGC CATGTCCAGG TCTCGTTTTC ACCTGTGCCA 46800
GAGAAGATCT ATCTGACTTT GGCCATGGAA GAGGTATAGC AGGTATCAGT TGGAGAGGGC 46860
TGGAAAAGCT CCCTGGTGCT AGATATGGAC GACCTGAGCT TCCAGTCCTG GCTCTTGCAG 46920
CCACCAGGCA TTTGACATGG GCAGAAGCAC TTTTCCTCAC TGAGCCTCTG TTTCCTCATC 46980
TGTAAAATGG GAATCATGGT GATGGTGTGA TATTTGAACA AGTTTTTTTT TTTTTTTCAA 47040
AATTGCTTTG TAAACTGCAA AGCTCTGAAT AAGTGTTTAT TTGGGATTAT TAGGAACTGC 47100
-89-
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
TTTGCTGGAA CAGTCTACCA GAGGGATGGA AGGAGAGGAA CTGAGAAATC GATTCTTTGA 47160
AATATTTTTA TCATATGAGA TACAAATATG TATCTATATA AATATAGATA TAAATATGAA 47220
CAAATATATC TGTCATAAAA TTTAAP~AAAG GATGAACCTT GCCCCCAATC TCACCCCTAG 47280
CAGCAACTAT TAATTTTTTG TTGTATATCT GCCCAGACAC ATATAAAATA TATATTCAAA 47340
CP~AAAAATAT AATCATATTA TAAACTTTGT TTTTTAGCTT GTTTATTCAC ATTACATGGA 47400
AATCTTTCAG CATCATGGCA TATAGATCTG TCTTTTTAAT ATTACTTCAT GGTCTAGGTG 47460
AACCATAGTT TATTTAGCAT TTTCCTTTTG GTAAACATTA AAGTTAGTTG CAATTTTTCA 47520
TCATATATTT TTTCTGGTCT TTTGTACATA TATCTATGAG AGAAATTCCT AGAAATAGGG 47580
TTGCTGACTC AAAGGATACC AGCATTTTAA ATTTTGGTAG GTACTACCAA ATTGCTCTTC 47640
ATAAAGAGTG TACAAATACA CCCTCCCACA AACAGAGTGC CTGCCTTCCA TGCCTGGACC 47700
AACCACAGGC ATTACCACCT CTGCTGAAGC TTTTTCATGA GACAAGGTCT TGCTCTGTTG 47760
CCCAGGCTGG AGTGCAGTGG CGTGATCTCT GCTCACTTCA ACCTCTGCCT CCCAGGTTCA 47820
AGTGACTGTC ATGTCTCAGC CTCTGGAGTA GCTGGGACTA CAGGTGCGTG CCACCAAACC 4?880
TGGCTAATTT TTGTATTTTT GGTAGAGATG GGGTTTTGCC ATGTTGGCCA GGCTGGTCTC 47940
GAACTCCTGG CCTCAAGTGA TTTGACTGCC TTGGCCTCCC AAAGTGCTGG AATTACAGGC 48000
GTGAGCCACC ATGTCTGGAC TGCTGAGGTT TTTTTTTTTT TTTTGAGACC AAGTTTCACT 48060
CTTGTAGCCC AGGCTGCAGT GCAATGGCAT GATCTTGGCT CACTGCAACC TCCGCCTCCC 48120
AGGTTCAAGG GATTCTCCTG CCTCAGCCTT CCAAGTAGCT GGGATTATAG GCATGTGCCA 48180
CCATGCCCAG CTAATTTTGT ATTTTTAGTA GAGATGGGGT TTCTTCATGT TGGTTAGGCT 48240
GGTCTCGAAC TCCCAACCTC AGGTGATCTG CCCGCCTTGG CCTCTCAAAG TGCTGGGATT 48300
ACAGGCATGA ACCACTGCGC CCAGCCTTGC TGAGGCTTTT AAAACCATGA AACGCTCCTC 48360
CTCCCTCAAA TGGTCATGTG GCCACTGCCT GCTTCATCAC ACTGCTCCTC TGTCTGACAA 48420
GCCTGTTCTT ATATAACACC AGTAGGTAGG GCCATCCGAG ACATGGTTAT CCAATAAAAT 48480
GGTAAGAACC AGCCCTAGGG TATTTGGGAA ACTGGCTGTG AGGGTTCAAT GGAATATTCA 48540
CATTTCCAAA CATAAAATCT AGCAGCAATG GAGAAACGTA CTTTAAGCAG AGAGTTTTGC 48600
GCCTGACACA AGAAATTATT ATTATTGTTG TTATTGAAAG TTCTGACACA CAGATCTCGG 48660
TTGTGTTTGG AAGGAGGATA GTCAGAGAGA GGAGGAAGGT ATGAAGAGGT CGAGGTGTTA 48720
GTTTTAAAAA GTGTGTCTTT GTCATTGTCG AGCTGTGGCT GGTCCCACAA CCTGGTTCTA 48780
TCAGGCCTTT GGTGTTACAA AATGCAAAAC ACCAGGCAAC CAAATAGCGT TTCCATGGAA 48840
GTATCCCATG ACCTCTGGTG CTGTGTACAG GTGAGACAGT GAGCACTCAG AAAGGGATGG 48900
CCTGGGTGGG GAGGGCGAAA GGGGCCTCTC CAGCCTCTGC AACATAAAAC AAGGGGCCAA 48960
TGGAAAGTTC TGGAACTGGA TCACTAAGAA GACAGGCCCC ACTGCTGGCA TGAGTGGGAT 49020
GACCAAAGAA TTAGGAAACT GAGATTGGAG TTGGTCACCA ATTCAACTGG CCCATTTAAA 49080
AATTTTCATA AGCAGGGACA GAGGATCAAG CCAAGAGCAC TAGGGAGATG GTGATGAATG 49140
GAAATTGTGT AAGGTAGATG GCTATGTGCC GGGGAAGGAG GAGAGAGGAT TCAGAATTAT 49200
AGGAATAATA CATGAAATGA CTGACAAAAG TAGCCTTTTA TGTGTGTTAT GTAATTTAAT 49260
CCTCTTAACC TTATAGAGTT AGCACTGTCA GGATCCACAT TAAAAAAAAA AAAGACGAAG 49320
CAGAAGCTCG GAGAAGTCAA ATTACTTGGC CAAGGTCAAG GTCACACAGC CACATGTGGC 49380
AAATCTGGAA TACAAACTTA GGTCTATCTG ACTTTAAACC AAAATGCTGC ATATAGCTTC 49440
GATTTCAGCA CAGCAGGGTT CAACTTGGAG ATAGAGGGTG GTGTTATAGA TTACCAGATA 49500
CGATAGTGGT AGGTTTTCTT CTGTCTTGAT GAAAGATGAG CTATTTTTAT CCTGTTGCAG 49560
GACAACGCGA AGGATCATGA CTTCCATTTT TGAACTGACA TTGTAGATTT GTGTATATTT 49620
GACAGCTCTA CCACATTCCC AACCCTATGC CCTCCTATCA CTCTTTTTGA GAATACTGGG 49680
CTAGTTGGGG GCAGTGTTGG GGGACTTGGG CCTGGGCGTA TGCTGGGAGG AAAGGCAAGG 49740
AGATTATGCA GTGTGGTAGT AGGGACTGGG GGAAGTTTTT TTGTTTTTTG TTTTTGTTTT 49800
TAAAATCCTA GTTGGTCCCC AGTGGAGCCT CCAGACCTCC TCAAAGTCTT TGAGGTTGTG 49860
ATTAATTACC ATATAAACTA GACAGTCCTT GGCCTTGGTG TTGCCATTCC AGCCTGTAAT 49920
TATCTTCATC ACAAGTTGCT GTCTGGCTTT GTTCTGTAGG TAGAGGCTCT TTCGTAGGTC 49980
CCTGCATGTC CCTGAGTCAC TAGCAGGCTC ACTTGTGCTT ATCCAAACTG GTGAATCATT 50040
AGCTGTCACC CTGGAGAGCA GTGCAGTTTG GGAAGGCGTG GGTGCGCCCA TGGAGAGGGT 50100
GATCCCCTCT CTCTTCTTTC CAGGCATGCG TAAGGAGCAG TGGCAGAGAA TTACGGAACA 50160
GAGGATGCTA TCATAGGTGA CCTATGAGCC AGGCACGTAC ATACGTGTCA TCTCAATGAA 50220
AGCTTTACAG CACAGGTTAT ACAAGTAGTA CACAGGGATA AACAGCAAGG TTCTTAGGTG 50280
GGTTTCAGAC CTGGCTCTGT CATTTATCTA GAGGTATGAC CTTGGCCCAA CCTTCCTAAC 50340
TTGTCTATGC CTTGATTTCC TCAACTATAA AATAGAGATA AAAATGGTAA CTGCATCCAA 50400
GAGCTTTTGA GAGGAATTGA TGCAAAGATG CAAGTACAGT GCCTAGCAAA CTGAAGCACT 50460
-90-
CA 02314677 2000-06-02
~WO 99/37809 PCT/US98/01260
CCATGAGGAG TGGTGATGCG GATGCTAATG CTGATGCTGG GACAAACTTA CACCCACTTT 50520
ACAGATGGGA GAACTGAGCC TCAAGTTGTT TAAAGTGGCA TAGCTAGTAA GTGGTAGACT 50580
TGGGATGAAA ACCCCAGTCT GTTTCCAAGT CAGGAACCCT TTCCTCCATA ATGCCGTCTG 50640
CATAAATTAG ACTGTTGGAC TGAAAAACAA TCCGTTCAAA CCACAAGGGT ACATTGGCCC 50700
AGGTTGCTTC TATGTTTTAT CCTCAATCTG AAGCAATATA ATGAGCAATG TAATGAGATT 50760
ATGTTAATAT TTACTCAGGG TTCTGGGAAA CCCAGAAGGG TTTCAGGGTA AACCATCTCC 50820
CAGCAAGCAA GGGCTCGCCC GCTAATTCCC CTTTCTTCCA AGACTGATCA GATTGCCCAG 50880
TGCCTAGTAA AATGCCAGTT TCCTTCTATG TGGAAGGGAG CAAAGCTGTC AGCTCCTGCT 50940
GGGGCACAGG GAGAGGATGT TTCTTGTGGA TAGGTAGGTG GTGCTTAGGG GTAGAGGCTC 51000
TGAGATCAGG CAGACATGGT TTCTATCTGT CCTCCCAGCA GTGTGTCCTT GGGTAAGTTA 51060
CTTAATGTTT CTCAGCTTCA ATGTCCTCAT CTTAAGATGA GGGATTATCA TGCTACTTTG 51120
TGGGGCCTTT GTGAGGATTA AATGAGATCT TAGTATCTGG CACATAGTAA GTGCTTAATA 51180
AAAATAATAA GGCAGAGCTG GGTAGATTGA GGGTTTGGTT TACAGCACTT TGACAGCAAG 51240
TTGCTTGTTT CCTGCCATTC AGAGACCCTG GCCAAACTAT GTCCATTGTG GCCACAAGAC 51300
CATTGGCATG TCAGCCTCCA AAAGAGAGAT GACTGCTCAG CAGGCATTAA CCAGATCAGA 51360
GGTTCTTTGA TTCAGCACAG TGCTCTCTTT TTGCACTGCT CTCAGTCTAC CAACAGTATC 51420
AATCACAGCA ACCATTCATG GTGCAAGGTG ATCTCCCTAA ACTTACATTA TATCTTTAAT 51480
CCTCACAGCA GCCTTGGGGG ATGGTATTAT TTCCATCTGT AGATGAGACA ATAGGGGCTC 51540
AGAGATGGTA GGTAATTGCC CAAGGACACA TAGCTGTTGG AGAAAGTAGT ATTGGAGCAA 51600
AATCTATGTG TGTGCATCTA GATTGACCAA CCTTCCTGGT TTGCCTGGGA ATATGGGGTT 51660
TTCTAGGATG TGGGGCATTC AGTGCTAAAA .TCAGGAAAGT CTAAGATGAG TTGGTTACTC 51720
TATATGCGGC CTCTCCGTGG AGGGTTGGTT GGTGGGCCTG GAAAAGGGAT AGGGATAAGA 51780
GAGAGAAGAG GAGGACGCAG AGAGAATGGC AGAAGCAACT CTGCACTGTT TCTTTCTGCA 51840
AAGATGTCTT TTCAATTCAA CCTGCTTGTT CAGTTCAACA AGCAGGTTTG AATGCCCTCG 51900
TCCTTGGAGG GAGTCACGTC AGGACTTTCC GGGTATTTGA CCGTGATGAA GAGCGCTGTC 51960
TGCCAGGGTT CGCCAGGCTG GGTGTGGAAA AATGGTGCCC CAAACCAGCC CCACATGGCA 52020
GAATAGGAAA CATGCTGTCA TCTTGCTTCA TCTGAATCTC CATTCCATGA GGGCAGGAAT 52080
TGTTTTCTTT TTTACTTCTA TAGCTGAAGC CCCAGTGCCC AGAATATGGC AGAAACTCCA 52140
GAAACATTGG TGGAATGTAG ACTATTGAAT AATTCCAAGT ACAAACCAAT GGTCCAGGGA 52200
GATTTAGATT CTGATGAAGG CAATCTGGGG AAGACTGAAT GGAGAAATAG CATTGGAAAC 52260
GGTTTGGATA CCACGTGTTG GGATCAGGAA GCAGAGGAGC ACAGAATGCT TGTGCAGAAG 52320
TGACATGGGC CCACTGCACC TGGGGTGGAC CCTGTGAGGT AGAGTTGGAG ACCAAGGGCC 52380
TGAGGACTGG ACATGTCGGT GGAGACCAGG TGGTGGAGGA TGGAGAATGC CATGCCCTCA 52440
GGGAGTTTGG ACTGCCTGTC GTTAAGCCAT TTTTTTCTCC AAATTTCAAT CCCCCTCATT 52500
CCATTGTCAC CATATTTGCC ATGTCTGTGT ACCTACCTAT ATTACTTATT TAACACTTTT 52560
CCTTCAAGTG ACTTACTTTT TAACTTTACA TTTGTTTTCA TATCAAACAC ACATGGCTGT 52620
TAAAATAAAA ATTACGATTT GAACTTAGAA TCATCTTGCC TACCACATGA GGTAGGTGTA 52680
CTTCCCTCTG AGGACCACAG CTCCAGCAAC TGGGGAACCG ACAAAGATTT TTGAAAGAAG 52740
AAATGATTCA GTTGCTTTTT GGGAAGACTA CACACGTGAG GAAGTACTGA GTGGAAGATA 52800
TGTGCATAAA ACATTGGCGC AATTGTGACT AACATGGTAA GAAATATTAT CAACGCAAGT 52860
TTGGGGGGCA TTTCAAAGTC TCTCAATGGT CATCCGGATG AAATATGCAA GAACTGCTCT 52920
CTCTCTCTCT CTCTCTGTCT TTTCTCTTCT TGGTCTCACT TTGCCCTCTT TCCCAGCAGC 52980
TCTGCCTTCT CCCCCATGCT TGCTGCCAAC AGCTCTGAGG AATGGGAGGG ATTGCAGTTC 53040
AAAGAGTAAA CAGGTCTACT CTGAGTAAGG CTGTGGGCTG TGCAGTGACC CCCAGTGGGT 53100
CTGGGTGCCT GGTAATGATG CCTGCACTGG CATGATGCTG TGGCTTTCCA GGCTTGTTTT 53160
ACCTGGTTGT GCAAAGAATG TTACCCCCAG CCAAGGCTCA AGTTCACAGA CCATTGGCCC 53220
ATCCCCTAAT AAGCATATTA TTCCCAGCTG GGCATTGAAC TTCCAAGTTA AGGTGACCTG 53280
CCAAACTGGA AAGAAAATGG ATTTGCAAAA ATCAGATGTT TGCCAACAGC ACCATCCCCC 53340
ACCACAACCA TAGACAATTG TGAGATCTAA AGTTGGACTC CCTGAGGTTT TCTGCCCTGG 53400
TGGTTCTGGC AACTCCTGGA GAGCCACAGA CTGATGAATT TGAGGATCAT AAACCTTAAG 53460
AAGACTTTAA AGTATTTTTG GCATTAATTG ACAAAGTCCA CAGCAAGCCA GGCATGCTCT 53520
TCTCTCCCAC TCCCCTTGTC AGAGATGTCT CTTTCCCCTT GCTCTTCTTA CCCCATTCTT 53580
TCCAGCATAA CCAAGCTTAA TAGCTTCCAT GTTTCCACTG TAAGGAAGTG AGCCGAGTGT 53640
GGTTGGTCTG TTTCACAGCA GGGCTATCCT CACACGAAAA GTTTTCAGAT GCATTGACTA 53700
TGCAGATTTT TGGCTCAGTT TGCAGAAGAC TTCCTTATTT CAGTTTTACT GTACACCCAC 53760
CTACATAATA CTTTTTGGTT CTTAGAATTT CAGAGCTATT AACCTCTAAA CTTAAATCAA 53820
-91-
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
AATTCTCATC AAACTTTCCT AGGGCCTTGT CATAAAAGAA ACTAAGTCTC AAAATAGGAC 53880
TTTTTGGCCT AATTTCCTTG TCCAGGAAGA CAGATTGACT AATTCCAAAC CCTGGACTCA 53940
CATGTGATTG CTAAGAATAG GGGTGGGGGA GAGAGAGGGG ACAAAAGTCA TAGACATGCC 54000
ATGACACATA TTGGGAGATT TCATCTGAAT TTCCCCTGAG TATGAAATTA TTCAGAAATA 54060
ATTCCAAGGG CTTCTTTTCT GACATTCCAC CAGTGTGCAG GTGCATATGT TTTGAATGAA 54120
CTGAATGGAT AATTTATTTA TACAAATGAG TCTTTTTGAA TAGTTGCAAT GGATGTGCTG 54180
TCAACTCTCC AATATCACTT CCAGGGGGTT TGTAGATGCA TTCTTTCCAT GGGCATCAGC 54240
AGGTCTGGAT CTCCTGCTTT CTATCTGAAA GGCACTGGTC TGGATCTCCT GCTTTCTATC 54300
TGAAAGGCAT TATGGGCAGC AGTCTGGTTG ATTTTATACT ACTTTGATAC ACTCTCAATT 54360
GCATACTAAG AATGAGGATG GAGAAACTGA TAGTGACCCT CACCCCAATT AGGTTTCACT 54420
ACTGCCCTTG ACCTTCATAT TTAATGCCTT TGTTATCACA GCAACTCTTT GCTCTATTTC 54480
TGGATCCAAA TGCCTAAGGA TCTCCCTGGG GTGTTAAGCT TGCTCAGTGC TATTAAACCT 54540
GGTGGGTGGC AGAGTGACCT TTGTATCACA AGAGCCTCAT GACTTCCCAG GCAAGACCAA 54600
GTCACAACTT TTCCAATGGA TTTCCCCTCG ATTCTTATTC TGAGCATTTA GCTTTTTAAA 54660
TATTTGGCTC TGAAGGGCAG GGGCTAAACA TTGTTCTGTA AGATCCAAAC CTGCTTGTAT 54720
ATTTTATACT TTTGTTTTTT CATTTCAACT TTCCGATCTC GCTCTTCTGA GAAACATTCA 5478 0
CATTTCCAAT TGCATTCCAG AACTGAGCTT GACTTTCCAT GTCCATGTAA GATCTTGTAA 54840
TTCAAATTTC AGCCAGCTGC TAAGCTTCTC TTTTCTGGAG GGGATTGTGG TTAAGAGATC 54900
TTGTTTTGCA ATGACGCTGT CTGGTCTGAG CTCCAGCTAC TTGTCTTATT TACTGTGCAA 54960
CCTTGGCCAT GTTAACTTAT CAGGCTCATG AGGCTCGGTT TTCTCATCTA TAAAGTGAGA 55020
AAATGAATAG TACCTATCTG ATGGAGTTTT TCTAAGGCTT AAATGAAGTA ATGCAAATTA 55080
AATTCTTAGT CTAGTCACTG GGAAAAGATG AAAACTTAAC GAATATGAAT AGTCACTATT 55140
CTGTTTCTTT TTTTCTATGC CATTCCGGCT TCACCTCCTT CTCTTACTTT TTCCCTTTCT 55200
TTTTTCATTT GTTTTCTTTT TTTTTTTTTT CTTTTTTGAG ATGGAGTTTC GCTCTTGTTG 55260
CCCAGACTGG AGTACAATGG CATGATCTTG GCTCGCTGCA ACCTCCACCT CCCAGGTTCA 55320
AGCGATTCTC CTGCCTCAGC CTCTCGAGTA CCTGGGATTA CAGGTGCCCA CCACCATGCC 55380
TGGCTAATTT TTGTATTTTT AGTAGAGATG GGGTTTCACC ATGTTGGCCA GGCTGGTCTT 55440
GAACTTCTGA CCTCAGGTGA TCCACCCACC TCAGCCTCCC AAAGTGCTGG GATAACAGGA 55500
TGCCGCACCA CTGTGCCTGG CCTCTTCTAC TTTTTCTTAG AAACATGGAG GGTTAGTTCT 55560
CTGGCCACTC ATATGAAACT TCATTCCCTG CTAAGGTGGA AGTATTGGAG TTCAAGCTCT 55620
ACACTTAGTG GAGGGAGTAA ATAAGCATTT CCAGAGAGCC CACCAAGTGC CATGCAATCT 55680
CCTAATGCTT TGTACTATTT CTCATTTAAC CCCCCAAACA GCTCACTGAG TATGTTAATA 55740
TCCCCAATAA ACAGATAGGG AAACTGAGAC CTAAAGTTTG AGCAAATATG GCAAAGTTTT 55800
CCTAGGCTGT CTGGCTTTAA AAACAATGTC CTTTCACCGC ATCAGGCTGC TTCTGAGGAG 55860
