Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
LYSTl AND ~LYST2 G~Nl~ COMPOSITIONS
AND METHODS OF USl~
1. Background of the Invention
The present application is a continuation in part of U. S. Provisional Patent Application
Serial No. 60/XXX,XXX, filed December 23, 1996 and of U. S. Provisional Patent
Application Serial No. 60/XXX,XXX, filed December 20, 1996, which is a contiuation in
o part of U. S . Provisional Patent Application Serial No 60/011,146, filed February 1, 1996,
the entire contents of which are specifically incorporated herein by reference. The United
States government has certain rights in the present invention pursuant to Grants AI 39651
and SP30-AR 41943 from the National Tn~tit~tes of Health.
15 1.1 Field ofthe Invention
The present invention relates generally to the field of molecular biology. More particularly,
certain embodiments concern methods and compositions comprising novel DNA segm~nt~, and
proteins derived from m~mm~ n species. More particularly, the invention provides Lystl and
Lyst2 gene compositions from murine origins and the homologous LYSTl and LYSTZ gene
2 o compositions from human origins. Various methods for making and using these LYST/Lyst DNA
~e~m~.ntc, native peptides and synthetic protein derivatives are disclosed, such as, for example, the
use of DNA segm~nts as diagnostic probes and temrl~t~s for protein production, and the use of
LYSTl, Lystl, LYST2, and Lyst2 proteins, fusion protein carriers and Lyst-derived peptides in
various pharrnacological and immnnc~logical applications.
1.2 Description of the Related Art
1.2.1 Chediak-l~ chi (C~) Syndrome
Chediak-Higashi syndrome (CHS) is an autosomal recessive, immlme deficiency disease
that maps on cl~ )su,~l~ (Chr~ 1q42-q43 (Goodrich and Holcombe, 1995; Barrat et al. 1996; Fukai
et al., 1996). Af~ected individuals have giant, perinuclear Iysosomes, defective granulocyte, NK
3 0 and cytolytic T cell function, and die prematurely of infection or mfl~;gn~nc y (Beguez Cesar, 1943;
Blume et al., }968; Wolffet al., 1972; Blume and Wolff, 1972; Root et al., 1972; Roder et al.,
1982; Baetz et aL, 1995). CHS patients also e~ibit partial oculo.,uL~Ieo-ls albinism, platelet storage
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pool deficiencv and neurologic defects such as periphe~al neuropathy and ataxia (Windhorst et al.,
1968;Meyersetal., 1974;Maedaetal., 1989;PettitandBerdal, 1984;MisraetaL, 1991). Recently
it was demonstrated that intr~c~ r protein transport to and from the lysosome is disordered in
CHs(Baetzefal.~ 1995;Brandtefal., 1975;Burkhardtetal., 1993;Zhaoetal., 1994). Such
5 functional defects in secretory Iysosomes of granular cells (leukocytes, melanocytes,
m.~g~k~ryocytes and cerebellar Purkinje cells) provide a unifying hypothesis that can explain the
diverse clinical features of CHS (Griffiths, 1996).
As an z~nfececl~nt to identification of the human CHS gene, the inventors undertook
positional cloning of the mouse mutation beige (bg), which had long been considered homologous
0 to CHS. The clinical and pathologic features of CHS and bg are very similar and bg maps on
proximal mouse Chr 13 within a linkage group conserved with human chromosome lq42-q43 (the
position of the CHS locus) (Jenkins etaL, 1991). Additional evidence that human C~IS and bg
mice were homologous disorders came from interspec;fic genetic complementation studies, which
demonstrated that fusion of bg mouse and human CHS fibroblasts failed to reverse Iysosomal
15 morphologic abnormalities (Penner and Prieur, 1987).
Recently the inventors' group and one other succeeded in identifying the gene that is
defective in bg mice (Perou etal., ~996a). However, the reported bg c~n~ te cDNA sequences
(Lyst and BG) were di~ . Both sequences were isolated from the same yeast artificial
chromosome (~AC) clone. This YAC had been authentic~ted by mapping within the bg critical
20 region and by restoration of normal Iysosomal morphology to bg fibroblasts upon transfection
(Perou et al., 1996a; Perou et aL, 1996b). Furthermore, both of the candidate gene sequences
cont~inf~d mutations in different bg alleles.
1.3 ~eficiencies in the Prior Art
Methods for the tre~tm~-nt and diagnosis of Chediak-Higashi Syndrome have not been
2 5 developed because the sequence of the CH gene has not been identified in mice or hnm~n~.
Despite some recent studies in mice, there is only speculation that a linkage similar to that found
in beige mice might exist in the human gene (Owen, ef al., 19~6). There is some evidence that
indicate that the CH mutation is located in the same gene in mouse, mink and human OEerou and
Kaplan, 1993); however, except for the beige mouse, the locus of the mutation has not been
3 Q identified.
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CHS patients have been reported to suffer from several serious medical conditions,
;n~ rling impaired natural killer cell activity (Haliotis et al., 1980) and defective Iymphocyte-
m~ ted antibody dependent cell medi~ted leukocyte mediated ADCC against tumor cell ta}gets
(Klein, et al., 19~0). Despite the recognition ofthese deficiencies, little progress in tre~tm~nt has
been achieved, mainly because the gene harboring the mutation leading to these impairments has
not yet been identified.
Che~ k-Higashi Syndrome occurs only in a small minority of the population. However,
there is a growing realization ofthe potential role ofthe CH gene product ~LYST1) in developing
tr~tment~ for conditions such as systemic autoimmlme disease and possibly certain types of
l o m~ n~ncy related to the regulation of protein trafficking within cells by the CH gene (LYSTl ).
Therefore, what is lacking in the prior art is the isolation and characterization of the CH gene
from mice and hnm~n~, useful in the development of trf~tm~nt~ and assays for autoimm-m~
diseases such as CHS and certain forms of cancer.
2. Summary of the Invention
Positional cloning ofthe mouse CHS homologous is f~.ilitslted by the existence of
numerous remutations at the bg locus. All have arisen spontaneously, with the exception of the
SB/LeJ-bg allele, which was indnc.ed by radiation. The present invention addresses one or more
of the foregoing or other problems associated with the detection of Chediak-Higashi Syndrome in
hllnnzln.~ Both the mouse gene and the homologous human have been cloned and sequenced.
2 o The isolation and sequencing of the Chediak-Higashi gene (LYST 1 ) from both murine and
human sources has now provided methods of detecting CHS at the gene level, such as by various
assays making use of the gene, gene segment.~ and/or the encoded proteins or polypeptides. In
addition to the practical value, the gene provides a tool for under~t~n-iing and controlling
me~h~ni~m~ of regulation of protein t}affic~king to Iysosomes, and particularly to the contribution
of vesicular sorting to diverse cellular functions. An immetli~tc result ofthe identification ofthe
LYSTl gene is the ability to perform linkage analysis and to identify individuals at risk to have
progeny carrying the mtlt~tecl gene. The inventors have shown that the murine gene, Lys~l, and
BG sequences are derived from a single gene with alternatively spliced mRNAs. In an important
embodiment, the inventors have also irl~ntified the human homolog of the bg gene ~Lyst 1), LYSTI .
3 0 LYSTl maps within the CHS critical region and is mllt~ted in several CHS patients.
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i:. l L YST and Lyst Gene Compositions
As used herein, the term "DNA segm~nt" refers to a DNA molecule that has been isolated
free of total genomic DNA of a particular species. Therefore, a DNA segment encoding
LYST/Lyst refers to a DNA se~m~nt that contains LYST or Lyst coding sequences yet is isolated
away from, or purified free from, total genomic DNA of the species from which the DNA
segm~.nt is obtained. Tncl~l-led within the term "DNA segment", are DNA se~m~ntc and smaller
fragments of such segment.c, and also recombinant vectors, inclT~rling, for example, pl~cmi(1.c,
cosmids, phagemids, phage, viruses, and the like. Preferred LYST genes are the T.YSI'l and
LYST2 genes from human origin, while p. t~rel I ~d Lyst genes are the Lystl and ~'yst2 genes from
murine origin.
Similarly, a DNA segrnent comprising an isolated or purified LYST/Lyst gene refers to a
DNA segment in~ rling a LYST or Lyst coding sequence and, in certain aspects, regulatory
sequences, isolated substantially away from other naturally occurring genes or protein encoding
sequences. In this respect, the terrn "gene" is used for simplicity to refer to a functional protein,
polypeptide or peptide encoding unit. As will be understood by those in the art, this functional
term in~ dçs both genomic sequences, extra-genomic and plasmid-encoded sequences and
smaller engineered gene segm~nt~ that express, or may be adapted to express, proteins,
polypeptides or peptides. Such segrnents may be naturally isolated, or modified synthetically by
the hand of man. Preferred DNAs are those which comprise one or more LYST genes, with human
2 o LYSTI and LYST2 genes being particularly pl ere-l ed, or one or more Lyst genes, with murine
I,ys~l and Lyst2 genes being particularly ple~f~lled.
"Isolated subst~nti~lly away from other coding sequences" means that the gene of interest,
in this case, a gene encoding a LYST/Lyst protein or peptide, forms the significant part of the
coding region of the DNA segment and that the DNA segrnent does not contain large portions of
2 5 naturally-occurring coding DNA, such as large chromosomal fr~grn~nt~ or other functional genes
or polypeptide coding regions. Of course, this refers to the DNA segm~nt as originally isolated,
and does not exclude genes or coding reg;ons later added to the segm~nt by the hand of man.
In particular embodim~-nt~, the invention concerns isolated DNA segm~.nts and
recombinant vectors incorporating DNA sequences that encode a LYST/Lyst species that includes
3 o ~,vithin its amino acid sequence an amino acid sequence ~c~nti~lly as set forth in SEQ ID NO:2, ..
SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: l 0, SEQ ID NO: 12, or SEO ID
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NO:14. In other particular emborTimentc~the invention concerns isolated DNA segment~ and
reconll,i..a..L vectors incorporating DNA seguences that include within their sequence a nucleotide
sequence ess~.nti~lly as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
SEQ ID NO:9, SEQ ID NO: 11, or SEQ ID NO: 13.
The term "a sequence essentially as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ~D NO:14" means that the
sequence substantially corresponds to a portion of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:8, SEQ I[) NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and has relatively few amino
acids that are not i(lenti~l to, or a biologically functional equivalent of, the amino acids of SEQ
1 0 ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ l:D NO: 12, or
SEQ ID NO: 14. The term "biologically functional equivalent" is well understood in the art and is
further defined in detail herein (for example, see Illustrative Embodiments). Accordingly,
se~l~nces that have between about 70% and about 80%; or more preferably, between about 81%
and about 90%; or even more preferably, between about 91% and about 99%; of amino acids that
are i~l~ntic~l or functionally equivalent to the amino acids SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14 will be sequences that
are "ec~enti~lly as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ
ID NO:10, SEQ ID NO:12, or SEQ ID NO:14".
In certain other embo~lim~nt~, the invention concerns isolated DNA segm-o.nt~ and
2 o recombinant vectors that include within their sequence a nucleic acid sequence css~nti~lly as set
forth in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, or SEQ ID NO:13. The term "ecs~nti~lly as set forth in SEQ ID NO:1, SEQ ID NO:3,
SEQIDNO:5, SEQIDNO:7, SEQIDNO:9, SEQIDNO:11, orSEQII~NO:13" isusedinthe
same sense as described above and means that the nucleic acid sequence subst~nti~lly corresponds
toaportionofSEQIDNO:1, SEQIDNO:3, SEQIDNO:5, SEQIDNO:7, SEQIDNO:9,
SEQ ID NO: 11, or SEQ ID NO: 13 and has relatively few codons that are not identical, or
functionally equivalent, to the codons of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO: 11, or SEQ ID NO: 13. Again, DNA segm~?nt~ that encode
proteins ~libi~ g LYST, Lyst, LYST-like, or Lyst-like activity will be most plere;ll~d.
3 o It will also be understood that amino acid and nucleic acid sequences may include
additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet
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still be essentially as set forth in one ofthe sequences disclosed herein, so long as the seq-lçnce
meets the criteria set forth above, including the mRintçnRn~e of biological protein activity where
protein expression is concerned. The addition of terminal sequences particularly applies to nucleic
acid sequences that may, for example, include various non-coding sequences flRnking either ofthe
5 5' or 3' portions ofthe coding region or may include various upstream or dowll~Ll~all, regulatory
or structural genes.
Naturally, the present invention also encompRc.~es DNA Segm~nt.s that are complf m~ntRry,
or e~ntiRlly complementary, to the sec~uence set forth in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID
NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, or SEQ ID NO:13. Nucleic acid
0 seqnçnces that are "complementary" are those that are capable of base-pairing according to the
standard Watson-Crick complementarity rules. As used herein, the term "complementary
se~uences" means nucleic acid sequences that are substantially complementary, as may be
slc~e~ed by the same nucleotide comparison set forth above, or as defined as being capable of
hybridizing to the nucleic acid segment of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO: 11, or SEQ ID NO: 13 under relatively stringent conditions
such as those described herein.
The nucleic acid ~e~mentc of the present invention, regardless of the length of the coding
sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation
signals, additional restriction enzyme sites, multiple cloning sites, other coding segmçnt~, and the
2 o like, such that their overall length may vary considerably. It is therefore contemplated that a
nucleic acid fragment of almost any length may be employed, with the total length preferably
being limited by the ease of 1~l ~al ~tion and use in the int~nde(i reconlb;llallL DNA protocol. For
example, nucleic acid fragm~nt~ may be prepared that include a short contiguous stretch identical
to or complementary to SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID
2 5 NO:9, SEQ ID NO: l l, or SEQ ID NO: 13, such as about 14 nucleotides, and that are up to about
10,000 or about 5,000 base pairs in length, with segment~ of about 3,000 being plt:rell~d in
certain cases. DNA segm~nts with total lengths of about 2,000, about 1,000, about 500, about
200, about 100 and about 50 base pairs in length (inn.ll~ding all intermediate lengths) are also
contemplated to be useful.
3 o It will be readily understood that "intern~eriiRte lengths", in these contexts, means any
length between the ~uoted ranges, such as 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23, e~c.; 30, 31,
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32, etc.; 50, 51, 52, 53, elc.; 100, 101, 10~, 103, etc.; 150, 151, 152, 153, etc.; inr.lllrling all
integers through the 200-500; 501-1,000; 1,001-2,000; 2,001-3,000; 3,001-5,000; 5,001-10,000
ranges, up to and including sequences of about 12,001, 12,002, 12,003, 13,001, 13,002 and the
like.
It will also be understood that this invention is not limited to the particular nucleic acid
sequences disclosed in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID
NO:9, SEQ ID NO;11, or SEQ ID NO:13, or to the particular amino acid sequences as disclosed
in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID
NO:12, or SEQ ID NO:14. RecolllbinallL vectors and isolated DNA Segmt~.nt~ may therefore
1 o variously include the LYST or Lyst coding regions themselves, coding regions bearing selected
alterations or modifications in the basic coding region, or they may encode larger polypeptides
that nevertheless include LYST, Lyst, LYST-like, or Lyst-like coding regions or may encode
biologically functional equivalent proteins or peptides that have variant amino acids sequences.
If desired, one may also prepare fusion proteins and peptides, e.g, where the LYST or
Lyst coding regions are aligned within the same expression unit with other proteins or peptides
having desired functions, such as for purification or imm-ln~detection purposes (e.g, proteins that
may be purified by affinity chromatography and enzyme label coding regions, respectively).
Recombinant vectors form further aspects of the present invention. Particularly useful
vectors are contemplated to be those vectors in which the coding portion of the DNA segment,
2 o whether encoding a full length protein or smaller peptide, is positioned under the control of a
promoter. The promoter may be in the form of the promoter that is naturally associated with a
LYSTI, Lystl, LYST2, or Lysf2 gene, as may be obtained by isolating the 5' non-coding sequences
located upstream of the coding segm~nt for example, using recombinant cloning and/or pC~M
technology, in connection with the compositions disclosed herein.
In other embo{limf?ntc, it is contemplated that certain advantages will be gained by
positioning the coding DNA segment under the control of a recombinant, or heterologous,
promoter. As used herein, a recombinant or heterologous promoter is intencled to refer to a
promoter that is not norrnally associated with a LYST/Lyst gene in its natural environment. Such
promoters may include LYST or Lyst promoters normally associated with other genes, and/or
3 o promoters isolated from any bacterial, viral, eukaryotic, or m~mm~ n cell. Naturally, it will be
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important to employ a promoter that effectively directs the c;~ ;ssion of the DNA segment in the
cell type, organism, or even animal, chosen for expression. The use of promoter and cell type
combinations for protein expression is generally known to those of skill in the art of molecular
biology, for example, see Sambrook et al., l 989. The promoters employed may be constitutive,
5 or inducible, and can be used under the appropriate conditions to direct high level ~ ssion of
the introduced DNA segmçnt such as is advantageous in the large-scale production of
recombinant proteins or peptides.
Prokaryotic expression of nucleic acid segments of the present invention may be
performed using methods known to those of skill in the art, and will likely comprise ~ ession
10 vectors and promotor sequences such as those obtained from tac, trp, lac, lacW5 or T7. When
expression of the recombinant LYST1 LYST2, Lystl or Lyst2 proteins is desired in eukaryotic
cells, a number of expression systems are available and known to those of skill in the art. An
exemplary eukaryotic promoter system contemplated for use in high-level expression is the Pichia
e,.~lc;ssion vector system (Pharmacia LKB Biotechnology).
In connection with c~y~ssion embodiments to prepare recombinant reco,,,blnallL LYSTl
LYST2, Lystl or Lyst2 proteins and peptides, it is contemplated that longer DN~ cegm~?nt.c will
most often be used, with DNA segment~ encoding the entire LYST1 LYST2, Lystl or Lyst2 or
functional domains, epitopes, ligand binding domains, subunits, etc. being most preferred.
~Iowever, it will be appreciated that the use of shorter DNA s~m~nt.c to direct the expression of
2 0 LYSTl LYST2, Lystl or Lyst2 peptides or epitopic core regions, such as may be used to
generate anti-LYST or Lyst antibodies, also falls within the scope of the invention DNA
se~n~nt~ that encode peptide ~nti~n,c from about 15 to about lOO amino acids in length, or more
preferably, from about 15 to about 50 amino acids in length are cont~n7~ ted to be particularly
useful.
2 ~ The ~YSl' or Lyst genes and DNA segmçntc may also be used in connection with somatic
res~ion in an animal or in the creation of a transgenic animal. Again, in such emborl;nt~nt~ the
use of a rec~ bina1ll vector that directs the ~ t;".,ion of the full length or active LYST/Lyst
protein is particularly contemplated. Expression of a LYS~/Lyst transgene in animals is
particularly contemplated to be useful in the production of anti-LYSTtLyst antibodies for use in
3 o passive immllni7~tinn methods, the detection of LYST/Lyst proteins, and the purification of
LYST/Lyst protein in large quantity.
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In addition to their use in directing the t;~ ssion of LYST/Lyst, the nucleic acid
sequences disclosed herein also have a variety of other uses. ~or example, they also have utility
as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that
nucleic acid segm~nt~ that comprise a sequence region that consists of at least a 14 nucleotide
long contiguous sequence that has the same sequence as, or is complementary to, a 14 nucleotide
long contiguous sequence of S~Q ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ
ID NO:9, SEQ 1~) NO: 11, or SEQ ID NO: 13 will find particular utility. Longer contiguous
identical or compl~m~.nt~ry sequences, e.g, those of about 20, 30, 40, 50, 100, 200, 500, 1000
(inchlfling all intermediate lengths) and even up to full length sequences will also be of use in
0 certain embo~iment~.
The ability of such nucleic acid probes to specifically hybridize to LYST/Lyst-encoding
sequences will enable them to be of use in detecting the presence of complem~.nt~ry sequences in
a given sample. However, other uses are envisioned, in~ ding the use of the sequence
h~ ldLion for the preparation of mutant species primers, or primers for use in pl ~I.al illg other
genetic constructions.
Nucleic acid molecules having sequence regions con~i.cting of contiguous nucleotide
stretches of 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or so, identical or
complelllellLaly to SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,
SEQ ID NO: 11, or SEQ ID NO: 13 are particularly contemplated as hybridization probes for use
2 o in, e.g, Southern and Northern blotting. This would allow LYST/~yst structural or regulatory
genes to be analyzed, both in diverse cell types and also in various bacterial cells. The total size
of fr~gm~nt, as well as the size of the complpm~nt~ry stretch(es), will ~ Iltim~tely depend on the
intçn~ed use or application of the particular nucleic acid segm~nt Smaller fr~gmçnt~ will
generally find use in hybridization embodiments, wherein the length of the contiguous
2 5 compl~ e.l~, y region may be varied, such as between about 14 and about 100 nucleotides, but
larger conti&~-Qus complementarity stretches may be used, according to the length complementary
seq~l~n-.~c one wishes to detect.
- The use of a hybridization probe of about 14-25 nucleotides in length allows the formation
of a duplex molecule that is both stable and selective. Molecules having contiguous
3 o compl~ ellL~Iy sequences over stretches greater than 14 bases in length are generally pre~ell~d,
though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality
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and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid
molecules having gene-complementary stretches of 15 to 25 contiguous nucleotides, or even
longer where desired.
Hybridization probes may be selected from any portion of any of the sequences disclosed
5 herein. All that is required is to review the sequ~nce set forth in SEQ ID NO:l, SEQ ID NO:3,
SEQ ID NO:S, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, or SEQ ID NO:13 and to select
any continuous portion of the sequence, from about 14-25 nucleotides in length up to and
in~ ing the full length sequence, that one wishes to utilize as a probe or primer. The choice of
probe and primer sequences may be governed by various factors, such as, by way of example
0 only, one may wish to employ primers from towards the termini of the total sequence.
The process of selecting and preparing a nucleic acid segment that in~ des a contiguous
sequence from within SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID
NO:9, SEQ ID NO:ll, or SEQ ID NO:13, may alternatively be described as p~ epa, i~lg a nucleic
acid fr~gm~nt Of course, fr~gm~n~.~ may also be obtained by other techniques such as, e.g, by
15 me~h~nical shearing or by restriction enzyme digestion. Small nucleic acid segments or fr~gm~nt,c
may be readily p~ al~d by, for example, directly synt~ ing the fragment by f~hemic~l means, as
is commonly practiced using an automated oligonucleotide synthesi7~or Also, fr~gm~nt.c may be
obtained by application of nucleic acid reproduction technology, such as the PCRTM technology of
U.S. Patent 4,683,202 (incorporated herein by reference), by introducing selected sequences into
2 o recombinant vectors for recomhin~nt production, and by other recombinant DNA techniques
generally known to those of skill in the art of molecular biology.
Accordingly, the nucleotide sequences of the invention may be used for their ability to
selectively form duplex molecules with complen~e,lL~, y stretches of the entire LYST/Lyst gene or
gene fragments. Depending on the application envisioned, one will desire to employ varying
2 5 conditions of hybridization to achieve varying degrees of selectivity of probe towards target
sequence. For applications re~llinng high selectivity, one will typically desire to employ
relatively stringent conditions to form the hybrids, e.g, one will select relatively low salt and/or
high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCI at
tenlpe~L~Ires of 50~C to 70~C. Such selective conditions tolerate little, if any, mi~m~h between
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the probe and the template or target strand, and would be particularly suitab}e for isolating LYST
or ~yst genes.
Of course, for some applications, for example, where one desires to prepare mllt~nt.c
employing a mutant primer strand hybridized to an underlying template or where one seeks to
5 isolate LYST or Lyst sequences from related species, functional equivalents, or the like, less
stringent hybridization conditions will typically be needed in order to allow formation of the
heteroduplex. In these circumstances, one may desire to employ conditions such as about 0.15 M
to about 0.9 M salt, at temperatures ranging from 20~C to 55~C. Cross-hybridizing species can
thereby be readily identified as positively hybridizing signals with respect to control
0 hybridizations. In any case, it is generally appreciated that conditions can be rendered more
stringent by the addition of increasing amounts of formz~micle, which serves to destabilize the
hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be
readily manipulated, and thus will generally be a method of choice depending on the desired
results.
In certain embo~limiont~, it will be advantageous to employ nucleic acid seq~nc~ of the
present invention in ~;o-llbillaLion with an a~prolJ~iate means, such as a label, for determining
hybridization. A wide variety of appropriate indicator means are known in the art, incl~l~ling
fiuorescent, radioactive, c;l~ylllaLic or other ligands, such as avidin/biotin, which are capable of
giving a detect~hle signal. In pl~rt;lled embo~limçnt~, one will likely desire to employ a
2 0 fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of
radioactive or other en~/hol-l"~ 1 undesirable reagents. In the case of enzyme tags, colorimetric
indicator substrates are known that can be employed to provide a means visible to the human eye
or spectrophotometrically, to identify specific hybridization with complf~. "e,~ , y nucleic acid-
co"~ i"g samples.
2 5 In general, it is envisioned that the hybridization probes described herein will be useful
both as reagents in solution hybridization as well as in embodiments employing a solid phase. In
embo-liment~ involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to
a s~1ecte~1 matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific
hybridization with selected probes under desired conditions. The selected conditions will depend
3 o on the particular circum~f~nc~es based on the particular criteria required (depending, for example,
on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization
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probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound
probe molecules, specific hybridization is detected, or even qll~nfit~te~1, by means ofthe label.
2.2 Recombinant ~ost Ce11s ~md Vectors
Particular aspects of the invention concern the use of plasmid vectors for the cloning and
5 expression of reco,nbi,lall~ peptides, and particular peptide epitopes comprising either native, or
site-speciffcally mllt~tec~ LYST or Lyst proteins, peptides, or epitopes. The generation of
recombinant vectors, transformation of host cells, and ~ ssion of recombinant proteins is well-
known to those of skill in the art. Prokaryotic hosts are p,er~, led for expression of the peptide
compositions of the present invention. An example of a pl t;~l l ed prokaryotic host is E. coli, and
in particular, E. coli strains JM101, XL1-Blue~, RR1, LE392, B, X1776 (ATCC3 1537), and
W3110 (F-, ~~, prototrophic, ATCC273325). Alternatively, otherE;nterobacteriaceae species
such as Salmonella ~yphimz~rizlm and Serratia marcescens, or even other Gram-negative hosts
inclll~ling various Pseudomonas species may be used in the recombinant expression of the genetic
constructs disclosed herein.
In general, plasrnid vectors cont~inin~ replicon and control sequences which are derived
from species compatible with the host cell are used in connection with these hosts. The vector
or~ alily carries a replication site, as well as marking sequences which are capable of providing
phenotypic selection in ll~nsrolllled cells. For example, ~. coli may be typically transformed
using vectors such as pBR322, or any of its derivatives (Bolivar ef al., 1977). pBR322 contains
2 o genes for ampicillin and tetracycline resistance and thus provides easy means for identifying
transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also
contain, or be modified to contain, promoters which can be used by the microbial organism for
t;ssion of endogenous proteins.
In addition, phage vectors co~ g replicon and control sequences that are compatible
2~ with the host microol~anislll can be used as transforming vectors in connection with these hosts.
For example, bacteriophage such as ~GEM~M-11 may be utilized in making a recombinant vector
which can be used to transform susceptible host cells such as E. coli LE392.
Those promoters most common~y used in recombinant DNA constTuction include the ,~
l~ct~m~e (penicillinase) and lactose promoter systems (Chang et al., 1978; Itakura et al., 1977;
3 0 Goeddel et al., 1979) or the tryptophan (~rp) promoter system (Goeddel et al., 1980). The use of
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13
recolllbh~allL and native microbial promoters is well-known to those of skill in the art, and details
concerning their nucleotide sequences and specific methodologies are in the public domain,
enabling a skilled worker to construct particular recombinant vectors and expression systems for
the purpose of producing compositions of the present invention.
In addition to the ~l ~r~ d embodiment ~lt;ssion in prokaryotes, eukaryotic microbes,
such as yeast cultures may also be used in conjunction with the methods disclosed herein.
Saccharomyces cerevisiae, or common bakers' yeast is the most commonly used among
eukaryotic microor~ni~mi, although a number of other species may also be employed for such
eukaryotic ~ ssion systems. For expression in Saccharo~nyces, the plasmid YRp7, for
example, is commonlyused (Stinchcomb etal., 1979; ~ingsm~n etal., 1979; Tschemperetal.,
1980). This plasmid already contains the trpL gene which provides a selection marker for a
mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC44076 or
PEP4-1 (Jones, 1977). The presence of the trpL lesion as a characteristic of the yeast host cell
genome then provides an effective environment for cletecting Ll~nsro--llation by growth in the
absence oftryptophan.
Suitable promoting sequences in yeast vectors include the promoters for 3-
phosphoglycerate kinase (~il ,(?~ et al., 1980) or other glycolytic enzymes (Hess et al., 1968;
Holland et al., 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,
pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-
2 o phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose
isomerase, and glucokinase. In constructing suitable expression plasmids, the tellllinalion
sequences associated with these genes are also ligated into the expression vector 3' ofthe
sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
Other promoters, which have the additional advantage of transcription controlled by growth
2 5 conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose
utilization. Any plasmid vector cont~ining a yeast-compatible promoter, an origin of replication,
and tellllill~Lion sequences is suitable.
3 o In addition to microorg~ni~m~, cultures of cells derived from multicellular org~ni~m~ may
also be used as hosts in the routine practice of the disclosed methods. In principle, any such cell
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culture is workable, whether from ve, Leb, ~e or invertebrate culture. ~owever, interest has been
greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture3 has a
become a routine procedure in recent years. Examples of such useful host cell lines are VERO
and HeLa cells, Chinese hamster ovary (CHO) cell lines, and Wl38, B~, COS-7, 293 and
MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of
replication, a promoter located in front of the gene to be expressed, along with any necessary
ribosome binding sites, 3~NA splice sites, polyadenylation site, and L,~lls~ Lional terminator
sequences.
For use in m~mm~ n cells, the control functions on the expression vectors are often
10 obtained from viral material. For example, commonly used promoters are derived from polyoma,
Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early and late promoters of
SV40 virus are particularly useful because both are obtained easily from the virus as a fragment
which also contains the SV40 viral origin of replication (Fiers et al., ~ 978). Smaller or larger
SV40 fr~,~m~nt~ may also be used, provided there is int~.lu(1ed the apploxill,ately ~50 bp sequence
15 ~ n~linp from the HindIII site toward the Bgll site located in the viral origin of }eplication.
Further, it is also possible, and often desirable, to utilize promoter or control sequences normally
associated with the desired gene sequence, provided such control sequences are compatible with
the host cell systems.
The origin of replication may be obtained from either construction of the vector to include
2 o an exogenous origin, such as may be derived from SV40 or other viral (e.g, Polyoma, Adeno,
VSV, BPV) source, or may be obtained from the host cell chromosomal replication mech~ni~m
If the vector is integrated into the host cell chromosome, the latter is often sufficient.
It will be further understood that certain ofthe polypeptides may be present in ql-~ntiti~
below the detection limits of the Coomassie brilliant blue staining procedure usually employed in
2 5 the analysis of SDS/PAGE gels, or that their presence may be masked by an inactive polypeptide
of sirnilar Mr Although not neceq~ . y to the routine practice of the present invention, it is
conteml lated that other detection techniques may be employed advantageously in the
vi.e ~ tion of particular polypeptides of interest. Immunologically-based techniques such as 5
Western blotting using enzymatically-, radiolabel-, or fluorescently-tagged antibodies described
3 o herein are considered to be of particular use in this regard. Alternatively, the peptides of the
present invention may be detectecl by using antibodies of the present invention in combination
,
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- with secondary antibodies having affinity for such primary antibodies. This secondary antibody
may be enzymatically- or radiolabeled, or a}ternatively, fluorescently-, or colloidal gold-tagged.
Means for the labeling and detection of such two-step secondary antibody techni~ues are well-
known to those of skill in the art.
5 2.3 Recombinarlt E~pression of one or more LYST Gene Products
As used throughout, a "LYST/Lysl" gene is int~ntiecl to mean a LYST or Lysf gene from a
m~mm~ n source, with human LYSTand murine Lyst genes being most pl~rel-ed. In keeping
with the genetic nom~n~l~tl-re sçhemçs known to those of skill in the art, 'CLYST' genes are those
genes derived from human sources while "Lyst" genes are those genes derived from murine
10 sources. Thus, LYS~l and LYST2 genes are two genes of the "LYST/Lyst" family which are
isolatedfromhlImzln~,while Lystl and Lyst2 l~lesc;llltwogenesofthe "LYST/Lyst" familywhich
are their murine homologs, respectively.
In analogous fashion, a "LYST/Lyst" protein is int~n~lcd to mean a LYST or Lyst protein
isolated from a m~mm~ n source, with human and murine peptides being most preferred. In
15 keeping with the genetic nomen~l~tl-re schemes known to those of skill in the art, "LYST"
proteins are those proteins encoded by LYSTgenes derived from human sources while "Lyst"
proteins are those proteins encoded by Lyst genes derived from murine sources. Thus, LYST1
and LYST2 are the proper design~tions of two proteins of the "LYST/Lyst" protein family which
are isolated from hl~m~nc7 while Lystl and Lyst2 represent the two homologous proteins ofthe
2 o LYST/Lyst protein family isolated from murines.
Because there are long and short isoforms of these proteins, the inventors have referred
throughout the specification to "Lystl isoform I," "Lyst1 isoform II," and so forth to tiictin~li.ch
between the two isoforms. Such isoform ~ie~ign~tions may also be abbreviated as "Lyst1-I" or
"Lystl-II," and so forth. Human protein isoforms may be referred to in corresponding manner:
2~ "LYST1-I" and "LYSTl-isoform I" describe the long isoforrn ofthe human protein, while
"LYSTl-II" and "LYSTl-isoform II" are terms used to described the short isoform ofthe human
proteins. Therefore, Lyst l -I and Lyst l -II are terms used to represent two isoforms of the murine
-- isoforms of Lystl, and LYSTl -I and LYSTl-II are terms used to represent two isoforms of the
human LYSTl. Similarly, Lyst2-I and Lyst2-II would represent two isoforms ofthe murine
3 o Lyst2 protein, while LYST2-I and LYST2-II would represent two isoforms of the human LYST2
protein.
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The present invention also concerns recombinant host cells for expression of an isolated
LYSTl, Lystl, LYST2, or Lyst2 gene. It is contemplated that virtually any host cell may be
ernployed for this purpose, but certain advantages may be found in using a bacterial host cell such
as E. coli, 5. typ~imurium, B. subfilis, or others. :~xpression in eukaryotic cells is also
5 contemplated such as those derived from yeast, insect~ or m~mm~ n cell lines. These
l~colllbillant host cells may be employed in connection with "ove ~ es~hlg" the LYSTl, Lystl,
LYST2, or Lyst2 protein, that is, increasing the level of ~l es~ion over that found naturally in
"~"~ n cells. As is well known to those of skill in the art, many such vectors and host cells
are readily available for the recolllbillallL c~ l ession of proteins, one particular detailed example of
a suitable vector for expression in m~mm~ n cells is that described in U. S. Patent 5,168,050,
incorporated herein by reference. However, there is no requirement that a highly purified vector
be used, so long as the coding segment employed encodes a protein or peptide of interest (e.g,
the LYST1, Lystl, LYST2, or Lyst2 protein) and does not include any coding or regulatory
seq~7~nc.os that would have an adverse effect on cells. Therefore, it will also be understood that
15 useful nucleic acid sequences may include additional residues, such as additional non-coding
sequ~nc~.~ fl~nkin~ either ofthe 5' or 3' portions ofthe coding region or may include various
regulatoly se~uences.
After identifying an applupli~LLe epitope-encoding nucleic acid molecule, it may be inserted
into any one of the many vectors currently known in the art, so that it will direct the expression
2 o and production of the protein or peptide epitope of interest (e.g., the LYST1, Lystl, LYST2, or
Lyst2 protein) when incorporated into a host cell. In a recombinant expression vector, the coding
portion of the DNA segment is positioned under the control of a promoter. The promoter may be
in the form ofthe promoter which is naturally associated with a LYST1-, Lystl-, LYST2-, or
Lyst2-encoding nucleic acid segment~ as may be obtained by isolating the 5' non-coding
25 sequ~nces located upstream ofthe coding segm~nt, for example, using recombinant cloning
and/or PCR~M technology, in connection with the compositions disclosed herein. Direct
amplification of nucleic acids using the PCRIM technology of U.S. Patents 4,683,195 and
4,683,202 (herein incorporated by reference) are particularly contemplated to be useful in such
methodologies.
3 o In certain embo~1im~nts, it is contemplated that particular advantages will be gained by
positioning the LYST1-, Lystl-, LYST2-, or Lyst2-encoding DNA segrn~?nt under the control of a
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1'1 '
recoll,L.hlalll, or heterologous, promoter. As used herein, a recombinant or heterologous
promoter is intended to refer to a promoter that is not normally associated with a LYSTl, Lystl,
LYST2, or Lyst2-encoding DNA segment in its natural environment. Such promoters may
include those norrnally associated with other genes, and/or promoters isolated from any other
bacterial, viral, eukaryotic, or m~mm~ n cell. Naturally, it will be important to employ a
promoter that effectively directs the e~ures~ion of the DNA segment in the particular cell
cont~ining the vector comprising the LYST1-, Lystl-, LYST2-, or Lyst2-encoding nucleic acid
se~ nt
The use of reco~ allL promoters to achieve protein expression is generally known to
those of skill in the art of molecular biology, for example, see Sambrook et al., (1989). The
promoters employed may be constitutive, or inducible, and can be used under the al)plc,~vliate
conditions to direct high level or re~-l~ted expression ofthe introduced DNA segment For
eukaryotic ~ l ession, the currently pr~l l ed promoters are those such as CMV, RSV LTR, the
SV40 promoter alone, and the SV40 promoter in combination with the SV40 çnh~nc~r In
certain embo(lim~nt~, the ~l ession of recombinant LYSTl, Lystl, LYST2, or Lyst2 protein is
carried out using prokaryotic expression systems, and in particular bacterial systems such as E.
~oli. Such prokaryotic expression of nucleic acid segment~ of the present invention may be
performed using methods known to those of skill in the art, and will likely comprise expression
vectors and promotor sequences such as those obtained from ~ac, t~p, lac, lacW5 or T7
2 o promotors.
For the expression of the LYSTl, Lystl, LYST2, or Lyst2 protein and LYST1-, Lystl-,
LYST2-, or Lyst2-derived epitopes, once a suitable clone or clones have been obtained, whether
they be native sequences or genetically-modified, one may proceed to prepare an expression
system for the recombinant plt;pal ~lion of the LYSTl, Lystl, LYST2, or Lyst2 protein or peptides
2 5 derived from one or more of the LYSTl, Lystl, LYST2, or Lyst2 proteins. The engineering of
DNA segrnent(s) for expression in a prokaryotic or eukaryotic system may be performed by
techniques generally known to those of skill in recombinant expression. It is believed that
virtually any expression system may be employed in the ~ s~ion of LYST1, Lystl, LYST2, or
- Lyst2 proteins or epitopes derived from such proteins.
A 3 0 Alternatively, it may be desirable in certain embodiments to express the gene products or
derived epitopes in eukaryotic expression systems. The DNA sequences encoding the desired
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epitope ~either native or mutagenized) may be separately expressed in various eukaryotic systems
as is well-known to those of skill in the art.
It is proposed that transformation of host cells with I:)NA se~m~nt~ encoding such
epitopes will provide a convenient means for obtaining the protein or peptide of interest.
5 Genomic sequences are suitable for eukaryotic expression, as the host cell will, of course, process
the genomic transcripts to yield functional mRNA for translation into protein.
It is similarly believed that almost any eukaryotic expression system may be utilized for the
c~ cssion of the proteins of the present invention, or of peptides or epitopes derived from such
proteins, e.g., baculovirus-based, gll~t~n7ine synthase-based or dihydrofolate redl~ct~ce-based
10 systems may be employed. In plerelled embodiments it is contemplated that plasmid vectors
incorporating an origin of replication and an efflcient eukaryotic promoter, as exemplified by the
eukaryotic vectors of the pCMV series, such as pCMV5, will be of most use.
For expression in this manner, one would position the coding sequences ~dj~c~nt to and
under the control of the promoter. It is understood in the art that to bring a coding sequence
15 under the control of such a promoter, one positions the 5' end ofthe transcription initiation site of
the transcriptional reading frame of the protein between about l and about 50 nucleotides
"downstream" of (i. e., 3' of) the chosen promoter.
Where eukaryotic expression is contemplated, one will also typically desire to incorporate
into the transcriptional unit which includes nucleic acid sequences encoding the LYSTlLsyt gene
20 product or LYST/Lyst-derived peptides, an appl~,pliate polyadenylation site ~e.g,
5'-AATAAA-3') if one was not contained within the original cloned se~mt?nt Typically, the
poly-A addition site is placed about 30 to 2000 nucleotides "do~ LIealll" of the tc~lllillaLion site
of the protein at a position prior to transcription terrnination.
It is contemplated that virtually any of the commonly employed host cells can be used in
2 5 connection with the expression of the LYST1, Lystl, LYST2, or Lyst2 proteins and epitopes
derived thel eL Ulll in accordance herewith. Examples include cell lines typically employed for
eukaryotic expression such as 239, AtT-20, HepG2, VERO, HeLa, CHO, WI 38, BHK, COS-7,
RIN and MDCK cell lines.
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- It is further contemplated that the proteins9 peptides, or epitopic peptides derived from
native or recolllbinalll LYSTl, Lystl, LYST2, or Lyst2 proteins may be "ovel~ ssed", i.e.,
expressed in increased levels relative to its natural expression in human cells, or even relative to
the t;x~ iOn of other proteins in a recombinant host cell co,~ LYSTl-, Lystl-, LYST2-, or
5 Lyst2-encoding DNA segmçntC Such ovel t;A~I ession may be ~ ~sesse~1 by a variety of methods,
incll-rling radiolabeling and/or protein purification. However, facile and direct methods are
e~lled, for example, those involving SDS/PAGE and protein st~ining or Western blotting,
followed by qU~ntit~tive analyses, such as densitometric sc:~nnin~ of the resultant gel or blot. A
specific increase in the level of the recolllbinal,L protein or peptide in comparison to the level in
10 natural LYSTl-, Lystl-, LYST2-, or Lyst2-producing cells is indicative of ov~ vl es~ion, as is a
relative ablmd~nçe ofthe specific protein in relation to the other proteins produced by the host
cell and, e.g, visible on a gel.
As used herein, the term "engineered" or "recombinant" cell is int~n(lecl to refer to a cell
into which a recombinant gene, such as a gene encoding LYST1, Lystl, LYST2, or Lyst2 has been
5 introduced. Therefore, çnginçt-,red cells are tli.~tin~li.ch~hle from naturally occurring cells which
do not contain a recombinantly introduced gene. Engineered cells are thus cells having a gene or
genes introduced through the hand of man. Recombinantly introduced genes will either be in the
form of a single structural gene, an entire genomic clone comprising a structural gene and fl~nking
DNA, or an operon or other functional nucleic acid segment which may also include genes
2 o positioned either upstream and/or dow~ l eanl of the promotor, regulatory elements, with or
without introns, or a cDNA clone comprising the structural gene itself, or even genes not
naturally associated with the particular gene of interest.
Where the introduction of a recombinant version of one or more of the foregoing genes is
required, it will be important to introduce the gene such that it is under the control of a promoter
2 5 that effectively directs the expression of the gene in the cell type chosen for engineering. In
general, one will desire to employ a promoter that allows constitutive (constant) expression of the
gene of interest. Commonly used constitutive eukaryotic promoters include viral promotors such
as the cytomegalovirus (CMV) promoter, the Rous sarcoma long-terminal repeat (LTR)
sequence, or the SV40 early gene promoter. The use of these constitutive promoters will ensure
3 o a high, constant level of expression of the introduced genes. The inventors have noticed that the
level of t;x~l e~sion from the introduced genes of interest can vary in dirr~l c;lIL clones, or genes
isolated from dirrel t;llL strains or bacteria. Thus, the level of expression of a particular
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'2~
recolllbhlallL gene can be chosen by evz~lu~ting di~ele-lL clones derived from each transfection
study; once that line is chosen, the constitutive promoter ensures that the desired level of
expression is perm~n~.ntly m~int~ined. It may also be possible to use p}omoters that are specific
for cell type used for engineering, such as the insulin promoter in insulinoma cell lines, or the
5 prolactin or growth hormone promoters in anterior pituitary cell lines.
2.4 Detection of LYST/Lyst Gene Products
A further aspect of the invention is the preparation of immlln~-logical compositions, and in
particular anti- LYST/Lyst antibodies for diagnostic and therapeutic methods relating to the
detection and diagnosis of CHS. Methods for diagnosing CHS and the detection of LYST/Lyst -
10 encoding nucleic acid segments in clinical samples using nucleic acid compositions are alsoobtained from the invention. The nucleic acid sequences encoding LYST/Lyst are useful as
diagnostic probes using conventional techniques such as in Southern hybridization analyses or
Northern hybridization analyses to detect the presence of LYST/Lysf nucleic acid ~e~mçntc within
a clinical sample from a patient suspected of having such a condition. In a p~ ed embodiment,
15 nucleic acid sequences as disclosed in SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO: 1 I and SEQ ID NO: 13 are preferable as probes for such
hybridization analyses.
2.!5 Methods for Producing an Immune Response
Also disclosed in a method of generating an immlme response in an animal. The method
2 o generally involves ~lmini~t~oring to an animal a pharm~reutical composition comprising an
imm1lnologically effective amount of a peptide composition disclosed herein. Preferred peptide
compositions include the peptide disclosed in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SLQ
ID NO:8, ~EQ ID NO:lO, SEQ ID NO:12, or SEQ ID NO:14.
The invention also encompasses LYST/Lyst and LYST/Lyst -derived peptide antigen
25 compositions together with pharmaceutically-acceptable excipients, carriers, diluents, adjuvants~
and other components, such as additional peptides, antigens, or outer membrane preparations, as
may be employed in the formulation of particular vaccines.
Antibodies may be of several types in~ ling those raised in heterologous donor animals
or human volunteers immlmi7ed with the LYST/~yst gene product, monoclonal antibodies
3 o (n~Abs) resulting from hybridomas derived from fusions of B cells from imml lni7~d animals or
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- humans with compatible myeloma cell line~s, so-called "hllm~ni7ed" mAbs reslllting from
expression of gene fusions of combinatorial determining regions of mAb-encoding genes from
heterologous species with genes encoding human antibodies, or LYST/Lyst -reactive
antibody-co.,~ illg fractions of plasma from human donors suspected of having CHS. It is
contemplated that any of the techniques described above might be used for the v~ccin~tion of
subjects for the purpose of antibody production. Optimal dosing of such antibodies is highly
dependent upon the pharmacokinetics of the specific antibody population in the particular species
to be treated.
Using the peptide antigens described herein, the present invention also provides methods
of generating an imrnune response, which methods generally comprise a-lmini.ctering to an animal,
a pharm~reutic~lly-acceptable composition comprising an immunologically effective amount of a
LYST/Lyst peptide composition. Preferred animals include m~mm~lc~ and particularly humans.
Other pler~lled animals include murines, bovines, equines, porcines, ~ninçc, and felines. The
composition may include partially or significantly purified LYST/Lyst peptide epitopes, obtained
from natural or recol,lbilla"~ sources, which proteins or peptides may be obtainable naturally or
either chemically synthesized, or alternatively produced in vitro from recombinant host cells
e~ s~ing DNA segmenfc encoding such epitopes. Smaller peptides that include reactive
epitopes, such as those between about l0 and about 50, or even between about 50 and about ~00
amino acids in length will often be pl ~rel I ed. The antigenic proteins or peptides may also be
2 o combined with other agents, such as other LYST/Lyst -related peptides or nucleic acid
compositions, if desired.
By "immlmologically effective amount" is meant an amount of a peptide composition that
is capable of generating an immune response in the recipient animal. This inçllldes both the
generation of an antibody response (B cell response), and/or the stim~ tion of a cytotoxic
2 5 immlme response (T cell response). The generation of such an immlme response will have utility
in both the production of useful bioreagents, e.g, CTLs and, more particularly, reactive
antibodies, for use in diagnostic embo-1imentc, and will also have utility in various prophylactic or
A therapeutic embodiments. Therefore, although these methods for the stim~ tion of an immnne
response include vaccination regimen-c and tre~tment regimenC, it will be understood that
3 o achieving either of these end results is not nec~c.c~ry for practicing these aspects of the invention.
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- Further means contemplated by the inventors for generating an imm-ln~ response in an
animal inc.lll~1çc ~lmini.ct~ring to the anirnal, or human subject, a pharm~cel1tic~lly-acceptable
composition comprising an immlln~logically effective amount of a nucleic acid composition
encoding a LYST/Lyst epitope, or an immunologically effective amount of an ~tt~n--~ted live
5 Ol~ i'.lll that includes and expresses such a nucleic acid composition. The "immlln~logically
effective amounts" are those amounts capable of s~im~ ting a B cell and/or T cell response.
Imrnunoformulations ofthis invention, whether int~n~led for vac~.;n~tion, L~e~l.,.~..l, or for
the generation of antibodies useful in the detection of ~HS, may comprise native, or synthetically-
derived ~ntig~nic peptide fr~gmPnt.~ from these proteins. As such, ~ntigPn;c functional equivalents
10 of the proteins and peptides described herein also fall within the scope of the present invention.
An "antigenically functional equivalent" protein or peptide is one that incorporates an epitope that
is immllnologically cross-reactive with one or more epitopes derived from any of the particular
proteins disclosed. Antigenically functional equivalents, or epitopic sequences, may be first
desi~ne~l or predicted and then tested, or may simply be directly tested for cross-reactivity.
The id~nti~tion or design of suitable epitopes, and/or their functional equivalents,
suitable for use in immunoformulations, vaccines, or simply as antigens (e.g., for use in detectit n
protocols), is a relatively straightforward matter. For example, one may employ the methods of
Hopp, as enabled in U.S. Patent 4,554,101, incorporated herein by reference, that teaches the
ntific~tion and preparation of epitopes from amino acid sequences on the basis of
2 0 hydrophilicity. The methods described in several other papers, and software programs based
thereon, can also be used to identify epitopic core sequences, for example, Chou and Fasman
(1974a,b; 1978a,b; 1979); Jameson and Wolf (1988); Wolf et al., (1988); and Kyte and Doolittle
(1982) address this subject. The amino acid sequence ofthese "epitopic core sequences" may
then be readily incorporated into peptides, e*her through the application of peptide synthesis or
2 5 l ecolllbillant technology.
It is proposed that the use of shorter antigenic peptides, e.g, about 25 to about 50, or
even about 15 to 25 amino acids in length, that incorporate epitopes of the LYST/Lyst protein
will provide advantages in certain circ.l-m~t~nces, for example, in the preparation of vaccines or in
immunologic detection assays. Exemplary advantages include the ease of p~ pal aLion and
3 0 purification, the relatively low cost and improved reproducibility of production, and advantageous
biodistribution .
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In still further embodiments, the present invention collcel"s immunndetection methods and
associated kits. It is contemplated that the proteins or peptides of the invention may be employed
to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in
accordance with the present invention, may be employed to detect LYST/Lyst proteins or
5 peptides. Either type of kit may be used in the imml-nf~detection of compounds, present within
clinical samples, that are indicative of CHS. The kits may also be used in antigen or antibody
purification, as ap~ lia~e.
In general, the plt:r~led imml~n~detection methods will include first obtaining a sample
suspected of co~ -g a LYST/Lyst -reactive antibody, such as a biological sample from a
10 patient, and cont~cfing the sample with a first LYST/Lyst protein or peptide under conditions
effective to allow the formation of an imm~-nt~complex (primary immnn(~. complex). One then
detects the presence of any primary imm~nt)complexes that are formed. Preferable LYST/LYsT
proteins include LYSTl and LYST2 from human origins, and Lystl and Lyst2 proteins derived
from murine origins.
Contacting the chosen sample with the LYST/Lyst protein or peptide under conditions
effective to allow the formation of (primary) imm--ne complexes is generally a matter of simply
adding the protein or peptide composition to the sample. One then incubates the mixture for a
period of t;me sufficient to allow the added antigens to form immnn~ complexes with, i.e., to bind
to, any antibodies present within the sample. After this time, the sample composition, such as a
2 0 tissue section, LLISA plate, dot blot or western blot, will generally be washed to remove any non-
specifically bound antigen species, allowing only those specifically bound species within the
immnne complexes to be detected
The detection of immnnocomplex formation is well known in the art and may be achieved
through the application of numerous approaches known to the skilled artisan and described in
various publications, such as, e.g, Nakamura et al., (19~7), incorporated herein by reference.
Detection of primary immune complexes is generally based upon the detection of a label or
marker, such as a radioactive, fluorescent, biological or enzymatic label, with enzyme tags such as
~' ~lkz~line phosphatase, urease, horseradish peroxidase and glucose oxidase being suitable. The
particular antigen employed may itself be linked to a (letect~ble label, wherein one would then
3 o simply detect this label, thereby allowing the amount of bound antigen present in the composition
to be determined.