CAGAGCCACC TTGCTTTTGT AAGTCTGTTG GAATAGGCTC TGAGATGCCA CACGTTATCC 55920
CAAATAATTA GGCATCTGGA TGGAGATTTT ATACATTTTC TACTTGGACC TGAGTTTGCT 55980
GTCTCTCATG GTTCCTGGGT GAAAGAGGCC AGGCCCTGAG ACCTTTACCC AAGGTTGGCT 56040
CTACCAAAAT ATCTTCTTGA GTGAGTTCTC TGGTTGATCA TCTGTGGAAC AATGTGGGAG 56100
CCTACTAAAT ATGAATGGAA AATGAGGAAT GCAAAATGGA TGGTTTTCTC CACTATCACC 56160
TCACCCTTGG AGGTGTTTGC TGATTTGGTA GATGTGTGGA GGAACTCAGG AGTCTGAATT 56220
TGTAAAGGTA ATTTGGATGC TTCATTAGCT TAGAAAGGAC ACAGCAGGGA GAACTATATA 56280
GCAGAGAAGG CTGGATGCCT ATGAGGGTAG GGAAGGGAAA ACAAGGGGGT GGGGCTGTAG 56340
CTGCCCTACC TCCGGTCCAT ATATGGCTGC ATTTCTTTAA TCTCTTTTAC TTTTGGGATT 56400
CCATGGTAGT AAACAAAGAG TTCTTATGTT AAAACAATTG CTATCTAATT GTACAGCATG 56460
GTGAATATAG TCAATAACAA TGTATCATGT ATTTGCAAAT TGCTAAGAGA GTAGATTGTG 56520
TTTTCACCAC ACACAAAAAT GGCAAGTATG TGAGGTAATG CCCATGTTAA TTAGCTCAAT 56580
TTAGCCACTC CACAATGTGT GTGTGTGTGT GTGTGTGTGT GTATATATAT ATATATGTTT 56640
ATGTATATAT ACACACACAC ATATATATGA CATGTCAGAA TGTCATGTTT TATTCCATAA 56700
ATATATACAA ATTTTATTTG TCAATATAAA AAGAATAATA CCTGGAAAAA Cp~AAAAAAAA 56760
ATCCTAAGTG CTATACTTAT AAAGAAATCT TCCTCATACA AAAAAGAAGA AATTCTGGCC 56820
ACAGGAAGGT TGCCTGAAAA TGGCCACCTT TTTCATGATT TTCCCTCCCT TTCTGAGACT 56880
GAGAAATGAG CCTTCTTGAA GACCCTGATG GAAATACTGT GAAGAAACTA AGACAGTTGG 56940
ATTCAAGAAC CAAAATGCTT ATCGTAGCAG TGAGGTTGGC TTGAAGTCAG GGAACAGTGT 57000
AAAGCTATTT GTGGGGAAAG ATAAGGCCAG AAAGAGATTG ATAAAATACA GGCGAGACCA 57060
AAGGAACAGG GCAGGGGCAA ATTAGTTTAG GCAAGAATAG AGGCGTCTTG ATATTAATTA 57120
AAATATGGAG GAGGAGTCCA GAAAATTCAT CCTTGGTGCT TGGGTAAGTT TAGCAACATG 57180
-92-
CA 02314677 2000-06-02
WO 99/37809 PC'T/US98/01260
TTCAGATGCC TGAGTTTTGT GTGTGTATGT GTGTGGGCAT GCACGTGTGT GTGTACACAG 57240
TGGGTCATTC TTCTCAGGAA GAGTGAGCCA CTCTCCCCTC CTCCAGCACC AAAGTGGCCC 57300
CCACCTTGGC ACGCCAGTGG CACATGCCAT TGGGCCAGGA TTTGCTCAGA ATGCAGGCAC 57360
ACAGACATAA TGTCAGGAGG CATTGCTGGT GTGTGTCACA TCAACCTGTT AGAACAACTG 57420
TCAACGTGTG ACCTCCCAAA CAGAACTCAG GTGCCCCCTT CAGAGACCGT AAAGCTTGTC 57480
CTTAGAGGAT AATGAAGATC CCCAGGAACC TCATCTAATC CAAAACCAAA AGATTTGGGA 57540
AATGTGACCT TTAGAGGGGA GTAGCATTAA GAAGCAAAAT GATACTTATT AATTCTGTTG 57600
CTTATTTGAC TGTAACCAGT ATAATAAATG ATCATATTCT GCTCGATTTA ATTCCCCCTC 57660
CCCATAAGTT TCACAAGACC AGAAGGAGTT TCTTCTTCCC ATTGGTCTTA CATTAATATT 57720
CTTGTACGGC TTTCACTAAA TAGATGCCGT GTTCTGCCCT GGAGGTAACA CCACGTCATT 57780
AGGAGGAGAT GATAGACAGA AATATATACA AACACACACT TGCTTTCAAA AATAAATATA 57840
GGCCCTCTAG TTAAAAGGTA TTGTGTAAAG TGTGTGAGCA TCCTCTTTCT TGCAAAGCAA 57900
GCACACAGCT TCCATTAATC TTGTAGCCAC AGCCTGTGTT GGTGTTAAGA CTCAGATTCC 57960
TTAACGCTTG ATACTTGGCT TAAAGAGATT CTTTGTCCTG GCCTTGATTT GGGAATTAAG 58020
ATCCCTAGGG TTTTTGGTTT TACAGTATGG ATCTTCTAGG AGACAACCCG ACTGACCTCC 58080
GGGTCTCCAG GCCACCACAC ACAACCTGGT TTGCTTTGCT CTGTTCCCCT TTTCCTCTGT 58140
GGGGACCAGC ACAGGACTCA ACTCAAGGGC TCTGTGTCTG TGCACAGGTT GGAGAGGGTG 58200
ATAGGGCCTT GACCTGTAGG GACAACCAGG AAGATTTCTA TGCAGAGTAA TTGGGTTTCT 58260
AGAGTTTGTT TCAGTTGATT TGAGGGCAAG CTGCTTGGCC TCTCTCTCTT GATTCTTCCC 58320
ATCCACAGAA TAAAGACAAT CAGCTTTGTT TATCACTCTG TTCATTTTGC TATGTCTTTA 58380
TCAGCCCCCC AGAGAATTCA GGAGCACAGA ACAAGTGCTG GAGGTCTCTC TTGCCAGAGT 58440
CCTCCTTGAG AACTTACAAT GTGTCCATAT TAAGGATCTG CTGTGTTTGA TGATTTTGTG 58500
ATTACACTTT AAACTTCTTA TCCATAAAGG ACATACTTGA TATATCTGAG ACTTGTAGTA 58560
GAAGGCCTTG AGACATCCAT CTCATCCCAT CATTATCTAT CTATCATCTA TCTATCTATC 58620
TATCTATCTA TCTATCTATC TATCTATCTA TCTATCATCT ATCTATCTAT CGCCAGTACT 58680
GTCTTGTTGA AGTTGGCAGT AGGGTGAAAG ACCTCAAACT CCAAAGGACT TTCCGTATGG 58740
ATGCAATATA CCTGCAATTC TAGCTTTTTT GTGTTTTTTT TTTTAGGTTG GGGGTGAGGG 58800
GTATTGTTTT CATTTTTGTT TTTCTTCTGG AAGGTTCAAC TAAGACCCAA GTAAAAAGAA 58860
GAATCAATAC TTAATAAGTA CCCAGCAAGT AGCAGGCACA CTTTTAGGTA CTTTATTTAC 58920
AAAAAAACCT CCACAAATAA AGTGGCTTGT GAGTATGAGG TGACATCTTT CCCTCCCCTC 58980
CCACCATCAC TACCCCAATA TGACTCGTCT CAATAGCCCT CCAATCTAAA ATGGACTAAA 59040
TACAAGTGGA TAAAGAAATG GAGATTTAAC CAGAATTCTT CAGCTATAAA TTACAGGGCC 59100
TATAATTAAA GGTGATTGGG ACTGGGTCAG AGAGCCACAT CACTTTTGTG GTTGCATTTG 59160
AAGTTCACTA TCTCTTGACC ACACAACCCT AGCCCTTCTA CTCCCACCCT GCTGTCTCAG 59220
GTTAATCTCA GGCAATGGTG TAAAGAAGGC CAAGTTTGTT TCCCTGGAGT CCCACGGGCT 59280
CTAGCAATAA TGCTTCCCTT TTCTCATGAG TGCCCCGCCA CCCACCCCCC TTCACCATCA 59340
CTACACACAA ATGCCCTGCA GTGGGTGGAA TGTAGTTACT TCAGGTTGTG CCTGATTTGT 59400
CTCTCAAGCA AAACTCCAGC AGGCCATTCC CTCAGGGCCC TGCTCTCAGA TCTGGAACTG 59460
ATAGACTAAT TGGGGCTAAT GTGATAATGG GAAATAATGA AATTTGTTGT TTTTATCAGT 59520
GTGTATATGG GGCGGGGTTT ACATTTGCAT TTTCACAGGG CCCTTGGCAA GTTCACAGGG 59580
TTGAACAGTT GGGAAGGGTG GGAATGTCTG GGGCAGGTTA GGGAGGCAGA GGGATTTATT 59640
AGAACTCCCC TAAACTGCAC TGACCAAAGC CTCAAGCCCT TCTTCAAGAC CTGCCCAGCT 59700
TCCAAGACCT TCCCAAGTCC ACCCTTGTTT TCCCACTGAG TCTTTTACAC TTTCAGAAAC 59760
CTCTGAATTT GTGTAGAAAC TAGAAAi~IAAT AAGTAAGAAA AGACTAATAC TACTGCACAC 59820
TCACTGTTCC CCCTTAATAT AATAACCAGT TTTTATTCTA TTCAGTCAGC CTTTGACCAT 59880
AAGCAGACCT TTTTTTTTTC TTTTTAACAC AAGTAACTTC TTGGTTTTGA TCACAAAATC 59940
TTTATCTCTG CCAAATCTCA ACTTCCCTTC CCTCTCCCAC AAAAGGGAGG CCCGTTGAGT 60000
CAAAGAAATC TGCTTAGACA CTTTGCTCAT GCCAGGCCAG~TGTCCTGGAA GGTTCAACAG 60060
AGAGAGTTAA TGGTTGGGGG ATGGTATTTT TCTTTGCTAG GAGCAGTCAT TCACCCGTAT 60120
GGGAGAAGGT ACATTTGTGA CCCAGTGAAG CAGGTACAGG TAACTCCCCA TATGTCCCTT 60180
GGCCCAAGGG AATAGAGGTT GCCTGGGTAT TTGAATCCGT AGATCCTCCC TAATATTCCA 60240
CCTTCTTCTT GTCCAAACTG TGCTTTTTTA TTTCCAGTTT CAGCATTTTG GTCTTCTCAT 60300
CTCTAACTCT TATAGGGAGT GTCAATAAAC CTTTTAAAAA AGATCATGTA AGTGTCAAGA 60360
GGAAGTGAAG AACCTAGATA ATCCACCAAC CGGATAATCA GCTCTTGCAT ATTTGAGAGT 60420
TGACTGCTTG ACCTAAGCAT CTCCTCATAA GGTACCCTCC CTCCCAGGAC CTTCCCTTTC 60480
AAACCTCTCA AGGCTCTTAC CTGGGGCCAG GGGAGATAGG CTTTTCAAAG TCCATTGAAT 60540
-93-
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
TGCCAAGAGT CTCTGTCAAG AAGGCAGTCA TGGTGCCTGG AGAGGGAACT TGCTGGGAGC 60600
CCCTTCAGAG CCTGGTACTT ATAGAGCTAG GGAAAAGATC TTGATGCCAA AGCAGGGTGG 60660
ACTAAATACA GACTAATAAA TGAGACAGGT GCTCAAGAGG GCCCCTCCAT ACCATCATCT 60720
CCTCCAGATT TGGACTTCTA CTCACTTTGC TTTTACATTC CCTCTTCCCG ATGGTGTCTT 60780
TGGTGAGCAG GGTGCTTTTC ACCTGAAACA GCCTCTGAGC TGAAAAGAAC AGTCACCACC 60840
AAATCAATTC CTCATCCATT AACAGGTTGT CTCTCTGTTC TTGAGACACA GGCATTACCT 60900
GGTTAGACCT GTTTTGTTTG AACACTAACG TGTGAGTTGG CCAAATGCAA ATGAGCCAAT 60960
GTTTGTAATC CTTTATTTTA TTTTTTTAAA GGGCTGGGTA GCCAATCAGA AGAGGGGGAA 61020
GTGACTTAGG GAATTCCCGG TTGGTGGCTT ATTGCTTAAC ATCCTACAAA ATGATTTAAA 61080
ATTATTGTTA TATGCATTTA TCTTCACTCT GATGAGGGCT CAGACTTGAT AACGCCCGTG 61140
GTGCCCCATC CCTATAGGAG CTGGTGAGAT TGCAGCCTGC TGCCTCCCCT CCATCAGCCA 61200
CAGCTATTGG ATTTCCCACC CAGAATCTTT AGGTAAATGA GGTAAGTCCT GATTTTTAAA 61260
ACTTCTTTTG AATCTGGAAT CCAAACACTT GAGTGGAAAG AGAAGCCTGC TTTAAACTGG 61320
ACAGATGAAA CTAGAACAGA CTCTTGGAGA CGGCTGGCAG GAAGTGAAGC TCACCTTACC 61380
TGGGCTTACC TCACTGGGTC AAATCAGAAT TTTATTTTGG AGGGCAGGTT GGCTACTTTG 61440
GATATTATCT GTGAATTTCC TGCATTGTCT GGACTTCTAA TCTCTGTGAA TTTAAAAGCC 61500
CCCTCGTTTC CCTATGCCTG GGTGGCAAAA CCATTCCCCT GGGTTGAATT CTTCTGGAAC 61560
AAATAGGCAG CTAGAGATAG GTGGCTCTGA TATAGCTCAG AGAAGAAGTG GTTGGCTAAG 61620
TAGCTGTTAG GGCTCAGAGT ACACGGTCTC GCTTTCTAGA GATGTCTTCT GCTGGTAATT 61680
TTTCTGACTT ATGAGCTACA TGGAAAGGCC AATTTGTTTT TAATATGTTC CAGGACTGGA 61740
AAATGGCTAG AAATAGGCAA GAACATACAC AATCACACTG GAAAAAGTGG CCAGGCAGCC 61800
AAGGCAGGCA GAGGTATTGG GGAGAGCTGA ATATCTACAA AAACAAAAAT TCAGAAAAAA 61860
CAAAAATCAA TTTTGGCAAA GGGCTTCACT GTATAACAAG GGGACAAACT AACCCTTTGT 61920
TTACAAACTA ACCCTTTGTT TACTCCATTT TGTCCAGAAA ATACAACAAT CAGTTTTGGC 61980
AAAGGGCTTC ACTGTGTAAC AAGGGGACAA ACTAACCCTT TGTTTACTCC ATTTTGGGAG 62040
ACTATGATCA GACAGGCAGT TGTGACTCAG CAGCAACAAA TGCCTTCTGA GACAGGGATT 62100
CTTTTGATTT TGCTTGGACA TTGTGGAGAA GTGTTAGCCC CAATGTGGAC TGATCTGGGA 62160
ACAGTGGGAA ATTAACTTCT TGTTGGCAAA TATCAGGCTG AGGTGAGAAA GCGACATTTT 62220
CACCGTCCAT CTTTGCTGAT TTACCGTGCT CCCAGGATGG TGGGAGTGTG TGTTTTTAAG 62280
ATGGAGAGTG TATGCTTCTG GGTTCAAGTT CACAGGTGTC TCTGCTGGTT ATCTGCACTC 62340
ACCTTGGTAA CAGGGAGAAA GTGAGTGAAT GGATTCCAAG AACTTACTGA TGGAAGTCTA 62400
ATTCAGGAGT TGGTTCTGCA GCCATGGAGG TAAAGATGTG TTGATAGTCT TTCAATGTGT 62460
AAAAGGGCAA TTAGAGATTC TGTGTGACTG TGTGTTAATT CCACTGGGGT CAGGGGAAAA 62520
ATTTATTTCT AACAGAAAAG AAGAAGATAC GTTATTAGGA AGAATTTCAT GGCTAGGAGA 62580
TACTATCAGA AAAGGCTCTT AAGAGATTTT AAGGATGACT TTAATAGCCG CATTTGAAGT 62640
TTGCAGAGGA TCCACTTTTC CTCTTTTTGT GACCTAAAAT TCTGGGATGA TGAAATAACT 62700
CACCAATTCC ATCTTCTTAT AATATGGAGT CATGTAGACA ACACCATTTT CACACAAATG 62760
GCTAATGGTA TTTAAAAACC ATGATGGAAT GTGAATTGGG AGTCATTTGG AGGTCTGTAG 62820
TTGAACTTGA AAAAATAATA AATGTAATGG AGACAATACT TCACCGTGTT TCCAAAATAT 62880
TTTACAGAGG CATTTTAAAT GAAAGTCACT TTGAGGGAAC AGCTGTGCTG TAAGTTCTCT 62940
TACATGACTG CGCAAGATGG TAGCCTTCAT CAAGACCTCT CAAGGTAGTG TGGGTAGGGT 63000
GACGTGTTTG ATTCAGGCCT CGTTTGTTAT GAAAAGGCTC AAATTCAATT GTATTTGTTA 63060
TTTTTTTGGT TAAAAAGCAC CTATTTGTTC AATTCAAACA ATCCTTTTTG GTTTTTTTTT 63120
GAGATGAAGT CTCCGTCGCC CAGCCTGGAG TGCAGTGGCA TGATCTTGGC TGACTGCAAC 63180
CTCCGCCTCC CAGGTTCAAG TGATTCTCCC AACTCAGCCC CCCGAGTAGC TGGGATTACA 63240
TGTGCTCGCC ACTATGCCCA GTTAAGTTTT GTATTTTTAG TAGAGACGGG GTTTTGCCAT 63300
GTCAGCCAGG CTGGTTTTGA ACTCCTGACC TCAGGTGATC CACCTGCCTC AGCCTCCCAA 63360
AGTGCTGGGA TTATAGGCTT CAGCCACCGT GCCCAGCCAT ATTGTTTTCA TTTTTAATCT 63420
ATTAGTCTAT CGTGATCTCC CAGTGGAAGT ATCTTTGGCC TTTGTGGACG TCAGGAAAGC 63480
CCTACATTCC CACTCGCGAT TCCATGTTTA TGGGTACCCT AAATGCTCCC ATTAATTGAC 63540
CAACTTTACC CTGATCTTCT TTCAATATCT TTCTGACTCC TTGAAGGTAT GAGACAAAAT 63600
GGAAACTGAG AGGTTAAAAG GTTTACTAGG TTGCATTCAA TTAGCGAATT GGAAACTGGA 63660
AGGAGCTCCT ATCGGGTCTC AGGTCAGAAC GTGAGTGCTT TTGGCCAAAG TTCACTTCTG 63720
AGGAAGTAGA ATTTCGCTTT CTGGAATCTT GCGATATTTT ATTTCCTCTA TATCTTTCCC 63780
ATGCCCCCGA CCCACCCAAT CTCCACAAAT TTGGGGATTT GAGCACTGGG TTGTGATCGT 63840
TAGACCATCT TGCTTTTCTG AAAGCCCAGG GCAAGACCCC TGCTTCATGT CACAGTATCA 63900
-94-
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
AACACAGACA TAGAAGCTTG TACAAATTAT TGAGAAGTTA TTGTCTTTTC TCCCTTCCTC 63960
CATATGGAGT CATCTCTATG CCCTTTCATA CAGATGTGAT TTACGAAGAC CTCTGGGTTA 64020
GGGGTGGGGT GGTGAGCAAG AATCCCGTGG CAGAATCTGC TAACACACTT GAGAAGCAAT 64080
GTTGTGGTTT TAAGGAACTC AATCTAAAGC TTGAACCTGA TTTTCAGGGA TACCATTTTG 64140
CTGCCGTTTC AGCCCATTTC TCTTGTTAAG ATCGCTCTCT GGTAGAGTTG ACGTGACACT 64200
CATTTCTGTT GTGGGTGGGG CCCTGGTTGG GAGGCATTGG CTCCACTGCA GCCTGGGTGT 64260
CTAGAGACCA CATTCTCACC CTGCCTTTGT TACTGGGAAA CCGAACGCGG CGCTGTGGCT 64320
TTCAGCTTGG GTAAGCCGGG TCTGCGGCGG GGATTGCCAT CTGAAGACAG AGGCAGGAGG 64380
GCAGCCACAC CTTGCCCAGG TTCTCTTAAA TCTCTTGCTC TATAACTGAA AGGAGGGCAT 64440
AGATAATTAA CTTTATTTGA CATTTTTCAT ATCTAATTTT TAAGAATATG ATTTTAAAAT 64500
AATAGATTTG TTCTAAAGAG CAAACAATCT TGCTGTTATT AAAAACGTGT TTACTTAAAT 64560
TGAACGGGGT TTCAAAGGGC CAAGCTACTA AGCTGTGCAG GAAACAAACA GTGCAGTGAG 64620
GAGAATGGCT CCTCACCACA GCTATTCTTA GGGTGGGACA TAGTTTCAAG CCAAATGACA 64680
TTGATGTCCG GAAACCAGGA TGTGCTGAAG TAGAAATTTC CAGGGATCCC TCAGAGTTAT 64740
TTGCTAAAAT GTTTATTATT CTTCAGAGGG GGGTGGAAAT ATTTCTTTAA GAGTCTTCCT 64800
TGAAGAATTT TGAACTCCAG CTTTGGAGTG ATGGGAGCAC AGTGCAGGGA AGGCGGGATG 64860
TGAGGTGGTG TGCTGGACGG CAGTCTAGGG ACCTGGTCTA GCACTGGCAG AGCTGTGTGT 64920
CCCAGAGCAC ACATTCCCCT TTGCCAGGCT TTAGTTTCCT CCTCTAGGCA AAAGGGTTTG 64980
AACCTGACCA TCTTTAAGAT CCATTTTAAC CCTCAGATTC TGTGGCTGTG GTGATTGGGG 65040