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Alternatively, the primary immllne complexes may be cletected by means of a second
binding ligand that is linked to a detect~hle label and that has binding affinity for the first protein
or peptide. The second binding ligand is itself often an antibody, which may thus be termed a
"secondary" antibody. The primary immllne complexes are contacted with the labeled, secondary
5 binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow
the formation of secondary immlme complexes. The secondary immune complexes are then
generally washed to remove any non-specifically bound labeled secondary antibodies and the
i";.,g bound label is then detecte~l
For diagnostic purposes, it is proposed that virtualiy any sample suspected of cont~inin~
0 the antibodies of interest may be employed. Exemplary samples include clinical samples obtained
from a patient such as blood or serum samples, bronchoalveolar fluid, ear swabs, sputum samples,
middle ear fluid or even perhaps urine samples may be employed. This allows for the diagnosis of
CHS and related disorders. Furthermore, it is cont~mplated that such embodiments may have
application to non-clinical samples, such as in the titering of antibody samples, in the selection of
15 hybridomas, and the like. Alternatively, the clinical samples may be from veterinary sources and
may include such domestic animals as cattle, sheep, and goats. Samples from feline, canine, and
equine sources may also be used in accordance with the methods described herein.
In related embo-liment~, the present invention contemplates the preparation of kits that
may be employed to detect the presence of LYST/Lyst -specific antibodies in a sample. Generally
2 0 speaking, kits in accordance with the present invention will include a suitable protein or peptide
together with an immllnr~detection reagent, and a means for co~ ing the protein or peptide and
reagent.
The imm-ln~detectif~n reagent will typically comprise a label associated with a LYST/Lyst
protein or peptide, or associated with a secondary binding ligand. F.~ pl~ry Iigands might
2 5 include a secondary antibody directed against the first LYST/Lyst or peptide or antibody, or a
biotin or avidin (or streptavidin) ligand having an associated label. Detectable labels linked to
antibodies that have binding affinity for a human antibody are also contemplated, e.g, for
protocols where the first reagent is a LYST/Lyst peptide that is used to bind to a reactive
antibody from a human sample. Of course, as noted above, a number of exemplary labels are
3 o known in the art and all such labels may be employed in connection with the present invention.
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The kits may contain antigen or antibody-label conjugates either in fully conjugated form, in the
form of intermediates, or as separate moieties to be con~ugated by the user of the kit.
The container means will generally include at least one vial, test tube, flask, bottle, syringe
or other container means, into which the antigen may be placed, and preferably suitably allocated.
5 VVhere a second binding ligand is provided, the Icit will also generally contain a second vial or
other container into which this ligand or antibody may be placed. The kits of the present
invention will also typically include a means for cont~ining the vials in close confinement for
cornmercial sale, such as, e.g, injection or blow-molded plastic containers into which the desired
vials are retained.
10 2.6 Formulation as Vaccines
It is expected that to achieve an "immlln~logically effective formulation" it may be
desirable to adrninister LYST- or Lyst-encoding proteins to the human or animal subject in a
pharm~ce-ltically acceptable composition comprising an immunologically effective amount of
LYST or Lyst proteins or peptides mixed with other excipients, carriers, or diluents which may
15 improve or otherwise alter stimulation of B cell and/or T cell responses, or immunologically inert
salts, organic acids and bases, carbohydrates, and the like, which promote stability of such
mixtures. Immunostim~ tory excipients, often referred to as adjuvants, may include salts of
~Illminllm (often referred to as Alums), simple or complex fatty acids and sterol compounds,
physiologically acceptable oils, polymeric carbohydrates, chemically or genetically modified
2 o protein toxins, and various particulate or emulsified combinations thereof. LYST or Lyst proteins
or peptides within these mixtures, or each variant if more than one are present, would be expected
to comprise about 0.0001 to 1.0 milligrams, or more preferably about O.OOl to 0.1 milligrams, or
even more preferably less than 0.1 rnilligrams per dose.
It is also contemplated that ~tt~u~ted or~ni~m~ may be ~ngineçred to express
25 recombinant LYST or Lyst proteins or peptides, and the org~ni.cm~ themselves be delivery
vehicles for the invention. Pox-, polio-, adeno-, or other viruses, and bacteria such as Salmorlella,
Shigella, Listeria, Streptococcus species may also be used in conjunction with the methods and
compositions disclosed herein.
-
The naked DNA technology, often referred to as genetic immllni~tion, has been shown to
3 o be suitable for protection against infectious olg~ Such DNA segment.~ could be used in a
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variety of forms inr;lllAing naked DNA and plasmid DNA, and may ~rlmini~t~?red to the subject in
a variety of ways in~ ling parenteral, mucosal, and so-called microprojectile-based "gene-gun"
inoculations. The use of LYST or Lyst nucleic acid compositions of the present invention in such
immllni7~tion te~.hniquçc is thus proposed to be useful as a vaccination strategy against Lyme
5 disease.
It is recognized by those skilled ~n the art that an optimal dosing schedule of a v~ccin~tion
regimen may include as many as five to six, but preferably three to five, or even more pre~erably
one to three ~mini.ctrations of the immllni~ing entity given at intervals of as few as two to four
weeks, to as long as five to ten years, or occasionally at even longer intervals.
2.7 USE OF LYST1 PEPTrDES/~PTAnIE~S ASE~AR MACEUTICALS1~IAT ~OCK O~ MI~C
LYST1 ~UNCTION
Lyst reg~ tes degranulation of lysosomes, late endosomes and acidic secretory granules
primarily in leukocytes. Blockade of such degranulation using dominant-negatively acting
trl-nc~ted Lyst peptides may reasonably be expected to be efficacious in infl~mm~tory and
autoimmlme diseases such as asthma, urticaria, infl~mm~tory bowel disease, systemic lupus
eryth~m~tosus, rhellm~toid arthritis, psoriasis, systemic vasculitis, glomerulonephritis, multiple
sclerosis, post-angioplasty restenosis. Proofofthis principal is docllm~nted in Clark etal., 1982,
who demonstrated that bg mice are protected from lupus nephritis.
2.8 USE OFPHk~RM~CEUTICAL CO~DPOUNDS1~T BLOCK ORn11MIC LYST1 ~ NCTION
Lyst n-,gl-l~tes degranulation of lysosomes, late endosomes and acidic secretory granules
primarily in leukocytes. Blockade of such degranulation using domil~ -negatively acting
tnlnç~ted Lyst peptides may reasonably be expected to be efficacious in infl~mm~tory and
autoimm--ne diseases such as asthma, urticaria, infl~mm~tory bowel disease systemic lupus
erythematosus, rh.Q.Ilm~toid arthritis, psoriasis, systemic vasculitis, glomerulonephritis, multiple
2 5 sclerosis, post-angioplasty restenosis. Proof of this plinci~al is docllm~nted in Clark et al., (1982)
who demonstrated that bg mice are protected from lupus nephritis.
Lyst peptides that mimic or augment Lyst fiJnction may reasonably be expected to be
efficacious in the treatment of neoplasia. Proof of this principle is docllmPnted in Aboud et al.
(l993~ and Hayakawa et al. (1986), who demonstrate that bg mice and CHS patients are
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'27
susceptible to development off neoplasia, and have more aggressive neoplasms with accelerated
met~st~es.
2.9 IJSE OF LYS~2 PEPT~ES/APrAMERS AS PHARMACEU rlcAL AGEN rS THAT B~ocK
LYST2 P UNCTION O~ REPRODUCE LYST2 FUNCTIONS
Lyst2 is thought to act to regulate degr~n~ tion of vesicles within cells in the brain and
kidney. Bblockade of such degr~n~ tion using dominant-negatively acting trllnc~ted Lyst2
peptides may reasonably be expected to be efficacious for the tre~tment of neurologic and renal
degenerative diseases such as ~l7heimer's disease, motor neuron disease, Parkinson's disease,
acute tubular necrosis, glomerulonephritis and glomerulosclerosis.
2.10 USE OF pHARMAcEurIcAL COMPOUNDS ~AT BLOCK OR MIMIC LYST2 FUNCTIONS
Drugs that mimic the action of dolllina~lL-negatively acting tr ln~ted Lyst2 peptides.
Lyst2 is thought to act to regulate degranulation of vesicles within cells in the brain and kidney.
Blockade of such degranulation using dominant-negatively acting trllnç~ted Lyst2 peptides may
reasonab}y be expected to be effîcacious for the treatment of neurologic and renal degenerative
~ e~es such as ~l~hçimer's disease, motor neuron disease, Parkinson's disease, acute tubular
necrosis, glomerulonephritis and glomerulosclerosis.
3. BRlEF DESCR~rION OF THE DR~WINGS
The drawings forrn part of the present specification and are inclucle~l to fi~rther
demonstrate certain aspects of the present invention. The invention may be better understood by
reference to one or more of these drawings in combination with the detailed description of
specific embodiments presented herein.
IilG. lA. Ethidium bromide-stained pulsed field gels of DNA from clones derived from a
mouse YAC library. YAC clone numbers are shown above each panel and molecular size
standards (in kilobases~ to the left of each panel. 1380 is the host S. cerevisiae strain and
does not contain a YAC. Sizes of YAC clones are: 151HI = 950-kb, 195A8 = 650-kb,- 64F5 = 580-kb, 93E4 = 370-kb, 68E12 = 500-kb, 55F3 = 550-kb, 135G3 = 750-kb,
148H8 = 1000-kb, 84A8 = 370-kb, 148E11 = 650-kb, 165F7 = 500-kb.
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FIG. lB. Autoradiographs of correspQnding Southern blots from the gels shown in FTG. lA
hybridized with pBR322 {which cross-hybridizes to pYAC4. YAC clone numbers are
shown above each panel and molecular size standards (in kilobases) to the left of each
panel. 1380 is the host S. cerevisiae strain and does not contain a YAC. Sizes of YAC
clones are: 151H1 = 950-kb, l95A8 = 650-kb, 64F5 = 580-kb, 93E4 = 370-kb, 68E12 =
500-kb, 55F3 = 550-kb, 135G3 = 750-kb, 148H8 = 1000-kb, 84A8 = 370-kb, 148E11 =
650-kb; 165F7 = 500-kb.
FlG. 2. STS content mapping of bg critical region YAC and P1 clones. The presence of
an STS (y-axis) in a YAC/Pl clone (x-axis) is indicated by a filled box Each contig is
0 i~l~ntified by the degree of shading of the box The bg critical region extends from
proximal to D13Mif~34 to the interval between D13Mit207 and D13Mitl62/D13Mif305
(crossover location indicated by a double line). STS used for isolation of YAC clones
were Ni~5' for 151H1, 195A8, 64F5, 93E4, 68E12, and 55F3, Estm9 for 148E11,
D13Mitl34 for 165F7, D13Sf/c13 for 84A8, and D13Mi~207 for 135G3 and 148H8. P1
clones 8591 and 8592 were identified with D135flc13. YAC clone 64F5 is chimeric; YAC
clone 84A8 has acquired an internal deletion which includes D13Sfk6. The relative
orientation with respect to the centromere of the contig composed of the 9 clones l 95A8-
55F3 has not been established; The position of clones 165F7 and 148E11 with respect to
this contig has not been established.
2 o FIG. 3A. Genetic mapping of bg on mouse Chr 1. Haplotype analysis of proximal mouse
Chr 1 genetic markers in 504 C57BL/6J-bg, X (C57BL/6J-bg i x CAST/EiJ)Fl backcross
mice. Closed boxes represent the homozygous C3H pattern and open boxes the F
pattern. Number of mice of each haplotype are indicated.
FIG. 3B. Genetic mapping of bg on mouse Chr 1. Haplotype analysis of proximal mouse
Chr 1 genetic markers in 111 (C57BL/6J -~h-bg' x Mus domes~icus PAC)Fl X
C57BL/6J-bg' backcross mice. Closed boxes represent the homozygous C3H pattern and
open boxes the Fl pattern. Number of mice of each haplotype are in-lic~ted
FIG. 3C. Genetic mapping of bg on mouse Chr 1. Haplotype analysis of proximal mouse
Chr 1 genetic markers in 111 (C57BL/6J-W~h-bgJ x Mi~s m~ PWK)FI X C57BL/6J-
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bg ~ backcross mice. Closed boxes rep~esent the homozygous C3H pattern and open
boxes the Fl pattern. Number of mice of each haplotype are indicated.
FIG. 3D. Genetic mapping of bg on mouse Chr 1. Composite linkage map of mouse Chr 13
q in the vicinity of bg Loci are positioned according to their Ap~ aLe relative positions
of loci were ascertained by integration of data from the above three backcrosses and from
DietrichetaL, (1994).
FIG. 4. Autoradiograph of a pulse field gel Southern blot of mouse DNA probed with Nid.
Restriction endonucleases are shown above the panel and molecular size standards (in
kilobases) to the left. +/+=DBA/2J DNA; bg=SB-bg/bg DNA. Upon reprobing this blot
wsth GZ~3 or Estm9 all DBA/2J and SB-bg bands were of identical size.
FIG. 5A. DNA sequence of the CH gene (LYSTl) from position 1 to position 1400. The
DNA sequence continues in FIG. 5B.
FIG. 5B. Continuation of the DNA sequence of the CH gene in FIG. 5A beginning at position 1401 and continllin~ to position 2800.
15 FIG. 5C. Continuation of the DNA sequence of the CH gene in FIG. SB beginning at
position 2801 and contimling to position 3514.
FIG. 6. Amino acid sequence of the CH protein.
FIG. 7A. Genetic mapping of the Bl gene.
FIG. 7B. Genetic mapping of the bg and Bl genes.
2 o FIG. 7C. Detailed map showing the localiztion of the bg and Bl genes.
FIG. 8. Bl cDNA clones.
FIG. 9A. Deletion of part of B1 in bgllJ. Probe used in Southern analysis is probe A from
FIG. 8.
FIG. 9B. Deletion of part of Bl in bgllJ. Probe used in Southern analysis is probe B from
-25 FIG. 8.
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FIG. 9C. Deletion of part of B1 in bgllJ. Probe used in Southern analysis is probe C from
FIG. 8. (In bpllJ a deletion from bp 1250 to 2400 was observed.
FIG. 10. Physical mapping of B1 gene within bg critical region.
FIG. 11. Genetic and physical map of the bg non-re~;o~l~binalll interval on mouse
chromosome 13 showing the location of Lyst. Mouse chromosome 13 is shown by the
ho,i~oll~l line with the centromere on the left. The bg critical region is delin~tec~ by
chromosome crossovers (denoted with an X~ in animals 134 and 137 of an interspecific
mouse backcross [C57BL/6-bg' x (C57BL/6J-bg' x CAST/Ei~)Fl3. Microsatellite
markers D13MitI 72 and D13Mit239 flank bg proximally; D13Mitl 62 and DI 3Mit305 lie
distal to bg ~indicated by turquoise circles). YAC and P1 clones identified by PCRT~q
screening (Kusurni et al., 1993; Pierce et al., 1992) with oligonucleotides corresponding
to Nfd or D13Sfk13 are shown above the chromosome. Novel sequence-tagged sites
(STS, indicated by dark blue circles), generated by inverse repetitive element PCR or
direct or direct cDNA selection, were used to order clones within the contiguous array.
Novel mouse chromosome 13 STSs are numbered 1-18, corresponding to D13Sfkl to
D135flc18, respectively. Lyst was isolated from YAC 195A8, a 650-kb clone, by using
direct cDNA selection. The physical location of Lyst-associated STSs on YAC and P1
clones are shown in red (MGD accession number MGD-PMEX-13).
FIG. 12A. Intragenic deletion of Lysf in bgt'J DNA. Southern blot identific~.on of an
intragenic Lyst deletion in bg"J DNA~ A Southern blot was sequentially hybridized
~Barbosa et al., 1995) with 3 Lyst probes; This panel shows the probe (nucleotides 1,262-
3,433 of Lysf cDNA) which extends upstream from the bg~l~ deletion. Restriction
endonucleases are intlic~ted at the bottom of the panel, and molecular size standards (in
kb) are shown to the left. Similar results were obtained with 3 additional restriction
endonucleases. The bgtt~ mutation was discovered in 1992 at The Jackson Laboratory in
a C57BL/6J-jb mouse at generation N4 afLer transfer ~om B6C3Fe a/a. The mutation jb
had, in turn, been discovered 14 generations earlier in B6C3Fe-a~a-hyh mice at generation
N3 after transfer from C57BL/lOJ. The hyh mutation arose in C57BL/IOJ mice, and was
l"~ J.;"ed in that strain until ~ re, at F15. Thus the possible contributors of genetic
3 0 inforrnation to bg~ include C57BL/6J, C3HeB/FeJ and C57BL/I OJ. Southern blots were
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prepared from genomic DNA o~ all potential progenitor mouse strains, but only
C57BL/lOJ, C57BL/6J and C57BL/6J-bg" are shown.
~IG. 12B. Intragenic deletion of Lyst in bg"J DNA. Southern blot identification of an
intragenic Lyst deletion in bg~J DNA. A Southern blot was sequentially hybridized
(Barbosa et al., 1995) with 3 Lyst probes. This panel shows the probe (nucleotides 2,835-
3,433 of Lyst cDNA) is completely deleted. Restriction endonucleases are indic~te~l at the
bottom of each panel, and molecular size standards (in kb) are shown to the left.
I~lG. 12C. Intragenic deletion of Lyst in bg~'J DNA. Southern blot i(1~ntific~tion of an
intragenic Lyst deletion in bg"J DNA. A Southern blot was sequentially hybridized
(Barbosa et al., 1995) with 3 Lyst probes; Shown in this panel are results when the probe
(nucleotides 3,594-4,237 of ~yst cDNA) extends downstream from the big bg"J deletion.
Restriction endonucleases are indicated at the bottom of each panel, and molecular size
standards (in kb) are shown to the left. Similar results were obtained with 3 additional
restriction endonucleases.
F~G. 12D. Intragenic deletion of Lyst in bg"J DNA. PCRTM analysis of the bg"J deletion.
C57BL/lOJ, C3HeB/FeJ, C57BL/6J and C57BL/6J-bg" genomic DNA and Lyst cDNA
were used as templates in the PCRTM reactions. Amplicons illustrated correspond to: Lyst
cDNA nucleotides 1,337-1,837, which represent exon ,13 and are upstream from thedeletion. No amplicon was observed in control PCRTM reactions performed without
2 o template. More than 30 other STSs that had been localized within the bg non-
reco.,lbillal~L interval PCRTM amplified norrnally from bg~J DNA
~IG. 12E. Intragenic deletion of Lyst in bg"J DNA. PCR~ analysis of the bg"J deletion.
C57BL/lOJ, C3HeB/FeJ, C57BL/6J and C57BL/6J-bg" genomic DNA and ryst cDNA
were used as templates in the PCRTM reactions. Amplicons illustrated correspond to
nucleotides 2,670-3,210, which represent exon ~, which is deleted in bg"J DNA. No
amplicon was obse~red in control PCRTM reactions performed without template. More
than 30 other STSs that had been localized within the bg non-recolllbi,lanl interval PCRTM
amplified normally from bg"J DNA.
-
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- FIG. 12F. Intragenic deletion of rys~ in bg"J DNA. PCRTM analysis of the bg"j deletion.
C57BL/lOJ, C3HeB/FeJ, C57BL/6J and C57BL/6J-bg" genomic DNA and Lyst cDNA
were used as templates in the PCR~M reactions. Amplicons illustrated correspond to
nucleotides 4,913-5,433, which represents an exon downstream from the deletion. No
amplicon was observed in control PCR~M reactions performed without template.
FIG. 12G. Intragenic deletion of Lysf in bg"J DNA. Genomic structure of Lyst in the vicinity
of the bg"J deletion. Lyst exons (oc, ,~ , ~, and O are depicted by black boxes, and
intervening introns by a solid line. Nucleotides of the mouse Lyst cDNA that correspond
to exonic boundaries are indicated above the boxes. The 3' end of exon ,B, and all of
exons ~ and o, are deleted in bgl~ DNA . The region of Lyst protein that is deleted in
bg"J contains a pair of helices with N-terminal phosphorylation sites. Genomic structure
and intronic sequences were ascertained by sequence analysis of nested PCRTM products,
perforrned with exonic primers and P1 clone DNA as template (Kin~.cmQre ef al., 1994).
Boundaries of the bg"J deletion were determined by pC~M of genomic DNA.
lilG. 13A. Northern blot analysis of mouse and human Lyst. Northern blots of 2 ~g poly(A)t
RNA from various mouse tissues (Clontech) hybr;dized with probes that correspond to
nucleotides 4,423-4,621 of mouse Lyst cDNA.
FIG. 13B. Northern blot analysis of mouse and human Lyst. Northem blots of 2 ,ug poly(A)~
RNA from various mouse tissues ~Clontech) hybridized with probes that correspond to
2 o nucleotides 1,430-2,457 (exon ,B)of mouse ~ysf cDNA. Molecular size standards (in kb)
are shown to the left. Hybridization of rnouse mRNA with probes from mouse Lyst exons
cc and ~ gave identical results to those shown with exon ,B, whereas probes from exons ~,
~, and ~ gave results icl-?n~ic~l to those shown in FIG. I3A.
FIG. 13C. Northern blot analysis of mouse and human Lyst. Northern blot of 2 ,ug poly(A)+
RNA from various human Iymphoid tissues, hybridized with a probe that corresponds to
nucleotides 357-800 of human LYST cDNA. Molecular size standards (in kb) are shown
to the left.
FIG. 13D. Northern blot analys;s of mouse and human Lyst. Northern blot of 2 ~g poly(A)~
RNA from human cancer cell lines, hybridized with a probe that corresponds to
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~3
nucleotides 357-800 of human LYST cDNA. Molec~lar size standards (in kb) are shown
to the left.
FIG. 14A. Mutation analysis of LYST cDNA from CHS patients. A Northern blot of 2 c,~g
aliquots of Iymphoblastoid poly(A)+ RNA from CHS patients and a control. The probe
used for hybridization corresponds to nucleotides 490 to 817 of LYST.
FIG. 14B. SSCP analysis of cDNA corresponding toLYSTnucleotides 439 to 806. Each lane
contains samples from individual patients as indic~te(l. Note the appearance of an extra
band in lanes corresponding to patients 371 and 373.
FIG. 14C. Sequence ch~ .atograms showing mutations in LYST cDNA clones from patients
0 371 and 373. The upper part is normal human LYST cDNA sequence. The arrows indicate
the positions of a G insertion (patient 371) and C to T substitution (patient 373). The
antisense strand of LYSTis shown.
FIG. 15A. Physical mapping of LYST Monochromosomal somatic cell hybrid blot (BIOS
La~bor2tor es, Ne~w E~aven., Cor.nerticut) Co~ , DNA frorr. 24 so.llatic cell lîybrid cell
lines and three control DNAs (human, hamster, or mouse) digested with EcoRI. The cell
line and chromosome number are indicated at the top of the figure. *Mix lane consists of
1.5 mg human DNA and 4.5 mg mouse DNA. **Human/hamster somatic cell hybrid. All
others are human/mouse hybrids. Molecular size standards (in kb) are shown to the right.
The blot was hybridized with a probe corresponding to nucleotides 2923-4865 of human
2 o LYST cDNA.
FIG. 15B. Southern blot of CHS critical region YACs digested with TaqI. The YAC
coordinates are indicated at the top and molecular size standards (in kb) are shown to the
left. The probe used for the hybridization corresponds to LYST nucleotides 490 to 817.
FIG. l~C. The same Southern blot shown in panel B, rehybridized with a probe
corresponding to LYST nucleotides 4551 to 4977. A third LSYT probe (corresponding to
nucleotides 3032-4722) also hybridized to the same YAC clones.
FIG. 15D. Physical map of human chromosome 1 showing the location of LYST within a
YAC contig of the CHS critical region (Barrat et al. 1996). The upper part represents
chromosome I . The microsatellite markers DISI 79 (centromeric) and ~7-12396
,
-- -- === ===== = . = = = =
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3 ~
(telomeric) ~ank the CHS locus. YAC clones are shown below the chromosome. The
figure is not drawn to scale.
~IG. 16A. Genomic o~an~Lion of LYST. Schematic representation of PCR~ clones
corresponding to the human LYST cDNA ~Genbank accession number U70064). The
solid and open bars represent the LYST coding region and the 5' UTR, respectively.
Nucleotide 5095 corresponds to the transition between sequences conserved with Lyst
(Barbosa et al., 1996) and BG (Perou et al., 1996a). The three human ESTs identified by
database searches with the mouse sequence (#1, #2 and ~3; Genbank accession numbers:
~1, L77889; #2, W26957; #3, H51623) are shown at the top. Clones #4, #5, #6 and #8
are RT-PCRTM products. Clone #7 is a 2 kb 5'RACE product.
FIG. 16B Alternative splicing of mouse Lyst. Solid boxes represent Lyst exons ~ and 1.
Splicing of exon c~ to exon ~ occurs in the Lyst-I mRNA (12 kb). The hatched box.epl~;selll~ the intronic region that forms the 3' end of the Lyst-II ORF (5.9 kb). An
asterisk indicates a stop codon and an 'A' indicates a polyadenylation signal within the
1 5 intron. Nucleotide positions indicated are from Genbank accession number L77884
(Lyst-II) and U70015 ~Lyst-I).
FIG. 16C. Detection of Lyst-I and Lyst-rf by RT-PCR~M and genomic PCRTM.
DNAse-treated mouse melanocyte RNA was reverse transcribed and amplified with
primers F1/Rl (expected amplicon size 273 bp) or Fl/R2 (expected amplicon size 560
bp). RNAse-treated C57BL/6J DNA was amplified with primers Fl/R1. The primer
sequences are:
Fl, 5'-TGTG&AATACATCCAATG~ATCCGAGAGTGC-3';
F2, 5'-GAGCCAAGAAAGAGGCTGAT-3';
Rl, 5'-GGTTTCGGACTCAAAAGTTTGTCGGAACTT-3';
R2, 5'-GAGACCCATATGGAGATTTC-3'.
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- 4. DESCRIPTION OFILLU~,TRATrV~ L~qBODIME~TS
4.1 L~ST ENCODING NUCLEIC ACLD SEGMENTS
As used herein, the term "LYSTl gene" is used to refer to a gene or DNA coding region
that encodes a Chediak-Higashi protein, polypeptide or peptide.
The definition of a "LYST~ gene", as used herein, is a gene that hybridizes, under
relatively stringent hybridization conditions ~see, e.g., Maniatis et al., 1982), to DNA sequences
presently known to include LYST1 gene sequences. It will, of course, be understood that one or
more than one genes encoding LYST1 proteins or peptides may be used in the methods and
compositions of the invention. The nucleic acid compositions and methods disclosed herein may
0 entail the ~lmini~tration of one, two, three, or more, genes or gene seEmen~.C. The maximum
number of genes that may be used is limited only by practical considerations, such as the effort
involved in ~imlllt~neously preparing a large number of gene constructs or even the possibility of
~liçjting a significant adverse cytotoxic effect.
As used herein, the term "LYST2 gene" is used to refer to a gene or DNA coding region
that encodes a LYST2 protein, polypeptide or peptide.
The definition of a "LYST2 gene", as used herein, is a gene that hybridizes, under
relatively stringent hybridization conditions (see, e.g, Maniatis et al., 1982), to DNA sequences
presently known to include LYST2 gene sequences. It will, of course, be understood that one or
more than one genes encoding L~ST2 proteins or peptides may be used in the methods and
2 o compositions of the invention. The nucleic acid compositions and methods disclosed herein may
entail the ~imini.~tration of one, two, three, or more, genes or gene segment.~. The m~xim-lm
number of genes that may be used is limited only by practical considerations, such as the effort
involved in simlllt~neously ple~ g a large number of gene constructs or even the possibility of
eliciting a significant adverse cytotoxic effect
In those embodiments involving multiple genes of the present invention, the LYST and
Lyst genes disclosed herein may be combined on a single genetic construct under control of one
or more promoters, or they may be prepared as separate constructs ofthe same of difrelellL types.
-
Thus, an almost endless combination of different genes and genetic constructs may be employed.
Certain gene combinations may be de~igned to, or their use may otherwise result in, achieving
3 o synergistic effects on formation of an immlme response, or the development of antibodies to gene
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products encoded by such nucleic acid segment~ or in the production of diagnostic and tre~tment
protocols for, among other things, Chediak-~igashi Syndrome. Any and all such combinations
are int~nrled to fall within the scope of the present invention. Indeed, many synergistic effects
have been described in the scientific literature, so that one of ordillal y skill in the art would readily
5 be able to identify likely synergistic gene combinations, or even gene-protein coll-billaLions.
It will also be understood tihat, if desired, the nucleic segm~nt or gene could be
~dl..;";~ d in combination with further agents, such as, e.g, proteins or polypeptides or various
pharm~ceutically active agents. So long as genetic material forms part of the composition, there
is virtually no limit to other components which may also be included, given that the additional
0 agents do not cause a significant adverse effect upon contact with the target cells or tissues.
4.2 I~IERAPEUTIC~D DIAGNOSTIC ~TS
Therapeutic kits comprising, in suitable container means, a LYST or Lyst composition of
the present invention in a pharm~ce~tically acceptable formulation represent another aspect of the
invention. The LYST or Lyst composition may be native LYST or Lyst protein, trlmc~ted LYST
15 or Lyst protein, site-specifically m~t~ted LYST or Lyst-encoding DNAs, or LYST- or Lyst-
derived peptide epitopes, or alternatively antibodies which bind the native LYST or Lyst gene
product, trl-nç~ted LYST or Lyst protein, site-specifically mut~tç~ LYST or Lyst protein, or
LYST- or Lyst-encoded peptide epitopes. In other embodiments, the LYST or Lyst composition
may be nucleic acid sçgment.~ encoding one or more native LYST or Lyst proteins, truncated
20 I_YST or Lyst proteins, site-specifically mTItated LYST or Lyst proteins, or peptide epitope
derivatives of LYST or Lyst. Such nucleic acid segm~nts may be DNA or RNA, and may be
either native, recolllbhlanL, or mutagenized nucleic acid segments.
The kits may comprise a single container means that contains the LYST or Lyst
composition. The container means may, if desired, contain a pharm~cç~ltically acceptable sterile
25 excipient, having associated with it, the LYST or Lyst composition and, optionally, a detectable
label or ;m~g;ng agent. The formulation may be in the form of a g~l~tinous composition, e.g, a
collagenous- LYST or Lyst composition, or may even be in a more fluid form. The container
means may itself be a syringe, pipette, or other such like apparatus, from which the LYST or Lyst
composition may be applied to a tissue site, injected into an animal, or otherwise a-lmin;~tered as
,
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needed. However, the single container mAeans may contain a dry, or Iyophilized, mi~Ature of a
LYST or Lyst composition, which may or may not require pre-wetting before use.
Alternatively, the kits of the invention may comprise distinct container means for each
component. In such cases, one container would contain the LYST or Lyst composition, either as
5 a sterile DNA solution or in a lyophilized form, and the other container would include the matrix,
whiGh m~~r or Amay not itse!f be pr~=~v~ .ted ~th a ste,lle sol..tion, or be in a gel~--inous, liquid or
other syringeable forrAnA.
The kits may also comprise a second or third container means for cont~ining a sterile,
pharmaceutically acceptable buffer, diluent or solvent. Such a solution may be re~uired to
10 formulate the LYST or Lyst component into a more suitable forr~A for application to the body,
e.g, as a topical preparation, or alternatively, in oral, parenteral, or intravenous forms. It should
be noted, however, that all components of a kit could be supplied in a dry form ~Iyophilized),
which would allow for "wetting" upon contact with body fluids. Thus, the presence of any type
of pharmaceutically acceptable buffer or solvent is not a requirement for the kits of the invention.
5 The kits may also comprise a second or third container means for cont~ining a pharms~c.e~lti~lly
acceptable detectable im~gin~ agent or compcsition.
The container means will generally be a con~ainer such as a vial, test tube, flask, bottle,
syringe or other co~llailAer means, into which the components of the kit may placed. The matrix
and gene components may also be aliquoted into smaller containers, should this be desired. The
20 kits ofthe present invention may also include a means for coni~ining the individual containers in
close confinP.m~nt for coA~nercial sale, such as, e.g, injection or blow-molded plastic containers
into which the desired vials or syringes are retained.
Irrespective of the number of containers, the kits of the invention may also comprise, or be
packaged with, an instrument for ~i~in~ with the pl~c~m~nt of the llltim~te LYST or Lyst
2 5 composition within the body of an animal. Such an instrument may be a syringe, pipette, forceps,
or any such medically approved delivery vehicle.
-
4.3 METHODS OF NUCLEIC ACATD DELIVERY A~ND DNA TRANSFECTION
In certain embo-lim~nts, it is contemplated that the nucleic acid segments disclosed herein
will be used to transfect ~ .pliaLe host cells. Technology for introduction of DNA into cells is
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well-known to those of skill in the art. Four ge~eral methods for delivering a nucleic segment into
cells have been described:
(1) rh~mic~l methods (Graham and VanDerEb, 1973);
(2) physical methods such as microinjection (Capecchi, 1980), electroporation (Wong
and Neum~nn 1982, Fromrn et al., 1985) and the gene gun (Yang et al., 1990);
(3) viral vectors (Clapp, 1993; Eglitis and Anderson, 1988); and
(4) receptor-m~ tec1 mçrh~ni~m~ (Curiel etal., 1991; Wagner ef al., 1992).
4.4 LnPosoMEs AND NANOCAPSULES
In certain emboriiment.~, the inventors contemplate the use of liposomes and/or
lo nanocapsules for the introduction of particular peptides or nucleic acid segment~ into host cells.
Such formulations may be preferred for the introduction of pharrnaceutically-acceptable
formulations of the nucleic acids, peptides, and/or antibodies disclosed herein. The formation and
use of liposomes is generally known to those of skill in the art (see for example, Couvreur et al.,
1977 which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy of
15 intracellular bacterial infections and diseases). Recently, liposomes were developed with
improved serum stability and circulation half-times (Gabizon and Papahadjopoulos, 1988; Allen
and Choun, 1987).
Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-
Mic~h~ nri etal., 1987). To avoid side effects due to intr~ce~ r polyrneric overlo~ ng~ such
2 o ultrafine particles (sized around 0.1 ~lm) should be decignçcl using polymers able to be degraded
in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are
contemplated for use in the present invention, and such particles may be are easily made, as
described (Couvreur et a~., 1977; 1988).
Liposomes are formed from phospholipids that are dispersed in an aqueous merlil~m and
25 spontaneously form mllltil~mellar concentric bi}ayer vesicles (also termed m~lltil~mellar vesicles
(MLVs). MLVs generally have diameters of from 25 nm to 4 ~Lm. Sonication of MLVs results in
the formation of small unil~m~ r vesicles (SUVs) with diameters in the range of 200 to 500 C,
cont~ining an aqueous solution in the core.
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In addition to the tear.hing~ of Couvreur el al. (1988), the following information may be
utilized in generating liposomal formulations. Phospholipids can form a variety of structures other
than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low
ratios the liposome is the p~er~ d structure. The physical characteristics of liposomes depend on
pH, ionic strength and the presence of divalent cations. Liposomes can show 1OW permeability to
ionic and polar substances, but at elevated temperatures undergo a phase transition which
markedly alters their permeability. The phase transition involves a change from a closely packed,
ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as
the fluid state. This occurs at a characteristic phase-transition temperature and results in an
o increase in permeability to ions, sugars and drugs.
Liposomes interact with cells via four di~e~ lL mech~ni~m.~: ~ndocytosis by phagocytic
cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell
surface, either by nonspecific weak hydrophobic or electrostatic forces~ or by specific interactions
with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid
bilayer of the liposome into the plasma membrane, with ~imlllt~neous release of liposomal
contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular
membranes, or vice versa, without any association of the liposome contents. It often is difficult to
determine which merl~ .,. is operative and more than one may operate at the same time.
4.5 M ETHODSFOR PREPARUNG A~NTIBODY CO~IPOSITIONS
In another aspect, the present invention contemplates an antibody that is immunoreactive
with a polypeptide of the invention. As stated above, one of the uses for LYST- or Lyst-derived
epitopic peptides according to the present invention is to generate antibodies. :~eference to
antibodies throughout the specification inr.l~lec whole polyclonal and monoclonal antibodies
(mAbs), and parts thereof, either alone or conjugated with other moieties. Antibody parts include
Fab and F(ab)2 fr~gmen~s and single chain antibodies. The antibodies may be made in vivo in
suitable laboratory animals or in vitro using recombinant DNA ter.hniq~es. In a preferred
embodiment, an antibody is a polyclonal antibody. Means for preparing and characterizing
antibodies are well known in the art (See, e.g, Harlow and Lane, 1988).
Briefly, a polyclonal antibody is prepared by immlmi7.in~ an animal with an immlmngen
comprising a polypeptide of the present invention and collecting antisera from that immlmi7ed
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- animal. A wide range of animal species can be used for the production of antisera. Typically an
animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea pig.
Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for
production of polyclonal antibodies.
Antibodies, both polyclonal and monoclonal, specific LYST- or Lyst-derived epitopes may
be prepared using convçnticn~l immlm;7~ti~n techniques, as will be generally known to those of
skill in the art. A composition co~ ini~I~ antigenic epitopes of particular proteins can be used to
immllni7e one or more experimental ~nim~l~, such as a rabbit or mouse, which will then proceed
to produce specific antibodies against LYST- or Lyst-derived peptides. Polyclonal antisera may
0 be obtained, after allowing time for antibody generation, simply by bleeding the animal and
pl ~aling serum samples from the whole blood.
The amount of immlmc-gen composition used in the production of polyclonal antibodies
varies upon the nature ofthe imm-lnogen, as well as the animal used for immllni7~tion A variety
of routes can be used to ~timini~t-~r the immllnogen (subcutaneous, intr~ml-,scl-t~r, intradermal,
intravenous and illLl~peli~oneal). The production of polyclonal antibodies may be monitored by
sampling blood of the immllni7ed animal at various points following immnni7~tion A second,
booster in~ection, also may be given. The process of boosting and titering is repeated until a
suitable titer is achieved. When a desired level of immunogenicity is obtained, the immllni~ed
animal can be bled and the serum isolated and stored, and/or the animal can be used to generate
mAbs (below).
One of the important features obtained from the present invention is a polyclonal sera that
is relatively homogenous with respect to the specificity of the antibodies therein. Typically,
polyclonal antisera is derived from a variety of different "clones," i.e., B-cells of di~el~l,L lineage.
mAbs, by contrast, are defined as corning from antibody-producing cells with a common B-cell
2 5 ancestor, hence their "mono" clonality.
When peptides are used as :~ntigl~n~ to raise polyclonal sera, one would expect
considerably less variation in the clonal nature of the sera than if a whole antigen were employed.
Unfortunately, if incomplete fragments of an epitope are presented, the peptide may very well
assume multiple (and probably non-native) conroll,.alions. As a result, even short peptides can
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- produce polyclonal antisera with relatively plural specificities and, unfortunately, an antisera that
does not react or reacts poorly with the native molecule.
Polyclonal antisera according to present invention is produced against peptides that are
predicted to comprise whole, intact epitopes. It is believed that these epitopes are, therefore,
5 more stable in an imrnunologic sense and thus express a more consistent immunologic target for
the immune system. Under this model, the number of potential B-cell clones that will respond to
this peptide is considerably smaller and, hence, the homogeneity of the resulting sera will be
higher In various embodiments, the present invention provides for polyclonal antisera where the
clonality, i.e., the percentage of clone reacting with the same molecular determinant, is at least
10 80%. Even higher clonality - 90%, 95% or greater - is contemplated.
To obtain mAbs, one would also initially immllni~e an experimental animal, oftenpreferably a mouse, with a LYST- or Lyst-cont~ining composition. One would then, after a
period of time sufficient to allow antibody generation, obtain a population of spleen or Iymph cells
from the animal. The spleen or Iymph cells can then be fused with cell lines, such as human or
15 mouse myeloma strains, to produce antibody-secreting hybridomas. These hybridomas may be
isolated to obtain individual clones which can then be screened for production of antibody to the
desired peptide.
Following immlmi~tion, sp}een cells are removed and fused, using a standard fusion
protocol with plasmacytoma cells to produce hybridomas secreting mAbs against the LYST or
20 Lyst protein. Hybridomas which produce mAbs to the selected antigens are identified using
standard techniques, such as ELISA and Western blot methods. Hybridoma clones can then be
cultured in liquid media and the culture supernatants purified to provide the LYST- or Lyst-
specific mAbs.
It is proposed that the mAbs of the present invention will also find useful application in
2 5 immllnochemical procedures, such as ELISA and Western blot methods, as well as other
procedures such as immunoprecipitation, immunocytological methods, etc. which may utilize
antibodies specific to the LYST or Lyst protein. In particular, anti-LYSTlLyst antibodies may be
used in immunoabsorbent protocols to purify native or recombinant LYST/Lyst proteins or
LYST/Lyst-derived peptide species or synthetic or natural variants thereof.
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The antibodies disclosed herein may be employed in antibody cloning protocols to obtain
cDNAs or genes encoding LYST/Lyst proteins from other species or o~ , or to identify
proteins having signific~nt homology to the LYST/Lyst gene product. They may also be used in
inhibition studies to analyze the effects of LYST/Lyst protein in cells, tissues, or whole animals.
5 Anti- LYST/Lyst antibodies will also be useful in immllnolocalization studies to analyze the
distribution of cells expressing LYST/Lyst protein during pa~ticular cellular activities, or for
example, to determine the cellular or tissue-specific distribution of LYST/Lyst under di~e~
physiological conditions. A particularly useful application of such antibodies is in purif3~ing native
or recombinant LYST/Lyst proteins, for example, using an antibody affinity column. The
10 operation of all such immllnological techniques will be known to those of skill in the art in light of
the present disclosure.
4.6 RECoMsiNANT EXPRESSION OF"LYST FA~nLY" P~PTnD~S
Recombinant clones expressing the "LYST family" nucleic acid segment.~ may be used to
prepare purified recombinant LYST protein (rLYST), purified rLYST-derived peptide antigens as
15 well as mutant or variant recombinant protein species in significant qu~ntities. The selected
antigens, and variants thereof, are proposed to have significant utility in diagnosing and treating
CHS. For example, it is proposed that rLYSTs, peptide variants thereof, andlor antibodies
against such rLYSTs may also be used in immlln(~assays to detect the presence of LYST or as
vaccines or immllnotherapeutics to treat CHS and related disorders. Additionally, by application
2 o of techniques such as DNA mnt~g~nesic, the present invention allows the ready preparation of so-
called "second generation" molecules having modified or simplified protein structures. Second
generation proteins will typically share one or more properties in common with the full-length
~ntig-~n, such as a particular antigeniC/imml1nngenic epitopic core sequence. Epitopic sequences
can be obtained from relatively short moi~cules prepared from knowledge of the peptide, or
25 encoding DNA sequence information. Such variant molecules may not only be derived from
s.?lected irnmunogenic/ antigenic regions of the protein structure, but may additionally, or
alternatively, include one or more functionally equivalent amino acids selected on the basis of
similarities or even differences with respect to the natural sequence.
. ~ -- =
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Y3
4.7 ANTIBODY CO~nPOS1TIONSA~D FO~nULATIONS~HEREOF
Means for ple~ hlg and characterizing antibodies are well known in the art (See, e.g,
Harlow and Lane (1988); incorporated herein by reference). The methods for generating mAbs
generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a
polyclonal antibody is prepared by imm~lni7:in~ an animal with an immllnogenic composition in
accordance with the present invention and collecting antisera from that immnni7ed animal. A
wide range of animal species can be used for the production of antisera. Typically the animal used
for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat.
Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for
production of polyclonal antibodies.
As is well known in the art, a given composition may vary in its immunogenicity. It is
often nec~ss~ry therefore to boost the host immune system, as may be achieved by coupling a
peptide or polypeptide immllnngen to a carrier. Exemplary and pleIèlled carriers are keyhole
limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin,
mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for
conjugating a polypeptide to a carrier protein are well known in the art and include
glutaraldehyde, m-maleimidobenzoyl-N-hydroxys~lc~inimide ester, carbodiimide and bis-biazotized
ben~i~1ine.
mAbs may be readily prepared through use of well-known techniques, such as thoseexemplified in U.S. Patent 4,196,265, incorporated herein by reference. Typically, this technique
involves immllni~in~ a suitable animal with a selected immlmQgen composition, e.g, a purified or
partially purified protein, polypeptide or peptide. The immllni7in~ composition is ~(1mini~t~red in
a manner effective to stiml-l~te antibody producing cells. Rodents such as mice and rats are
pl ert~ ;d ~nims~l.c7 however, the use of rabbit, sheep or frog cells is also possible. The use of rats
may provide certain advantages (Goding, 1986), but rnice are pl~re~-~;d, with the BALB/c mouse
being most preferred as ~his is most routinely used and generally gives a higher percentage of
stable fusions.
Following immllni~tion, somatic cells with the potential for producing antibodies,
specifically B-lyrnphocytes (B-cells), are selected for use in the mAb generating protocol. These
3 o cells may be obtained from biopsied spleens, tonsils or Iymph nodes, or from a peripheral blood
sarnple. Spleen cells and peripheral blood cells are plt:rell~d, tlle former because they are a rich
t . ,.
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source of antibody-producing cells that are in the dividing plasmablast stage, and the latter
because peripheral blood is easily ~cc~scihle. O~en, a panel of animals will have been immllni7:ed
and the spleen of animal with the highest antibody titer will be removed and the spleen
lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an
5 immllni7:ed mouse contains approximately about 5 x 107 to about 2 X108 Iymphocytes.
The antibody-producing B Iymphocytes from the imml~ni7ed animal are then fused with
cells of an immortal myeloma cell, generally one of the same species as the animal that was
imm~lni~ed Myeloma cell lines suited for use in hybridoma-producing fusion procedures
preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that
o r.ender them incapable of growing in certain selective media which support the growth of only the
desired fused cells (hybridomas).
Any one of a number of myeloma cells may be used, as are known to those of skill in the
art (Goding, 1986; Campbell, 1984). For example, where the immlmi7ed animal is a mouse, one
may use P3-X63/Ag8, X63-Ag8.653, N~1/1.Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-11,
MPCll-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3,
IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-~y2 and UC729-6 are all useful
in connection with human cell fusions.
One pl~rell~d murine myeloma cell is the NS-l myeloma cell line (also termed P3-NS-l-
Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by
20 requesting cell line repository number GM3573. Another mouse myeloma cell line that may be
used is the 8-~7~ nine-resistant mouse murine myeloma SP2/0 non-producer cell line.
Methods for generating hybrids of antibody-producing spleen or Iymph node cells and
myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2: 1 ratio, though the
ratio may vary from about 20: 1 to about 1: 1, respectively, in the presence of an agent or agents
2 5 (chemical or electrical) that promote the filsion of cell membranes. Fusion methods using Sendai
virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol
(PEG), such as 37% (v/v) PEG, by Ge~er et al. (1977). The use of electrically incluced fusion
methods is also app-op,iate ~Goding, 1986).
Fusion procedures usually produce viable hybrids at low fre~uencies, about 1 x 10-6 to
30 about 1 x 10-8. However, this does not pose a problem, as the viable, fused hybrids are
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.li~e~ ted from the parental, l~nfil~e-l cclls (particularly the llnfil~e(l myeloma cells that would
normally continue to divide in~çfinitely) by culturing in a selective medium. The selective me~ m
is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the
tissue culture media. Exemplary and pl ~r~l I ed agents are aminopterin, methotrexate, and
5 azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and
pyrimi~lin~s~ whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate
is used, the media is supplem~ntecl with hypc~xn~ and thymidine as a source of nucleotides
(HAT me-lillm). Where ~aserine is used, the media is suppl~mented with hypox~nthine.
The pl~rel,~d selection medium is HAT. Only cells capable of operating nucleotide
0 salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key
enzymes of the salvage pathway, e.g, hypoxanLhil~e phosphoribosyl transferase (E~RT), and they
cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture
and generally die within about two weeks. Therefore, the only cells that can survive in the
selective media are those hybrids formed from myeloma and B-cells.
This culturing provides a population of hybridomas from which specific hybridomas are
selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone
dilution in microtiter plates, followed by testing the individual clonal supelllalanLs (after about two
to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as
radioimmunoassays, enzyme immlmoassays, cytotoxicity assays, plaque assays, dot
2 o imml~n~binding assays, and the like.
The selected hybridomas would then be serially diluted and cloned into individual
antibody-producing cell lines, which clones can then be propagated in(i~finitç]y to provide mAbs.
The cell lines may be exploited for mAb production in two basic ways. A sample of the
hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the
2 5 type that was used to provide the somatic and myeloma cells for the original fusion. The injected
animal develops tumors secreting the specific mAb produced by the fused cell hybrid. The body
fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high
concel~ Lion. The individual cell lines could also be cultured in vi~ro, where the mAbs are
.~ naturally secreted into the culture me~lillm from which they can be readily obtained in high
30 concentrations. mAbs produced by either means may be further purified, if desired, using
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filtration, centrifugation and various chroma~tographic methods such as HPLC or affinity
chromatography .
4.8 I~U~UNOASSAYS
As noted, it is proposed that native and synthetically-derived peptides and peptide
5 epitopes of the invention will find utility as immun~gens, e.g, in connection with vaccine
development, or as antigens in immllnr~assays for the detection of reactive antibodies. Turning
first to immllnnassays, in their most simple and direct sense, preferred immunoassays of the
invention include the various types of enzyrne linked immllnosorbent assays (ELISAs), as are
~nown to those of skill in the art. However, it will be readily appreciated that the utility of
10 I,YST-derived proteins and peptides is not limited to such assays, and that other useful
embodiments include RIAs and other non-enzyme linked antibody binding assays and procedures.
In ~lerell~;d El~I~A assays, proteins or peptides incorporating LYST, rLYST, or LYST-
derived protein antigen sequences are immobilized onto a selected surface, preferably a surface
e~ibiLillg a protein affinity, such as the wells of a polystyrene microtiter plate. After washing to
15 rernove incompletely adsorbed material, one would then generally desire to bind or coat a
nonspecific protein that is known to be ~ntig~nically neutral with regard to the test antisera, such
as bovine serum albumin (BSA) or casein, onto the well. This allows for blocking of nonspecific
adsorption sites on the immobilizing surface and thus reduces the background caused by
nonspecific binding of antisera onto the surface.
Af~er binding of antigenic material to the well, coating with a non-reactive material to
reduce background, and washing to remove unbound material, the immobilizing surface is
c~ nt~cted with the antisera or clinical or biological extract to be tested in a manner conducive to
immlln~ complex (antigen/antibody) forrnation. Such conditions preferably include diluting the
antisera with diluents such as BSA, bovine gamma globulin ~BGG) and phosphate buffered saline
2 5 (PBS)/TweenTM. These added agents also tend to assist in the reduction of nonspecific
background. The layered antisera is then allowed to incubate for, e.g., from 2 to ~ hours, at
temperatures preferably on the order of about 2~~ to about 27~. Following inc~1b~tion, the
antisera-contacted surface is washed so as to remove non-immlln~complexed material. A
pl~;rell~d washing procedure includes washing with a solution such as PBS/Tween~M, or borate
3 o buffer.
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Following formation of specific imrmunocornplexes between the test sample and the bound
antigen, and subsequent washing, the occurrence and the amount of immlmocomplex formation
may be determined by subjecting the complex to a second antibody having specificity for the first.
Of course, in that the test sample will typically be of human origin, the second antibody will
5 preferably be an ant;body having specificity for human antibodies. To provide a detecting means,
the second antibody will plc;rel~ly have an associated detectable label, such as an enzyme label,
that will generate a signal, such as color development upon incubating with an approl)~iate
chromogenic substrate. Thus, for example, one will desire to contact and incubate the antisera-
bound surface with a urease or peroxidase-con)~ ted anti-human IgG for a period of time and
0 under conditions that favor the development of immunocomplex formation (e.g., incubation for 2
hours at room temperature in a PBS-cont~inin~ solution such as PBS-TweenIM).
After incubation with the second enzyme-tagged antibody, and subsequent to washing to
remove unbound material, the amount of label is quantified by incubation with a chromogenic
substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-b~n~thi~7oline)-6-sulfonic
15 acid (ABTS) and ~ O2, in the case of peroxidase as the enzyme label. Q~l~ntit~tion is then
achieved by measuring the degree of color generation, e.g, using a visible spectrum
spectrophotometer.
ELISAs may be used in conjunction with the invention. In one such ELISA assay,
proteins or peptides incorporating antigenic sequences of the present invention are immobilized
20 onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a
polystyrene microtiter plate. After washing to remove incompletely adsorbed material, it is
desirable to bind or coat the assay plate wells with a nonspecific protein that is known to be
antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein
or solutions of powdered milk This allows for blocking of nonspecific adsorption sites on the
25 irnmobilizing surface and thus reduces the background caused by nonspecific binding of antisera
onto the surface.
4.9 1M~UNOPRECIPITATION
The ant;-LYST protein antibodies of the present invention are particularly useful for the
isolation of LYST protein antigens by imml-noprecipitation. Tmmlmnprecipitation involves the
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separation ofthe target antigen component*om a~complex mixture, and is used to discriminate or
isolate rninute amounts of protein.
In an alternative embodiment the antibodies of the present invention are useful for the
close juxtaposition of two antigens. This is particularly useful for increasing the localized
concentration of antigens, e.g, enzyme-substrate pairs.