GTGGTGGGAG TACCTGGGGG TCAGCAGGAT AAGCACGAAT CTGTGAGAGC TGAGAACAGG 65100
TGGGAGAAGC CTTCTAAGGA TGAGGCAGGA AAGATTAGCA AGAGCCCTTA AATGGATTCT 65160
TTAGGGCCTT CAGAATTTTG GCTAAAGGCT ATACTAGTGG AGGTACTAAG ACCTGACACC 65220
TGGAGCCTTT ATTAAGGATG TTAGAATCCA CTCCCATGAC AACATCCCAG CTTTGCCAAT 65280
TTGCCTCATG TGTCTCAAGC TGGTGGGAAT GTAGAAGTGG ATGAAACAGA CTGTTTTGTG 65340
ATGGCAGGGA ACAGCCTATG CACAGGGGCA GGTGCTCTAC TGGTGTCTTC TATAAAACGC 65400
CAAAGCAGCC CGCCAGAAAA TGGACATTTA GGCACTCGTG GTGTCTACTG AGTTTGTATG 65460
GTACTGATGA GCTTGCTTGA CTGATTATCC ATGACTTACT GAGTAGATCG AACGTATGTG 65520
GACTCACTTC TCCTAGAGGA AGACCCTGTG GCTGCCCCAG CCACTGAGCA GCCTAACCTG 65580
GAGACCCTGA TGTGCCCAGA AAGCGTCAAC CTTGTATCTG GAGAAACCAG AACTTGCAAC 65640
AGGGCCAAGC AGGGTGGCCC ATTTAAAGAG GCTCCTAGGG TTTTAATTGA CCTTGTTTTA 65700
AAAGAGACAC CCTGTAAAAT ACTCCTATGA AAACTTATTT CACAAGCACC TAACCGCATT 65760
CTGTCTTTGG TTTGTTTTAC GGGGCCGGGC CCCTTGTTCT GGTCAATTGG TCTGCATTAT 65820
CTCTCCTCCT CCAATCTCAC CACACACCCT GGCCTCTGGG AGGCTTCCTC CCTTCTTTTT 65880
TTTTGTTTGT TTTGTTTTTT TAGCATCTTA GTTGTTACTA GGGGTACTTG CCTACTTATT 65940
TAAAATATGG CCAGTATAGG TGCATACAAA ATGTGCTTTC TGATTAAAAC AAAGCCAAAA 66000
ATAAAAAGAA ACCAAAATGC CTATTATAGT AGTTGGATTT TTAGACTAAC AGACCACCTC 66060
ATTAACCCTG TCATTTTACC ATAACAACTT ATTTTTATCT TTGTATGACC TTGTCTCAAT 66120
GTCCTTTTTC TTTGATGTTG TTGCAATTAT GAACATCAAA TTTCATAGCT GCTTTTCCAC 66180
CCCACTTTCT ATCACAGAAG CACAATAAAT AATCTTGGGG GCTGGGCTCT TGTTGGCCCA 66240
ACTGTGGCTT CAAAACATTT CAGTTGCCTG TCCAGCCCTT TCTTAGCCTG ATACAACATC 66300
CCCCAAAAGT CTGTTGAGCT TTTCCTGGAA TAAGAAGAGG GTCTTCTACT TTTTGAATAG 66360
AGCAATGGAG ATTGGAGAAT ATGGTCATCT TGTGGAGGTT ATTCCAGGCT TCTTCTTAGG 66420
AACCTTAAAA AAAATCTCCT CAGTAGGGCT GATGATATAT TCTGGACAAT AAGGTGAGCA 66480
GAGTCTGAAA GATGAGAGCA ATTTTCAATC TTGTCATGAT TTCATCTAGT CAGCCTCATT 66540
TCATCTAGTC ATGAGGCTGA CTAATGATAA GACTTGCTTT GTCTTTGCAG TGTACTCTAG 66600
ATTTGACTCT AAATTCAGCC TCTGTCTTGA TCATGCCCAC TTAGAAAATT AGAGTGCAGC 66660
TAGCTCACCT TTTAGTCATC TTAATTCCAC TAGGCAGAAG GCTGTGGGTC AAGGAATGTT 66720
GATGGAGTAA AATTTGACTG CATGTGTATC TGAAGGGGTA GGAGGCTAAG AGATTTTATG 66780
GCTTGGAAGC TGCTGAGATG TGGTGTAAAG AACACTGGAC TTAGAGTCCA GACACCTGAG 66840
TTTAAGCTGG ACTCTACCAC TGGGTAGTTG AATGACTTTG AGTGAGTTAT ATAAGCTCTA 66900
GCATCTAAGT TTTCTCATCT GGAAAATGGA GTTAATAACA TCTACTGCAT TGGGCTGTTG 66960
TAAAGATTAA ATTAACAAAG AATGTGAAAG CACCTGAACA AAAGCTTGTG AGTAAATAAT 67020
TAGTAATTTG TGGAATGAAC ATCAAGGGAA GTCTTCAATT TGGGTGTTTT CAGTGAGTTT 67080
CTGTTGGGTC AGAGTGAATG GATATTAAAT TCTGGGATTT TGGTTTGTGT GTGTGTGTGT 67140
GTGTGTGTGT GTGTGTGTGT GGCGATCAAC ATTGGTTCTT CACTGTGACC TTAGGAAAGA 67200
AATGCAATAG GGTTTTTATT GGGAAGGTGG GTAGCAGGGA GATGCATGAA CCATATTAAG 67260
-95-
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
GGGGGACCTC CAAATTGAAC CTTGTTTTGA GTCAACTGCA AACCACAACC RAGGAGGTCC 67320
TGGGAGACCT GGGGTGACTT GGGGTGATTG GGTATGCAGC ACATTCCTGT TCTTGTGTCC 67380
TGATGCCTGG CAAGTAGGGA CCTGCAGAAA ATACTGATTC TCCTCCAGGC AGTTCACATG 67440
ACTAGCTTTT AGGAGTGAGT ATACCGTTGC CCACCCCTAA AATTCTTGAT CATGTCTCCA 67500
GATGTCTACT GACCACTGAT GCTGAGGTCA TGAATCTTGG GCATTCTAGA GGCTTTGGGA 67560
AAAAAAATTC TACTTACTTC TTTTGCCCAG ACACTCTGGG GTCTACCTCT TGGTAAATTA 67620
TTCAAATGAG GTTTCTGGTC ATGCAAATGT GGTTTCTAGA GCCTATTTGA ATTGAACAAG 67680
TAGTTCTTAT TATTAGTAAA ACAGCAAGGA TCCCTAACTT GGGGTCCAAG GGTAAATTCA 67740
GGGTTTCTGT GAACTTGGAT GTAAAAAAAA ATTGTGTTTA TTTTCAATAA TCTCTAACTA 67800
GAATTTAACA TTTTCTTTCA ATATGAATGT AGGCAAAACT CCATGGTAGT ATTAGCTGCA 67860
ATTGTGACTA TCACCAGGAT AAATCACATT TTCATGTCTT ATTACACCTA TTACATATAT 67920
CACAAAAAGT GGGTATTTGA TATCAAGTTA GATCTGCACT AGGTAGATAT TCTTATTTAA 67980
TGTATTAACA AGGAAGCACA TATATTGTTA TCAGGTTGGT GCAAAAGTAA TTGTGGTTCT 68040
TGCCATTAAA AATAATTACA AAAACAGCCA GTCTGGCCAA CATGGCGAAA CCCCATCTCT 68100
ACTAAAAATA CAGGTGTGGT AGCACACACC TGTAATCCCA GCTACTTGGG AGGCTGAGGC 68160
AGGAGAATCA TTTGAACCTG GGAAGCAGAG GCTGCAGTGA GCCAAGATCA CACCACTGCA 68220
CTCTAGCCTG AGCAACAGAG TGAGACTCTG TCTCAAAAAA ATTAAAAAAT p~AAAAAAAAC 68280
TCTGTAATTA CTTTTGCACC AACATAATAT GATATCACAC ATTTATTTTA AAAAGTATTT 68340
TGACATTGTC TTTTAATATA AATTTTTTTA AATCTTATAA TATTTTAATT TGTCATGTAA 68400
AAATATTATT TTGAGAAGAG GCCTGTAGGC CTCACTAGAT TACAAAACAG ATCCATCGTA 68460
CAGATGAAAG GTTAAGAACA CCTCATTTAC AGCATTCTCT CACACACGAC TAACGAAATG 68520
ACTTCTGAAC AGCGCCAGTT GATAGATGTT CTCTGCCAAA AGGGGAATAT GATCTTCCCA 68580
TATGTTCCTG CCTATGGGTA GCCTTGGAGT TGTGAAGGGA CTTTGGCATA ATGAAGATGA 68640
TAATAAGAAT GATAATGGTA ATTTGTTGAG TGCCTGCTGT AAGCCAGGTG GTTACAGTCC 68700
TGTTCAATGT CATGTTTAGT TTAATCCTCC CAATGACCTC AGGAGGTAGT GCATGGAACA 68760
AAGACAGAAG AGATCCCCTG CCCACCCACG GTAATGAAAC ATGGGTACAG GTGAAGGCAA 68820
AAGTGGGGAC TGACCCTTTG GAGATGGCTG ATGTCACGAG TGTGCAACCT GTGCAGTTCC 68880
ACGGGGCCCC ATGCTTAGAA AGGTTCCATG TTTGGTTTAA GGCTCTGCTG TTGCCATCTT 68940
AAAATTCTTC GTAAGTTTTG AACAAAGGGC CCTGCATGTT CCTTTTACAC TGAGCTCTGC 69000
AAATGATGTA GCTGGTCCTG CCTCTGGTTA TGGTGAAATG GAATGTATGA CAACTCCTGA 69060
GACTGGGAGT CTGGGAAGCT GCTGCGGAGA GCCCTCTCCT CATTTTCATC AGGCTCAGCT 69120
ACGCAACCTC TGGTGGAAAG CTATGGCCTG TTGAGGAGGG AGGATGTCGT TTTTGAGTTA 69180
GTGAGTTTTC CAGTTTTGTT TGAGCTCCAA AGCTTTCCTC CAAACAACTG GAAAGATGGC 69240
TGAATAATTG GCTGAAAGGG ATTTAATCCC TTGAAAAACC TTTCTGGTAG GGAGTTGCTG 69300
GCAATACTGG TGGGTTTTTC ATGATTTTAT TTTACAGAGG GCTTGCTACG TAAACCAGTG 69360
AGCCAGGAGA AACAGAATAA AGTCTGTTCT GGAAGGAAAA ATGAGACCTG GTGTGCCACG 69420
AGTCTAGTGT TCTCATAGGA AGGCTCTAAA AACAAACTCA GCTTTCCTGC TATTGAATGA 69480
TTATCTCTAT AAAAGGAAAC TTTACTTCTT CTAAAGGAGA GGTCGTCTAA TTTGTGAGAA 69540
AATTCAGATG TTATTTGCTT CTTAAGCTGC AAGGATGCTA ATGAAATAAT TCTCATGAAG 69600
TTCTGTTGGT GTTTTAGGGC TAAGTTTTTA TAGACTGTTC CAAAATTCAA AACAGGGATG 69660
TGGACGTAGT GATGGTGGAA GAGGGGAAGA CTTTTCCTCG ATTTCTTTGC CTGAGGGATG 69720
GAATTCAGGC TCCCCCAATA ACATATTCAT GGTCTTTCTC TGGTCAGTCA GTGATGTTCA 69780
TAACACAAGC AAGCCTGTCA TCAGGACCAA TCTGTGATGG CTGAGACATC AGGTGCTCTT 69840
CCAAAAGAGC CATAATTCAC CCTTCATTTC CCAAGGTTTT TTTTTTCTTG CTGTTATTAC 69900
TGCTCTTTTA TCATGGTTAA TAAGTCTGAG GTGGCTTCAG ACAGCCAGTC CTAACCCCTG 69960
AGTCAATCTG GGGCCTCTAA CAGGAAGCCA GACTGAAGTT CTGATAGATG GGTTTGAGTG 70020
GCTGTGAACT GTGTTTCTGT AGCATCCAGA CTGATTTGCA CTGAAAGGGA GCTTCCATAT 70080
TAGGGTACAA GGATGATCAA TATGTCTCCT GTTTATATTT GGTGGAAAAA GTTGTGGGAA 70140
TCGTGCTTAA AGGATCTCAA CTTTGAAATT AAAAGTATAA CGTCCTAACA GACATCCTCC 70200
TTCTCTTTAG AAACACAAGG ATCCATTTTC AAGTAATTTC AAAAGAACTA TGTTGCTTTC 70260
CCCACCCCTT CCCAAGTACA CTTATTATAA TATATCCAGT CCATTTGCTA GCTTTGTGTC 70320
TTTAGAAAAG TTGCTTAACC TCTCTCTGTA AAATGGTGCT TATATTAGTA CTAACATTCA 70380
GGGTTATTGT GAGGATTAAA TGAGGTAATT CATGTAATGA CTAGTTCTAT TTCTAGCACA 70440
ATTTAAACCC TCAACAAATA TGAACTATTA TCACTGTCAT AGTTTTTGTT GTTGTTTTCT 70500
AATTATATAA TCTTCAAGAT TCTGAGATGG GGGCTGTTGC TCTTTCCTTG ACTTGAACAT 70560
CTTGGTCTTT TCCTAGGAGG AAACTTGACT CTTGAAATGG TCAAATCCAT TGTCCTAGTT 70620
-96-
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
CATCCTGACC CCTCCCTGGC TCCAATCCCC ACCCCTTACC GTCCTCCACC CTTCCTACAT 70680
TCCTGCACAG TTGGTCTTAT TTATTTTTCA GTCAACTAAG GGTGTTGTTA AATCTTTTAT 70740
TTTTCTGCTG CCCGATTTGG TTCTAAGCAC TCCACTCCCT ACGCTGCTCA TAACAAGAAT 70800
GCCTGGGAAC GCTCAGTCAG CCATATCCCT CCCCTGTCGG AACACCCAGT TCTTAATGCT 70860
CCTGGAGAGG CAACATTTCT GAGGCCCCAC TGCCATAAGC CCCCCTCCCC CATGAAGCCA 70920
GTGGTCTGGT AGTAATGAAC CCCCAACGGC CCGGAGAAAA CTGGGGCAAG GTGTTTGTCT 70980
GGGGAAATGT TGCATGTTGC CTTGACTGTG CTTTCTTCTA CAAAGCTTAA AAAGAGATAT 71040
TATATTATTT TATTTTTATT TTTATTTTTG AGATGGAGTC TTACTTGGTT GCCCAGGCTG 71100
GGTGTGCAGT GGCACAGTCA TGGCTCACTG CAACCTCCAC CTCCTGGGTT CAAGTGATTC 71160
TCCTGCCTCA GCCTCCCAAG TAGCTGGGAC TACAGGCACA TGCCACCATG CCTGGCTAAT 71220
TTGTATATTT TTAGTAGAGA CGGGGTTTCA CCATATTAAC TACATTGGTC TTGAACTCCT 71280
GACCTCAAGT GATATGCCCG CCTCGGACTC CCAAAGTGCT GGGATTAAAA GCATGAGCCA 71340
CTGCGCCCGG CCAAAAAGAG ATATTCAAAA GCTCCCTCTG ACTGTGTGTG CTGAAGGCTG 71400
AGTGCTGATG CCATTGCTTA ATTAATGTTG TTCATGATCT CCATTTGGGC GATTTGTTTA 71460
GCTCCTTGTG GCCCTTTTTG GACTTAGCTT ATCATGTGAC ATTGACAAAT TAATGAGAAG 71520
TGAGCATGTG ATGATGCTTG GATTAGGACA GAAATCACAT CTAGGACATC TCAGGCCCTT 71580
TCCACCTGGG ACCTGAGACC TCAAATCTCT TGGCAGGAGA TGAGTGGGTC TACACAGCCC 71640
GATTTTGAGG TAGGTGTGGC TAGCCTCATT TATGCGATGG GAAAACTGTG GTCCGGGAAC 71700
CAGGGGTTTT CAAATTATGC TTTTTGCCCA GGGCTGGATG TAGGATGTCT GGGGGAGAGG 71760
CTTGACTGAG ATCTGGGTAC ACTGAGCCTC CACTTTAGGA GGTAACCTAG AGACTACACC 71820
TACTCCCTAA ACTGTATTGA CTTTTGGAAG TCAACCATTT AGAAGAGTGT GGTTTTGGTT 71880
TCGATCGTAT CCCAGCAGTC TTTTCTCTGC CCTTGTTAAT CTGATTCATG ATCTGAACCT 71940
GGGCTGGCTG GAGGCTGGCC ATGTCACTTT GCAGACCATG GACACCCCTG AGTGCCCTCA 72000
CAGAACCAGC CAATGGAAAA GTACAACGTC TTCTGGCTTC TCAGCCTTGC CATCTCCCTC 72060
TGGCCTATTT GATACCCCCT TTTATATTGA GGGAGTGAAA ATGTAGCATC CAAACTGAAA 72120
ACGCAGGTTT TTCTTTGGTT TTTATAGGAA AAACAAATTG GCATGAACAC TCAGTCAAAC 72180
CAGCTCAGGC TGTTTGGGCA GATGCCTTTC TTTGCTTTTT TCTGTTTATT TTCCTACAAA 72240
TCAATGCTTA ACTGCGTTGT TATCGGAGCA GAGCAACAGG TGCAAAAAAA TAACTCTGCT 72300
GCCAACTCAA ATGAAAAGGT AGGGCTTATA CCCTCTGGGA GGTATTCAGA AGATAACAGA 72360
AGCCCCTGCC AGCAACTGAA TTAACAGCTC TGTTTACGGT GGGTTTTATG TTAACAACCT 72420
GCTCCTGACC CTCCTACACA TAAACACACC ATTGTCTCAG AGAGAGACAT TCAGCCATCC 72480
AGACAACCCA CTGCTTTATT CTGCCCTGAG TGGAGATTGG TTTTGGCTCA GGCTGCTTTG 72540
TGAAACTCAG AAGCATTATC CTCTCTGCCA ACTCCACGTC CTAGTCAGAG TTTTCTGTGA 72600
AGGCAAGGGC ATGGGGTTGC CGGAGAGAAG AGGATTGGTC CTGCTTTTAA GCCTAGCTGA 72660
AATTCTTTTC AAGGTTGGTC ATTCTCAAAT GCCAGAGAGG GTTGCCCGGC TCTCTCTGCT 72720
CTTGCCCCAT TCCATTCACA ACAGGAGGTG GGGAATGAGC TCAGATGACT TTGGAAGGAG 72780
CCACTATTAT TTTGGAAGCC GTGTCCTTGT GAATAGTCCA TCAGGGTAGG GCAGCGTCTA 72840
TGTTTTGTTA ACTATTGTAT CGCCAGCACC TAGCAAAGTG CCCAGCATCT AGTAGACACT 72900
TGGTAAATAT GTATGAATTA CAGAGGGT 72928
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5427 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
ATTTATCTTC ACTCTGATGA GGGCTCAGAC TTGATAACGC CCGTGGTGCC CCATCCCTAT 60
AGGAGCTGGT GAGATTGCAG CCTGCTGCCT CCCCTCCATC AGCCACAGCT ATTGGATTTC 120
CCACCCAGAA TCTTTAGGTA AATGAGATCA TGATTCTGGA AGGAGGTGGT GTAATGAATC 180
TCAACCCCGG CAACAACCTC CTTCACCAGC CGCCAGCCTG GACAGACAGC TACTCCACGT 240
-97-
CA 02314677 2000-06-02
WO 99/37809 PC'T/US98/01260
GCAATGTTTCCAGTGGGTTTTTTGGAGGCCAGTGGCATGA 300
AATTCATCCT
C31GTACTGGA
CCAAGTACCAGGTGTGGGAGTGGCTCCAGCACCTCCTGGACACCAACCAG CTGGATGCCA360
ATTGTATCCCTTTCCAAGAGTTCGACATCAACGGCGAGCACCTCTGCAGC ATGAGTTTGC420
AGGAGTTCACCCGGGCGGCAGGGACGGCGGGGCAGCTCCTCTACAGCAAC TTGCAGCATC480
TGAAGTGGAACGGCCAGTGCAGTAGTGACCTGTTCCAGTCCACACACAAT GTCATTGTCA540
AGACTGAACAAACTGAGCCTTCCATCATGAACACCTGGAAAGACGAGAAC TATTTATATG600
ACACCAACTATGGTAGCACAGTAGATTTGTTGGACAGCAAAACTTTCTGC CGGGCTCAGA660
TCTCCATGACAACCACCAGTCACCTTCCTGTTGCAGAGTCACCTGATATG AAAAAGGAGC720
AAGACCCCCCTGCCAAGTGCCACACCAAAAAGCACAACCCGAGAGGGACT CACTTATGGG780
AATTCATCCGCGACATCCTCTTGAACCGAGACAAGAACCCAGGATTAATA AAATGGGAAG840
ACCGATCTGAGGGCGTCTTCAGGTTCTTGAAATCAGAGGCAGTGGCTCAG CTATGGGGTA900
AAAAGAAGAACAACAGCAGCATGACCTATGAAAAGCTCAGCCGAGCTATG AGATATTACT960
ACAAAAGAGAAATACTGGAGCGTGTGGATGGACGAAGACTGGTATATAAA TTTGGGAAGA1020
ATGCCCGAGGATGGAGAGAAAATGAAAACTGAAGCTGCCAATACTTTGGA CACAAACCAA1080
AACACACACCAAATAATCAGAAACAAAGAACTCCTGGACGTAAATATTTC AAAGACTACT1140
TTTCTCTGATATTTATGTACCATGAGGGGAAAAAGAAACTACTTCTAACG GGAAGAAGAA1200