4.10 W ESTERN BLOTS
The compositions of the present invention will find great use in immlln~blot or western
blot analysis. The anti-LYST antibodies may be used as high-affinity primary reagents for the
identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or
0 combinations thereof. In coniunction with imml~noprecipitation, followed by gel electrophoresis,
these may be used as a single step reagent for use in detecting antigPn~ against which secondary
reagents used in the detection of the antigen cause an adverse background. This is especially
useful when the anti~n~ studied are immllnoglobulins (preçlu-ling the use of immllnoglobulins
binding bacterial cell wall components~, the antigens studied cross-react with the detecting agent,
or they rnigrate at the same relative molecular weight as a cross-reacting signal. Immunologically-
based detection methods in conjunction with Western blotting (inçlntlin~ enzymatically-,
radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety~ are considered
to be of particular use in this regard.
4.11 ~ARU~ACEUTICAL CO~IPOSITIONS
2 o The pharm~ce -tical compositions disclosed herein may be orally ~rlmini~tf red, for
example, with an inert diluent or with an s~c.~imil~hle edible carrier, or they may be enclosed in
hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be
incorporated directly with the food of the diet. For oral therapeutic ~(lmini~tration, the active
compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal
2 5 tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and
preparations should contain at least 0.1% of active compound. The percentage of the
compositions and preparations may, of course, be varied and may conveniently be between about
2 to about 60% of the weight of the unit The amount of active compounds in such
therapeutically useful compositions is such that a suitable dosage will be obtained.
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~)
The tablets, troches, pills, capsules and thé like may also contain the following: a binder,
as gum tr~g~c~nth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a
t1i~int~.grating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such
as magnesium stearate; and a sweet~ning agent, such as sucrose, lactose or saccharin may be
5 added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the
dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid
carrier. ~arious other materials may be present as coatings or to otherwise modify the physical
form of the dosage unit. For in~t~nc~, tablets, pills, or capsules may be coated with shellac, sugar
or both. A syrup of elixir may contain the active compounds sucrose as a sweetf ninp agent
10 methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
Of course, any material used in preparing any dosage unit forrn should be pharm~ce~ltically pure
and substantially non-toxic in the amounts employed. In addition, the active compounds may be
incorporated into sustained-release plepaldlion and formulations.
The active compounds may also be ~lminict~red parenterally or intraperitoneally.5 Solutions of the active compounds as free base or pharmacologically acceptable salts can be
prepared in water suitabIy mixed with a surfactant, such as hydroxypropylcellulose. Dispersions
can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
Under ordinary conditions of storage and use, these preparations contain a preservative to prevent
the growth of microor~;~ni.cm.~.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions
or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy
syringability exists. It must be stable under the conditions of m~m-f~ctllre and storage and must
be preserved against the co~ g action of microo~ , such as bacteria and fungi. The
carrier can be a solvent or dispersion merlillm cont~ining~ for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable
mixtures thereof, and vegetable oils. The proper fiuidity can be ",~ ;..e~l; for example, by the
use of a coating, such as lecithin, by the m~int~n~nce of the required particle size in the case of
dispersion and by the use of surf~t~ntc. The prevention of the action of microorg~ni~m~ can be
3 o brought about by various antibacterial and ~ntifilng~l agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to
include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the
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injectable compositions can be brought about by the use in the compositions of agents delaying
absorption, for e7~ample, ahlmimlm monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the
required amount in the appropriate solvent with various of the other ingredients enumerated
5 above, as required, followed by filtered sterilization. Generally, dispersions are prepared by
incorporating the various sterilized active ingredients into a sterile vehicle which contains the
basic dispersion merlillm and the required other ingredients from those enumerated above. In the
case of sterile powders for the preparation of sterile injectable solutions, the ple~r~ d methods of
plepal~lion are vacuum-drying and freeze-drying techniques which yield a powder of the active
0 ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
As used herein, "pharm~elltically acceptable carrier" includes any and all solvents,
dispersion media, coatings, ~ntihact~rial and ~ntifimg~l agents, isotonic and absorption delaying
agents and the like. The use of such media and agents for pharm~ce~ltical active substances is well
known in the art. Except insofar as any conventional media or agent is incompatible with the
15 active ingredient, its use in the therapeutic compositions is contemplated. Suppl~ment~ry active
ingredients can also be incorporated into the compositions.
For oral prophylaxis the polypeptide may be incorporated with excipients and used in the
form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared
incorporating the active ingredient in the required amount in an a~ liate solvent, such as a
2 o sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be
incorporated into an antiseptic wash co"l~;";l-g sodium borate, glycerin and potassium
bicarbonate The active ingredient may also be dispersed in dentifrices, in~ tling gels, pastes,
powders and slurries. The active ingredient may be added in a therapeutically effective amount to
a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and
2 5 h1lmf~ct~nt~.
The phrase "pharm~ce~ltic~lly-acceptable" refers to molecular entities and compositions
that do not produce an allergic or similar untoward reaction when ~(lmini~tered to a human. The
preparation of an aqueous composition that contains a protein as an active ingredient is well
understood in the art. Typically, such compositions are prepared as injectables, either as liquid
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solutions or suspensions; solid forrns suitable for solution in, or suspension in, liquid prior to
injection can also be prepared. The preparation can also be ~m~ ified.
The composition can be formnl~te~l in a neutral or salt form. Pharm~centically-acceptable
salts, include the acid addition salts (formed with the free amino groups of the protein) and which
are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such
organic acids as acetic, oxalic, tartaric, m~n(lelic, and the like. Salts forrned with the free carboxyl
groups can also be derived from inorganic bases such as, for example, sodium, pot~ m,
arnmonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be ~rlmini~t~red
o in a manner compatible with the dosage formulation and in such amount as is therapeutically
effective. The forrnulations are easily ~rimini~t~red in a variety of dosage forms such as injectable
solutions, drug release capsules and the like.
For parenteral ~imini~tration in an aqueous solution, for example, the solution should be
suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or
glucose. These particular aqueous solutions are especially suitable for intravenous, intram-lcclll~r,
subcutaneous and intraperitoneal ~lmini~tration. In this connection, sterile aqueous media which
can be employed will be known to those of skill in the art in light of the present disclosure. For
example, one dosage could be dissolved in 1 ml of isotonic NaCI solution and either added to
1000 ml of hypodermoclysis fluid or injected at the proposed site of infilsion, (see for example,
"Remington's Pharm~cell~ical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some
variation in dosage will necessarily occur depending on the condition of the subject being treated.
The person responsible for ~t1mini.~tration will, in any event, determine the apprc,pliate dose for
the individual subject. Moreover, for human ~lmini.~tration, preparations should meet sterility,
pyrogenicity, general safety and purity standards as re~uired by FDA Office of Biologics
2 5 standards.
4.12. ~PITOPIC CORE ~EQUENCES
The present invention is also directed to protein or peptide compositions, free from total
cells and other peptides, which comprise ~ purified protein or peptide which incorporates an
epitope that is immnnologically cross-reactive with one or more of the antibodies of the present
3 o invention.
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As used herein, the terrn "incorporatmg an epitope(s) that is imrnunologically cross-
reactive with one or more anti-LYST protein antibodies" is int~n~led to refer to a peptide or
protein antigen which in~ (lçc a primary, secondary or tertiary structure similar to an epitope
located within a LYST polypeptide. The level of similarity will generally be to such a degree that
5 monoclonal or polyclonal antibodies directed against the LYST polypeptide will also bind to,
react with, or otherwise recognize, the cross-reactive peptide or protein ~ntigçn Various
immllnn~c.Say methods may be employed in conjunction with such antibodies, such as, for
example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in
the art.
0 The identification of LYST-derived epitopes such as those derived from the LYST gene
or LYST-like gene products and/or their functional equivalents, suitable for use in vaccines is a
relatively straightfor~vard matter. For example, one may employ the methods of ~opp, as taught
in U.S. Patent 4,554,101, incorporated herein by reference, which teaches the identification and
pl~al~LLion of epitopes from amino acid seq~l~nces on the basis of hydrophilicity. The methods
described in several other papers, and software programs based thereon, can also be used to
identify epitopic core seq~lçnçes (see, for exarnple, Jameson and Wolf, 1988; Wolf et al., 1988;
U.S. Patent Number 4,554,101). The amino acid sequence of these "epitopic core sequences"
may then be readily incorporated into peptides, either through the application of peptide synthesis
or recombinant technology.
2 o Preferred peptides for use in accordance with the present invention will generally be on the
order of about 5 to about 25 amino acids in length, and more preferably about 8 to about 20
arnino acids in length. It is proposed that shorter antigenic peptide sequences will provide
advantages in certain CilCl~ CÇ':, for example, in the prt;~al~lion of vaccines or in
~mmlmQlogic detection assays. Exemplary advantages include the ease of plt;~al~lion and
2 5 purification, the relatively low cost and improved reproducibility of production, and advantageous
biodistribution.
It is proposed that particular advantages of the present invention may be realized through
the pl ~al ~Lion of synthetic peptides which include modified and/or extended
epitopic/immlmQgenic core sequences which result in a "universal" epitopic peptide directed to
3 o the LYST gene product or LYST-related sequences. It is proposed that these regions represent
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those which are most likely to promote T-cell or B-cell stim~ tion in an animal, and, hence, elicit
specific antibody production in such an animal.
An epitopic core sequence, as used herein, is a relatively short stretch of amino acids that
is "complementary" to, and therefore will bind, antigen binding sites on LYST protein epitope-
specific antibodies. Additionally or alternatively, an epitopic core sequence is one that will elicit
antibodies that are cross-reactive with antibodies directed against the peptide compositions of the
present invention. It will be understood that in the context of the present disclosure, the term
"complementary" refers to amino acids or peptides that exhibit an attractive force towards each
other. Thus, certain epitope core sequences of the present invention may be operationally defined
in terms of their ability to compete with or perhaps displace the binding of the desired protein
antigen with the corresponding protein-directed antisera.
In general, the size of the polypeptide antigen is not believed to be particularly crucial, so
long as it is at least large enough to carry the identified core sequence or sequences. The smallest
useful core sequence expected by the present disclosure would generally be on the order of about
5 amino acids in length, with sequences on the order of 8 or 25 being more plerelled. Thus, this
size will generally correspond to the smallest peptide antigens prepared in accordance with the
invention However, the size of the antigen may be larger where desired, so long as it contains a
basic epitopic core sequence.
The id~ntific~tion of epitopic core sequences is known to those of skill in the art, for
example, as described in U.S. Patent 4,554,101, incorporated herein by reference, which teaches
the i~entific~tion and preparation of epitopes from amino acid sequences on the basis of
hydrophilicity. Moreover, numerous computer programs are available for use in predicting
antig~nic portions of proteins (see e.g, Jameson and Wolf, 1988, Wolf et al., 1988).
Computerized peptide sequence analysis programs (e.g, DNAStar(~) software, DNAStar, ~nc.,
Madison, WI) may also be useful in designing synthetic LYST peptides and peptide analogs in
accordance with the present disclosure.
To confirm that a protein or peptide is immllnologically cross-reactive with, or a biological
functional equivalent of, one or more epitopes of the disclosed peptides is also a straightforward
matter. This can be readily determined using specific assays, e.g., of a single proposed epitopic
3 o sequence, or using more general screens, e.g, of a pool of randomly generated synthetic peptides
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5~
or protein fr~gment~. The screening assaysmay be employed to identify either equivalent antigens
or cross-reactive antibodies. In any event, the principle is the same, i.e., based upon competition
for binding sites between antibodies and antigens.
Suitable competition assays that may be employed include protocols based upon
5 immllnohistochemical assays, ELISAs, RlAs, Western or dot blotting and the like. In any of the
competitive assays, one of the binding components, generally the known element, such as the
LYST gene product or LYST-derived peptides, or a known antibody, will be labeled with a
detectable label and the test components, that generally remain unlabeled, will be tested for their
ability to reduce the amount of label that is bound to the corresponding reactive antibody or
1 o antigen.
As an exemplary embodiment, to conduct a competition study between a LYST protein
and any test antigen, one would first label LYST with a detect~ble label, such as, e.g, biotin or an
enzymatic, radioactive or fluorogenic label, to enable subsequent iclentific~tion. One would then
incubate the labeled antigen with the other, test, antigen to be examined at various ratios (e.g.,
5 l~ l0 and l:l00) and, after mixing, one would then add the mixture to an antibody of the
present invention Preferably, the known antibody would be immobilized, e.g., by ~tt~t-.hing to an
ELISA plate. The ability of the mixture to bind to the antibody would be determined by detecting
the presence of the specifically bound label. This value would then be compared to a control
value in which no potentially competing (test) antigen was in~lucled in the incubation.
2 o The assay may be any one of a range of imml-nological assays based upon hybridization,
and the reactive antigens would be detected by means of detecting their label, e.g, using
streptavidin in the case of biotinylated antigens or by using a chromogenic substrate in connection
with an enzymatic label or by simply cletecting a radioactive or fluorescent label. An antigen that
binds to the same antibody as LYST, for example, will be able to effectively compete for binding
to and thus will si~nific~ntly reduce LYST binding, as evidenced by a reduction in the amount of
label detected.
The reactivity of the labeled ~ntip~n, e.g, a LYST composition, in the absence of any test
antigen would be the control high value. The control low value would be obtained by incubating
the labeled antigen with an excess of unlabeled LYST antigen, when competition would occur and
reduce binding. A ~ignific~nt reduction in labeled antigen reactivity in the presence of a test
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antigen is indicative of a test antigen that is "cross-reactive", i.e., that has binding affinity for the
same antibody. "A significant reduction", in terms of the present application, may be defined as a
reproducible (i.e., con~i~tçntly observed) reduction in binding.
In addition to the peptidyl compounds described herein, the inventors also contemplate
5 that other sterically similar compounds may be fo~ tecl to mimic the key portions of the
peptide structure. Such compounds, which may be termed peptidomimetics, may be used in the
same manner as the peptides of the invention and hence are also functional equivalents. The
generation of a structural functional equivalent may be achieved by the techniques of modelling
and chemical design known to those of skill in the art. It will be understood that all such sterically
0 similar constructs fall within the scope of the present invention.
Syntheses of epitopic sequences, or peptides which include an antigenic epitope within
their sequence, are readily achieved using conventional synthetic techniques such as the solid
phase method (e.g., through the use of a commercially-available peptide synthçsi7e~r such as an
Applied Biosystems Model 430A Peptide Syntheci7çr) Peptide antigens synthç~i7ed in this
15 manner may then be aliquoted in predetermined amounts and stored in conventional manners,
such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending
use.
In general, due to the relative stability of peptides, they may be readily stored in aqueous
solutions for fairly long periods of time if desired, e.g, up to six months or more, in virtually any
20 aqueous solution without appreciable degradation or loss of antigenic activity. However, where
~xtP.ntlçd aqueous storage is contemplated it will generally be desirable to include agents including
buffers such as Tris or phosphate buffers to m~int~in a pH of about 7 0 to about 7.5. Moreover,
it may be desirable to include agents which will inhibit microbial growth, such as sodium azide or
Merthiolate. For çxt~.nded storage in an aqueous state it will be desirable to store the solutions at
25 4~C, or more preferably, frozen. Of course, where the peptides are stored in a Iyophilized or
powdered state, they may be stored virtually intl~finitçly, e.g, in metered aliquots that may be
, t:llydl ~led with a predetermined amount of water (preferably distilled) or bu~er prior to use.
.
~ 4.13 SITE_SPECIFIC MUTAGENESIS
- Site-specific mllt~gçn~.si.~ is a technique useful in the preparation of individual peptides, or
3 o biologically functional equivalent proteins or peptides, through specific mutagenesis of the
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underlying DNA. The technique, well-known to those of skill in the art, further provides a ready
ability to prepare and test sequence variants, for example, incorporating one or more of the
foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
Site-specific mutagenesis allows the production of mllt~nt.~ through the use of specific
5 oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a
sufficient number of a-ljac~nt nucleotides, to provide a primer sequence of sufficient size and
sequence complexity to form a stable duplex on both sides of the deletion junction being
traversed. Typically, a primer of about 14 to about 25 nucleotides in length is preferred, with
about 5 to about l 0 residues on both sides of the junction of the sequence being altered.
0 In general, the technique of site-specific mutagenesis is well known in the art, as
exemplified by various publications. As will be appreciated, the technique typically employs a
phage vector which exists in both a single stranded and double stranded form. Typical vectors
useful in site-directed mutagenesis include vectors such as the ~13 phage. These phage are
readily commercially-available and their use is generally well-known to those skilled in the art.
Double-stranded plasmids are also routinely employed in site directed mutagenesis which
tolimin~tes the step of L~ rellhlg the gene of interest from a plasmid to a phage.
In general, site-directed mutagenesis in accordance herewith is performed by first
obtaining a single-stranded vector or melting apart of two strands of a double-stranded vector
which inclndes within its sequence a DNA sequence which encodes the desired peptide. An
2 0 oligonucleotide primer bearing the desired mnt~ted sequence is prepared, generally synthetically.
This primer is then annealed with the single-stranded vector, and sub~ected to DNA polymerizing
enzymes such as E. colf polymerase I Klenow fragment, in order to complete the synthesis of the
mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original
non-mnt~ted seql-enc~e and the second strand bears the desired mutation. This heteroduplex
vector is then used to Ll~lsf~ applopliaLe cells, such as E colf cells, and clones are selected
which include recombinant vectors bearing the mllt~ted sequence arr~n~çm~nt
The ~ palaLion of sequence variants of the selected peptide-encoding DNA segment~
using site-directed mutagenesis is provided as a means of producing potentially useful species and
is not meant to be limiting as there are other ways in which sequence variants of peptides and the
3 o DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the
desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain
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57
sequence variants. Specific details reg~r~1ing these methods and protocols are found in the
teaching~ of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis
et QI~1982~ each incorporated herein by reference, for that purpose.
.1
4.14 BIOLOGICAL ~ NCTIONAL EQU~VALENTS
Modification and ch~nges, may be made in the structure of the peptides of the present
invention and DNA segm.?nt~ which encode them and still obtain a functional molecule that
encodes a protein or peptide with desirable characteristics. The following is a rii~c~ls~ion based
upon ~h~nging the arnino acids of a protein to create an equivalent, or even an improved, second-
generation molecule. The amino acid changes may be achieved by c~h~ngin~ the codons of the
DNA sequence, according to the codon chart listed in TABLE 1.
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TABLEl
Amino Acids Codons
Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU
Aspartic acid Asp D GAC GAU
Glutamic aci~ Glu E GAA GAG ~.
Phenylalanine Phe F UUC UUU
Glycine Gly G GGA GGC G~G GGU
Histidine His H CAC CAU
Isoleucine Ile I AUA AUC AUU
Lysine Lys K A~A AAG
Leucine Leu L UUA WG CUA CUC CUG C W
Methionine Met M AUG
Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU
Glutamine Gln Q GAA CAG
Arginine Arg R AGA AGG CGA CGC CGG CGU
Serine Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU
Valine Val V GUA GUC GUG GW
Tryptophan ~rp W UGG
Tyrosine Tyr Y UAC UAU
For example, certain amino acids may be substituted for other amino acids in a protein
structure without appreciable loss of interactive binding capacity with structures such as, for
5 exa~nple, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is
the interactive capacity and nature of a protein that defines that protein's biological functional
activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of
course, its underlying DNA coding seqll~nce7 and nevertheless obtain a protein with like
~lope,Lies. It is thus contemplated by the inventors that various changes may be made in the
10 peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode
said peptides without appreciable loss of their biological utility or activity.
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5~
In making such changes, the hydro~athic index of amino acids may be considered. The
irnportance of the hydropathic amino acid index in conferring interactive biologic ~unction on a
protein is generally understood in the art (Kyte and Doolittle, 19~2, incorporate herein by
reference). It is accepted that the relative hydropathic character of the amino acid contributes to
5 the secondary structure of the resultant protein, which in turn defines the interaction of the protein
with other molecules, for example, enzyrnes, substrates, receptors, DNA, antibodies, antigens, and
the like. Each arnino acid has been ~igned a hydropathic index on the basis of their
hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine
(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine
(+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine
(--1.3); proline (--1.6); hi~titline (--3.2); ~lut~m~te (--3.5); gl-lt~mine (--3.5); aspartate (--3.5);
asparagine (-3.5); Iysine (-3.9); and arginine (-4.5).
It is known in the art that certain amino acids may be substituted by other amino acids
having a similar hydropathic index or score and still result in a protein with sirnilar biological
15 activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the
substitution of amino acids whose hydropathic indices are within +2 is pl~rt;ll ~d, those which are
within _1 are particularly preferred, and those within +0.5 are even more particularly pref~lled. It
is also understood in the art that the substitution of like amino acids can be made effectively on
the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that
20 the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its
c~nt arnino acids, correlates with a biological property of the protein.
As clet~iled in U.S. Patent 4,554,101, the following hydrophilicity values have been
ssi~n~d to amino acid residues: arginine (+3.0); Iysine (+3.0); aspartate (+3.0 _ 1); glllt~m~te
(+3.0 + 1); serine (+0.3); asparagine (+0.2); glllt~mine (+0.2); glycine (0); threonine (-0.4);
proline (--0.5 + 1); alanine (-0.5); hicti(line (--0.5); cysteine (-1.0); methionine (--1.3); valine (--
}.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.33; phenylalanine (--2.5); tryptophan (-3.4). It
is understood that an amino acid can be substituted for another having a similar hydrophilicity
value and still obtain a biologically equivalent, and in particular, an irnmunologically equivalent
protein. In such changes, the substitution of arnino acids whose hydrophilicity values are within
30 +2 is plere.l~d, those which are within ~1 are particularly p.ere-l~d, and those within _0.5 are
even more particularly pl~L~Iled.
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As outlined above, amino acid substitutions are generally therefore based on the relative
similarity of the amino acid side-chain substit~ nt.c, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the
foregoing characteristics into consideration are well known to those of skill in the art and include:
arginine and Iysine; ~ t~m~te and aspartate; serine and threonine; g1--t~mine and asparagine; and
valine, leucine and isoleucine.
* * * * * * * ~ * *
. EXA~rPLES
The following exarnples are inç1~-(1ed to demonstrate p~ ed embodiments of the
invention. It should be appreciated by those of skill in the art that the techniques disclosed in the
examples which follow represent techniques discovered by the inventors to function well in the
practice of the invention, and thus can be considered to constitute ple;rc~ d modes for its
practice. However, those of skill in the art should. in light of the present disclosure, appreciate
that many changes can be made in the specific embodiments which are disclosed and still obtain a
like or similar result without departing from the spirit and scope of the invention.
.1 EXA~DPLEI- M APPLNG OF1~nE BG CRUTICALI~EGION ON M OUSE CEDR13
Three mouse mutations whose molecular basis is unknown, beige (bg), crinkled (cr), and
progressive motor neuronophathy C,~mn), are clustered within 2 cM on proximal mouse Chr 13.
As part of a regional positional cloning effort, a high resolution physical map has been established
2 o of a 0.24 cM interval of mouse Chr 13 which corresponds to the bg critical region. l 1 Yeast-
artificial chromosomes (YACs) and 2 Pl clones, isolated using bg critical region STS, were
characterized by STS-content mapping. This was achieved using existing microsatellite markers
and 20 novel sequence tagged sites (STS) which were generated from critical region YAC clone
DNA by inverse-repetitive element PCRTM and direct selection. 2400-kb of the bg-critical region
was isolated in YAC and Pl clones. Expressed sequence tags were identified from a bg-critical
region YAC clone by direct selection, and 1 ~ st;ll~ potential c~ntlid~tes for bg and cr.
Positional cloning represents an approach to disease gene icl~.ntific.zl~ion based solely upon
chromosomal location. In the l 0 years since its inception, positional cloning has become
established as a general, relatively efficient mode of identification of genes causing m~mm~ n
CA 02244744 1998-07-29
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M~nrl~ n disorders (Collins, 1995). Recently developed techniques and resources have both
n~ mhcred and codified positional cloning; precise genetic mapping of a locus is followed by
physical mapping and cloning of the resultant nonrecombinant interval in overlapping genomic
clones (contigs) constructed using vectors which accommodate large DNA inserts. Transcribed
sequences are then systematically identified from contig genomic clones and screened for
mutations in affected individuals. An additional advantage of positional cloning is that it
represents a regional, rather than disease-specific, approach. Thus reagents and resources
developed for the purpose of cloning a specific disease gene, such as novel sequence tagged sites
(S~r~), precise genetic maps, and establishrnent of relationships among clones in a contig, are also
useful in positionally cloning other loci mapping within the same genomic region.
The region of proximal mouse Chr 13 ~ <c~nt to the extra-toes (Xt) locus is rich in
mutant phenotypes, and 1 epresc~ an interval where a regional approach to disease gene
identification may be synergistic. Xf is homologous to the human disorder Greig
cephalopolysyndactyly; using a positional ç~n~ te approach, mutations in a zinc-finger gene
(Gli3) were shown to underlie X~ (Vortkamp et al., 1992; Hui and Joyner, 1993). Very close to
Xt lies the recessive mutation progressive motor neuronopathy (pmn), a model for Werdnig-
Hoffmann spinal mn~c~ r atrophy (0 recombinants in 246 meioses, Brunialti et al., 1995). The
recessive mutation crinkled (cr) maps approximately 2 cM proximal to Xt (23 recombinants in
1197 meioses; Swank et al., 1991; Lyon e~ al., 1967). Finally, beige (bg), the homolog of human
2 o Chediak-Higashi syndrome, maps between cr and Xt (Lane, 1971, Lyon and Meredith, 1969). bg
is particularly amenable to a positional cloning approach for 3 additional reasons:
(1.) the Pxi~t~nce of numerous bg alleles f~.il;t~tes c~n~litl~te gene mutation analysis;
(2.) bg is associated with a characteristic cellular phenotype (giant, perinuclear,
dysfunctional lysosomes) offering the possibility of screening c~n-1i<i~te. genes by genetic
2 5 compl~m.o.nt~tion; and
(3.) direct selection can be utilized to identify transcribed sequences which are
c~n-litl~tes for bg from YAC clones since all cell types are affected in bg homozygotes.
Positional cloning of bg has been performed as an ~ntececlf-nt to id~ntific.~tion of the
- homologous human gene, which is probably defective in human ~hediak-Higashi syndrome.
3 o Using backcross rnice, bg was previously located to a 0.24 cM interval on Chr 13. The example
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illustrates the further characterization of the bg critical region with 20 novel sequence tagged sites
(STS), and the isolation of overlapping YAC and Pl clones which encompass most of this region
of mouse Chr l 3 .
~ M ATERLALSAI~D METHODS
5 5.1.1.1 YAC MA*nPULATION
A mouse genomic DNA library constructed in the vector pYAC4 (Kusumi et al.,l994;Research Genetics Inc.~ was screened by PCRTM with primers derived from STS fl~nking bg
False positive PCR~M products were lllin;~";~d by raising ~nne~lin3~ temperatures, and addition of
an enhancer of polymerase specificity as necessary (Perfect Match, Str~t~ne7 La Jolla, CA).
0 Veracity of PCRlM products was checked by product digestion with suitable restriction
endonucleases, and by inclusion of control yeast DNA in all PCR~ reactions. Individual colonies
of yeast clones col"~;",ng YACs of interest were isolated on plates and frozen in 50% glycerol to
prevent occurrence of microdeletions. YAC clones were grown in liquid YPD merlinm,
converted to spheroplasts at exponential growth using Zymolase (ICN Pharmaceuticals, Costa
15 Mesa, CA), and chromosomal DNA purified in agarose. YAC ~NA was separated from host
yeast chromosomes using preparative pulsed field electrophoresis (PFGE) with low melting point
agarose ~SeaPla~lue~M GTG, FMC Bioproducts, Rockland, ME), and excised with a sterile blade.
5.1.1.2 Pl CLONES
A mouse genomic DNA library constructed in the vector Pl (Pierce ef al., 1992; Genome
2 o Systems Inc., St. Louis, MO) was screened by PCR~M with primers derived from STS fl~nkin~ bg
Stabs corresponding to positive clones were streaked on kanamycin plates, and DNA prepared
from individual colonies as described (Pierce et al., 1992).
5.1.1.3 PULSED ~IEL1~ ELECTROPHORESIS
Preparation of high molecular weight DNA in agarose blocks, restriction enzyme
25 digestion, PFGE, and Southern transfer were performed as previously described (~ing~more et
al., 1989). In brief, mouse splenocytes, Iymph node cells, or yeast spheroplasts, were suspended
in 0.5% low-melting point agarose (InCert~, F~C BioProducts) at 1-2 x 107 cells per ml
(m~mmz~ n cells) or 1-2 x 1 ol'' cells per ml (yeast). DNA was prepared by inc~lb~fion of agarose
blocks in SoO m~ EDTA (pH 9.0), 1% sodium lauroyl sarcosinate, 2% proteinase K at 50~C
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~3
twice for 24 h. Blocks were then wash~d, treated with phenylmethylsulfonylfluoride, washed
again, and digested with 2-10 units/~gDNA of restriction endonucleases (Boehringer-M~nnh~im
Biochemicals, Tn~i~n~rolis, IN), if necessary. PFGE was carried out in 1% agarose gels
~Fastlane, FMC BioProducts) at 14~C in lX TBE using a Gene Navigator unit (Pharmacia,
Piscataway, NJ). Separation of 50-1500 kb DNA molecules was achieved using pulses ramped
from 70-145 sec at 145 V for 46 h. Gels were stained with ethic~ m bromide to visualize
molecular size standards (oligomers of ~ phage, and chromosomes of Saccharomyces cerevisiae
[FMC BioProducts]). Southern ~l~llsre. of DNA onto Zeta-probeTM membranes (Bio-Rad
Laboratories), and filter hybridizations were performed as previously described (King.cmQre ef al.,
1989). ~.~.cignmerlt of two probes to a common restriction fragment was based on se~uential
hybridization of a filter and exhibition of identity by double or partial digests.
5.1.1.4 MOLECULAR PROBES
All probes were labeled by the hexanucleotide technique with "-[3ZP]dCTP as previously
described ~Kingsmore e~ al., 1989). Restriction endonuclease fr~gment~ representing ends of
1~; YAC clones were identified by Southern blot hybridization with pBR322 (which hybridizes
efficiently to pYAC4); YAC clone internal restriction endonuclease fr~gm.?nt~ were identified by
hybridization with a mouse B1 repetitive element probe.
5.1.1.~ IN rERspERsED REPETITIVE ELEMENT-POLYMERASE CHAIN REACTION
Il~-PCRTM was performed e.~.ct~nti~lly as described using mouse Bl repetitive element
primers and PFGE-purified YAC DNA as template (Hunter et aL, 1993; Simmler et al., 1991).
The B1 repetitive element-specific primers used were 5'-CCAGGACACCAGGGCTACAGAG-3'
(SEQ ID NO:75) (forward primer, derived from the 3'-end of B1) and /or 5'-
CCCGAGTGCTGGGATTAAAG-3' (SEQ ID NO:76) (reverse primer, derived from the 5'-end
of Bl). Inter-Bl PCRTM was performed with the forward primer alone, the reverse primer alone,
or both primers together. PCRIM amplification reactions were performed using 40 ng of YAC
DNA, 1 ~LM of each primer, and 200 ,uM of each dNTP in a 20 lal reaction. Cycling parameters
were 95~C for 2 min, followed by 32 cycles of 94~C for 20 sec, 55~C for 30 sec, and 72~C for 2
min. IRE-PCR~M products were isolated either by band excision from low-melting agarose gels,
or by TA subcloning (Invitrogen). IRE-PCRrM products were se~uenced, screened for the
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C~
presence of common mouse repetitive element sequences, and nonrepetitive regions of the
sequence used to design oligonucleotides suitable for sequence tagged sites (STS).
5.1.1.6 DIR~CT SELECTION
Direct selection was performed as previously described (Lovett et al., 1991, Lovett,
5 1994). Briefly, cDNA was generated from mouse spleen by reverse transcription using random-
and oligo(dT)-priming, ligated to amplification ~ceettec, and PCRTM amplified. Preparative
PFGE was used to purify YAC 1 95A8 DNA, which was biotin-labelled, denatured, and
hybridized in solution to the denatured cDNA pool. Repetitive elements, cDNA corresponding to
rRNA, and yeast genes were blocked to Cot=20. YAC DNA (w~th ~nne~le~l cDNAs) waslo captured on streptavidin-coated beads, washed at high stringency, and encoded cDNAs eluted.
Eluted cDNAs were PCRTM-amplified, and subjected to a further round of direct selection.
Selected cDNAs were reamplified by PCRTM, subcloned into ~gtlO, and individual clones picked
into SM buffer in 96-well plates. Direct selection products were amplified from phage-co~ i..g
supernatents by PCRlM with the following primers:
5'-GTTGTAAAACGACGGCCAGTGGGAAGTTCAGCCTGGTTAAG-3' (SEQ ID NO:77); and
5'-GACAGGAAACAGCTATGACCAGAGTATTTCTTCCAGGGTA-3' (SEQ ID NO:78).
Direct selection amplicons were cycle sequenced with standard M13 fonvard and reverse
primers. Oligonucleotides suitable for STS were designed using direct selection product
sequences.
20 5.1.1.7 STS PCRTM
PCRTM amplification reactions were performed using 40 ng of template DNA (YAC clone,
Pl clone, S. cerevisiae strain 1380, or C57BL/6J genomic DNA), 1 ~M of each primer, and 200
llM of each dNTP in a 20 ~11 reaction as described (Barbosa et al., 1995). Cycling parameters
were 95~C for 2 min, followed by 34 cycles of 94~C for 20 sec, 45-58~C for 30 sec, and 72~C for
25 20 sec. Amplification products were separated on 3% agarose gels, and vieu~ e~l by ethidium
bromide st~inin~ or by end-labeling one of the primers using [~ 32PlATP and T4 polynucleotide
kinase, and separation of products on 6% denaturing polyacrylamide gels, with autoradiographic
vi.ell~li7~tion. Simple sequence length polymorphism (SSLP) primers were as described (Dietrich
,
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et al., 1994; Research Genetics Inc., Hunstsville, AL). Novel STS primer sequences, arnplicon
sizes, and ~nne~ling temperatures are s~-mm~rized in Table 2.
5.1.2 RESULTS AN1D DISCUSSION
5.1.2.1 ISOLATION OF YACS AND P1S
11 YAC clones and 2 P1 clones were isolated from mouse YAC and P1 libraries by
PCRTM using markers g~netic~lly mapped within the bg critical region. YAC clone sizes, as
determined by PFGE, Southern blotting and hybridization with pBR322, are illustrated in FIG. 1.
YAC clones were e7~mined for chimerism, microdeletions, and overlaps by STS content
mapping. Previously described SSLP were the first source of STS to be ~ti~i7ed The genomic
0 region encompassing bg is particularly rich in such SSLP (38 have been localized within a 2 cM
interval Cont~ining bg; Dietrich et al., 1994). Additional proximal chromosome 13 STS were
generated using IRE-PCR~M and direct selection.
5.1.2.2 NOVEL CHR 13 STS DERIVED BY ~RE_PCRTM
IRE-PCRTM represents a rapid and facile method with which to saturate a genomic region
15 with novel STS for initial characterization of YAC clones and contig development (Hunter et al.,
1993; Simmler et al., 1991). IRE-PCRTM was performed using YAC DNA as template and
primers derived from ends of the mouse repetitive element B1 which were oriented in opposite
directions. IRE-PCRTM products were subcloned, sequenced, and nonrepetitive regions used to
design oligonucleotides suitable for sequence tagged sites. 12 novel STS fDI35f7c1-D135f7c12)
20 were developed by this method (Table 2), and physically ~e~i~ned to Chr 13 YAC and Pl clones
by PCRTM (FIG. 2).
5.1.2.3 NOVEL C~R 13 STS DERIVED BY DIRECT SELECTION
Direct selection was performed with YAC 195A8, a 650-kb YAC which was easily
purified from preparative pulsed field gels since it did not comigrate with host yeast
25 chromosomes. 192 ~ntli(l~te cDNA fr~gmentc were eluted from YAC19SA8 following two
rounds of direct selection with mouse splenocyte cDNA. 56 of these direct selection products
were se~uenced. Comparison with DNA se~uence databases revealed 2 (4%) nidogen (Nid), 32
(57%) novel, 12 (21%) repetitive elements (B1=2, B2=1, LINE1=4, IAP=2, XL30=1, MT=1, (
satellite=1), and 9 (16%) co.. l~",i~ s (rRNA=3, actin=1, Nip2=1, plasmid=4). The presence of
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l\~id cDNA fr~gment~ among these products confirmed the efficacy of the selection procedure in
enriching for YAC 195A8-encoded genes. Furthermore, of 8 STS corresponding to novel direct
selection products, 7 mapped back to YAC195A8 by PCRTM analysis (D13Sf*13-D13SfkI9; Table
2,1~IG. 2). D135f~cI3 and DI3Sp~I8 also hybridized sufficiently well to Southern blots to permit
5 physical mapping adjacent to Nid on a polymorphic NotI fragment (1100-kb in DBA/2J DNA and
1150-kb in SB/LeJ DNA). D135fk13 was also genetically mapped within the bg critical region in
504 backcross mice ~C57BL/6J-bg' X (C57BLJ6J-bg' x CAST~Ei3Fl] using a TaqI polymorphism.
5.1.2.4 ARRANGEMENI'OFPROXIMAL CHR 13 YAC AND Pl CLONESIN CONTIGS
YAC and P1 clones were typed for the presence or absence of STS derived from SSLP,
10 Il~E-PCRTM amplicons, and direct selection products. STS content mapping enabled ~x~min~tion
of clones for chimerism and microdeletions. One YAC clone, 64F5, was chimeric. This YAC,
while 580-kb in size (FIG. 1), contained only D13Mit44, and not STS derived from the 5'- or 3'-
ends of Nid (FIG. 2). Since the latter two STS are separated by less than 65-kb in mouse
genomic DNA (Durkin et aL, 1995), and since DI3Mit44 is located within the Nid gene, the
1~; portion of YAC 64F5 derived from Chr 13 was con~ clçd to be less than 80-kb.
YAC clone (84A8) contained an internal deletion which inçlllded D13Sf~c6 (FIG. 2).
Furthermore, the physical size of 84A8 ~370-kb) was considerably smaller than expected: the
~ii.ct~nçe between the other genetic markers it encomr~e~l was apploxilllately 600-kb,
CO~lllllillg a substantial genomic deletion within this YAC. Some YAC clones have been
20 reported to be unstable in culture, and become progressively smaller with time (Nehls et al.,
1995). YAC 84A8 may exhibit such instability.
STS content mapping also enabled ordering of YAC and P1 clones within the bg critical
region and integration of clones into 2 contigs (FIG. 2). Contig 1 comprised 7 YAC and 2 Pl
clones, extended from D135fk19 to D13Sflc2, and was applu~lllately 1150-kb in length. The
25 orientation this contig with respect to c~ lllere was not established. The second contig 2
consisted of 2 YAC clones. It extended ~om D13Mit207 (proximal) to D13SfklO (distal), and
was approx;,.,~ly 1000-kb in length. Contig 2 spanned the crossover dçf;ning the distal border
of the bg critical region (:FIG. 2). Despite STS content mapping, 2 additional critical region YAC
clones rçm~inçc1 llnlinke(l with these contigs (165F7 and 148E11). Isolation of YAC end clones
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~r)
will be necess~ry to definitively evaluate whether overlaps exist between these YACs and contig 1
or2.
Efforts to identify YAC clones corresponding to one critical region genetic marker
(D13Mi~114) and the two STS which define the proximal border of the bg critical region
(~D13Mifl 72 and D13Mit239J were l~n.~lcces.~fi-l; furthermore, these STS were not present in any
of the Chr 13 YAC/P 1 clones identified. These data suggest that a region of the nonrecombinant
interval remains unrepresented in the present YAC and P I clones, or, alternatively, that
additional microdeletions exist in the YAC clones. Based upon evaluation of overlaps between
YAC and P1 clones, the bg critical region was estim~ted to be at least 2400-kb in length.
0 Direct selection products identified from ~AC 195A8 using splenocyte cDNA not only
allowed STS content mapping of Chr 13 YACs, but also constitute candidate genes for bg and cr.
Both of these mouse mutations appear to result from defects in constitutively expressed genes by
virtue of abnormal phenotypes in all organs examined The large number of bg alleles available
enables effective screening of candidate genes by a combination of Southern and northern
hybridization and RT- PCRTM, using nucleic acid from multiple bg alleles and coisogenic controls.
While such studies are inefficient methods for detection of point mutations, they are highly
effective in detection of intragenic deletions, l~L~L~nspositions, and genomic rearrangements,
which together account for a large enough proportion of spontaneous mouse mutations to make
likely the detection of a mutation in one of the bg alleles. While only one allele of cr exists, it
2 o arose in offspring of a mouse treated with nitrogen mustard, and therefore is more likely to be
associated with a genomic rearrangement detectable using the same screening techniques.
In summary, app~ dLely 2400-kb of the bg critical region has been physically mapped
and isolated in the form of YAC and Pl clones. These studies represent an nec~ss~ry interm~ te
step in positional cloning of bg, and may also be of value in positional cloning of cr andpmn.
CA 02244744 1998-07-29
W 097/28262 PCT~US97/01748
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CA 02244744 1998-07-29
W O 97/28262 PCTrUS97/01748
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WO 97/28262 PCT~US97/01748
5.2 EXA~IPLE 2 - MAPP~NG OF THE BEIGE LOCUSTO M OUSE L~ST 13
This example illustrates the generation of a high resolution genetic map of proximal Chr
13 in the vicinity of bg, and the identification of two genes which are tightly linked to bg These
5 studies precisely localize bg on Chr 13, and provide a foundation for YAC contig development
and efficient screening of ~nt1~ te genes for bg.
S.2. 1 MATERIALS AND METHODS
.2.1,1 MICE
C57BL/6J-bg~ X (C57BL/6J-bgJ x CAST/EiJ)Fl backcross mice were bred and m~int~ined
as described (Barbosa et al., 1995). (C57BL/6J-bg' x PWK~ X C57BL/6J-bg' backcross mice,
and (C57BL/6J-bg' x PAC)FI X C57BL/6J-bg ~ backcross mice used have been described
(Holcombe et al., 1991).
.2.1.2 SOUrHERN~YBRIDIZATION
DNA was isolated from mouse organs using standard techniques and digested with
5 restriction endonucleases, and 10 ~g samples were subjected to electrophoresis on 0.9% agarose
gels. DNA was ll~n:,rellt;;d to Zeta-probe membranes (Bio-Rad Laboratories, Hercules, CA), and
filter hybri~li7~tions were performed as prev~ously described (Barbosa et aL, 1995).
5.2.1.3 NORTHERN BLOT ANALYSIS
20 ~g of total RNA prepared from liver, spleen and kidney of C57BL/6J-+/+, C57BL/6~-
2 o bg', SB/LeJ-bg, and C3H/EleJ-bg~ mice using standard techniques, was separated on
forrn~lclehyde agarose gels, transferred to Zeta-probe mel.lbl~lles ~Bio-Rad Laboratories), and
hybridized as previously described (KingemQre et al., 1994).
5.2.1.4 RT- PCRTM ASSAYS
Total RNA was prepared from liver of C57BL/6J-+/+, C57BL/6J-bg', SB/LeJ-bg, and C3H/HeJ-
25 bg~ rnice by extraction with phenol / ~l~niriine isothiocyanate (TRIzol7, Gibco BRL,Gaithersburg, MD). The template for qu~ntit~tive RT- PCR~M assays was 1-10 ng of first-strand
CA 02244744 1998-07-29
W O 97/28262 PCT~US97/01748
cDNA, which had been syntheci7ed from total RNA with an oligo(dT) primer and Moloney
murine leukemia virus reverse transcriptase (Stratagene, La Jolla, CA). The nidogen ~Nid)
primers used for RT- PCRTM correspond to bp 3805-3822, and bp 3938-3955 of the mouse Nid
cDNA (Durkin et al., 1988). The Es~m9 primers used were:
5'-CAGGTGGAGATGCTGTTC-3' (F1) (SEQ II) NO:59)
5'-GAGATGCCTTCAGGCAGT-3' (R1) (SEQ ID NO:60)
5'-CCGTTAGTGTGTAGTCTC-3' (F2) ~SEQ ID NO:61)
5'-CTTGCTCTCACTGTTCTC-3' (R2) (SEQ ID NO:62).
0 These correspond to the S' and 3' ends, respectively, of an Estm9 cDNA (Bett~nh~ns~.n and
Gossler, 1995). RT-PCR7 products were amplified from bg, ~g', bf~, and +/+ RNA with Nid
primers or Estm9 primers Fl-Rl or F2-R2. Qr1~ntit~tive RT-PCR7 of aldolase A, which is
constitutively expressed, was also performed, to ensure that e~,ual amounts of bg, bg', b~, and
+,'+ template were used
(Aldolase A primer 1: 5'-TGGATGGGCTGTCTGAACGC-3', (SEQ ID NO:63);
primer 2: 5'-TGCTGGCAGATGCTGGCATA-3', (SEQ ID NO:64).
PCR~M reactions were performed in a 50 1ll volume cont~ining 1-20 ng of cDNA, 1 ,uM of
each primer, 200 ,uM each dNTP, 10 rnM Tris-HCI, pH 8.8, 50 mM KCl, 1.5 mM MgCI2, and
1.25 U AmpliTaq7 DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT). Cycling profiles
consisted of an initial denaturation (94EC for 2 min) followed by 25 cycles of 94EC for 30 sec,
55-58EC for 30 sec, and 72EC for 1 minute per kb of expected product length. PCR~M products
were separated by electrophoresis on agarose gels, and quantified by intensity of ethi~ m
brornide st~ining
5.2.1.5 SSLP PCRTM
- 25 PCRTM amplification reactions were performed using 40 ng of genomic DNA, 1 ~M of
each primer (Dietrich et al., 1994; Research Genetics, Inc., Huntsville, AL), and 200 ,uM of each
dNTP in a 20 ,ul reaction as described (Barbosa et al., 1995). Cycling parameters were 95EC for
2 min, followed by 36-38 cycles of 94EC for 20 sec, 58EC for 30 sec, 72EC for 10 sec. Where
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possible, amplification products (20 ~LI) were separated on 3% agarose gels, and vt~ T-7~d by
ethi~lillm bromide staining. SSLP with allele sizes differing among strains by less than 8 bp were
typed by end-labeling one of the primers using [y32P}ATP and T4 polynucleotide kinase,
separation of amplification products (4 ~l) on 6% denaturing polyacrylamide gels, and
5 vi~u~li7~tion by autoradiography. SSLP allele sizes are sl-mm~3ri7ed in FIG. 3A, FIG. 3B, FIG.
3C and FIG. 3D.
5.2.1.6 PULSED FIELD ELECTROPHORESIS
Preparation of high molecular weight DNA in agarose blocks, restriction enzyme
digestion, pulsed field electrophores;s (PFGE~, and Southern transfer were performed as
0 previously described (~ing~mQre et al., 1989). In brief, mouse splenocytes or lymph node cells
were suspended in 0 5% low-melting point agarose ~InCert, FMC BioProducts, Rockland, ME) at
1-2 H 107 cells per ml. DNA was prepared by incubation of agarose blocks in 500 mM EDTA
(pH 9.0), 1% sodium lauroyl sarcosinate, 2% proteinase K at 50EC twice for 24 h. Blocks were
then washed, treated with phenylmethylsulfonylfluoride, washed again, and r~ sted with 2-10
15 units/~Lg DNA of restriction endon~lf.l~ces ~Boehringer Mannheim Bior.hçmic.~l~). PFGE was
carried out in 1% agarose gels (Fastlane, FMC BioProducts) at 14EC in lX TBE using a Gene
Navigator system (Pharmacia, Piscataway, NJ). Separation of 50-1500 kb DNA molecules was
achieved using pulses ramped from 70-145 sec at 145 V for 46 hr, 1000-6000 kb DNA was
resolved by pulses of 15-90 min at 50 V for 6 or 10 days. Gels were stained with ethi~ m
20 bromide to visualize molecular size standards (oligomers of ~ phage, and chromosomes of
Saccharomyces cerevisiae and Schizosaccharomyces pombe [FMC BioProducts]). Southern
transfer of DNA onto Zeta-probe'l9 membranes ~Bio-Rad Laboratories), and filter hybridizations
were performed as previously described (Kingsmore et al., 1989). ~iEnm~nt of two probes to a
cormnon restriction fragment was based on sequential hybridization of a filter and exhibition of
2 5 identity by double- or partial-digests.
5.2.1.7 M OLECULAIR ~ OBES
All probes were labeled by the hexanucleotide technique with "-[32P]dCTP as previously
described (King~more et a~., 1989). The nidogen f~Ni~ probe used was pN-5 (Jenkins et
al., 1991). The glioblastoma oncogene homolog-3 ~GIi3) probe was derived from pGli3a (Hui and
30 Joyner, 1993). The probes used for the T cell receptor ( chain ~Tcrg), and the mid-gestation
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~<~
embryo cDNA ESTM9, have been described previously (Holcombe et aL,1991). Informative
CAST/EiJ RFLV sizes are sumrnarized in ~IG. 3D; informative PAC and PWK RFLV for Tcrg
were as described (Holcombe et al., 1991).
-
5.2.2 RESULTS
Previous mapping studies, using 3 separate backcrosses segregating for the bg locus {2
intraspecific backcrosses ~(C3H/3~IeJ x C57BL/6J-bgJ)Fl X C57BL/6J-bg J ], and [(C57B~/6J -
~h-bgJ x Mus domesticus PAC)FI X C57BL/6J-bgJ ], and an intersubspecific backcross
[(C57BL/6J-~h-bg' x Mus ml~c7~17~s PWK)FI X C57BL/6J-bgJ] ~, have shown bg to lie pl o~ ,al
to Tcrg on mouse Chr 13 (Holcombe et al., 19~7, 1991). In order to assess c.~n~ te genes for
0 linkage to bg and as a precedent to positional cloning, the inventors have now generated a high-
resolution linkage map of ploxhllal mouse Chr 13 using the latter 2 backcrosses and a third,
novel backcross.
5.2.2.1 PHENOTYPIC ANALYSIS OF BG BACKCROSS MICE
Th}ee backcrosses segregating for bgwere lltili7ed; Phenotypic analysis of 109 (C57BL/6J
_~h-bgJ x Mus domesticus PAC)FI X C57BL/6J-bgJ backcross mice, and 111 (C57BL/6J-W~h-
bg3 x Mus m7~c7,~ PWK)Fl X C57BL/6J-bg J backcross mice has been reported previously
(Holcombe et al., 1991). The third backcross was established between C57BL/6J-bg' mice and
M2ls castaneus (CAST/EiJ), and 504 [C57BL/6J-bgJ X (C57BL/6J-bgJ x CAST/EiJ)FI ] progeny
were generated. Mus castaneus was chosen as the second parent in the latter intrasubspecific
backcross due to the increased likelihood of ~letecti~n of DNA polymorphism in comparison to
intraspecific crosses. Mice were phenotyped for the presence or absence of a beige-colored coat;
Penetrance of bg in all of the crosses was complete (359 of 726 backcross mice [49%~ exhibited a
beige-colored coat).
5.2.2.2 IDENTIFICATION OF INFORMATIVE RFLV AND SSLP
2 s Inro- Il.aLi~e RFLV were ascertained by hybridizing gene probes to Southern blots
- co"~ g genomic DNA from C57BL/6J-bg~ and CAST/EiJ, PAC, or PWK parental mice
digested with various restriction endonucleases. Table 3 lists the sizes of unique CAST/EiJ RFLV
for Gli3 and Nid. PV~K and PAC RFLV for Tcrg have been described previously (Holcombe et
al., 1991); CAST/EiJ RFLV for Estm9 have been described previously. Inrul~ i\re SSLP were
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ascertained by PCRTM of genomic DNA from C57BL/6J-bg' and CAST/EiJ, PAC, and PWKparental mice. A,~plo~ .aLe sizes of SSLP- PCRTM products are listed in Table 3.
5.2.2.3l~UECISE GENETIC ~IAPP~NG OFBG ON PROXIMAL M OUSE CEIR13
111 (C57BL/6J ~h-bg' x Mus domesticus PAC)Fl X C57BL/6J-bg~ backcross mice,
111 (C57BL/6J-~h-bgJ x Mus m~cl~2~ PWK)FI X C57BI16J-bg ~ backcross mice, and 504
[C57BL/6J-bg' X (C57BL/6J-bgJ x CAST/EiJ)Fl ] backcross mice were genotyped for a total of
23 SSLPs and 3 RFLVs known to map to ploxil~lal mouse Chr 13. At each locus, backcross
DNA displayed either the homozygous or heterozygous Fl pattern. Linkage relationships were
determined using segregation analysis (Green, 1981), and the best gene order decided by
.I,il~ill.;,,.l,on of crossover events and l~.limin~tion of double crossover events (Bishop, 1985).
Haplotype analysis for each cross is shown in FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D.
Upon retyping of previously published genotypes of the PAC and PWK backcrosses
(Holcombe et aL, 1991), 4 errors were cletecte~l In each case, the coat-color had been
incorrectly assigned, resulting in the generation of a double crossover within a genetic interval of
less than 0.5 cM; since such events are predicated against by positive interference, these animals
were ~ cluded from subsequent analysis. Upon exclusion of these ~nim~1~, no significant
dirrelt;llces in gene order or l~coll~ lation frequencies were found among the three crosses.