ACACTACAGTCGATTAAAAAAATTATTTTGTTACTTCGAAGTATGTCCTA TATGGGGAAA1260
AAACGTACACAGTTTTCTGTGAAATATGATGCTGTATGTGGTTGTGATTT TTTTTCACCT1320
CTATTGTGAATTCTTTTTCACTGCAAGAGTAACAGGATTTGTAGCCTTGT GCTTCTTGCT1380
AAGAGAAAGAAAAACAAAATCAGAGGGCATTAAATGTTTTGTATGTGACA TGATTTAGAA1440
AAAGGTGATGCATCCTCCTCACATAAGCATCCATATGGCTTCGTCAAGGG AGGTGAACAT1500
TGTTGCTGAGTTAAATTCCAGGGTCTCAGATGGTTAGGACAAAGTGGATG GATGCCGGGA1560
AGTTTAACCTGAGCCTTAGGATCCAATGAGTGGAGAATGGGGACTTCCAA AACCCAAGGT1620
TGGCTATAATCTCTGCATAACCACATGACTTGGAATGCTTAAATCAGCAA GAAGAATAAT1680
GGTGGGGTCTTTATACTCATTCAGGAATGGTTTATCTGATGCCAGGGCTG TCTTCCTTTC1740
TCCCCTTTGGATGGTTGGTGAAATACTTTAATTGCCCTGTCTGCTCACTT CTAGCTATTT1800
AAGAGAGAACCCAGCTTGGTTCTTTTTTGCTCCAAGTGCTTAAAAATAAG TTGGAAAAAG1860
GAGACGGTGGTGTGGAAATGGCTGAAGAGTTTGCTCTTGTATCCCTATAG TCCAAGGTTT1920
CTCAATCTGCACAATTGACATTTTTGGCCGGAGTGTTCTTTGTGGTGAGG GCTTTCCTGT1980
GCATTGTAAGATGTTCAGCAGTATCCACTCATGGTCTCTAACCACTTGAC ACCAGAAACC2040
CCCCAGCTGTGATAACGCAAAATGTCTCTAGACATCACCAAATGTTCCCT GGGGGTGGCA2100
AATTTGCCCTTGATTGAGAACCACCAGTTTAGCTAGTCAATATGAGGATG GTGGTTTATT2160
CTCAGAAGAAAAAGATATGTAAGGTCTTTTAGCTCCTTAGAGTGAAGCAA AAGCAAGACT2220
TCAACCTCAACCTATCTTTATGTTTTAAATATTAGGGACAATAAGTTGAA ATAGCTAGAG2280
GAGCTTCTTTTCAGAACCCCAGATGAGAGCCAATGTCAGATAAAGTAAGC ATAGCAATGT2340
AGCAGGAACTACAATAGAAGACATTTTCACTGGAATTACAAAGCAGAATT AAAATTATAT2400
TGTAGAAGGAAACACCAAGAAAAGAATTTCCAGGGAAAATCCTCTTTGCA GGTATTAATT2460
CTTATAATTTTTTGTCTTTTGGATTATCTGTTTACTGTCTCATCTGAACT GATCCCAGGT2520
GAACGGTTTATTGCCTAGATTTGTACTCAGAGGAATTTTTTTTGTTTTGT TTTGTCTTTT2580
AAGAAAGGAAAGAAAGGATGAAAAAAATAAACAGAAAACTCAGCTCAGGC ACAATTGTCA2640
CCAAGGAGTTAAAAGCTTCTTCTTCAATAGAGGAATTGTTCTGGGGGTCC TGGAGACTTA2700
CCATTGAGCCATGCAATCTGGGAAGCACAGGAATAAGTAGACACTTTGAA AATGGATTTG2760
AATGTTCTCATCCCTTTTGCAGCTTTTCTTTTTGGCTCTCTCATGTCCTT GGCTTGCTCC2820
TCTATTCTACCTCTCTTTCTCCAGCAATAATATGCAAATGAAGACATGTA TCCATAAGAA2880
GGAGTGCTCTTCATCAACTAATAGAGCACCTACCACAGTGTCATACCTGG TAGAGGTGAG2940
CAATTCATATTCAAAGGTTGCAAAGTGTTTGTAATATATTCATGAGGCTG GAAGTAAGAA3000
GAATTAAAAATTTGTCCTAATTACAATGAGAACCATTCTAGGTAGTGATC TTGGAGCACA3060
CATGAATAACTTTCTGAAGGTGCAACCAAATCCATTTTTATTTCTGCCTG GCTTGGTCAC3120
CTCTGTAAAGGTTTAACTTAGTGTTGTCAAGTAACAGTTACTGAAAGAGC TGAGAAAAAG3180
AACAATGAACAGCAACGATCTTGACTGTGCAACTCAGACATTCCTGCAGA AAAGACATAT3240
GTTGCTTTACAAGAAGGCCAAAGAACTATGGGGCCTTCCCAGCATTTGAC TGTTCATTGC3300
ATAGAATGAATTAAATATCCAGTTACTTGAATGGGTATAACGCATGAATA TTTGTGTGTC3360
TGTGTGTGTGTCTGAGTTGTGTGATTTTATTAGGGGCATCTGCCAATTCT CTCACTGTGG3420
TTCCTTCTCTGACTTTGCCTGTTCATCATCTAAGGAGGCTAGATCCTTCG CTGACTTCAC3480
CATTCCTCAAACCTGTAAGTTTCTCACTTCTTCCAAATTGGCTTTGGCTC TTTCTTCAAC3540
CTTTCCATTC.AAGAGCAATCTTTGCTAAGGAGTAAGTGAATGTGAAGAGT ACCAACTACA3600
-98-
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
ACAATTCTACAGATAATTAG TGGATTGTGTTGTTTGTTGAGAGTGAAGGTTTCTTGGCAT3660
CTGGTGCCTGATTAAGGCTT GAGTATTAAGTTCTCAGCATATCTCTCTATTGTCTTGACT3720
TGAGTTTGCTGCATTTTCTA TGTGCTGTTCGTGACTTGGAGAACTTAAAGTAATCGAGCT3780
ATGCCAACTTGGGGTGGTAA CAGAGTACTTCCCACCACAGTGTTGAAAGGGAGAGCAAAG3840
TCTTATGGATAAACCCTCCT TTCTTTTGGGGACACATGGCTCTCACTTGAGAAGCTCACC3900
TGTGCTGAATGTCCACATGG TCACTAAACATGTTATCCTTAAACCCCCCGTATGCCTGAG3960
TTGAAAGGGCTCTCTCTTAT TAGGTTTTCATGGGAACATGAGGCAGCAAATCTATTGCTA4020
AGACTTTACCAGGCTCAAAT CATCTGAGGCTGATAGATATTTGACTTGGTAAGACTTAAG4080
TAAGGCTCTGGCTCCCAGGG GCATAAGCAACAGTTTCTTGAATGTGCCATCTGAGAAGGG4140
AGACCCAGGTTATGAGTTTT CCTTTGAACACATTGGTCTTTTCTCAAAGTTCCTGCCTTG4200
CTAGACTGTTAGCTCTTTGA GGACAGGGACTATGTCTTATCAATCACTATTATTTTCCTG4260
TTACCTAGCATGGGACAAGT ACACAACACATATTTGTTCAATGAATGAATGAATGTCTTC4320
TAAAAGACTCCTCTGATTGG GAGACCATATCTATAATTGGGATGTGAATCATTTCTTCAG4380
TGGAATAAGAGCACAACGGC ACAACCTTCAAGGACATATTATCTACTATGAACATTTTAC4440
TGTGAGACTCTTTATTTTGC CTTCTACTTGCGCTGAAATGAAACCAAAACAGGCCGTTGG4500
GTTCCACAAGTCAATATATG TTGGATGAGGATTCTGTTGCCTTATTGGGAACTGTGAGAC4560
TTATCTGGTATGAGAAGCCA GTAATAAACCTTTGACCTGTTTTAACCAATGAAGATTATG4620
AATATGTTAATATGATGTAA ATTGCTATTTAAGTGTAAAGCAGTTCTAAGTTTTAGTATT4680
TGGGGGATTGGTTTTTATTA TTTTTTTCCTTTTTGAAAAATACTGAGGGATCTTTTGATA4740
AAGTTAGTAATGCATGTTAG ATTTTAGTTTTGCAAGCATGTTGTTTTTCAAATATATCAA4800
GTATAGAAAAAGGTAAAACA GTTAAGAAGGAAGGCAATTATATTATTCTTCTGTAGTTAA4860
GCAAACACTTGTTGAGTGCC TGCTATGTGCACGGCATGGGCCCATATGTGTGAGGAGCTT4920
GTCTAATTATGTAGGAAGCA ATAGATCTCGGTAGTTACGTATTGGGCAGATACTTACTGT4980
ATGAATGAAAGAACATCACA GTAATCACAATATCAGAGCTGAATTATCCTCAGTGTAGCT5040
TCTTGGAATTCAGTTTCTGG AACTAGAGATAGAGCATTTATTAAAAAAAACTCCTGTTGA5100
GACTGTGTCTTATGAACCTC TGAAACGTACAAGCCTTCACAAGTTTAACTAAATTGGGAT5160
TAATCTTTCTGTAGTTATCT GCATAATTCTTGTTTTTCTTTCCATCTGGCTCCTGGGTTG5220
ACAATTTGTGGAAACAACTC TATTGCTACTATTTAAAAAAAATCAGAAATCTTTCCCTTT5280
AAGCTATGTTAAATTCAAAC TATTCCTGCTATTCCTGTTTTGTCAAAGAATTATATTTTT5340
CAAAATATGTTTATTTGTTT GATGGGTCCCAGGAAACACTAATAAAAACCACAGAGACCA5400
GCCTGGAAAA~~AAAA AAAAAAA 5427
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5510 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
ATCGCTCTCTGGTAGAGTTGACGTGACACTCATTTCTGTTGTGGGTGGGGCCCTGGTTGG 60
GAGGCATTGGCTCCACTGCAGCCTGGGTGTCTAGAGACCACATTCTCACCCTGGCTTTGT 120
TACTGGGAAACCGAACGCGGCGCTGTGGCTTTCAGCTTGGGTAAGCCGGGTCTGCGGCGG 180
GGATTGCCATCTGAAGACAGAGGCAGGAGGGCAGCCACACCTTGCCCAGATCATGATTCT 240
GGAAGGAGGTGGTGTAATGAATCTCAACCCCGGCAACAACCTCCTTCACCAGCCGCCAGC 300
CTGGACAGACAGCTACTCCACGTGCAATGTTTCCAGTGGGTTTTTTGGAGGCCAGTGGCA 360
TGAAATTCATCCTCAGTACTGGACCAAGTACCAGGTGTGGGAGTGGCTCCAGCACCTCCT 420
GGACACCAACCAGCTGGATGCCAATTGTATCCCTTTCCAAGAGTTCGACATCAACGGCGA 480
GCACCTCTGCAGCATGAGTTTGCAGGAGTTCACCCGGGCGGCAGGGACGGCGGGGCAGCT 540
CCTCTACAGCAACTTGCAGCATCTGAAGTGGAACGGCCAGTGCAGTAGTGACCTGTTCCA 600
GTCCACACACAATGTCATTGTCAAGACTGAACAAACTGAGCCTTCCATCATGAACACCTG 660
GAAAGACGAGAACTATTTATATGACACCAACTATGGTAGCACAGTAGATTTGTTGGACAG 720
-99-
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
CAAAACTTTCTGCCGGGCTC AGATCTCCATGACAACCACCAGTCACCTTC~TGTTGCAGA780
GTCACCTGATATGAAAAAGG AGCAAGACCCCCCTGCCAAGTGCCACACCAAAAAGCACAA840
CCCGAGAGGGACTCACTTAT GGGAATTCATCCGCGACATCCTCTTGAACCCAGACAAGAA900
CCCAGGATTAATAAAATGGG AAGACCGATCTGAGGGCGTCTTCAGGTTCTTGAAATCAGA960
GGCAGTGGCTCAGCTATGGG GTAAAAAGAAGAACAACAGCAGCATGACCTATGAAAAGCT1020
CAGCCGAGCTATGAGATATT ACTACAAAAGAGAAATACTGGAGCGTGTGGATGGACGAAG1080
ACTGGTATATAAATTTGGGA AGAATGCCCGAGGATGGAGAGAAAATGAAAACTGAAGCTG1140
CCAATACTTTGGACACAAAC CAAAACACACACCAAATAATCAGAAACAAAGAACTCCTGG1200
ACGTAAATATTTCAAAGACT ACTTTTCTCTGATATTTATGTACCATGAGGGGAAAAAGAA1260
ACTACTTCTAACGGGAAGAA GAAACACTACAGTCGATTAAAAAAATTATTTTGTTACTTC1320
GAAGTATGTCCTATATGGGG AAAAAACGTACACAGTTTTCTGTGAAATATGATGCTGTAT1380
GTGGTTGTGATTTTTTTTCA CCTCTATTGTGAATTCTTTTTCACTGCAAGAGTAACAGGA1440
TTTGTAGCCTTGTGCTTCTT GCTAAGAGAAAGAAAAACAAAATCAGAGGGCATTAAATGT1500
TTTGTATGTGACATGATTTA GAAAAAGGTGATGCATCCTCCTCACATAAGCATCCATATG1560
GCTTCGTCAAGGGAGGTGAA CATTGTTGCTGAGTTAAATTCCAGGGTCTCAGATGGTTAG1620
GACAAAGTGGATGGATGCCG GGAAGTTTAACCTGAGCCTTAGGATCCAATGAGTGGAGAA1680
TGGGGACTTCCAAAACCCAA GGTTGGCTATAATCTCTGCATAACCACATGACTTGGAATG1740
CTTAAATCAGCAAGAAGAAT AATGGTGGGGTCTTTATACTCATTCAGGAATGGTTTATCT1800
GATGCCAGGGCTGTCTTCCT TTCTCCCCTTTGGATGGTTGGTGAAATACTTTAATTGCCC1860
TGTCTGCTCACTTCTAGCTA TTTAAGAGAGAACCCAGCTTGGTTCTTTTTTGCTCCAAGT1920
GCTTAAAAATAAGTTGGAAA AAGGAGACGGTGGTGTGGAAATGGCTGAAGAGTTTGCTCT1980
TGTATCCCTATAGTCCAAGG TTTCTCAATCTGCACAATTGACATTTTTGGCCGGAGTGTT2040
CTTTGTGGTGAGGGCTTTCC TGTGCATTGTAAGATGTTCAGCAGTATCCACTCATGGTCT2100
CTAACCACTTGACACCAGAA ACCCCCCAGCTGTGATAACGCAAAATGTCTCTAGACATCA2160
CCAAATGTTCCCTGGGGGTG GCAAATTTGCCCTTGATTGAGAACCACCAGTTTAGCTAGT2220
CAATATGAGGATGGTGGTTT ATTCTCAGAAGAAAAAGATATGTAAGGTCTTTTAGCTCCT2280
TAGAGTGAAGCAAAAGCAAG ACTTCAACCTCAACCTATCTTTATGTTTTAAATATTAGGG2340
ACAATAAGTTGAAATAGCTA GAGGAGCTTCTTTTCAGAACCCCAGATGAGAGCCAATGTC2400
AGATAAAGTAAGCATAGCAA TGTAGCAGGAACTACAATAGAAGACATTTTCACTGGAATT2460
ACAAAGCAGAATTAAAATTA TATTGTAGAAGGAAACACCAAGAAAAGAATTTCCAGGGAA2520
AATCCTCTTTGCAGGTATTA ATTCTTATAATTTTTTGTCTTTTGGATTATCTGTTTACTG2580
TCTCATCTGAACTGATCCCA GGTGAACGGTTTATTGCCTAGATTTGTACTCAGAGGAATT2640
TTTTTTGTTTTGTTTTGTCT TTTAAGAAAGGAAAGAAAGGATGAAAAAlAATAAACAGAAA2700
ACTCAGCTCAGGCACAATTG TCACCAAGGAGTTAAAAGCTTCTTCTTCAATAGAGGAATT2760
GTTCTGGGGGTCCTGGAGAC TTACCATTGAGCCATGCAATCTGGGAAGCACAGGAATAAG2820
TAGACACTTTGAAAATGGAT TTGAATGTTCTCATCCCTTTTGCAGCTTTTCTTTTTGGCT2880
CTCTCATGTCCTTGGCTTGC TCCTCTATTCTACCTCTCTTTCTCCAGCAATAATATGCAA2940
ATGAAGACATGTATCCATAA GAAGGAGTGCTCTTCATCAACTAATAGAGCACCTACCACA3000
GTGTCATACCTGGTAGAGGT GAGCAATTCATATTCAAAGGTTGCAAAGTGTTTGTAATAT3060
ATTCATGAGGCTGGAAGTAA GAAGAATTAAAAATTTGTCCTAATTACAATGAGAACCATT3120
CTAGGTAGTGATCTTGGAGC ACACATGAATAACTTTCTGAAGGTGCAACCAAATCCATTT3180
TTATTTCTGCCTGGCTTGGT CACCTCTGTAAAGGTTTAACTTAGTGTTGTCAAGTAACAG3240
TTACTGAAAGAGCTGAGAAA AAGAACAATGAACAGCAACGATCTTGACTGTGCAACTCAG3300
ACATTCCTGCAGAAAAGACA TATGTTGCTTTACAAGAAGGCCAAAGAACTATGGGGCCTT3360
CCCAGCATTTGACTGTTCAT TGCATAGAATGAATTAAATATCCAGTTACTTGAATGGGTA3420
TAACGCATGAATATTTGTGT GTCTGTGTGTGTGTCTGAGTTGTGTGATTTTATTAGGGGC3480
ATCTGCCAATTCTCTCACTG TGGTTCCTTCTCTGACTTTGCCTGTTCATCATCTAAGGAG3540
GCTAGATCCTTCGCTGACTT CACCATTCCTCAAACCTGTAAGTTTCTCACTTCTTCCAAA3600
TTGGCTTTGGCTCTTTCTTC AACCTTTCCATTCAAGAGCAATCTTTGCTAAGGAGTAAGT3660
GAATGTGAAGAGTACCAACT ACAACAATTCTACAGATAATTAGTGGATTGTGTTGTTTGT3720
TGAGAGTGAAGGTTTCTTGG CATCTGGTGCCTGATTAAGGCTTGAGTATTAAGTTCTCAG3780
CATATCTCTCTATTGTCTTG ACTTGAGTTTGCTGCATTTTCTATGTGCTGTTCGTGACTT3840
GGAGAACTTAAAGTAATCGA GCTATGCCAACTTGGGGTGGTAACAGAGTACTTCCCACCA3900
CAGTGTTGAAAGGGAGAGCA AAGTCTTATGGATAAACCCTCCTTTCTTTTGGGGACACAT3960
GGCTCTCACTTGAGAAGCTC ACCTGTGCTGAATGTCCACATGGTCACTAAACATGTTATC4020
CTTAAACCCCCCGTATGCCT GAGTTGAAAGGGCTCTCTCTTATTAGGTTTTCATGGGAAC4080
-1 ~~-
CA 02314677 2000-06-02
WO 99/37809 PCTNS98/01260
ATGAGGCAGCAAATCTATTGCTAAGACTTTACCAGGCTCA GGCTGATAGA4140
AATCATCTGA
TATTTGACTTGGTAAGACTTAAGTAAGGCTCTGGCTCCCAGGGGCATAAGCAACAGTTTC4200
TTGAATGTGCCATCTGAGAAGGGAGACCCAGGTTATGAGTTTTCCTTTGAACACATTGGT4260
CTTTTCTCAAAGTTCCTGCCTTGCTAGACTGTTAGCTCTTTGAGGACAGGGACTATGTCT4320
TATCAATCACTATTATTTTCCTGTTACCTAGCATGGGACAAGTACACAACACATATTTGT4380
TCAATGAATGAATGAATGTCTTCTAAAAGACTCCTCTGATTGGGAGACCATATCTATAAT4440
TGGGATGTGAATCATTTCTTCAGTGGAATAAGAGCACAACGGCACAACCTTCAAGGACAT4500
ATTATCTACTATGAACATTTTACTGTGAGACTCTTTATTTTGCCTTCTACTTGCGCTGAA4560
ATGAAACCAAAACAGGCCGTTGGGTTCCACAAGTCAATATATGTTGGATGAGGATTCTGT4620
TGCCTTATTGGGAACTGTGAGACTTATCTGGTATGAGAAGCCAGTAATAAACCTTTGACC4680
TGTTTTAACCAATGAAGATTATGAATATGTTAATATGATGTAAATTGCTATTTAAGTGTA4740
AAGCAGTTCTAAGTTTTAGTATTTGGGGGATTGGTTTTTATTATTTTTTTCCTTTTTGAA4800
AAATACTGAGGGATCTTTTGATAAAGTTAGTAATGCATGTTAGATTTTAGTTTTGCAAGC4860
ATGTTGTTTTTCAAATATATCAAGTATAGAAAAAGGTAAAACAGTTAAGAAGGAAGGCAA4920
TTATATTATTCTTCTGTAGTTAAGCAAACACTTGTTGAGTGCCTGCTATGTGCACGGCAT4980
GGGCCCATATGTGTGAGGAGCTTGTCTAATTATGTAGGAAGCAATAGATCTCGGTAGTTA5040
CGTATTGGGCAGATACTTACTGTATGAATGAAAGAACATCACAGTAATCACAATATCAGA5100
GCTGAATTATCCTCAGTGTAGCTTCTTGGAATTCAGTTTCTGGAACTAGAGATAGAGCAT5160
TTATTAAAAAAAACTCCTGTTGAGACTGTGTCTTATGAACCTCTGAAACGTACAAGCCTT5220
CACAAGTTTAACTAAATTGGGATTAATCTTTCTGTAGTTATCTGCATAATTCTTGTTTTT5280
CTTTCCATCTGGCTCCTGGGTTGACAATTTGTGGAAACAACTCTATTGCTACTATTTAAA5340
AAAAATCAGAAATCTTTCCCTTTAAGCTATGTTAAATTCAAACTATTCCTGCTATTCCTG5400
TTTTGTCAAAGAATTATATTTTTCAAAATATGTTTATTTGTTTGATGGGTCCCAGGAAAC5460
ACTAATAAAAACCACAGAGACCAGCCTGGA AAAAAAAAAA 5510
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5667 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:4:
ATCGCTCTCTGGTAGAGTTGACGTGACACTCATTTCTGTTGTGGGTGGGGCCCTGGTTGG60
GAGGCATTGGCTCCACTGCAGCCTGGGTGTCTAGAGACCACATTCTCACCCTGCCTTTGT120
TACTGGGAAACCGAACGCGGCGCTGTGGCTTTCAGCTTGGGTAAGCCGGGTCTGCGGCGG180
GGATTGCCATCTGAAGACAGAGGCAGGAGGGCAGCCACACCTTGCCCAGCTGCACACCCA240
GTAACAAGTTTCCTCAGTGCGGGTATCTGCCACAGGCTGGGCTGGTCATCAAAGGGCCTC300
AGTCATATTTTAATAGAGCTCTTCAAGTATCTGGCTTTGTGATAATATCAGGAATCAGTT360
GGTTTCTCTGACAGACACTGCCCATTATCATGATTCTGGAAGGAGGTGGTGTAATGAATC420
TCAACCCCGGCAACAACCTCCTTCACCAGCCGCCAGCCTGGACAGACAGCTACTCCACGT480
GCAATGTTTCCAGTGGGTTTTTTGGAGGCCAGTGGCATGAAATTCATCCTCAGTACTGGA540
CCAAGTACCAGGTGTGGGAGTGGCTCCAGCACCTCCTGGACACCAACCAGCTGGATGCCA600
ATTGTATCCCTTTCCAAGAGTTCGACATCAACGGCGAGCACCTCTGCAGCATGAGTTTGC660
AGGAGTTCACCCGGGCGGCAGGGACGGCGGGGCAGCTCCTCTACAGCAACTTGCAGCATC720
TGAAGTGGAACGGCCAGTGCAGTAGTGACCTGTTCCAGTCCACACACAATGTCATTGTCA780
AGACTGAACAAACTGAGCCTTCCATCATGAACACCTGGAAAGACGAGAACTATTTATATG840
ACACCAACTATGGTAGCACAGTAGATTTGTTGGACAGCAAAACTTTCTGCCGGGCTCAGA900
TCTCCATGACAACCACCAGTCACCTTCCTGTTGCAGAGTCACCTGATATGAAAAAGGAGC960
AAGACCCCCCTGCCAAGTGCCACACCAAAAAGCACAACCCGAGAGGGACTCACTTATGGG1020
AATTCATCCGCGACATCCTCTTGAACCCAGACAAGAACCCAGGATTAATAAAATGGGAAG1080
ACCGATCTGAGGGCGTCTTCAGGTTCTTGAAATCAGAGGCAGTGGCTCAGCTATGGGGTA1140
-1~1-
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
AAAAGAAGAACAACAGCAGCATGACCTATG CCGAGCTATGAGATATTACT1200
AAAAGCTCAG
ACAAAAGAGAAATACTGGAGCGTGTGGATGGACGAAGACTGGTATATAAATTTGGGAAGA1260
ATGCCCGAGGATGGAGAGAAAATGAAAACTGAAGCTGCCAATACTTTGGACACAAACCAA1320
AACACACACCAAATAATCAGAAACAAAGAACTCCTGGACGTAAATATTTCAAAGACTACT1380
TTTCTCTGATATTTATGTACCATGAGGGGAAAAAGAAACTACTTCTAACGGGAAGAAGAA1440
ACACTACAGTCGATTAAAAAAATTATTTTGTTACTTCGAAGTATGTCCTATATGGGGAAA1500
AAACGTACACAGTTTTCTGTGAAATATGATGCTGTATGTGGTTGTGATTTTTTTTCACCT1560
CTATTGTGAATTCTTTTTCACTGCAAGAGTAACAGGATTTGTAGCCTTGTGCTTCTTGCT1620
AAGAGAAAGAAAAACAAAATCAGAGGGCATTAAATGTTTTGTATGTGACATGATTTAGAA1680
AAAGGTGATGCATCCTCCTCACATAAGCATCCATATGGCTTCGTCAAGGGAGGTGAACAT1740
TGTTGCTGAGTTAAATTCCAGGGTCTCAGATGGTTAGGACAAAGTGGATGGATGCCGGGA1800
AGTTTAACCTGAGCCTTAGGATCCAATGAGTGGAGAATGGGGACTTCCAAAACCCAAGGT1860
TGGCTATAATCTCTGCATAACCACATGACTTGGAATGCTTAAATCAGCAAGAAGAATAAT1920
GGTGGGGTCTTTATACTCATTCAGGAATGGTTTATCTGATGCCAGGGCTGTCTTCCTTTC1980
TCCCCTTTGGATGGTTGGTGAAATACTTTAATTGCCCTGTCTGCTCACTTCTAGCTATTT2040
AAGAGAGAACCCAGCTTGGTTCTTTTTTGCTCCAAGTGCTTAAAAATAAGTTGGAAAAAG2100
GAGACGGTGGTGTGGAAATGGCTGAAGAGTTTGCTCTTGTATCCCTATAGTCCAAGGTTT2160
CTCAATCTGCACAATTGACATTTTTGGCCGGAGTGTTCTTTGTGGTGAGGGCTTTCCTGT2220
GCATTGTAAGATGTTCAGCAGTATCCACTCATGGTCTCTAACCACTTGACACCAGAAACC2280
CCCCAGCTGTGATAACGCAAAATGTCTCTAGACATCACCAAATGTTCCCTGGGGGTGGCA2340
AATTTGCCCTTGATTGAGAACCACCAGTTTAGCTAGTCAATATGAGGATGGTGGTTTATT2400
CTCAGAAGAAAAAGATATGTAAGGTCTTTTAGCTCCTTAGAGTGAAGCAAAAGCAAGACT2460
TCAACCTCAACCTATCTTTATGTTTTAAATATTAGGGACAATAAGTTGAAATAGCTAGAG2520
GAGCTTCTTTTCAGAACCCCAGATGAGAGCCAATGTCAGATAAAGTAAGCATAGCAATGT2580
AGCAGGAACTACAATAGAAGACATTTTCACTGGAATTACAAAGCAGAATTAAAATTATAT2640
TGTAGAAGGAAACACCAAGAAAAGAATTTCCAGGGAAAATCCTCTTTGCAGGTATTAATT2700
CTTATAATTTTTTGTCTTTTGGATTATCTGTTTACTGTCTCATCTGAACTGATCCCAGGT2760
GAACGGTTTATTGCCTAGATTTGTACTCAGAGGAATTTTTTTTGTTTTGTTTTGTCTTTT2820
AAGAAAGGAAAGAAAGGATGAAAAAAATAAACAGAAAACTCAGCTCAGGCACAATTGTCA2880
CCAAGGAGTTAAAAGCTTCTTCTTCAATAGAGGAATTGTTCTGGGGGTCCTGGAGACTTA2940
CCATTGAGCCATGCAATCTGGGAAGCACAGGAATAAGTAGACACTTTGAAAATGGATTTG3000
AATGTTCTCATCCCTTTTGCAGCTTTTCTTTTTGGCTCTCTCATGTCCTTGGCTTGCTCC3060
TCTATTCTACCTCTCTTTCTCCAGCAATAATATGCAAATGAAGACATGTATCCATAAGAA3120
GGAGTGCTCTTCATCAACTAATAGAGCACCTACCACAGTGTCATACCTGGTAGAGGTGAG3180
CAATTCATATTCAAAGGTTGCAAAGTGTTTGTAATATATTCATGAGGCTGGAAGTAAGAA3240
GAATTAAAAATTTGTCCTAATTACAATGAGAACCATTCTAGGTAGTGATCTTGGAGCACA3300
CATGAATAACTTTCTGAAGGTGCAACCAAATCCATTTTTATTTCTGCCTGGCTTGGTCAC3360
CTCTGTAAAGGTTTAACTTAGTGTTGTCAAGTAACAGTTACTGAAAGAGCTGAGAAAAAG3420
AACAATGAACAGCAACGATCTTGACTGTGCAACTCAGACATTCCTGCAGAAAAGACATAT3480
GTTGCTTTACAAGAAGGCCAAAGAACTATGGGGCCTTCCCAGCATTTGACTGTTCATTGC3540
ATAGAATGAATTAAATATCCAGTTACTTGAATGGGTATAACGCATGAATATTTGTGTGTC3600
TGTGTGTGTGTCTGAGTTGTGTGATTTTATTAGGGGCATCTGCCAATTCTCTCACTGTGG3660
TTCCTTCTCTGACTTTGCCTGTTCATCATCTAAGGAGGCTAGATCCTTCGCTGACTTCAC3720
CATTCCTCAAACCTGTAAGTTTCTCACTTCTTCCAAATTGGCTTTGGCTCTTTCTTCAAC3780
CTTTCCATTCAAGAGCAATCTTTGCTAAGGAGTAAGTGAATGTGAAGAGTACCAACTACA3840
ACAATTCTACAGATAATTAGTGGATTGTGTTGTTTGTTGAGAGTGAAGGTTTCTTGGCAT3900
CTGGTGCCTGATTAAGGCTTGAGTATTAAGTTCTCAGCATATCTCTCTATTGTCTTGACT3960
TGAGTTTGCTGCATTTTCTATGTGCTGTTCGTGACTTGGAGAACTTAAAGTAATCGAGCT4020
ATGCCAACTTGGGGTGGTAACAGAGTACTTCCCACCACAGTGTTGAAAGGGAGAGCAAAG4080
TCTTATGGATAAACCCTCCTTTCTTTTGGGGACACATGGCTCTCACTTGAGAAGCTCACC4140
TGTGCTGAATGTCCACATGGTCACTAAACATGTTATCCTTAAACCCCCCGTATGCCTGAG4200
TTGAAAGGGCTCTCTCTTATTAGGTTTTCATGGGAACATGAGGCAGCAAATCTATTGCTA4260
AGACTTTACCAGGCTCAAATCATCTGAGGCTGATAGATATTTGACTTGGTAAGACTTAAG4320
TAAGGCTCTGGCTCCCAGGGGCATAAGCAACAGTTTCTTGAATGTGCCATCTGAGAAGGG4380
AGACCCAGGTTATGAGTTTTCCTTTGAACACATTGGTCTTTTCTCAAAGTTCCTGCCTTG4440
CTAGACTGTTAGCTCTTTGAGGACAGGGACTATGTCTTATCAATCACTATTATTTTCCTG4500
-102-
CA 02314677 2000-06-02
WO 99/37809 PCT/US98/01260
TTACCTAGCA ACACAACACA ATGAATGAATGAATGTCTTC4560
TGGGACAAGT TATTTGTTCA
TAAAAGACTCCTCTGATTGGGAGACCATATCTATAATTGGGATGTGAATCATTTCTTCAG4620
TGGAATAAGAGCACAACGGCACAACCTTCAAGGACATATTATCTACTATGAACATTTTAC4680
TGTGAGACTCTTTATTTTGCCTTCTACTTGCGCTGAAATGAAACCAAAACAGGCCGTTGG4740
GTTCCACAAGTCAATATATGTTGGATGAGGATTCTGTTGCCTTATTGGGAACTGTGAGAC4800
TTATCTGGTATGAGAAGCCAGTAATAAACCTTTGACCTGTTTTAACCAATGAAGATTATG4860
AATATGTTAATATGATGTAAATTGCTATTTAAGTGTAAAGCAGTTCTAAGTTTTAGTATT4920
TGGGGGATTGGTTTTTATTATTTTTTTCCTTTTTGAP.AAATACTGAGGGATCTTTTGATA4980
AAGTTAGTAATGCATGTTAGATTTTAGTTTTGCAAGCATGTTGTTTTTCAAATATATCAA5040
GTATAGAAAAAGGTAAAACAGTTAAGAAGGAAGGCAATTATATTATTCTTCTGTAGTTAA5100
GCAAACACTTGTTGAGTGCCTGCTATGTGCACGGCATGGGCCCATATGTGTGAGGAGCTT5160
GTCTAATTATGTAGGAAGCAATAGATCTCGGTAGTTACGTATTGGGCAGATACTTACTGT5220
ATGAATGAAAGAACATCACAGTAATCACAATATCAGAGCTGAATTATCCTCAGTGTAGCT5280
TCTTGGAATTCAGTTTCTGGAACTAGAGATAGAGCATTTATTAAAAAAAACTCCTGTTGA5340
GACTGTGTCTTATGAACCTCTGAAACGTACAAGCCTTCACAAGTTTAACTAAATTGGGAT5400
TAATCTTTCTGTAGTTATCTGCATAATTCTTGTTTTTCTTTCCATCTGGCTCCTGGGTTG5460
ACAATTTGTGGAAACAACTCTATTGCTACTATTTAAAAiAAAATCAGAAATCTTTCCCTTT5520
AAGCTATGTTAAATTCAAACTATTCCTGCTATTCCTGTTTTGTCAAAGAATTATATTTTT5580
CAAAATATGTTTATTTGTTTGATGGGTCCCAGGAAACACTAATAAAAACCACAGAGACCA5640
GCCTGGAAAAPu~AAAAAAAAAAAAAAA 5667
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 300 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Met Ile Leu Glu Gly Gly Gly Val Met Asn Leu Asn Pro Gly Asn Asn
1 5 10 15
Leu Leu His Gln Pro Pro Ala Trp Thr Asp Ser Tyr Ser Thr Cys Asn
20 25 30
Val Ser Ser Gly Phe Phe Gly Gly Gln Trp His Glu Ile His Pro Gln
35 40 45
Tyr Trp Thr Lys Tyr Gln Val Trp Glu Trp Leu Gln His Leu Leu Asp
50 55 60
Thr Asn Gln Leu Asp Ala Asn Cys Ile Pro Phe Gln Glu Phe Asp Ile
65 70 75 80
Asn Gly Glu His Leu Cys Ser Met Ser Leu Gln Glu Phe Thr Arg Ala
85 90 95
Ala Gly Thr Ala Gly Gln Leu Leu Tyr Ser Asn Leu Gln His Leu Lys
100 105 110
Trp Asn Gly Gln Cys Ser Ser Asp Leu Phe Gln Ser Thr His Asn Val
115 120 125
Ile Val Lys Thr Glu Gln Thr Glu Pro Ser Ile Met Asn Thr Trp Lys
130 135 140
Asp Glu Asn Tyr Leu Tyr Asp Thr Asn Tyr Gly Ser Thr Val Asp Leu
145 150 155 160
Leu Asp Ser Lys Thr Phe Cys Arg Ala Gln Ile Ser Met Thr Thr Thr
165 170 175
Ser His Leu Pro Val Ala Glu Ser Pro Asp Met Lys Lys Glu Gln Asp
-103-
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180 185 190'
Pro Pro Ala Lys Cys His Thr Lys Lys His Asn Pro Arg Gly Thr His
195 200 205
Leu Trp Glu Phe Ile Arg Asp Ile Leu Leu Asn Pro Asp Lys Asn Pro
210 215 220
Gly Leu Ile Lys Trp Glu Asp Arg Ser Glu Gly Val Phe Arg Phe Leu
225 230 235 240
Lys Ser Glu Ala Val Ala Gln Leu Trp Gly Lys Lys Lys Asn Asn Ser
245 250 255
Ser Met Thr Tyr Glu Lys Leu Ser Arg Ala Met Arg Tyr Tyr Tyr Lys
260 265 270
Arg Glu Ile Leu Glu Arg Val Asp Gly Arg Arg Leu Val Tyr Lys Phe
275 280 285
Gly Lys Asn Ala Arg Gly Trp Arg Glu Asn Glu Asn
290 295 300
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2428 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
CAGGGGTGCCGGGTTGCTCA GCCACACCTGTTATTGCTGCCTCTGATTTG60
GGCCATGGGA
TGTGACACTGAGAAGCCCACAGGCCTGTCCCTCCAACTCGGTGGACCCTCTCTGTGTGCA120
TTTGGTGTGTGAGCCAGCTCTGAGAAGGGTTCAGAAGCCACTGGAGGCATCTGGGGACCT180
CAGCTTCCATGCCATCTCTGCCTCACTCCCACAGGGTAATGTTGGACTCGGTGACACACA240
GCACCTTCCTGCCTAATGCATCCTTCTGCGATCCCCTGATGTCGTGGACTGATCTGTTCA300
GCAATGAAGAGTACTACCCTGCCTTTGAGCATCAGACAGCCTGTGACTCATACTGGACAT360
CAGTCCACCCTGAATACTGGACTAAGCGCCATGTGTGGGAGTGGCTCCAGTTCTGCTGCG420
ACCAGTACAAGTTGGACACCAATTGCATCTCCTTCTGCAACTTCAACATCAGTGGCCTGC480
AGCTGTGCAGCATGACACAGGAGGAGTTCGTCGAGGCAGCTGGCCTCTGCGGCGAGTACC540
TGTACTTCATCCTCCAGAACATCCGCACACAAGGTTACTCCTTTTTTAATGACGCTGAAG600
AAAGCAAGGCCACCATCAAAGACTATGCTGATTCCAACTGCTTGAAAACAAGTGGCATCA660
AAAGTCAAGACTGTCACAGTCATAGTAGAACAAGCCTCCAAAGTTCTCATCTATGGGAAT720
TTGTACGAGACCTGCTTCTATCTCCTGAAGAAAACTGTGGCATTCTGGAATGGGAAGATA780
GGGAACAAGGAATTTTTCGGGTGGTTAAATCGGAAGCCCTGGCAAAGATGTGGGGACAAA840
GGAAGAAAAATGACAGAATGACGTATGAAAAGTTGAGCAGAGCCCTGAGATACTACTATA900
AAACAGGAATTTTGGAGCGGGTTGACCGAAGGTTAGTGTACAAATTTGGAAAAAATGCAC960
ACGGGTGGCAGGAAGACAAGCTATGATCTGCTCCAGGCATCAAGCTCATTTTATGGATTT1020
CTGTCTTTTAAAACAATCAGATTGCAATAGACATTCGAAAGGCTTCATTTTCTTCTCTTT1080
TTTTTTAACCTGCAAACATGCTGATAAAATTTCTCCACATCTCAGCTTACATTTGGATTC1140
AGAGTTGTTGTCTACGGAGGGTGAGAGCAGAAACTCTTAAGAAATCCTTTCTTCTCCCTA1200
AGGGGATGAGGGGATGATCTTTTGTGGTGTCTTGATCAAACTTTATTTTCCTAGAGTTGT1260
GGAATGACAACAGCCCATGCCATTGATGCTGATCAGAGAAAAACTATTCAATTCTGCCAT1320
TAGAGACACATCCAATGCTCCCATCCCAAAGGTTCAAAAGTTTTCAAATAACTGTGGCAG1380
CTCACCAAAGGTGGGGGAAAGCATGATTAGTTTGCAGGTTATGGTAGGAGAGGGTGAGAT1440
ATAAGACATACATACTTTAGATTTTAAATTATTAAAGTCAAAAATCCATAGAAAAGTATC1500
CCTTTTTTTTTTTTTTGAGACGGGTTCTCACTATGTTGCCCAGGGCTGGTCTTGAACTCC1560
TATGCTCAAGTGATCCTCCCACCTCGGCCTCCCAAAGTACTGTGATTACAAGCGTGAGCC1620
ACGGCACCTGGGCAGAAAAGTATCTTAATTAATGAAAGAGCTAAGCCATCAAGCTGGGAC1680
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TTAATTGGATTTAACATAGG TTCACAGAAA GTTTCCTAACCAGAGCATCTTTTTGACCAC1740
TCAGCAAAACTTCCACAGAC ATCCTTCTGG ACTTAAACACTTAACATTAACCACATTATT1800
AATTGTTGCTGAGTTTATTC CCCCTTCTAA CTGATGGCTGGCATCTGATATGCAGAGTTA1860
GTCAACAGACACTGGCATCA ATTACAAAAT CACTGCTGTTTCTGTGATTCAAGCTGTCAA1920
CACAATAAAATCGAAATTCA TTGATTCCAT CTCTGGTCCAGATGTTAAACGTTTATAAAA1980
CCGGAAATGTCCTAACAACT CTGTAATGGC AAATTAAATTGTGTGTCTTTTTTGTTTTGT2040
CTTTCTACCTGATGTGTATT CAAGCGCTAT AACACGTATTTCCTTGACAAAAATAGTGAC2100
AGTGAATTCACACTAATAAA TGTTCATAGG TTAAAGTCTGCACTGACATTTTCTCATCAA2160
TCACTGGTATGTAAGTTATC AGTGACTGAC AGCTAGGTGGACTGCCCCTAGGACTTCTGT2220
TTCACCAGAGCAGGAATCAA GTGGTGAGGC ACTGAATCGCTGTACAGGCTGAAGACCTCC2280
TTATTAGAGTTGAACTTCAA AGTAACTTGT TTTAAAAAATGTGAATTACTGTAAAATAAT2340
CTATTTTGGATTCATGTGTT TTCCAGGTGG ATATAGTTTGTAAACAATGTGAATAAAGTA2400
TTTAACATGTTC1?~AAAAAAA AAAAAAAA 2428
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 265 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Met Pro Ser Leu Pro His Ser His Arg Val Met Leu Asp Ser Val Thr
1 5 10 15
His Ser Thr Phe Leu Pro Asn Ala Ser Phe Cys Asp Pro Leu Met Ser
20 25 30
Trp Thr Asp Leu Phe Ser Asn Glu Glu Tyr Tyr Pro Ala Phe Glu His
35 40 45
Gln Thr Ala Cys Asp Ser Tyr Trp Thr Ser Val His Pro Glu Tyr Trp
50 55 60
Thr Lys Arg His Val Trp Glu Trp Leu Gln Phe Cys Cys Asp Gln Tyr
65 70 75 80
Lys Leu Asp Thr Asn Cys Ile Ser Phe Cys Asn Phe Asn Ile Ser Gly