The best gene order and recolllbillalion frequency ~ standard deviation) for the[C57BL/6J-bgJ X (C57BL/6J-bg' x CAST/EiJ)Fl ] backcîoss was: centromere - D13Mitl58,
D13Mifl72, D13Mit205, D13Mit206, D13Mit239 - 0 20 ~t 0.20 cM - bg', Nid, Estm9,
D13Mif44, D13Mitll4, D13Mitl34, D13Mit207 - 0 20 ~ 0.20 cM - Gli3, D13Mit56,
D13Mitl62, D13Mitl74, D13Mit237, D13Mit240, D13Mit305 - 0.20 ~ 0.20 cM - D13Mit218,
D13Mit219, D13Mit271 - 0.40 + 0.28 cM - D13Mit3, D13Mitl33 - telomere.
The best gene order and recombination frequency (~ standard deviation) for the
2 5 [(C57BL/6J WSh bg' x Mus domes~icus PAC)FI X C57BL/6J-bg ~] backcross was: c~llLI ulllere -
D13Mit79 - 5.4 ~ 2.1 cM - D13Mitl - 0.9 + 0.9 cM - bg~, D13Mif44, D13Mitl34, D13Mitl74,
D13Mif205 - 0.9 + 0.9 cM - ~crg, D13Mit218, D13Mit219 - 3.6 + 1.8 cM - D13Mit3 - telomere.
The best gene order and recoll~ aLion frequency ~t standard deviation) for the
~(C57BL/6~- ~h-bg~X Mus m~cul7~ PWK)FI X C57BL/6J-bgJ ] backcross was: centromere -
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D13Mit~9 - 5.4 ~ 2.1 cM - D13Mitl - 0.~ ~t 0.9 cM - bg', D13Mit44, Dl3Mitl34, Dl3Mit205,
Dl3Mit237 - 0.9 ~ 0.9 cM - Dl3Mitl 74 - 0.9 ~ 0.9 cM - ~crg, D13Mit218, D13Mit219 - 0.9
0.9 cM - D13Mit3 - telomere.
A composite linkage map of proximal mouse Chr 13, derived by integration of these 3
crosses, is shown in FIG. 3D. The combined results delimit the region cont~ining bg to a 0.24 +
0.17 interval on Chr 13, fianked proximally by the genetic markers D13Mitl 72 and D13Mit239,
and distally by Gli3, D13MitS6, D13Mitl62, D13Mit237, D13Mit240, and D13Mit305. bg
cosegregated with 6 genetic markers (Nid, Estm9, D13Mit44, D13Mitl l 4, D13Mitl 34 and
D13Mit207). Backcross mice with recombination events which define the bg nonrecombinant
0 interval were derived from the [C57BL/6J-bg~ X (C57BL/6J-bg~ x CAST/EiJ)FI ] backcross.
5.2.2.4 EVALUATION OFTHE CA~DID~CY OF NID AND ESTM9 FOR CAUSALITY ~NBG
Given the availability of numerous bg alleles, it was reasoned that northern, Southern, and
~T- PCRIM analyses would be effective modalities for initial evaluation of the ç~n~lirl~cy of Nid
and Estm9 for causality in bg
Southern blots were generated with DNA from 6 bg alleles: SB/LeJ-bg, C57BL/6J-bg ',
C3H/HeJ-bg ~', DBA/2J-bg ar, C57BL/6J-bg lar, C57BL/6J-bg "~, and from appropriate +/+
coisogenic controls using 5 restriction endonucleases (Eco~, HindIII, BamHI, MspI, and TaqI).
No restriction fragment length differences were observed between bg alleles and coisogenic
controls upon hybridization with Nid or Estm9, exc.~ in~ a deletion or insertion in these genes
2 o from causality in these bg alleles.
Expression of Nid and Estm9 in bg mice was exarnined by northern blot analysis and
qu~ntit~tive RT-PCR7. Hybridization of northern blots of liver and kidney RNA from +/+, bg,
bg', and bg~ with probes for Nid and Es~m9, yielded signals of similar size and intensity in bg
and +/+ RNA. Furthermore, no difference in amplicon size or amount was observed upon
qn~ e RT-PCR7 using liver or kidney RNA from +/+, bg, bg', and bg 2'mice and
olig~mllrleotides for Nid or Estm9, indicating expression of Nid and Estm9 to be grossly intact in
bg.
,.
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7~
5.2.2.5 ~ YSICAL ~L~PPING OFPROXInL~L MOUSE CEnR13 ~NTHE VIC~TY OF B~
Cytogenetic and physical mapping studies have demonstrated mouse mutations induced by
gonadal x-irradiation to be frequently associated with genomic rearrRng~.rnent.c (typtcally deletions
or translocations). The SB/LeJ-bg allele was discovered among the offspring of a male which had
received such tre~tment In order to e~amine SB/LeJ-bg DNA for a genomic rearrangement,
physical mapping studies were undertaken by pulsed field gel electrophoresis using high molecular
weight DNA and restriction endonucleases which cleave infre~uently. PFGE-Southern blots were
generated using DNA from DBA/2, C57BL/6J-bgJ, CAST/EiJ and SB/LeJ-bg splenocytes, and
probed sequentially with the 3 genes which map in the vicinity of bg ~Nid, Estm9, and Gli3).
0 Physical linkage of these genes was not possible, since hybridization with Estm9, Gli3 and Nid
gene probes revealed no bands of identical size (Table 4).
No differences were observed in the sizes of bands identified in SB./LeJ-bg and control
D~A upon hybridization with Gli3 or Estm9 ~Table 4). However, hybridization of the same blots
with an Nid gene probe did reveal band size disparities. With 5 restriction endon--çle~c~s (NotI,
l~z~I, lVruI, and SrfI complete digests, NaeI partial digest, and NotI/MI2~I double digest),
differences were observed between DBA/2 and the other DNAs ~C57BL/6J-bg', CAST/EiJ and
SB/LeJ-bg). In each case, the DBA/2 fragment was 25-50kb smalier than the band identified in
C57BL/6J-bg', SB/LeJ-bg, or CAST/EiJ DNA (FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D; Table
4). No differences in Nid band sizes were evident among other mouse strains Px~minecl
(C57BL/6J-bgJ, SB/LeJ-bg, and CAST/EiJ). Other restriction endonucleases, which identify
smaller fr~gm~.nte when probed with Nid (BssHII, ClaI, NaeI, ~maI, XhoI) were identical in all
strains tested (FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, Table 4). Nidfragment size differences were
observed using both methylation-sensitive and -ine~n.citive restriction endom-~le~e~c.
~.2.3 DISCUSSION
Previous studies have localized bg to ~roxi.l.al Chr 13. Lyon et al.,(l 969) demonstrated
bg to be 0.5 cM ploxi~llal to the mutation Xt, which corresponds to the Gli3 gene. Several
groups have demonstrated tight linkage between bg and Tcrg (Holcombe et al.,l987, 1991;
Justice et al., 1990). Jenkins et al.,(l991) found bg to cosegregate with Nid in 123 meiotic
events. Precise genetic mapping of bg has been undertaken with respect to these genes and
3 o recently identified SSLP markers (Dietrich e~ aL, 1994) as an antecedent to generation of a YAC
contig of the genomic region encompassing bg These results are in agreement with previous
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q7
studies of genetic marker order on chromosome 13, although the greater number of meioses
utilized in the present study permitted separation of loci which cosegregated in previous studies,
and enabled localization of bg to a 0.24 cM interval on proximal mouse Chr 13. No statistically
significant differences in genetic ~ t~nces between ll-alkel~ were observed among the present
5 crosses or between them and previous studies. Cosegregation of bg and Nid was observed in 504
meiotic events, suggesting bg to map within a linkage group conserved between proximal mouse
Chr 13 and the distal long arm of human Chr 1 (Jenkins et al.,1991). By implication, the
homologous human locus, CHS, may be expected to lie on human Chr lq42.1-lq43, which
represent the appl~xh.late limits of this conserved linkage group (Jenkins et aL, 1991; Mattei et
lO al., 1994). Localization of bg to a 0.24 cM interval will enable the generation of a YAC contig
encompassing bg. Those genetic markers which cosegregate with bg will serve as nucleation
points for rapid contig assembly.
If it is ~csllmed that a haploid mouse genome is 1500cM in size and contains 60,000,
randomly distributed genes, it would be expected that the 0.24 cM bg critical region should
15 contain 10 genes. In the present report, two genes, Nid and Estm9, were localized within this
interval, and thereby represent candidate genes for the bg locus Nidogen, however, can be
excluded from candidacy for bg for functional reasons. While bg mice exhibit a constitutive
intracellular defect in Iysosomal trafficking, nidogen is a component of basement membranes, a
sper.i~li7ed extr~r.~ r matrix structure limited to certain tissues (Durkin et al., 1988). The
e~ntli~ y of Estm9 cannot yet be evaluated on functional grounds. Estm9 is a novel mouse
expressed sequence which was recently identified from a day 10.5 p.c. mouse embryo cDNA
library (Bett~nh~ns~n and Gossler, 1995). Comparison of partial Estm9 cDNA sequences with
DNA and peptide databases demonstrate ~ignifiç~nt sequence similarity only with uncharacterized
human ESTs. While the function of Estm9 is unknown, ~ ,-t;ssion analysis reveals it to be
25 ct n~titutively expressed, temporally and spatially, in the mouse (Bett~nh~llsen and Gossler, 1995).
Initial genetic evaluation of the c~ncii~1~cy of Nid and Estm9 for bg by northern and Southern blot
hybridization or qll~n~it~tive RT- PCR~M, revealed no differences between several bg alleles and
coisogenic controls. These studies do not definitively exclude Nid or Estm9 from ~n~ cy for
bg. A more robust method of evaluation for bg c~n~ te genes would be genetic
3 o complementation. Cell lines derived from bg mice exhibit pathognomonic phenotypes (Burkhardt
et al., 1993; Gow et al., 1993; Baetz et al., 1995), which can be abrogated by genetic
complementation (Perou and Kaplan, 1993; Penner and Prieur, 1987; Gow et al., 1993). Studies
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to P.Y~mine the ability of Nid or Estm9 to complement bg-associated phenotypes in vitro are being
pursued.
Physical mapping studies of the bg critical region were undertaken to evaluate the
radiation-induçecl SB-bg allele for the presence of a gross genomic rearrangemel.t. SB-bg -
specific restriction fragment length differences were not observed with Nid, Estm9, or Gli3 gene
probes. Furthermore, all critical region SSLP arnplicons (D13Mit44, D13Mitll4, DI3Mitl34
and DI3Mif207) were present in SB-bg DN~. Together, these data preclude the existence of a
gross genomic rearrangement in SB-bg DNA. However, DBA/2-specific pulsed-field
electrophoresis RFLPs were observed with Nid using 5 restriction endonucleases. In each case,
0 the DBA/2 fragment identified with Nid was 25-50 kb smaller than the corresponding band
identified in control DNA (FIG. 3A, FIG 3B, FIG. 3C, and FIG. 3D). No difference in band
sizes were observed among other strains or upon reprobing of PFGE-Southem blots with Gli3 or
Estm9. Since fragment size differences were observed with many rare-cutting restriction
endonucleases, inclu-ling several which are methylation-insensitive, it is unlikely that they are
merely interstrain differences in Dl~A methylation or point mutations. Instead, it is suggested that
a genomic rearr~ng~.mçnt has occurred in the DBA/2 mouse at a distance of less than 900 kb from
Nid (FIG. 3D). ~he rearrangement may represent a small (25-50 kb) genomic deletion in the
OBA/2 mouse. The functional significance of such a putative rearrangement is uncertain.
Intt;~ Lillgly, a similar phenomenon was recently described in the vicinity of the human nidogen
gene (Goodrich and Holcombe, 1995) upon hybridization to pulsed field gel electrophoresis
Southern blots of human genomic DNA digested with Sall, nidogen identified polyrnorphic band
sizes in C~lç~ n populations. In 2 CHS patients that have been e7c~min~d to date,
homozygosity for one NID allele was observed, s~lggesting the possibility of linkage of human
CHS and NID (Goodrich and Holcombe, l995). Definitive mapping of human CHS, however,
2 5 must await identiffcation of the mouse bg gene. On a practical note, the interstrain differences in
pulsed field restriction fragment length provide a physical l~n-1m~rk within the bg nonrecolllbillallL
interval. Thus bg ç~n~ qte genes can be easily screened for physical linkage of with Nid as a
means of determining whether or not they lie within the bg nonrecollll)hlallt interval.
In s-lmm~ry, the bg locus has been localized, which is the mouse homolog of human CHS,
3 o to a ~n~mic interval corresponding to approximately one four-hundredth of mouse Chr 13. This
represents an important intermediate step in the positional cloning of bg, and thereby human CHS.
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r7q
T~BLE 3
Informative Proximal Chr 13 SSLP Allele Sizes
SSLPAllele Size (bp) C57BL/6J-bg~ CASTlEiJPAC PWK
~ D13MitI 147 149 137* 151*
D13Mit3 159 196 240* 178*
D13Mit44(Nid:~ 124 116 116* 116*
D13MitS6 226 260 226* 228*
D13Mit79 107 NllLL 103* 91*
DI3Mitll4 149 137 ND 149*
D13Mitl33 150 181 150 181
D13Mitl34 123 137 137* 153*
D13MitlS8 144 135 ND ND
D13Mitl62 105 135 ND 105*
D13Mitl 72 146* 154 ND 158*
D13MitI74 123 135 129* 131*
D13Mit205 120 140 128* 132*
D13Mit206 146 168 ND ND
D13Mit207 136 148 ND ND
D13Mit218 178 194 186* 186*
D13Mit219 266 282 270* 282*
D13Mit237 114 102 118* 92*
D13Mit239 99 116 120* 90*
D13Mit240 166 124 124* 130*
D13Mit271 127 135 ND ND
D13Mit305 118 144 ND ND
*=F~tim~tefl size ND=not done
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TA:BLE 4
Restriction Fragment Length Polymorphisms Used To Genetically Map Nid And Gli3 In
S04 lC57BL/6J-B~ X (CS7BL/6J-B~1 X CAST/Eij)Fll Mice
Gene Probe Strain Informative Band Sizes (kb)
Rest. Endo.
Nid C57BL/6J-bg ~ MspI 3 .2, 2.0
CAST/EiJ ~L, 0 8
Gli3 C57BL/6J-bg' MspI 5.0, 2 4, l.S, 1 2
CAST/EiJ 5.0 3.0, 2.4, 0 9
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w 097/28262 PCTrUS97/01748
$1
TABLE 5
PF G E restriction r. ~ l~nt sizes ~in kb) of Nid and Esfm9 in
S13~LeJ-bg and D B A/2J D N A
Restriction Nid Estm9
Endonuclease D B A/2 S B-bg/bg* D B A/2 SB-bg/bg
NotI 1100 1150 670,300 670,300
MluI 1800,975 1850,1025770,670 770,670
NotI/MluI 97~ 1025 N ot done Not done
SrJI 1000 1050 N ot done Not done
NaeI 900,400 950,400 280,250 280,250
ClaI 925,650 925,650 630,500 630,500
SmaI 200,150 200,150 N ot done N ot done
BssllII 250,25 250,25 Not done N ot done
*SB/LeJ-bg band sizes were also observed using CAST/EiJ and C57BL16J genomic DNAs.
5.3 EXAMPLE 3 -- ~ENTIFICATION OF THE ~IOMOLOGOUS BEIGE AND C~s GENES
As described above, the inventors have localized the bg locus within a 0.24 centimorgan
interval on mouse chromosome 13, and isolated contiguous arrays of YACs that cover 2,400 kb
of this interval. C~n~ te cDNAs for bg were isolated from Y A C 195 A8, which contains 650 kb
of the bg non-recombinant interval using direct cDNA selection with mouse spleen cDNA
(FIG. I l). Of 56 ~nrlid~te cDNA clones analyzed from a direct-selection study, evidence for
causality in bg was found in one (see below), and this gene was ~lesign~ted Lyst (lysosomal
trafficking regulator). As this clone was 132 nucleotides long, additional Lyst sequences were
sought by s~;lee~ g three mouse cDNA libraries and pe-r(~----illg polymerase chain reaction
~PCRTM) amplification of cDNA ends (King~mQre et al., 1994). Ten overlapping Lyst clones
were identified, representing ~7 kb (Genbank accession number, L77889). These were physically
~ign~d to mouse chromosome 13 with pulsed field gel electrophoresis (PFGE) Southern blots,
confirming that they were all derived from a single gene (mouse genome database accession
e number, MGD-PMEX-14). The Lyst probes identified the same polymorphic PFGE restriction
fra~m~nt~ as nidogen (Nid), indicating that Lyst and Nid are clustered within 650 kb. Lyst was
also mapped genetically in 504[C57B L/6-b ~ x (C57BL~6J-b ~ x CAST/EiJ)FI] backcross mice
by means of three TaqI restriction fragment length polymorphisms (RFLPs). The Lyst RFLPs
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g'~
cosegregated with bg (and Nid:), confirming their colocalization on proximal mouse chromosome
13 (MGD accession number, MGD-CREX-615).
Evidence for Lys~ mutations was found in two bg alleles. A 5-kb genomic deletion that
contained the 3' end of Lyst exon ,B, and exons ~ and o, was identified in bg"~ DNA (FIG. 12).
The bg"J deletion corresponds to the loss of ~00 internal amino acids of the predicted Lyst
peptide. Furthermore, whereas the 5' end of the bgl'J deletion occurs within Lyst exon 13, the 3'
end is intronic. Therefore the trl-nr.~ted Lyst mRNA in bg~J mice is also anticipated to splice
incorrectly, terminate prematurely, and lack polyadenylation.
Qll~ntit~tive reverse transcription (RT)-PCR~M demonstrated a moderate decrease in Lyst
mRNA in bg and bg~ liver, and a gross reduction in bg~ (Lyst ~OD afl[er norm~li7~tion for
,B-actin mRNA; +/+, 1.00; ~g~/bg~, 0.19; bg/~g, 0.2~; bg:t/bg', 0.40). A comm~n~--rate reduction
in b~ transcript abundance was noted by using several primer pairs derived from di~lt:nL
regions of the Lyst cDNA. Aberrant Lyst RT-PCRTM products were not observed. Theparticularly striking (more than fivefold) reduction in Lyst expression evident in bg~ homozygotes
s~ggested the existence of a mutation in hg~ Lyst that results in decreased transcription or mRNA
instability. The molecular basis of the decrease in Lyst mRNA in bg2J is not yet known, but it is
reminiscent ofthe leaky ablation of mature message associated with an intronic I~Lloll~n~l,osition
event (King~mnre ef al., 1994).
The predicted open reading frame (ORF) of Lyst was 4,635 nucleotides, encoding aprotein of 1,545 amino acids and relative molecular mass 172,500 (M~ 172.5K) (FIG. 13a).
Nucleotides 51-74 are rich in CG nucleotides, a comrnon feature of the S' region of ho~ keeping
genes. Comparison with DNA databases indicated that Lyst is novel, and resembles only
uncharacterized human-expressed sequence tags (ESTs). The sequence of a cDNA clone
corresponding to one such human EST (Genbank accession number L77889) matched the 5'
region of mouse Lyst (nucleotide identity was 76% in the 5' untr~n~l~tetl region (U~I~), 91% in
the ORF, and amin-acid identity was 97~/O; FIG. 13c); another human EST matched the 3' region
of the mouse Lyst coding domain (Genbank accession number W26957). On hybridization to
PFGE Southern blots of mouse DNA, the human clones i~l~ntified restriction fr~gm~nt~ that were
in~ tin~1ich~1e from mouse Lystl; physical mapping of the human clones to the same region of
3 o the mouse genome as Lyst indica~es that they are indeed homologous to Lyst.
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~3
It has been suggested that CHS and bg represent homologous disorders, as their clinical
features(Blume and Wolff, 1972) and defects in Iysosomal transport (Burkhardt et al., 1993) are
identical. Homology of bg and CHS is supported by genetic compl~ment~tion studies; fusion of
fibroblasts from bg rnice and CHS patients failed to reverse Iysosomal abnormalities, in contrast to
fusions with normal cells (Perou and Kaplan, 1993). Furthermore, recent genetic linkage studies
have shown that CHS maps within a linkage group conserved between human Chromosome lq43
and the bg region on mouse Chromosome 13. Therefore LYST mutations in CHS patients were
sought by seq~n~ing LYST lymphoblast and fibroblast cDNAs corresponding to these 3~STs
from 10 CHS patients. In one patient, a single-base insertional mutation was found at nucleotides
0 117-118 of the LYST coding domain, res~ in~ in a frame shift and tei,iiinalion after amino acid
62 (FIG. 13c).
Previous studies showing spontaneous aggregation of membrane-bound concanavalin A
(capping) suggest that there is a defect in microtubule dynamics in bg cells (Oliver, Zurier and
Berlin, 1975; Oliver and Zurier, 1976). In a search of the SWISSPROT database, using Blitz and
BLASTP, a similarity was found between a domain in Lyst and st~thmin (oncoprotein 18), a
phosphoprotein that may regulate polymeration of microtubules (Belmont and Mitchison, 1996)
(27% identity from residues 463 to 536; best expected occurrence by chance, 4.36 x 10~). The
domain is ~t~thmin that m~t~.hec Lyst is helical and has heptad repeats that participate in coiled-
coil interactions with other proteins (Sobel, 1991; Maucuer et al., 1995). The st~thmin-like
2 o region of Lyst is also predicted to be helical and formed coiled coils. However, it is the charged
residues, rather than the hydrophobic ones, that are conserved between Lyst and st~thmin,
~lg~estin~ that the sequence similarity is not primarily due to conserved secondary structure.
Thus this region of Lyst potentially encodes a coiled-coil protein-interaction domain that may
regulate microtubule-me~i~ted Iysosome transport. Although Lyst is no predicted to have
tr~n~me.mhrane helices, the C-terminal tetrapeptide (CYSP; amino acids 1,542-1,545) is strikingly
similar to known prenylation sites, which could provide ~tt~c.hment to lysosomal/late endosomal
membranes through thioester linkage with the cysteine.
Previous studies of bg leukocytes have shown correction of microtubule function (as
- ~sessed by Concavalin A capping) and natural killer activity when treated with inhibitors of
3 o protein kinase C (PKC) breakdown (Sato et al., 1990; Ito et al., 1989), suggesting that bg might
be re~ll~te-1 by phosphorylation. Lyst contains 25 sites of potential phosphorylation by PKC, 36
by casein kinase II (CKII~ (many of which overlap those of PKC), two by cAMP-dependent
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protein kinase, and one by tyrosine kinase (FIG. 13b). Almost half of the predicted helices
outside the st~thmin-like region (14 of 30) have a PKC- or CKII-phosphorylation signal at their
amino terminus, and eight of them form consecutive helical pairs. Thus Lyst seems to contain
helical bundles with clusters of phosphorylation sites at either end. Stathmin also has an N-
terminal phosphorylation site and helix motif, and these Lyst domains may have a similar 'signal
relaying' function to ~lal~,.l;" (Sobel, 1991; Maucuer ef aL, 1995). Furthermore, phosphorylation
of these positions could provide a control meçh~ni~m by causing a collrvll~laLional shift in the
bundles, thereby affecting interactions with other molecules.
Northern analysis and RT-PCRTM indicated that Lyst is ubiquitously transcribed, both
temporally and spatially, in mouse arld human tissues (FIG. 14). Northern blot analysis also
revealed complex alternative splicing of Lyst mRNA, with both constitutive and anatomically
restricted Lyst mRNA isoforms. The largest Lyst transcript in human and mouse was 12-14 kb,
but this transcript was not constitutively expressed. In mRNA from mouse spleen, human
peripheral blood leukocytes, promyelocytic le~lk~em;~ HL-60, and several le~lk~t-rni~ lines, the
12-14 kb isoform was either lm~letect~hle or barely detectable, but smaller Lyst transcripts were
a~undant (FIG. 14) Given the significance for bg mice and CHS patients of defects in the
Iysosomal and late-endosomal coln~a~ ~.llents of granulocytes, NK cells and cytolytic T
Iymphocytes (Gallin et al., 1974; Roder and Duwe, 1979; Saxena et aL, 1982; Baetz et aL, 1995),
it is likely that these Lyst mRNAs of ~3 kb and 4 kb represent the transcripts of primary functiona]
2 o siEnific.~nce Probes derived from the 5' or 3' ends of the Lyst
5.4 EXAMPLE 4 -- MU rATIoN ANALYSIS Ar~D PHYSICAL AND GENETIC MAPPING
ESTABLISH E[UMANLYSTAS THE CE~S GENE
5.4.1 MATERIAL~ AND METEIODS
5.4.1.1 CLONINGOFTHE~UMANLYS~GENE
Segm~nt~ of the human LYST sequence were obtained by an anchored, nested PCR~
(5' RACE-PCRTM) using liver cDNA as a t-omrl~te (Clontech Laboratories, Palo Alto, CA), by
RT-PCRTM using total RNA and by seq-len~tn~ of human ESTs similar in sequence to mouse Lyst.
For the 5' RACE-PCR~M two nested primers were used that were derived from a human EST
(GenBank accession number W26957) and had the following nucleotide sequence:
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g~
5'-CCAAGATGAAAGC~GCCGATGGGGAAAACT-3' (SEQ ID N0:65) and
5'-TCAGCCTCTTTCTTGCTCCGTGAAACTGCT-3' (SEQ ID N0:66).
. For RT-PCRTM experiments, total RNA was prepared from the promyelocytic ~L-60 cell
line. Reverse transcription was performed with Expand (Boehringer l\/r~nnheim, Meylan France)
5 with the following primer pairs:
5'-AGTTTATGAGTCGAAATGAT-3' (SEQ ID N0:67) and
5'-GAATGATGAAGTTGCTCTGA-3' (bp 490-2034) ~SEQ ID NO:68),
5'-CAGGAGTTCTTGAGATGGA-3' (SEQ ID NO:69) and
5'-ATCTTTCTGTTGTTCCCCTA-3' (bp 1,891-3050) (SEQ ID NO:70),
5'-TAGGGGAGCAACAGAAAGAT-3' (SEQ I~ NO:71)and
5'-GCTCATAGTAGTATCACTTT-3' (bp 3320-4722) (SEQ ID NO:72).
The primers used to amplify the cDNA between bp 1891 and 3050 were derived from the
mouse Lyst sequence. Human primers were ~e.eignecl from the sequence of the PCRTM product
15 (1159 bp) and used to amplify the fl~nking sequences.