B5 90 95
Leu Gln Leu Cys Ser Met Thr Gln Glu Glu Phe Val Glu Ala Ala Gly
100 105 110
Leu Cys Gly Glu Tyr Leu Tyr Phe Ile Leu Gln Asn Ile Arg Thr Gln
115 120 125
Gly Tyr Ser Phe Phe Asn Asp Ala Glu Glu Ser Lys Ala Thr Ile Lys
130 135 140
Asp Tyr Ala Asp Ser Asn Cys Leu Lys Thr Ser Gly Ile Lys Ser Gln
145 150 155 160
Asp Cys His Ser His Ser Arg Thr Ser Leu Gln Ser Ser His Leu Trp
165 170 175
Glu Phe Val Arg Asp Leu Leu Leu Ser Pro Glu Glu Asn Cys Gly Ile
180 185 190
Leu Glu Trp Glu Asp Arg Glu Gln Gly Ile Phe Arg Val Val Lys Ser
195 200 205
Glu Ala Leu Ala Lys Met Trp Gly Gln Arg Lys Lys Asn Asp Arg Met
210 215 220
Thr Tyr Glu Lys Leu Ser Arg Ala Leu Arg Tyr Tyr Tyr Lys Thr Gly
225 230 235 240
-i as-
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Ile Leu Glu Arg Val Asp Arg Arg Leu Val Tyr Lys Phe Gly-Lys Asn
245 250 255
Ala His Gly Trp Gln Glu Asp Lys Leu
260 265
(2) INFORMATION FOR SEQ ID NO: B:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2280 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
CTGGGAGCGCCTGCCTTCTCTTGCCTTGAA TTGGACCTAGCCACCGCTGC60
AGCCTCCTCT
CCTCACGGTAATGTTGGACTCGGTGACACACAGCACCTTCCTGCCTAATGCATCCTTCTG120
CGATCCCCTGATGTCGTGGACTGATCTGTTCAGCAATGAAGAGTACTACCCTGCCTTTGA180
GCATCAGACAGCCTGTGACTCATACTGGACATCAGTCCACCCTGAATACTGGACTAAGCG240
CCATGTGTGGGAGTGGCTCCAGTTCTGCTGCGACCAGTACAAGTTGGACACCAATTGCAT300
CTCCTTCTGCAACTTCAACATCAGTGGCCTGCAGCTGTGCAGCATGACACAGGAGGAGTT360
CGTCGAGGCAGCTGGCCTCTGCGGCGAGTACCTGTACTTCATCCTCCAGAACATCCGCAC420
ACAAGGTTACTCCTTTTTTAATGACGCTGAAGAAAGCAAGGCCACCATCAAAGACTATGC480
TGATTCCAACTGCTTGAAAACAAGTGGCATCAAAAGTCAAGACTGTCACAGTCATAGTAG540
AACAAGCCTCCAAAGTTCTCATCTATGGGAATTTGTACGAGACCTGCTTCTATCTCCTGA600
AGAAAACTGTGGCATTCTGGAATGGGAAGATAGGGAACAAGGAATTTTTCGGGTGGTTAA660
ATCGGAAGCCCTGGCAAAGATGTGGGGACAAAGGAAGAAAAATGACAGAATGACGTATGA720
AAAGTTGAGCAGAGCCCTGAGATACTACTATAAAACAGGAATTTTGGAGCGGGTTGACCG780
AAGGTTAGTGTACAAATTTGGAAAAAATGCACACGGGTGGCAGGAAGACAAGCTATGATC840
TGCTCCAGGCATCAAGCTCATTTTATGGATTTCTGTCTTTTAAAACAATCAGATTGCAAT900
AGACATTCGAAAGGCTTCATTTTCTTCTCTTTTTTTTTAACCTGCAAACATGCTGATAAA960
ATTTCTCCACATCTCAGCTTACATTTGGATTCAGAGTTGTTGTCTACGGAGGGTGAGAGC1020
AGAAACTCTTAAGAAATCCTTTCTTCTCCCTAAGGGGATGAGGGGATGATCTTTTGTGGT1080
GTCTTGATCAAACTTTATTTTCCTAGAGTTGTGGAATGACAACAGCCCATGCCATTGATG1140
CTGATCAGAGAAAAACTATTCAATTCTGCCATTAGAGACACATCCAATGCTCCCATCCCA1200
AAGGTTCAAAAGTTTTCAAATAACTGTGGCAGCTCACCAAAGGTGGGGGAAAGCATGATT1260
AGTTTGCAGGTTATGGTAGGAGAGGGTGAGATATAAGACATACATACTTTAGATTTTAAA1320
TTATTAAAGTCAAAAATCCATAGAAAAGTATCCCTTTTTTTTTTTTTTGAGACGGGTTCT1380
CACTATGTTGCCCAGGGCTGGTCTTGAACTCCTATGCTCAAGTGATCCTCCCACCTCGGC1440
CTCCCAAAGTACTGTGATTACAAGCGTGAGCCACGGCACCTGGGCAGAAAAGTATCTTAA1500
TTAATGAAAGAGCTAAGCCATCAAGCTGGGACTTAATTGGATTTAACATAGGTTCACAGA1560
AAGTTTCCTAACCAGAGCATCTTTTTGACCACTCAGCAAAACTTCCACAGACATCCTTCT1620
GGACTTAAACACTTAACATTAACCACATTATTAATTGTTGCTGAGTTTATTCCCCCTTCT1680
AACTGATGGCTGGCATCTGATATGCAGAGTTAGTCAACAGACACTGGCATCAATTACAAA1740
ATCACTGCTGTTTCTGTGATTCAAGCTGTCAACACAATAAAATCGAAATTCATTGATTCC1800
ATCTCTGGTCCAGATGTTAAACGTTTATAAAACCGGAAATGTCCTAACAACTCTGTAATG1860
GCAAATTAAATTGTGTGTCTTTTTTGTTTTGTCTTTCTACCTGATGTGTATTCAAGCGCT1920
ATAACACGTATTTCCTTGACAAAAATAGTGACAGTGAATTCACACTAATAAATGTTCATA1980
GGTTAAAGTCTGCACTGACATTTTCTCATCAATCACTGGTATGTAAGTTATCAGTGACTG2040
ACAGCTAGGTGGACTGCCCCTAGGACTTCTGTTTCACCAGAGCAGGAATCAAGTGGTGAG2100
GCACTGAATCGCTGTACAGGCTGAAGACCTCCTTATTAGAGTTGAACTTCAAAGTAACTT2160
GTTTTAAAAAATGTGAATTACTGTAAAATAATCTATTTTGGATTCATGTGTTTTCCAGGT2220
GGATATAGTTTGTAAACAATGTGAATAAAGTATTTAACATGTTCAAAAAAAAAAAAAAAA2280
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(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 255 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Met Leu Asp Ser Val Thr His Ser Thr Phe Leu Pro Asn Ala Ser Phe
1 5 10 15
Cys Asp Pro Leu Met Ser Trp Thr Asp Leu Phe Ser Asn Glu Glu Tyr
20 25 30
Tyr Pro Ala Phe Glu His Gln Thr Ala Cys Asp Ser Tyr Trp Thr Ser
35 40 45
Val His Pro Glu Tyr Trp Thr Lys Arg His Val Trp Glu Trp Leu Gln
50 55 60
Phe Cys Cys Asp Gln Tyr Lys Leu Asp Thr Asn Cys Ile Ser Phe Cys
65 70 75 80
Asn Phe Asn Ile Ser Gly Leu Gln Leu Cys Ser Met Thr Gln Glu Glu
85 90 95
Phe Val Glu Ala Ala Gly Leu Cys Gly Glu Tyr Leu Tyr Phe Ile Leu
100 105 110
Gln Asn Ile Arg Thr Gln Gly Tyr Ser Phe Phe Asn Asp Ala Glu Glu
115 120 ' 125
Ser Lys Ala Thr Ile Lys Asp Tyr Ala Asp Ser Asn Cys Leu Lys Thr
130 135 140
Ser Gly Ile Lys Ser Gln Asp Cys His Ser His Ser Arg Thr Ser Leu
145 150 155 160
Gln Ser Ser His Leu Trp Glu Phe Val Arg Asp Leu Leu Leu Ser Pro
165 170 175
Glu Glu Asn Cys Gly Ile Leu Glu Trp Glu Asp Arg Glu Gln Gly Ile
180 185 190
Phe Arg Val Val Lys Ser Glu Ala Leu Ala Lys Met Trp Gly Gln Arg
195 200 205
Lys Lys Asn Asp Arg Met Thr Tyr Glu Lys Leu Ser Arg Ala Leu Arg
210 215 220
Tyr Tyr Tyr Lys Thr Gly Ile Leu Glu Arg Val Asp Arg Arg Leu Val
225 230 235 240
Tyr Lys Phe Gly Lys Asn Ala His Gly Trp Gln Glu Asp Lys Leu
245 250 255
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2498 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
-1 ~7-
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GAGGGGCTGACAGCGGCGTCCCTCGTCTGGGCAGCCTCCGCTCTGCCACTCTCCTCCCGT60
CCTGAGGATGGGACCCCCGGAAAAGCGGCCTCTGGAGGCCTGCCATGGCACCCAGAGCAG120
CCATTTTCCTCCCAGTTCTGGGGCTTTGGAAGGAGCTTGCGGATGAGGAGAGGGAGCCTC180
CGCAGGGCTCTGGCTCCCCTCCAGGGGCCGAGGCCGCACACAAAGCCGCTCTGTGGCCCA240
ATTACACCTACTGGATAGGATTGTTGAGGGGACCTGAGAAACTTGAGACGACAAGAACGC300
GTAGCGCCTCGGCTGGCTGAGGGTGCTGAGCCCTCGTGTTGTGTTCTCTCCAGCTTTCCC360
CGTGCCTCAGCCACTCTTCACGTTCCATCTGTGCTCTGTGCTGACCCGCCTGTGACTCAT420
ACTGGACATCAGTCCACCCTGAATACTGGACTAAGCGCCATGTGTGGGAGTGGCTCCAGT480
TCTGCTGCGACCAGTACAAGTTGGACACCAATTGCATCTCCTTCTGCAACTTCAACATCA540
GTGGCCTGCAGCTGTGCAGCATGACACAGGAGGAGTTCGTCGAGGCAGCTGGCCTCTGCG600
GCGAGTACCTGTACTTCATCCTCCAGAACATCCGCACACAAGGTTACTCCTTTTTTAATG660
ACGCTGAAGAAAGCAAGGCCACCATCAAAGACTATGCTGATTCCAACTGCTTGAAAACAA720
GTGGCATCAAAAGTCAAGACTGTCACAGTCATAGTAGAACAAGCCTCCAAAGTTCTCATC780
TATGGGAATTTGTACGAGACCTGCTTCTATCTCCTGAAGAAAACTGTGGCATTCTGGAAT840
GGGAAGATAGGGAACAAGGAATTTTTCGGGTGGTTAAATCGGAAGCCCTGGCAAAGATGT900
GGGGACAAAGGAAGAAAAATGACAGAATGACGTATGAAAAGTTGAGCAGAGCCCTGAGAT960
ACTACTATAAAACAGGAATTTTGGAGCGGGTTGACCGAAGGTTAGTGTACAAATTTGGAA1020
AAAATGCACACGGGTGGCAGGAAGACAAGCTATGATCTGCTCCAGGCATCAAGCTCATTT1080
TATGGATTTCTGTCTTTTAAAACAATCAGATTGCAATAGACATTCGAAAGGCTTCATTTT1140
CTTCTCTTTTTTTTTAACCTGCAAACATGCTGATAAAATTTCTCCACATCTCAGCTTACA1200
TTTGGATTCAGAGTTGTTGTCTACGGAGGGTGAGAGCAGAAACTCTTAAGAAATCCTTTC1260
TTCTCCCTAAGGGGATGAGGGGATGATCTTTTGTGGTGTCTTGATCAAACTTTATTTTCC1320
TAGAGTTGTGGAATGACAACAGCCCATGCCATTGATGCTGATCAGAGAAAAACTATTCAA1380
TTCTGCCATTAGAGACACATCCAATGCTCCCATCCCAAAGGTTCAAAAGTTTTCAAATAA1440
CTGTGGCAGCTCACCAAAGGTGGGGGAAAGCATGATTAGTTTGCAGGTTATGGTAGGAGA1500
GGGTGAGATATAAGACATACATACTTTAGATTTTAAATTATTAAAGTCAAAAATCCATAG1560
AAAAGTATCCCTTTTTTTTTTTTTTGAGACGGGTTCTCACTATGTTGCCCAGGGCTGGTC1620
TTGAACTCCTATGCTCAAGTGATCCTCCCACCTCGGCCTCCCAAAGTACTGTGATTACAA1680
GCGTGAGCCACGGCACCTGGGCAGAAAAGTATCTTAATTAATGAAAGAGCTAAGCCATCA1740
AGCTGGGACTTAATTGGATTTAACATAGGTTCACAGAAAGTTTCCTAACCAGAGCATCTT1800
TTTGACCACTCAGCAAAACTTCCACAGACATCCTTCTGGACTTAAACACTTAACATTAAC1860
CACATTATTAATTGTTGCTGAGTTTATTCCCCCTTCTAACTGATGGCTGGCATCTGATAT1920
GCAGAGTTAGTCAACAGACACTGGCATCAATTACAAAATCACTGCTGTTTCTGTGATTCA1980
AGCTGTCAACACAATAAAATCGAAATTCATTGATTCCATCTCTGGTCCCAGATGTTAAAC2040
GTTTATAAAACCGGAAATGTCCTAACAACTCTGTAATGGCAAATTAAATTGTGTGTCTTT2100
TTTGTTTTGTCTTTCTACCTGATGTGTATTCAAGCGCTATAACACGTATTTCCTTGACAA2160
AAATAGTGACAGTGAATTCACACTAATAAATGTTCATAGGTTAAAGTCTGCACTGACATT2220
TTCTCATCAATCACTGGTATGTAAGTTATCAGTGACTGACAGCTAGGTGGACTGCCCCTA2280
GGACTTCTGTTTCACCAGAGCAGGAATCAAGTGGTGAGGCACTGAATCGCTGTACAGGCT2340
GAAGACCTCCTTATTAGAGTTGAACTTCAAAGTAACTTGTTTTAAAAAATGTGAATTACT2400
GTAAAATAATCTATTTTGGATTCATGTGTTTTCCAGGTGGATATAGTTTGTAAACAATGT2460
GAATAAAGTATTTAACATGTTCAAAAAAAAAAAAAAAA 2498
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 164 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Met Thr Gln Glu Glu Phe Val Glu Ala Ala Gly Leu Cys Gly Glu Tyr
-108-
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1 5 10 _15
Leu Tyr Phe Ile Leu Gln Asn Ile Arg Thr Gln Gly Tyr Ser Phe Phe
20 25 30
Asn Asp Ala Glu Glu Ser Lys Ala Thr Ile Lys Asp Tyr Ala Asp Ser
35 40 45
Asn Cys Leu Lys Thr Ser Gly Ile Lys Ser Gln Asp Cys His Ser His
50 55 60
Ser Arg Thr Ser Leu Gln Ser Ser His Leu Trp Glu Phe Val Arg Asp
65 70 75 80
Leu Leu Leu Ser Pro Glu Glu Asn Cys Gly Ile Leu Glu Trp Glu Asp
85 90 95
Arg Glu Gln Gly Ile Phe Arg Val Val Lys Ser Glu Ala Leu Ala Lys
100 105 110
Met Trp Gly Gln Arg Lys Lys Asn Asp Arg Met Thr Tyr Glu Lys Leu
115 120 125
Ser Arg Ala Leu Arg Tyr Tyr Tyr Lys Thr Gly Ile Leu Glu Arg Val
130 135 140
Asp Arg Arg Leu Val Tyr Lys Phe Gly Lys Asn Ala His Gly Trp Gln
145 150 155 160
Glu Asp Lys Leu
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: ~Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
AAATGAGCCA ATGTTTGTAA T 21
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
AAATGAGCCA GTGTTTGTAA T 21
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 736 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
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(D) TOPOLOGY: linear _
(ii} MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
AGGAAGTGAA AGCTCTTGCA TATTTGAGAG60
GAACCTAGAT
AATCCACCAA
CCGGATAATC
TTGACTGCTTGACCTAAGCA TCTCCTCATA AGGTACCCTCCCTCCCAGGA CCTTCCCTTT120
CAAACCTCTCAAGGCTCTTA CCTGGGGCCA GGGGAGATAGGCTTTTCAAA GTCCATTGAA180
TTGCCAAGAGTCTCTGTCAA GAAGGCAGTC ATGGTGCCTGGAGAGGGAAC TTGCTGGGAG240
CCCCTTCAGAGCCTGGTACT TATAGAGCTA GGGAAAAGATCTTGATGCCA AAGCAGGGTG300
GACTAAATACAGACTAATAA ATGAGACAGG TGCTCAAGAGGGCCCCTCCA TACCATCATC360
TCCTCCAGATTTGGACTTCT ACTCACTTTG CTTTTACATTCCCTCTTCCC GATGGTGTCT420
TTGGTGAGCAGGGTGCTTTT CACCTGAAAC AGCCTCTGAGCTGAAAAGAA CAGTCACCAC480
CAAATCAATTCCTCATCCAT TAACAGGTTG TCTCTCTGTTCTTGAGACAC AGGCATTACC540
TGGTTAGACCTGTTTTGTTT GAACACTAAC GTGTGAGTTGGCCAAATGCA AATGAGCCAA600
TGTTTGTAATCCTTTATTTT ATTTTTTTAA AGGGCTGGGTAGCCAATCAG AAGAGGGGGA660
AGTGACTTAGGGAATTCCCG GTTGGTGGCT TATTGCTTAACATCCTACAA AATGATTTAA720
AATTATTGTTATATGC 736
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 333 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D} TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
GCCAGAGTCCTCCTTGAGAACTTACAATGTGTCCATATTAAGGATCTGCTGTGTTTGATG60
ATTTTGTGATTACACTTTAAACTTCTTATCCATAAAGGACATACTTGATATATCTGAGAC120
TTGTAGTAGAAGGCCTTGAGACATCCATCTCATCCCATCATTATCTATCTATCATCTATC180
TATCTATCTATCTATCTATCTATCTATCTATCTATCATCTATCTATCTATCGCCAGTACT240
GTCTTGTTGAAGTTGGCAGT.AGGGTGAAAGACCTCAAACTCCAAAGGACTTTCCGTATGG300
ATGCAATATACCTGCAATTCTAGCTTTTCTGTG 333
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
ACAGAATGAC RTATGAAAAG T 21
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
-110-
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
GTAACCAAGC KCAAGCCACC C 21
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
AAGGAGCCCA YCTGAGTGCA G 21
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
CGTTCCATCT STGCTCTGTG C 21
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STR.ANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
AGCGCCTCGG YTGGCTGAGG G 21
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STR.ANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21: .
TGTATTCAAG YGCTATAACA C 21
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C} STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
CACTGAGAAG CCNACAGGCC TGT 23
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
CCCACAGGCC WGTCCCTCCA A 21
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
CGTCCATCTC YAGCTCCAGG G 21
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
GACTTGATAA YGCCCGTGGT G 21
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(2) INFORMATION FOR SEQ ID N0:26: _
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
ACTTGATAAC RCCCGTGGTG C 21
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
CTCCCCTCCA WGAGCCACAG C 21
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
ATTTCCTGCA TNGTCTGGAC TT 22
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
ATCCAAACAC YTGAGTGGAA A 21
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
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(B) TYPE: nucleic acid _
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
AGTTTCCTCA RTGCGGGAGC T 21
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
GCGAGCACCT YTGCAGCATG A 21
(2). INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDBDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
TTCACCCGGG YGGCAGGGAC G 21
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
CTGGGGAAAA NNGATCGCTG AC 22
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
GTCAATTAAA YGGCTCTCAT T 21
(2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
TAGATCATTC RTAACCTGCC T 21
(2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
AAAGAGAAAT WCTGGAGCGT G 21
(2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:
ATGAGGGGAA MAAGAAACTA C 21
(2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:
TTTTGTATGT KACATGATTT A 21
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(2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
AGCTTGGTTC YTTTTTGCTC C 21
(2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
TTGACACCAG RAACCCCCCA G 21
(2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
AAATGAGCCA RTGTTTGTAA T 21
(2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
ATCCATTTTG YATTCCTCAT T 21
(2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
CTGGAGCTCA RACCAGACAG C 21
(2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
GCCAGTGCAG SCATCATTAC C 21
(2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
AGTTCAAATC RTAATTTTTA T 21
{2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:
TCATCAGAAT YTAAATCTCC C 21
(2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:47: _
GGAGATTCAG NTGAAGCAAG A 21
(2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:
TTTTTCCACA YCCAGCCTGG C 21
(2) INFORMATION FOR SEQ ID N0:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
CCCAGCCTGG YGAACCCTGG C 21
{2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:
CTCTTCATCA YGGTCAAATA C 21
(2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
CAACTTGCTG YCAAAGTGCT G 21
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(2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:
TACTATGTGC YAGATACTAA G 21
(2) INFORMATION FOR SEQ ID N0:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B} TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
ATGCCACTTT RRGACAACTT GAG 23
(2) INFORMATION FOR SEQ ID N0:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:54:
CGCATGCCTG KAAAGAAGAG A 21
(2} INFORMATION FOR SEQ ID N0:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
GGATAAGCAC MAGTGAGCCT G 21
(2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:56:
AAAGCCAGAC RGCAACTTGT G 21
(2) INFORMATION FOR SEQ ID N0:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STR.ANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:57:
TCTCAAAAAG RGTGATAGGA G 21
(2) INFORMATION FOR SEQ ID N0:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs '
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:58:
TCTGAATCCT STCTCCTCCT T 21
(2) INFORMATION FOR SEQ ID N0:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:59:
TAGAACCAGG WTGTGGGACC A 21
(2) INFORMATION FOR SEQ ID N0:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:60:
TTCTTGTGTC RGGCGCAAAA C 21
(2) INFORMATION FOR SEQ ID N0:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:61:
AACCAACATG RAGAAACCCC A 21
(2) INFORMATION FOR SEQ ID N0:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:62:
AATAAACTAT RGTTCACCTA G 21
(2) INFORMATION FOR SEQ ID N0:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:63:
ACATATTTGT RTCTCATATG A 21
(2) INFORMATION FOR SEQ ID N0:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:64:
CAAAGCAGTT YCTAATAATC C 21
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(2) INFORMATION FOR SEQ ID N0:65: .-
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:65:
AGATCCTAAC YGGGGCCTCC T 21
(2) INFORMATION FOR SEQ ID N0:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:66:
CTCTTTCTCT YTGCTTCCTC C 21
(2) INFORMATION FOR SEQ ID N0:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:67:
TTAGGAATCC WCAAATATGT A 21
(2) INFORMATION FOR SEQ ID N0:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:68:
GTCTGACTCC RCCTCCCTCA T 21
(2) INFORMATION FOR SEQ ID N0:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:69:
GAATCACATC RTGAGAAATG T 21
(2) INFORMATION FOR SEQ ID N0:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:70:
AATTCAATCC YTCACAGACT T 21
(2) INFORMATION FOR SEQ ID N0:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:71:
GTGTAGCCAG RGTTGCTAAT T 21
(2) INFORMATION FOR SEQ ID N0:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:72:
CCTAGAAATA SCCAAGGGCA C 21
(2) INFORMATION FOR SEQ ID N0:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:73:
AAATTCTCAT RCCTCACCCT C 21
(2) INFORMATION FOR SEQ ID N0:74:
(i} SEQUENCE CHARACTERISTICS:
(A} LENGTH: 21 base pairs
(B) TYPE: nucleic acid w
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:74:
TCCCACCCCT RTCACCTTCA T 21
(2) INFORMATION FOR SEQ ID N0:75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:75:
CCTCATTCTC RGAAGCCAAC A 21
(2) INFORMATION FOR SEQ ID N0:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:76:
GAAGAGCCGT YCAGTCCCTT T 21
(2) INFORMATION FOR SEQ ID N0:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:77:
TCCATAGGCT YTTTATTTGG C 21
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(2) INFORMATION FOR SEQ ID N0:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:78:
TCGTTTAGTA YACAGGCTTT G 21
(2) INFORMATION FOR SEQ ID N0:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs ,
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:79:
GCCTCAGTTG YCCCAGCTAT A 21
(2) INFORMATION FOR SEQ ID N0:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:80:
AGCAAAATGC WCTATGCACT G 21
(2) INFORMATION FOR SEQ ID N0:81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:81:
GTGTCCTGAC rf111~TNNNNNNN NACACTGCCT G 31
(2) INFORMATION FOR SEQ ID N0:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:82:
ATCAGATAAC RCCTACACTT A 21
(2) INFORMATION FOR SEQ ID N0:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C} STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:83:
TCTCTCTTCT SCCTGCCCTG T 21
(2) INFORMATION FOR SEQ ID N0:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:84:
TGGACACAGG KAGGGGAATA T 21
(2) INFORMATION FOR SEQ ID N0:85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:85:
TGTCACTTGC RCATACAAGG C 21
(2) INFORMATION FOR SEQ ID N0:86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:86:
ATCATCAGAT YAGCCCAGAA T 21
(2) INFORMATION FOR SEQ ID N0:87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:87:
TCAACAGAGA RAGTTAATGG T 21
(2) INFORMATION FOR SEQ ID N0:88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:88:
AGCAATAATG YTTCCCTTTT C 21
(2) INFORMATION FOR SEQ ID N0:89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:89:
TCTAGCTTTT YTGTGTTTTT T 21
(2) INFORMATION FOR SEQ ID N0:90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C} STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:90:
GATTCCTTAA YGCTTGATAC T 21
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(2) INFORMATION FOR SEQ ID N0:91: -
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:91:
CCTCCTCCAG YACCAAAGTG G 21
(2) INFORMATION FOR SEQ ID N0:92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B} TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:92:
ATGGCCACAG RTCAAATCCT G 21
(2) INFORMATION FOR SEQ ID N0:93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:93:
ACTGAGTGTT YATGCCAATT T 21
(2) INFORMATION FOR SEQ ID N0:94:
(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:94:
GACAAGCCCT RTCTGACACA C 21
(2) INFORMATION FOR SEQ ID N0:95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:95:
TGAAAAGCCT YCTTGCTGCC T 21
(2) INFORMATION FOR SEQ ID N0:96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:96:
TCCTGGAGTT YCTTTGCTCC C 21
(2) INFORMATION FOR SEQ ID N0:97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:97:
GATTCCAAAT WAACTAAAGA T 21
(2) INFORMATION FOR SEQ ID N0:98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:98:
GACCTCAAGT CRTCCACCCG CC 22
(2) INFORMATION FOR SEQ ID N0:99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:99: ' -
AACAAATACT MCCCCGCAAC CC 22
(2) INFORMATION FOR SEQ ID NO:100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:
ATTTTTTTTT NAAGGAAAAT A 21
(2) INFORMATION FOR SEQ ID NO:101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:
AAATTTCCCC MAAACAAGCA G 21
(2) INFORMATION FOR SEQ ID N0:102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:102:
GAGAAAGGGT RTGTGTGTGT G 21
(2) INFORMATION FOR SEQ ID N0:103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:103:
GTGTGTGTGT NNNNGTATGT GCGCGTG 27
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(2) INFORMATION FOR SEQ ID N0:104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:104:
ATCGGGAACC YCATACCCCA A 21
(2) INFORMATION FOR SEQ ID N0:105:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:105:
TTTGTTTCGC MATGAGGTAC G 21
(2) INFORMATION FOR SEQ ID N0:106:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:106:
TGAGGGTGTT STGGGCTGGA C 21
(2) INFORMATION FOR SEQ ID N0:107:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:107:
TCTTCATTGG YATCTGAATG T 21
(2) INFORMATION FOR SEQ ID N0:108:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
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(B) TYPE: nucleic acid _
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:108:
GCGAGCACCT YTGCAGCATG A 21
(2) INFORMATION FOR SEQ ID N0:109:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:109:
AACCCCCCCC MCACACACAC A 21
(2) INFORMATION FOR SEQ ID NO:110:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:
TCAGTGCTCT STAATCAGTC A 21
(2) INFORMATION FOR SEQ ID NO:111:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:
TCTTTGTGAA ANNAATTAGT CTG 23
(2) INFORMATION FOR SEQ ID N0:112:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:112: -
GCTGCCCTGA SAGCTGGGCC A 21
(2) INFORMATION FOR SEQ ID N0:113:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:113:
CCTTCTGATC YTTGTTTGCT G 21
(2) INFORMATION FOR SEQ ID N0:114:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS:.single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:114:
GGAACACTGA KTCTTGATTA G 21
(2) INFORMATION FOR SEQ ID N0:115:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:115:
TAGGCTTCTC YTGATAATTG A 21
(2) INFORMATION FOR SEQ ID N0:116:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:116:
TCTTAAAATA MTTGGCTTGT A 21
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(2) INFORMATION FOR SEQ ID N0:117: -
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:117:
TAGATCATTA RTAACCTGCC T 21
(2) INFORMATION FOR SEQ ID N0:118:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:
ATGAGGGGAA MAAGAAACTA C 21
(2) INFORMATION FOR SEQ ID N0:119:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:119:
TTGACACCAG RAACCCCCCA G 21
(2) INFORMATION FOR SEQ ID N0:120:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:120:
TGTTTTAAAT RTTAGGGACA A 21
(2) INFORMATION FOR SEQ ID N0:121:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:121:
GTAAGCATAG YAATGTAGCA G 21
(2) INFORMATION FOR SEQ ID N0:122:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:122:
GGCTCTTTCT KCAACCTTTC C 21
(2) INFORMATION FOR SEQ ID N0:123:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:123:
GACCCAGGTT RTGAGTTTTC C 21
(2) INFORMATION FOR SEQ ID N0:124:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:124:
GACAGAATGA YATATGAAAA G 21
(2) INFORMATION FOR SEQ ID N0:125:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
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(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:125:
TGTGTGACAC YGAGAAGCCC A 21
(2) INFORMATION FOR SEQ ID N0:126:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:126:
AGTACTGGAC MAAGTACCAG G 21
(2) INFORMATION FOR SEQ ID N0:127:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:127:
CCTGGGAGCA RGTATTGCAT T 21
(2) INFORMATION FOR SEQ ID N0:128:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:128:
AGATTTGAGG YCTCAGGTCC C 21
(2) INFORMATION FOR SEQ ID N0:129:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:129:
TGTCAATGTC RCATGATAAG C 21
(2) INFORMATION FOR SEQ ID N0:130:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:130:
TTGCCCCAGT KTTCTCCGGG C 21
(2) INFORMATION FOR SEQ ID N0:131:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:131:
TATGAGCAGC RTAGGGAGTG G 21
(2) INFORMATION FOR SEQ ID N0:132:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C.) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:132:
AGTTGACTGA AAAANTAAAT AAGAC 25
(2) INFORMATION FOR SEQ ID N0:133:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: Other .
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:133:
ATTCAAATAG SCTCTAGAAA C 21
(2) INFORMATION FOR SEQ ID N0:134:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:134:
CCCAGAATTT MATATCCATT C 21
(2) INFORMATION FOR SEQ ID N0:135:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:135:
TGACCCAACA RAAACTCACT G 21
(2) INFORMATION FOR SEQ ID N0:136:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:136:
CCAGAATATA WCATCAGCCC T 21
(2) INFORMATION FOR SEQ ID N0:137:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:137:
CATCAGCCCT WCTGAGGAGA T 21
(2) INFORMATION FOR SEQ ID N0:138:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:138:
CCAGAACAGA YTTTATTCTG T 21
(2) INFORMATION FOR SEQ ID N0:139:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:139:
TTCAGCCATC YTTCCAGTTG T 21
(2) INFORMATION FOR SEQ ID N0:140:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:140:
TCACTAACTC WAAAACGACA T 21
(2) INFORMATION FOR SEQ ID N0:141:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:141:
AACTCAAAAA YGACATCCTC C 21
(2) INFORMATION FOR SEQ ID N0:142:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xiy SEQUENCE DESCRIPTION: SEQ ID N0:142:
GAACTGCACA RGTTGCACAC T 21
(2) INFORMATION FOR SEQ zD N0:143:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:143:
TTGTTCCATG SACTACCTCC T 21
(2) INFORMATION FOR SEQ ID N0:144:
(iy SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:144:
ACAGCAGGCA YTCAACAAAT T 21
(2) INFORMATION FOR SEQ ID N0:145:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: Other -
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:145:
TTATTTTTGG STTTGTTTTA A 21
(2) INFORMATION FOR SEQ ID N0:146:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:146:
TAGGCTGTTC YCTGCCATCA C 21
(2) INFORMATION FOR SEQ ID N0:147:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:147:
GTGCTCTGGG MCACACAGCT C 21
(2) INFORMATION FOR SEQ ID N0:148:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D} TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:148:
AGACCCGATA RGAGCTCCTT C 21
(2) INFORMATION FOR SEQ ID N0:149:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:149:
CATCTTGCGC RGTCATGTAA G 21
(2) INFORMATION FOR SEQ ID N0:150:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:150:
CAGCACAGCT RTTCCCTCAA A 21
(2) INFORMATION FOR SEQ ID N0:151:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:151:
TTTGGAAACA YGGTGAAGTA T 21
(2) INFORMATION FOR SEQ ID N0:152:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:152:
ACACGGTGAA RTATTGTCTC C 21
(2) INFORMATION FOR SEQ ID N0:153:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:153:
AAAAGTGGAT MCTCTGCAAA C 21
(2) INFORMATION FOR SEQ ID N0:154:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:154:
CTTCAAATGC RGCTATTAAA G 21
(2) INFORMATION FOR SEQ ID N0:155:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:155:
CCTGGGAGCA YGGTAAATCA G 21
(2) INFORMATION FOR SEQ ID N0:156:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:156:
TGAAAATGTC RCTTTCTCAC CT 22
(2) INFORMATION FOR SEQ ID N0:157:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:157:
CCTGATATTT RCCAACAAGA A 21
(2) INFORMATION FOR SEQ ID N0:158:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:158:
AAAGGGTTAG YTTGTCCCCT T 21
(2) INFORMATION FOR SEQ ID N0:159:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:159:
TGAAAATAAA ASACAATTTT TT 22
(2) INFORMATION FOR SEQ ID N0:160:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:160:
CTGCTGTGGA CGAATAGG 18
(2) INFORMATION FOR SEQ ID N0:161:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:161:
TCAATATAAT CTTGCTTAAC TTGG 24
(2) INFORMATION FOR SEQ ID N0:162:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:162:
GACCTGTTTG GGTTGATTTC AG 22
(2) INFORMATION FOR SEQ ID N0:163:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:163:
GTTTCTTACA GTGTCTTGCT ATCACATCAC C 31
(2) INFORMATION FOR SEQ ID N0:164:
(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:164:
GAGGACTGGC AGTACCAAGT AAAC 24
(2) INFORMATION FOR SEQ ID N0:165:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:165:
GTTTCTTTGG TTCATTCTAA GATGGCTGG 29
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(2) INFORMATION FOR SEQ ID N0:166:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:166:
GCTGAGGCAG GAGAAAAGAC AAG 23
(2) INFORMATION FOR SEQ ID N0:167:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:167:
GTTTCTTCAT GCAAAGGTCA GGAGGTAGG 29
(2) INFORMATION FOR SEQ ID N0:168:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:168:
GTTGCTTCCA GACGAGGTAC ATG 23
(2) INFORMATION FOR SEQ ID N0:169:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:169:
GTTTCTTCAA TGGCTCCACA AACATCTCTG 30
(2) INFORMATION FOR SEQ ID N0:170:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:170:
AGGTTTAGGG GACAGGGTTT GG 22
(2) INFORMATION FOR SEQ ID N0:171:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:171:
GTTTCTTTCC TGGCTAACAC GGTGAAATC 29
(2) INFORMATION FOR SEQ ID N0:172:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:172:
GTTTCTTATT GCCTCCTCCC AAAATTC 27
(2} INFORMATION FOR SEQ ID N0:173:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:173:
AGAGGCCACT GGAAGACGAA 20
(2} INFORMATION FOR SEQ ID N0:174:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:174:
AACTGGAGTC AGGCAAAACG TG 22
(2) INFORMATION FOR SEQ ID N0:175:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:175:
GTTTCTTTGG CTGGTAAGGA AAGAAACCAC 30
(2) INFORMATION FOR SEQ ID N0:176:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:176:
GGCTAGGTTC ATAAACTCTG TGCTG 25
(2) INFORMATION FOR SEQ ID N0:177:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:177:
GTTTCTTGAT TGTTTGAGAT CCTTGACCCA G 31
(2) INFORMATION FOR SEQ ID N0:178:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:178:
GCCGAAATCA CAACACTGCA TC 22
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(2) INFORMATION FOR SEQ ID N0:179: w
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:179:
GTTTCTTGAT TCTGCTCTTA CTCTTGCCCC 30
(2) INFORMATION FOR SEQ ID N0:180:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:180:
GTAATAGAAC CAAAGGGCTG AGAC 24
(2) INFORMATION FOR SEQ ID NO:181:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:181:
GTTTCTTCGG AGTCAGACCT TACATTGTTG AG 32
(2) INFORMATION FOR SEQ ID N0:182:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:182:
ATCTCCCTGC TACCCACCTT 20
(2) INFORMATION FOR SEQ ID N0:183:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:183:
GTTTCTTGTT TTCAGTGAGT TTCTGTTGGG 30
(2) INFORMATION FOR SEQ ID N0:184:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:184:
GTGTGCCAAA CAACATTTGC 20
(2) INFORMATION FOR SEQ ID N0:185:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:185:
GTTTCTTCAA GCCATCAAGC TAGAGTGG 28
(2) INFORMATION FOR SEQ ID N0:186:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:186:
GGGCTTTTAA ACCCTTATTT AACC 24
(2) INFORMATION FOR SEQ ID N0:187:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
-1$~-
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:187: -
GTTTCTTAGG TGATCTCAGA GCCACTCA 28
(2) INFORMATION FOR SEQ ID N0:188:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:188:
AGGGCAGGTG GGAACTTACT 20
(2) INFORMATION FOR SEQ ID N0:189:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:189:
GTTTCTTTGG AGTCAGTTGA GCTTTCTACC 30
(2) INFORMATION FOR SEQ ID N0:190:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:190:
TGAACTTGCC TACCTCCCAG 20
(2) INFORMATION FOR SEQ ID N0:191:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:191:
GTTTCTTAGC ATATATCCTT ACACAAGCAC A 31
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(2) INFORMATION FOR SEQ ID N0:192: -
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:192:
CATGGTTCCA AAGGCAAGTT 20
(2) INFORMATION FOR SEQ ID N0:193:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:193:
GTTTCTTTTG AGGCTGAATG AGCTGTG 27
(2) INFORMATION FOR SEQ ID N0:194:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:194:
ACAGGTGGGA AGACTGAATG TC 22
(2) INFORMATION FOR SEQ ID N0:195:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:195:
GTTTCTTGCA GTACACATCA CATGACCTTG 30
(2) INFORMATION FOR SEQ ID N0:196:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
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(B} TYPE: nucleic acid _
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:196:
GAAATAGGCG GAAACTGGTT C 21
(2) INFORMATION FOR SEQ ID N0:197:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:197:
GTTTCTTCGT TGTGGTTGTT CAGAAAGG 28
(2) INFORMATION FOR SEQ ID N0:198:
(i} SEQUENCE CHARACTERISTICS:
(A} LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:198:
GGTCAAGTGT TCAGAACGCA TC 22
(2) INFORMATION FOR SEQ ID N0:199:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:199:
GTTTCTTGCA GGGATTATGC TAGGTCTGTA G 31
(2) INFORMATION FOR SEQ ID N0:200:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:200: _
AGCACTTCTG AGGAAGGGAC AC 22
(2) INFORMATION FOR SEQ ID N0:201:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:201:
GTTTCTTAGG GCAGGCAGAC ATACAAAC 28
(2) INFORMATION FOR SEQ ID N0:202:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:202:
GCCAATGTGT TCCTAGAGCG AC 22
(2) INFORMATION FOR SEQ ID N0:203:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STR.ANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:203:
GTTTCTTTTA AAGGGGGTAG GGTGTCACC 29
(2) INFORMATION FOR SEQ ID N0:204:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:204:
GGAAGGGAAA AGGACAAGGT TTTG 24
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(2} INFORMATION FOR SEQ ID N0:205:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:205:
GTTTCTTAGC AAGAGCACTG GTGTAGGAGT C 31
(2) INFORMATION FOR SEQ ID N0:206:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:206:
GCTTTTCAAG CACTTGTCTC 20
(2) INFORMATION FOR SEQ ID N0:207:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:207:
TGGGATTGTG ACTTACCATG 20
(2) INFORMATION FOR SEQ ID N0:208:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:208:
ACTTGGTGTC TTATAGAAAG GTG 23
(2) INFORMATION FOR SEQ ID N0:209:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
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(B) TYPE: nucleic acid _
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:209:
GTTTCTTAGC TGTGTTTGCT GCATC 25
(2) INFORMATION FOR SEQ ID N0:210:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:210:
AGATGTGTGA TGAGATGCAG 20
(2) INFORMATION FOR SEQ ID N0:211:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:211:
GTTTCTTCAA ATAGTGCAAC AAACCC 26
(2) INFORMATION FOR SEQ ID N0:212:
(i) SEQUENCE,CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:212:
TGTCATTCTG AAAGTGCTTC C 21
(2) INFORMATION FOR SEQ ID N0:213:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:213: ,
GTTTCTTCTG TAACTAACGA TCTGTAGTGG TG 32
(2) INFORMATION FOR SEQ ID N0:214:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:214:
TATCAAGGTA ATATAGTAGC CACGG 25
(2) INFORMATION FOR SEQ ID N0:215:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:215:
AGGTCTTTCA TGCAGAGTGG 20
(2) INFORMATION FOR SEQ ID N0:216:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:216:
ATTGCCAAAA CTTGGAAGC 19
(2) INFORMATION FOR SEQ ID N0:217:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:217:
AGGTGACATA TCAAGACCCT G 21
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(2) INFORMATION FOR SEQ ID N0:218: _
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:218:
TTGTCAACGA AGCCCAC 17
(2) INFORMATION FOR SEQ ID N0:219:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:219:
GTTTCTTGCA AGATTGTGTG TATGGATG 2g
(2) INFORMATION FOR SEQ ID N0:220:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:220:
GCTCTCTATG TGTTTGGGTG 20
(2) INFORMATION FOR SEQ ID N0:221:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:221:
AAGAGTACGC TAGTGGATGG 20
(2) INFORMATION FOR SEQ ID N0:222:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 19 base pairs
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(B) TYPE: nucleic acid _
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:222:
TCCATTAGAC CCAGAAAGG 1g
(2) INFORMATION FOR SEQ ID N0:223:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:223:
GTTTCTTCAC CAGGCTGAGA TGTTACT 27
(2) INFORMATION FOR SEQ ID N0:224:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:224:
AATCGTTCCT TATCAGGTAA TTTGG 25
(2) INFORMATION FOR SEQ ID N0:225:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:225:
GTTTCTTCAA AGAAAGCAAT TCCATCATAA CA 32
(2) INFORMATION FOR SEQ ID N0:226:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:226: _
GCATTTGTTG AAGCAAGCGG 20
(2) INFORMATION FOR SEQ ID N0:227:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:227:
CTTTGTTCCT TGGCTGATGG 20
(2) INFORMATION FOR SEQ ID N0:228:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:228:
AATAGTACCA GACACACGTG 20
(2) INFORMATION FOR SEQ ID N0:229:
(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:229:
CAATGGTTCA CAGCCCTTTT 20
(2) INFORMATION FOR SEQ ID N0:230:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:230:
AGCCTGGGAG ACAGAGTGAG 20
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(2) INFORMATION FOR SEQ ID N0:231: _
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:231:
GTTTCTTGCA CTTTTTGGGG AAGGTG 26
(2) INFORMATION FOR SEQ ID N0:232:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:232:
GTTCCTCCCT TCCCTCTCC 19
(2) INFORMATION FOR SEQ ID N0:233:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:233:
GTTTCTTTCA GGGACTGGAT TGTAG 25
(2) INFORMATION FOR SEQ ID N0:234:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:234:
GTGTTCTTTA TGTGTAGTTC 20
(2) INFORMATION FOR SEQ ID N0:235:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:235:
GTTTCTTGGC AACAGAGTGA GACTCA 26
(2) INFORMATION FOR SEQ ID N0:236:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:236:
GTGACATCCA GTGTTGGGAG 20
(2) INFORMATION FOR SEQ ID N0:237:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:237:
GTTTCTTCCT AAGCAAGCAA GCAATCA 27
(2) INFORMATION FOR SEQ ID N0:238:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:238:
AAAGGCAATT GGTGGACA 18
(2) INFORMATION FOR SEQ ID N0:239:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:239: -
GTTTCTTTTC AATCCTTGAT GCAAAGT 27
(2) INFORMATION FOR SEQ ID N0:240:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:240:
GGTGACAGAG CAAGATTTCG 20
(2) INFORMATION FOR SEQ ID N0:241:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:241:
GTTTCTTGTA GAGTTGAGGG AGCAGC 26
(2) INFORMATION FOR SEQ ID N0:242:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C} STRANDEDNESS: single
(D} TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:242:
CATCCATCTC ATCCCATCAT 20
(2) INFORMATION FOR SEQ ID N0:243:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:243:
GTTTCTTTTC ACCCTACTGC CAACTTC 27
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(2) INFORMATION FOR SEQ ID N0:244:
(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:244:
CCGCCATTTT AGAGAGCATA 20
(2) INFORMATION FOR SEQ ID N0:245:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:245:
GTTTCTTTTC TGGGACAATT GGTAGGA 27
(2) INFORMATION FOR SEQ ID N0:246:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:246:
TTTGTGTTAT TATTTCAGGT GC 22
(2) INFORMATION FOR SEQ ID N0:247:
(i} SEQUENCE CHARACTERISTICS:
(A} LENGTH: 30 base pairs
(B} TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:247:
GTTTCTTGTT TTTTGTTTCA GTTTAGGAAC 30
(2) INFORMATION FOR SEQ ID N0:248:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:248:
CATACCCAAA TCGTTCTCTT CCTC 24
(2) INFORMATION FOR SEQ ID N0:249:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:249:
GTTTCTTGGA AAAGCAAAGG CATCGTAGAG 30
(2) INFORMATION FOR SEQ ID N0:250:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:250:
TACTAACCAA AAGAGTTGGG G 21
(2) INFORMATION FOR SEQ ID N0:251:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:251:
CTATCATTCA GAAAATGTTG GC 22
(2) INFORMATION FOR SEQ ID N0:252:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:252: _
GTATGGCAGT AGAGGGCATG 20
(2) INFORMATION FOR SEQ ID N0:253:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:253:
AAGGTTACAT TTCAAGAAAT AAAGT 25
(2) INFORMATION FOR SEQ ID N0:254:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:254:
CTGTTCAGGC CTCAATATAT ACC 23
(2) INFORMATION FOR SEQ ID N0:255:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:255:
AAGAGGATAG GTGGGGTTTG 20
(2) INFORMATION FOR SEQ ID N0:256:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:256:
CCTCCCACCT AGACACAAT 19
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(2) INFORMATION FOR SEQ ID N0:257: -
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:257:
ATATGATCTT TGCATCCCTG 20
(2) INFORMATION FOR SEQ ID N0:258:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:258:
AAGAAAGACC TGGAAGGAAT 20
(2) INFORMATION FOR SEQ ID N0:259:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:259:
AAACAGCAAA ACCTCATCTC 20
(2) INFORMATION FOR SEQ ID N0:260:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: sirigle
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:260:
CCACCACTTA TTACCTGCAT 20
(2} INFORMATION FOR SEQ ID N0:261:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:261:
TGAATGAATG AATGAACGAA 20
(2) INFORMATION FOR SEQ ID N0:262:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:262:
AACTGTGATT GTGCCACTGC ACTC 24
(2) INFORMATION FOR SEQ ID N0:263:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:263:
GTTTCTTCAC CGCCTTTATC CCTCAAATG 29
(2) INFORMATION FOR SEQ ID N0:264:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:264:
GATGGGTGGA GGGCAGTTAA AG 22
(2) INFORMATION FOR SEQ ID N0:265:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:265:
GTCAAGCAAC TTGTCCAAGG CTAC 24
(2) INFORMATION FOR SEQ ID N0:266:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:266:
CAGGCTATCA GTTTCCTTTG GAG 23
(2) INFORMATION FOR SEQ ID N0:267:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:267:
GGCAGGTAAT ACTGGAGAAT TAGG 24
(2) INFORMATION FOR SEQ ID N0:268:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:268:
GACGGATCTC AGAGCCACTC 20
(2) INFORMATION FOR SEQ ID N0:269:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:269:
GTTTCTTAAA AGATAAGGGC TTTTAAACC 29
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(2) INFORMATION FOR SEQ ID N0:270:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY; linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:270:
AGTTTCACAG CTTGTTATGG 20
(2) INFORMATION FOR SEQ ID N0:271:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:271:
GGTTGATGAA GTGAGACTTT 20
(2) INFORMATION FOR SEQ ID N0:272:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:272:
ATGGTGGATG CATCCTGTG 19
(2) INFORMATION FOR SEQ ID N0:273:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:273:
GTTTCTTGTA TTGACTCCTC CTCTGC 26
(2) INFORMATION FOR SEQ ID N0:274:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
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(B) TYPE: nucleic acid -
(C) STR.ANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:274:
CAGTAAACAT 10
(2) INFORMATION FOR SEQ ID N0:275:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:275:
TGTTGAGTGG 10
(2) INFORMATION FOR SEQ ID N0:276:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:276:
TCTCCTCAAT GTGCATGT 18
(2) INFORMATION FOR SEQ ID N0:277:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(By TYPE: nucleic acid
(Cy STRANDEDNESS: single
(D) TOPOLOGY: linear
(iiy MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:277:
ATTCTACATA 10
(2) INFORMATION FOR SEQ ID N0:278:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
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(B} TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:278:
GTGTTTGCAT 10
(2) INFORMATION FOR SEQ ID N0:279:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:279:
ACAAGTTGGC 10
(2) INFORMATION FOR SEQ ID N0:280:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:280:
TAGTACCAGA 10
(2) INFORMATION FOR SEQ ID N0:281:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:281:
TACATCCAAG AAAA 14
(2) INFORMATION FOR SEQ ID N0:282:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
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(B) TYPE: nucleic acid -
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:282:
GAGACTCTGA CAAATATATA TA 22
(2) INFORMATION FOR SEQ ID N0:283:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:283:
TGTTGATCGC CAAACCAAAA TC 22
(2) INFORMATION FOR SEQ ID N0:284:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:284:
AATGCATGTA TGTATATGGT GTGGTATGTG TACATATG 38
(2) INFORMATION FOR SEQ ID N0:285:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:285:
CCTCCCAGAA CAATCATGAT AA 22
(2) INFORMATION FOR SEQ ID N0:286:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 86 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:286: --
AGACAGTCTC AAAAAATATT TTAAAGAAAA AGCTGGATAA ATAACTAGCT TTAAGAAAAT 60
AAGAAGAAAA AGAAAGAAGA AAGTAA 86
(2) INFORMATION FOR SEQ ID N0:287:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 86 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:287:
AACTAGCTTT AAGAAAATAA GAAGAAAAAG AAAGAAGAAA GTAAGAAAGA GAAAGAAAAG 60
AAAGAAAAGA AAGAGGAATG ATTGAC 86
(2) INFORMATION FOR SEQ ID N0:288:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:288:
CGCGCACATA CACCCTTTCT CT 22
(2) INFORMATION FOR SEQ ID N0:289:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:289:
CAGTAAACAT CATGTTGAGT GG 22
(2) INFORMATION FOR SEQ ID N0:290:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:290: -
TCTCCTCAAT GTGCATGTGT GCATGAGTGC ACATTCTACA TA 42
(2) INFORMATION FOR SEQ ID N0:291:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:291:
GTGTTTGCAT GTTGTACAAG TTGGC 25
(2) INFORMATION FOR SEQ ID N0:292:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
{B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:292:
TAGTACCAGA CACGTGCAGG CAAGCGCACC ATACATCCAA GAAAA 45
(2) INFORMATION FOR SEQ ID N0:293:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:293:
GGAGGCTGAG CAGGGGTGCC 20
(2) INFORMATION FOR SEQ ID N0:294:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:294:
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ACTCCCACAG GTACCTGCAG -. 20
(2) INFORMATION FOR SEQ ID N0:295:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:295:
CTGCCCTCAC GTAAGCGCCT 20
(2) INFORMATION FOR SEQ ID N0:296:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:296:
GCTGTTGCAG GGTAATGTTG 20
(2) INFORMATION FOR SEQ ID N0:297:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:297:
CATCAGACAG GTGCGTACA 19
(2) INFORMATION FOR SEQ ID N0:298:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:298:
GGCTGGTGAG GAGGGGCTGA 20
(2) INFORMATION FOR SEQ ID N0:299:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:299:
CGCTCTGTGG GTGAGCTTCA 20
(2) INFORMATION FOR SEQ ID N0:300:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:300:
TGTGGAATAG CCCAATTACA 20
(2) INFORMATION FOR SEQ ID N0:301:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:301:
AGGGTGCTGA GTGAGTAGTA 20
(2) INFORMATION FOR SEQ ID N0:302:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:302:
TTCTTTTCAG GCCCTCGTGT 20
(2) INFORMATION FOR SEQ ID N0:303:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
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(D} TOPOLOGY: linear -
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:303:
TGCTGACCCG GTATGGTGGT 20
(2) INFORMATION FOR SEQ ID N0:304:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:304:
TTTGGTGCAG CCTGTGACTC 20
(2) INFORMATION FOR SEQ ID N0:305:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:305:
CGCACACAAG GTCAGTGTTC 20
(2) INFORMATION FOR SEQ ID N0:306:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:306:
TCTTTCCCAG GTTACTCCTT 20
(2) INFORMATION FOR SEQ ID N0:307:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B} TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:307:
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ATCAAAGACT GTAAGTAACC - 20
(2) INFORMATION FOR SEQ ID N0:308:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:308:
TCTATTTCAG ATGCTGATTC 20
(2} INFORMATION FOR SEQ ID N0:309:
(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:309:
AGTAGAACAA GTAAGTGCAG 20
(2) INFORMATION FOR SEQ ID N0:310:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:310:
TTTTCAAAAG GCCTCCAAAG 20
(2) INFORMATION FOR SEQ ID N0:311:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:311:
GAGCCCTGAG GTAAGTTAAT 20
(2) INFORMATION FOR SEQ ID N0:312:
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(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 20 base pairs
(B} TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:312:
GCTTTTTCAG ATACTACTAT 20
(2) INFORMATION FOR SEQ ID N0:313:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:313:
TAACATGTTC AACTGTCTGT 20
(2) INFORMATION FOR SEQ ID N0:314:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:314:
TGTTATATGC ATTTATCTTC 20
(2) INFORMATION FOR SEQ ID N0:315:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:315:
GGTAAATGAG GTAAGTCCTG 20
(2) INFORMATION FOR SEQ ID N0:316:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
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(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:316:
TCTTGTTAAG ATCGCTCTCT 20
(2) INFORMATION FOR SEQ ID N0:317:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:317:
CCTTGCCCAG GTTCTCTTAA 20
(2) INFORMATION FOR SEQ ID N0:318:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:318:
GCAATCGCAC CTGCACACCC 20
(2) INFORMATION FOR SEQ ID N0:319:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:319:
ACTGCCCATT TCTGGTAAAG 20
(2) INFORMATION FOR SEQ ID N0:320:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:320:
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CCCCTAACAG ATCATGATTC - 20
(2) INFORMATION FOR SEQ ID N0:321:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B} TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:321:
ACGTGCAATG GTAAGAGGGC 20
(2) INFORMATION FOR SEQ ID N0:322:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:322:
TGTTTTGCAG TTTCCAGTGG 20
(2) INFORMATION FOR SEQ ID N0:323:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:323:
AAGTGGAACG GTGACTCTCT 20
(2) INFORMATION FOR SEQ ID N0:324:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:324:
TCCTTCACAG GCCAGTGCAG 20
(2) INFORMATION FOR SEQ ID N0:325:
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(i) SEQUENCE CHARACTERISTICS: -
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:325:
GAACAAACTG GTGAGTAGTA 20
(2) INFORMATION FOR SEQ ID N0:326:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:326:
TTTTTTGTAG AGCCTTCCAT 20
(2) INFORMATION FOR SEQ ID N0:327:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:327:
AGCACAGTAG GTAACTAACT 20
(2) INFORMATION FOR SEQ ID N0:328:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:328:
ATGGCCACAG ATTTGTTGGA 20
(2) INFORMATION FOR SEQ ID N0:329:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
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(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:329:
CTTCCTGTTG GTAAGCTGTC 20
(2) INFORMATION FOR SEQ ID N0:330:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:330:
TTCTCCTTAG CAGAGTCACC 20
(2) INFORMATION FOR SEQ ID N0:331:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:331:
AAAAAGCACA GTAAGTTGGC 20
(2) INFORMATION FOR SEQ ID N0:332:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:332:
TTTTCATCAG ACCCGAGAGG 20
(2) INFORMATION FOR SEQ ID N0:333:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:333:
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GAGCTATGAG GTGAGGAGTT _ 20
(2) INFORMATION FOR SEQ ID N0:334:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:334:
TTTGTTACAG ATATTACTAC 20
(2) INFORMATION FOR SEQ ID N0:335:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:335:
AGCCTGGAAA TGCGTGTTTC 20
(2) INFORMATION FOR SEQ ID N0:336:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:336:
CGAGAATTCA CTCGAGCATC AGG 23
(2) INFORMATION FOR SEQ ID N0:337:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STR.ANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:337:
CCTGATGCTC GAGTGAATTC T 21
-ig$-
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(2) INFORMATION FOR SEQ ID N0:338: _
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 848 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii} MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 1...848
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:338:
ATG ATT CTG GAA GGA AGT GGT GTA ATG AAT CTC AAC CCA GCC AAC AAC 48
Met Ile Leu Glu Gly Ser Gly Val Met Asn Leu Asn Pro Ala Asn Asn
1 5 10 15
CTC CTT CAC CAG CAA CCA GCC TGG CCG GAC AGC TAC CCC ACA TGC AAT 96
Leu Leu His Gln Gln Pro Ala Trp Pro Asp Ser Tyr Pro Thr Cys Asn
20 25 30
GTT TCC AGC GGT TTT TTT GGA AGC CAG TGG CAT GAA ATC CAC CCT CAG 144
Val Ser Ser Gly Phe Phe Gly Ser Gln Trp His Glu Ile His Pro Gln
35 40 45
TAC TGG ACC AAA TAC CAG GTG TGG GAA TGG CTG CAG CAC CTC CTG GAC 192
Tyr Trp Thr Lys Tyr Gln Val Trp Glu Trp Leu Gln His Leu Leu Asp
50 55 60
ACC AAC CAG CTA GAC GCT AGC TGC ATC CCT TTC CAG GAG TTC GAC ATT 240
Thr Asn Gln Leu Aap Ala Ser Cys Ile Pro Phe Gln Glu Phe Asp Ile
65 70 75 80
AGC GGA GAA CAC CTG TGC AGC ATG AGT CTG CAG GAG TTC ACG AGG GCA 288
Ser G1y Glu His Leu Cys Ser Met Ser Leu Gln Glu Phe Thr Arg Ala
85 90 95
GCA GGC TCA GCT GGG CAG CTG CTC TAC AGC AAC CTA CAG CAT CTC AAG 336
Ala Gly Ser Ala Gly Gln Leu Leu Tyr Ser Asn Leu Gln His Leu Lys
100 105 110
TGG AAC GGC CAA TGC AGC AGT GAC CTT TTC CAG TCC GCA CAC AAT GTC 384
Trp Asn Gly Gln Cys Ser Ser Asp Leu Phe Gln Ser Ala His Asn Val
115 120 125
ATT GTC AAG ACT GAA CAA ACC GAT CCT TCC ATC ATG AAC ACA TGG AAA 432
Ile Val Lys Thr Glu Gln Thr Asp Pro Ser Ile Met Asn Thr Trp Lys
130 135 140
GAA GAA AAC TAT CTC TAT GAT CCC AGC TAT GGT AGC ACA GTA GAT CTG 480
Glu Glu Asn Tyr Leu Tyr Asp Pro Ser Tyr Gly Ser Thr Val Asp Leu
145 150 155 160
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TTGGACAGT AAG TTC TGCCGG GCTCAG TCC ACA_ACC TCC 528
ACT ATC ATG
LeuAspSer Lys Phe CysArg AlaGln SerMet ThrThrSer
Thr Ile
165 170 175
AGTCACCTT CCA GCA GAGTCA CCTGAT AAAAAG GAGCAAGAC 576
GTT ATG
SerHisLeu Pro Ala GluSer ProAsp LysLys GluGlnAsp
Val Met
180 185 190
CACCCTGTA AAG CAC ACCAAA AAGCAC CCAAGA GGCACTCAC 624
TCC AAC
HisProVal Lys His ThrLys LysHis ProArg GlyThrHis
Ser Asn
195 200 205
TTATGGGAG TTC CGA GACATT CTCTTG CCAGAC AAGAACCCA 672
ATC AGC
LeuTrpGlu Phe Arg AspIle LeuLeu ProAsp LysAsnPro
Ile Ser
210 215 220
GGGCTGATC AAA GAA GACCGT TCGGAA ATCTTC AGGTTCCTG 720
TGG GGC
GlyLeuIle Lys Glu AspArg SerGlu IlePhe ArgPheLeu
Trp Gly
225 230 235 240
AAGTCAGAA GCT GCT CAGCTG TGGGGG AAGAAA AATAACAGT 768
GTG AAA
LysSerGlu Ala Ala GlnLeu TrpGly LysLys AsnAsnSer
Val Lys
245 250 255
AGCATGACA TAC AAG CTCAGC CGGGCT AGATAT TACTACAAA 816
GAG ATG
SerMetThr Tyr Lys LeuSer ArgAla ArgTyr TyrTyrLys
Glu Met
260 265 270
CGAGAAATC CTG CGT GTGGAT GGACGA 848
GAA CG
ArgGluIle Leu Arg ValAsp GlyArg
Glu Arg
275 280
(2) INFORMATION FOR SEQ ID N0:339:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 283 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:339:
Met Ile Leu Glu Gly Ser Gly Val Met Asn Leu Asn Pro Ala Asn Asn
1 5 10 15
Leu Leu His Gln Gln Pro Ala Trp Pro Asp Ser Tyr Pro Thr Cys Asn
20 25 30
Val Ser Ser Gly Phe Phe Gly Ser Gln Trp His Glu Ile His Pro Gln
35 40 45
Tyr Trp Thr Lys Tyr Gln Val Trp Glu Trp Leu Gln His Leu Leu Asp
5p 55 60
Thr Asn Gln Leu Asp Ala Ser Cys Ile Pro Phe Gln Glu Phe Asp Ile
65 70 75 80
Ser Gly Glu His Leu Cys Ser Met Ser Leu Gln Glu Phe Thr Arg Ala
-1 g~-
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85 90 95
Ala Gly Ser Ala Gly Gln Leu Leu Tyr Ser Asn Leu Gln His Leu Lys
100 105 110
Trp Asn Gly Gln Cys Ser Ser Asp Leu Phe Gln Ser Ala His Asn Val
115 120 125
Ile Val Lys Thr Glu Gln Thr Asp Pro Ser Ile Met Asn Thr Trp Lys
130 135 140
Glu Glu Asn Tyr Leu Tyr Asp Pro Ser Tyr Gly Ser Thr Val Asp Leu
145 150 155 160
Leu Asp Ser Lys Thr Phe Cys Arg Ala Gln Ile Ser Met Thr Thr Ser
165 170 175
Ser His Leu Pro Val Ala Glu Ser Pro Asp Met Lys Lys Glu Gln Asp
180 185 190
His Pro Val Lys Ser His Thr Lys Lys His Asn Pro Arg Gly Thr His
195 200 205
Leu Trp Glu Phe Ile Arg Asp Ile Leu Leu Ser Pro Asp Lys Asn Pro
210 215 220
Gly Leu Ile Lys Trp Glu Asp Arg Ser Glu Gly Ile Phe Arg Phe Leu
225 230 235 240
Lys Ser Glu Ala Val Ala Gln Leu Trp Gly Lys Lys Lys Asn Asn Ser
245 250 255
Ser Met Thr Tyr Glu Lys Leu Ser Arg Ala Met Arg Tyr Tyr Tyr Lys
260 265 270
Arg Glu Ile Leu Glu Arg Val Asp Gly Arg Arg
275 280
-1 gg-