5.4.1.2 DNA SEQUENCING AND SEQUENCE ANALYSIS
PCRTM products were cloned using a TA cloning kit (Invitr~gen Corporation, San Diego
California) and both strands were cycle sequenced. The sequences were analyzed with the GCG
Package (Devereux et al., 1984) and searches of the National Center for Biotechnology
20 Information database were performed using the BLAST network server (Altschul etal., 1990)
~National Library of Medicine, via INTERNET) and the Whiteh~ Tn.etit~te Sequence Analysis
Programs (MIT, Cambridge, ~ee~chl~sette)
5.4. 1 .3 SOU rHERN AND NORTH3 :RN BLOT ANALYSIS
Preparation of mouse, human and yeast DNA samples, digestion with restriction
25 endonucleases, agarose gel electrophoresis and Southern II~Ln~r~ls were performed using standard
techniques (Maniatis et al., 1984). The EcoR~ monochromosomal somatic cell hybrid blot was
obtained from BIOS Laboratories (New Haven, Connecticut). Isolation of poly(A)+RNA from
fibroblast and EBV-transformed B Iymphoblast cell lines, formaldehyde agarose gel
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electrophoresis and Northern blotting were perfo~e)d according to standard procedures (Maniatis
etaL, 1984). Membranes were hybridization with various LYS~ or actin probes labeled with
a32P-dCTP. Mouse genetic mapping analyses were performed as described (Barbosa ef al.,
1 995).
5.4.1.4 SSCP ANALYSIS
Detection of nucleotide c~ geo, by SSCP was performed as described by Orita etal.
(1989). Briefly, each PCR~ product was mixed with an equal volume of denaturing buffer and
heated to 95~C for 3 rnin., after which the samples were loaded onto 0.8 rnm thick, 10% native
polyacrylamide gels. Ge}s were run at ambient temperature at 9 W for 6-10 hours, depending on
10 the size of the PCR~ product. Bands were vi~ ecl by silver-st~inin~ (Beidler et al., 1982).
5.4.1.5 ~T.T~r~-SPECIFIC OLIGONUCLEOTIDE ANALYSIS
PCRTM products s~alll~llg the mutation site in patient 371 were Llallsr~lled to nylon
membranes using a slot blot appal~L~Is. A~pl oxilllately 5 ng of each PCR~M product was treated
~,vith a denaturing solution (0.5 M NaOH, 1.5 M NaCl), split in half and loaded in duplicate. Two
l5 17 mer oligonucleotides were synthesi7ed that span the region cont~ining the mutation. One
contained the sequence ofthe normal allele (S'-CGCACATGGCAACCCTT-3')(S~Q ID NO:73),
while the other contained the sequence of the mutant allele (5'-GCACATGGGCAACCCTT-3')
(SEQ ID NO:74). These were end-labeled with ~r'2P-dATP using T4 polynucleotide kinase and
hybridized to the membranes at 50~C. Hybridization and wash buffers were as described (Church
20 and Gilbert, 1984). Membranes were sequentially washed at 45~C, 55~C and 65~C for 10 min
each and exposed to X-ray film.
~.4.2 RESULTS
5.4.2. 1 A QUESTION OF TwO BG GENE:S
In order to resolve the dilemma created by the .o.~ci.ct~nce of two different bg ç~n~id~te
25 genes (Lyst and BG), the inventors isolated and sequenced additional mouse cDNA and genomic
clones collesponding to the 3' end of Lyst. An anchored, nested PCRTM (3'RACE-PCRTM) from
this region yielded two fr~n~.nts (1.25 kb and 2 kb). The 1.25 kb clone c~-nt~in~?(l the previously
published 3' end of Lyst, while the 2 kb clone contained sequences derived from Lyst (at the S' end)
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and from BG (at the 3' end). Reverse ~ s~,l?~ion and PCRTM (RT-PCRTM) confinned that
nucleotides 1-4706 of Lyst also represent the previously undetermined 5' end of the BG open
reading frame (FIG. 15c). A full length cDNA was assembled from nucleotides 1~706 of Lyst, the
2 kb 3'RACE-PCRTM clone and 6824 nucleotides of BG cDNA. This 11,817 bp cDNA seq~ nce
5 (Lyst-I, Genbank accession number U70015) corresponds to the largest mRNA observed in
Northern blots (~12 kb) (Goodrich and Holcombe, 1995).
Analysis of a Pl genomic clone (number 8592) co..li.;..;..~ Lyst and BG revealed that the
11,817bp Lys~-I cDNA results from splicing of Lyst exon (J (Cont~ining nucleotide 4706) to
downstream exon ~ (FIG. 15b). Incomplete splicing and reading through the intron c~' interposed
o between exons a and ~ yields the 5893 bp cDNA described by Barbosa ef aL (1996) (Lyst-rJ,
FIG. 15b, Genbank accession number L77884). Intron ~' encodes 37 in-frame amino acids
followed by a stop codon and a polyadenylation signal. Lyst-II corresponds to a smaller (~kb)
rnRNA observed on Northern blots. Lyst-I and Lyst-II are both present in poly(A)+ RNA from
many mouse tissues (FIG. 15b). The putative Lyst-I protein is of relative molecular mass 425,287
(Mr 425K) while that of Lyst-II is predicted to be of Mr 172.5K.
5.4.2.2 SEQUENCE OF HU MAN LYSTl AND LYST2 cDNAs
cDN~s corresponding to LYSTl, the human homolog of Lystl-isoforrn I (which is the
largest mRNA isoform of the bg gene) were obtained by identification of human expressed
sequence tags (ESTs) similar in sequence to mouse Lystl by database searches (Genbank
2 o accession numbers L77889, W26957 and ~51623). Intervening cDNA seq~lence~ were isolated
using RT-PCRTM with primers derived from mouse Lystl sequence and ~ cent ESTs. The
partial LYSTl cDNA sequence (Genbank Accession number U70064; 7.1 kb) was assembled by
~lignm~nt of these clones with mouse Lystl cDNA. Human LYST1 has 82% predicted amino
acid identity with mouse Lystl over 1,990 amino acids The predicted human LYST1 amino acid
sequence contains a 6 amino acid insertion relative to mouse Lystl at residue 1,039. Recently,
another group has published the sequence of the human LYSTl cDNA (Nagle et al., 1996). The
cDNA sequence of the present invention differs in at 4 nucleotides and 3 predicted amino acids
- from that of Nagle et aL (1996). This 13.5 kb cDNA sequence corresponds to the largest mRNA
(LYSTI-isoform 1[) observed on northern blots of human tissues (caption in FIG. 2). These
3 o northern blots also demonstrated the çxi~t~nce of a smaller LYST isoforrn (~4.5 kb, ~ç~i~n~ted
LYST-isoform II) that was similar in size to the smaller mouse Lystl mRNA, and that appeared to
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differ in distribution of ~ sion in human tissues from LYSTl-isoform I. ~s~lmin~ that the
genomic derivation of human LYSTl-isoform II was the same as mouse LystI-isoform II, the
sequence of the 3' end of the human LYST1 -II isoform was sought by cloning human LYSTl
intron F' using PCR~ of human genomic DNA with primers derived from LYSTl exon F and
5 mouse intron F' (caption in FIG. 2). The sequ~n~e of the S' end of human LYSTl intron F'
contained 17 codons in frame with LYSTl exon F, followed by a stop codon. By amplification of
a LYST1-isoforrn II cDNA from human peripheral blood RNA by RT-PCRIM with primers from a
5' LYSTl exon and ~ YSTl intron F', it was demonstrated that this intron was indeed retained in
human LYSTl-isoform II mRNA. Nucleotides 1-5905 of human LYSTl-isoform II cDNA are
0 identical to LYSTl-isoform I, and are followed by intron F' sequence (Genbank ~çcee~ion number
U~4744)(FIG. 2). The predicted intron-encoded amino termini of the mouse Lystl -isoforrn II
and human LYSTl- isoforrn II peptides shared 65% identity.
The only significant ~equ~n~e similarity of LYST1- isoforrn II to known proteins was with
the st~thmin family. Identity with mouse Lystl-isoform II in this region ~amino acids 376-540)
15 was g2% (and similarity was 99%)(FIG. 5).
.4.2.3 GENETIC AND PHrYSICAL MAPP~NG OF L YST
A 2 kb human LYST probe was ~si~ne~ to human chromosome 1 by hybridization to
human-rodent somatic cell hybrid DNA (FIG. 16). All of the bands that segregated with human
DNA hybridized only to somatic cell hybrids cont~ining human chromosome 1 DNA.
2 o In order to precisely map LYST on human chromosome 1, LYST probes were hybridized to
YAC clones encomr~c~ing the CHS critical region (FIG. 16b and FIG. 16c) (Barrat et al. 1996).
Three probes, derived from dirrelel~l segm~ntc of the LYST cDNA each hybridized to five ChrS
critical region YACs (FIG. 16d), co~ fillg loç~ tinn to the correct interval.
Genetic mapping in 504 [C57BL/6J-bg' x (C57BL/6J-bg' x CAST/EiJ)F,] backcross rnice
was used to determine whether LYST was the human homolog of the mouse bg gene. Using one
XbaI and two TaqI RFLPs, LYST was shown to coseg~egale with bg and Lyst on mouseChromosome 13.
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~Y
5.4.2.4 M UT~TION ~NALYSIS
As an initial screen for LYST mutations in CHS patients, we analyzed northern blots of
poly(A)+ RNA from CHS patients. The largest LYST rnRNA species (LYST-I, approximately 12
kb~ was greatly reduced in abundance or absent in Iymphoblastoid mRNA of patients Pl and P3,
5 respectively (FIG. 4a), while the smallerLYSTtranscript (LYST-II, a~uploxilllately 4.4 kb) was
both present and lln~l;"""i.clled in abundance. Rehybridization ofthis blot with an actin probe
confirmed that absence of the larger transcript was not due to uneven gel loading or RNA
degradation. Fibroblast poly(A)+ RNA from three other CHS patients (369, 371 and 373)
showed a moderate reduction in LYST-I mRNA (51-60% of control by densitometry), while the
~.YST-I~ rnRNA was çssenti~lly unaltered in ablln~l~nre (103-147% of control).
Single-strand confor~nation polymorphism (SSCP~ analysis was undertaken using cDNA
samples derived from Iymphoblastoid or fibroblast cells lines from CHS patients. Anomalous
bands were detccted in PCR~ products from the 5' end of the LYST OR~; in two unrelated CHS
patients dilTelenL from those with aberrant northern blot patterns (371 and 373, FIG. 4b).
15 Subsequent sequence analysis identified a C to T transition at nucleotide 148 ofthe coding
domain in patient 373 (FIG. 4c). Four of nine cDNA clones derived from patient 373 contained
this mutation. Rcstriction enzyme digestion confirmed this mutation. TaqI digestion of LYST
cDNA ~nucleotide 520 to 808) showed loss ofthis restriction site in patient 373 to be
heterozygous. The C to T substitution creates a stop codon at amino acid 50 (R50X).
Patient 371 had previously been shown to have a frame-shift mutation with a G insertion
at nucleotide 118 of the coding domain (FIG. 4c)~Barbosa ef al., 1996]. Each of five cDNA
clones isolated from lymphoblasts of patient 371 were found to contain this mutation.
Allele-specific oligonucleotide hybridization of cDNA from this patient failed to detect a signal
with an oligonucleotide corresponding to the normal allele, suggesting that the patient is either
2 5 homozygous or hemizygous for this mutation.
Mutations were identified in three other CHS patients: cDNA isolated from
EBV-transformed Iymphoblasts from patient 372 (deposited at the Coriell Institute as GM03365)
contained a homozygous C to T transition at nucleotide 3310 of the coding domain, that created a
stop codon at amino acid 1104 (Rl 104X)[Nagle et al., 1996]. Patient 370 contained a
homozygous C to T transition at nucleotide 3085 ofthe coding domain, that created a stop codon
at amino acid 1029 (Q1029X). Patient 369 had a hetelo~y~ s frame shift mutation. Nucleotides
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WO 97/28262 PCTAUS97/01748
q~
3073 and 3074 of the coding domain were deleted in two of five cDNA clones isolated from this
patient. The deletion results in a frame shift at codon 1026 and termination at codon 1030.
Lymphoblasts from all ofthese patients (369, 370, 371, 372, 373, P~ and P3) contain the 0
giant perinuclear Iysosomal vesicles that are the hallmark of CHS. Patients 369, 370, and 371 had
typical clinical presentations of CHS, with recurrent childhood infections and oculocutaneous
albinism. The parents of patients 369 and 370 are known not to have been cos~n~-inous. In
contrast, the clinical course of patients 372 and 373 was milder: Lymphoblasts were irnmortalized
from patient 372 at 27 years of age. He had oculocutaneous albinism, recurrent skin infections,
and peripheral neuropathy. Patient 373 has not had systernic infections and is alive at age 37.
0 Patient 373 does, however, have hypopigm~nted hair and irides as well as peripheral neuropathy.
5.4.2.5 EXPRESSION OF L YS~-I AND L YST-II IN HUMAN TISSUES
Analysis of northern blots of mouse mRNA had suggested that the relative abllnr~nce of
mouse Lys~-I and Lys~-II transcripts differed from tissue to tissue (Barbosa et al., 1996). The
relative abundance of LYSTmRNA isoforms in human tissues at difrel~llL developmental stages
was Px~min~cl by sequential hybridization of a poly(A)+ RNA dot blot with several LYSTcDNA
probes. The quantity of poly(A)+ RNA loaded on the blot was normalized to eight housekeeping
genes (phospholipase, ribosomal protein S9, tubulin, a highly basic 23 kD protein,
glyceraldehyde-3-phosphate dehydrogenase, hypo;~a-lLhille guanine phosphoribosil transferase,
$-actin, and ubiquitin) to allow estimation of the relative abundance of LYST mRNA isoforms in
dirrelellL tissues.
Using a probe that hybridized only to LYST-I transcripts (the largest LYST isoform) on
northern blots (Barbosa et al., 1 g96), LYST-I mRNA was found to be most abundant in thymus
(adult and fetal), peripheral blood leukocytes, bone marrow, and several regions of the adult
brain. In contrast, no LYST-I mRNA was cletected in fetal brain. Negligible LYST-I transcription
w--as also ap~a, enL in heart, lung, kidney, or liver at any developmental stage.
A somewhat di~l t;lIL pattern of expression was evident upon rehybridization of the blot
with a probe derived from the 5' end ofthe coding domain of LYST, a region that hybridized to
both LYST-I and LYST-II mRNAs on northern blots (Barbosa e~ al., 1996). Consonant with the
pattern of LYST-I transcription was abundant expression detected with this probe in peripheral
3 o blood leukocytes, thymus (adult and fetal), and bone marrow, and negligible expression detec.ted
CA 02244744 1998-07-29
VVO 97128262 PCT~US97/01748
91
in skeletal muscle. However, several tissues with abundant LY3T-I transcripts, exhibited
considerably less hybridization signal with the 7.YST-I + LYST-II probe, including most regions of
the adult brain, fetal and adult thymus, and spleen. Furthermore, several tissues with negligihle
LYST-I transcription exhibited intense hybridization with the LYST-I + LYST-~I probe, including
adult and fetal heart, kidney, liver, and lung, and adult aorta, thyroid gland, salivary gland,
appendix, and fetal brain.
5.4.3 DISCUSSION
As described above, the novel mouse gene, Lyst (T~osomal traff}cking regulator), was
identified from a bg critical region YAC and showed that it was mllt~te-l in two bg alleles. The
1 o inventors also identified two human ESTs similar in sequence to mouse Lyst and identified a
mutation in one of these ESTs in a CHS patient. Siml-lt~neously, another group published a
partial cDNA sequence (BG) that had been isolated from the same YAC ~Perou et al., 1996a).
This partial cDNA was mllt~ted in two other bg alleles, but was di~el ~.lL in sequence from Lyst.
The inventors have resolved this bg gene dilemma by demonstrating that Lysf and BG sequences
are derived from a single gene with alternatively spliced mRNAs. The unrelated cDNA sequences
that had been reported are derived from non-overlapping parts of two Lyst isoforms with di~el en~
predicted C-terminal regions. The inventors described a 5893 bp cDNA (Lyst) while Perou et aL
reported a partial cDNA sequence (BG) without a 5' end (Perou et al., 1996a). By sequencing
additional RT-PCRTM products, the inventors have shown that nucleotides l -4706 of Lyst also
represent the previously undetermined 5' region of BG. Alternative splicing at nucleotide 4706,
however, results in bg gene isoforms that contain the 3 ' region of BG or Lyst. Splicing of Lyst
exon ~ (co~ g nucleotide 4706) to exon ~ results in an rnRNA (Lyst-I) that corresponds to
the largest band observed on Northern blots and that contains BG sequence at the 3' end.
Incomplete splicing at nucleotide 4706 results in the 5893 bp cDNA (Lyst-II) described by
2~i Barbosa et al. (1995) and contains intron-derived sequence at the 3' end. Lyst-II corresponds to a
smaller mRNA observed on Northern blots. While several other genes generate an alternative
C-terminus by incomplete splicing (Myers et al., 1995; Sugimoto et al., 1995; Sygiyama ef aL,
1996; Zhao and Manlley, 1996; Van De Wetering et al., 1996), the bg gene is unique in that the
predicted structures of the two C-termini are quite difr~ t. The C-terminus of Lyst-I contains a
3 o 'WD'-repeat domain that is similar to the ,~-subunit of heterotrimeric G proteins and which may
assume a propeller-like secondary structure (Lambright el al., 1996). In contrast, Lyst-II has a
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~'~
C-terminal prenylation motif that could prQvide ~tt~çhment to the Iysosomal membrane.
Although the prenylation signal is absent from Lyst-I, it contains a hydrophobic region that is
predicted to be membrane associated. The significance of these divergent features is increased by
the fact that Lyst is not predicted to have tr~n.~m~mkrane helices.
Identification of the human homolog of the bg gene, LYST, provided a second line of
evidence that Lyst and BG are derived from a single gene, since the LYST sequence overlaps both
Lyst and BG. The LYS~ cDNA identified corresponds to the mouse Lyst-I isoform. Northern
blots of human tissues had suggested that a similar complexity exists in the transcription of LYST,
the homologous human gene (Barbosa et al., 1996). We recently id~ntified two human ESTs
1 o homologous to mouse ~yst and described a mutation in one of these ESTs in a CHS patient
(Barbosa et al., 1996). Subsequently, another group published the cDNA sequence of the largest
LYSTisoforms (LYST-I), and iciçntified mutations in this gene in 2 additional patients with CHS
(Nagle et al., 1996) . Here we have described the identification of a second isoform of human
LYST. This cDNA, de~i~n~ted l.YST-rl, encodes a protein of 1531 amino acids that is
homologous to mouse Lysf-l:f . Like the latter, human ~YST-II n3RNA arises through incomplete
splicing and retention of a transcribed intron that encodes the C-terminus of the predicted
L~ST-II protein. The mouse and human LYST-J;~ -specific codons share 65 % predicted amino
acid identity. The stop codon, however, is not precisely conserved between human and mouse
LYSl-J~f . While mouse Lyst-II is predicted to contain a C-terminal prenylation motif (CYSP),
2 o translation of human LYST-~ is predicted to terminate 22 codons earlier and to lack this motif.
Several of the predicted structural features of mouse Lyst were conserved in human. The
most notable of these was a region similar in sequence to st~thmin (amino acids 376-540). While
mouse and human LYST had an overall amino acid identity of 81 %, identity in the st~1hmin-like
domain was 92% (and similarity was 99%). Stathmin is a coiled-coil phosphoprotein thought to
regulate microtubule polymerization and to act as a relay for intrac~ r signal tran.~ lction
(Sobel 1991; Belmont and Mitchison, 1996). This region of LYST may encode a coiled-coil
protein interaction domain and may regulate rnicrotubule-m~.fli~ted Iysosome trafficking.
Intriguingly, a defect in microtubule dynamics has previously been doGllmf~.nte(l in CHS (Oliver ,,
et al., 1975) and intact rnicrotubules are required for m~ n/;e of Iysosomal morphology and
tr~ffickin~ (MatteoniandKreis, 1987;SwansonetaL, 19~7;Swansonetal., 1992;0kaand
Weigel, 1983).
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~3
Other putative structural featu}es 4f LYST that are conserved between human and mouse
are several pairs of predicted helices with a protein kinase C- or casein kinase II-phosphorylation
signal at their N-terminus. These helical bundles have been hypoth~i7ed to have a signal
tr~n~-luction function similar to ~ l"";~ The conserved phosphorylation sites have been
hypotheci7ed to affect interactions of LYST with other molecules through phosphorylation ---
dependent conformational shifts in the helical bundles. The conservation of these features
between human and mouse lends credence to their biological relevance.
In order to evaluate the ç~n~ cy of LYST for CHS, segments of the LYST sequence were
mapped in the human genome. The CHS locus was recently ~csigned to human chromosome
0 lq42-43 (Goodrich and Holcombe, 1995; Barrat et al. 1996; Fukai et al., 1996), a result that had
been expected based on linkage conservation between the mouse chromosome 13 region
Col~ gthebglocusandhumanchromosome lq42-q43 (Beguez-Cesar, 1943). DIS2680 and
DIS163 were previously shown to represent the telomeric and centromeric limits, respectively, of
the CHS critical region (Barrat et al. 1996). Human LYST mapped within this CHS critical
region. The localization of all LYSTPCRTM products to CHS critical region YACs also precluded
the possibility that the LYST seqtlf?nce had been assembled from segments of closely related genes.
Northern blots demonstrated a 12 kb mRNA (corresponding to LYST-I) to be severely
reduced in abundance in two CHS patients. A 4.4 kb band (corresponding to LYS~-II), however,
was present in mRNA from these patients in normal abundance. These results suggest that, at
2 o least in some patients, CHS results from loss of the protein encoded by LYST-I rather than
LYST-rl. This result is surprising since previous Northern blots had suggested that the major
LYSTmRNA in granular cells was LYST-II, while LYST-I was either lln~etect~kle or barely
detect~ble in these cells. Because lysosomal trafficking defects in granular cells account for the
clinical features of CHS (Griffiths, 1996), it had been hypothesized that the 4.4 kb LYST-II
2 5 mRNA represented the Llans~;lip~ of primary functional significance. In this context, it is
interesting to note that the bg~' mutation results in the generation of a premature stop codon in
Lyst-I that is unlikely to affect Lyst-II mRNA processing (Perou et al., 1996a). These results
suggest that defects in LYST-I alone can elicit CHS and that LYST-rJ~ es~ion alone cannot
compensate for loss of LYST-I
3 o Mutations were identified within the coding domain of LYSTin five CHS patients, two of
which have been reported previously ~Barbosa et al., 1996, Nagle et al., 1996). The genetic
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lesions in three CHS (patients 370, 372 and373) were C to T transitions that resulted in
premature t~ il,aLion (Q102gX, R1104X and RSOX, respectively)[Nagle et a~ 996~. Two
other patients had coding domain frame shif't mutations that indllce~l premature t~lll~in~LLion One
of these, patient 371, had a G insertion at nucleotide 118 of the coding domain, leading to
premature termination at codon 63 (Barbosa e~ al., 1996). Allele-specific oligonucleotide analysis
indicated that this mutation was either homozygous or that mRN~ corresponding to this region is
not produced from the other allele (he~ y~osity). Patient 369 was heterozygous for a
dinucleotide deletion that results in premature termination at codon 1030. Interestingly, all bg
and CHS mutations identifiecl to date are predicted to result in the production of either trllnc~tecl
0 or absent LYST proteins (Barbosa et al., 1996; Nagle et al., 1996). Unlike Fanconi anemia, type
C, there does not appear to be a correlation between the length of the tnlnf~te(l LYST proteins
(which may or may not be sta~le) with clinical features or disease severity in C~S patients.
However, until the other mutant allele in patients 369 and 373 are identified, and the exact effects
of each mutation at the protein level are characterized, such correlation is imprecise.
Comparison of transcription of LYST-I and LYST-II in human tissues at diLrel ~
developmental stages revealed an overlapping but distinct pattern of expression. A qll~ntit~tive
estim~te ofthe expression ofthe smaller LYST m~NA isoforms was obtained by subtraction of
the relative hybridization intensity obtained with an LYST-I specific probe from that obtained with
a probe that hybridizes to all LYSTtranscripts. LYST-I transcripts predomin~tecl in thymus, fetal
2 o thymus, spleen, and brain (with the exception of amygdala, occipital lobe, putamen, and pituitary .
gland). Both LYST-I and LYST-II transcripts were abundant in the latter brain tissues, peripheral
blood leukocytes, and bone marrow. Only the smaller LYSTisoforms were expressed in several
tissues, in~ln~1ing heart, fetal heart, aorta, thyroid gland, salivary gland, kidney, liver, fetal liver,
appendix, lung, fetal lung, and fetal brain. The developmental pattern of LYSTmRNA isoform
ex~res~ion in brain was particularly interesting, since only the smallerLYSTisoforrns were
expressed in fetal brain, whereas the largest isoforrn (LYST-I) predomin~ed in many regions of
the adult brain.
In summary, the inventors have shown that the same gene is mllt~ted in human CHS and
bg mice. Without bone marrow transplantation, CHS patients typically die in childhood of
3 o infection and m~lign~ncy. The existence of an animal model of CHS with a similar genetic lesion
will assist efforts to develop novel therapies for this disease.
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5.5 EXAnIPLE5-- D NA SEQUENCES OF Mo~sE LYS~I
5.5.1CDNA SEQUENCE OF LONG ISOFORM (SE Q ID N 0:3)
1 TGAGAGCTCA CGCTGGCCTG GCAGCCTTGG TGAGTCGGGA TTCTCCTGCA
51 CCGGCGGGCG AGAGCGCGCG GCGGACCACA GAGCGGAGGT GAAGCCTTAT
- 5101 GCTGAGACAG TTTTATCTAG TTCATGAACC CA~ATTATAT ACAAGCTGAA
151 TGTTACAGAA GTGCTGA~AG ACTGCTCTGT CATGAGCACG GACAGCAACT
201 CATTGGCACG TGAGTTTCTG ATTGATGTCA ACCAGCTTTG CAATGCAGTG
251 GTCCAGAGGG CAGAAGCCAG GGAAGAAGAA GAAGAGGAGA CACACATGGC
301 AACTCTTGGA CAGTACCTTG TCCATGGACG AGGATTTCTG TTACTTACCA
10351 AACTA~ATTC TATCATTGAT CAGGCCCTGA CATGCAGAGA AGAACTCCTG
401 ACTCTTCTTC TGTCGCTCCT TCCCTTGGTG TGGAAGATAC CTGTCCAGGA
451 ACAGCAGGCA ACAGATTTTA ACCTGCCACT GTCATCTGAT ATAATCCTGA
501 CCA~AGA~AA GAACTCAAGT TTGCA~AAAT CAACTCAGGG A~AATTATAT
551 TTAGAAGGAA GTGCTCCATC TGGTCAGGTT TCTGCA~AAG TA~ACCTTTT
15601 TCGA~AAATC AGGCGACAGC GTA~AAGTAC CCATCGTTAT TCTGTAAGAG
651 ATGCAAGA~A GACACAGCTC TCCACCTCTG ACTCCGAAGG CAACTCAGAT
701 GA~AAGAGTA CGGTTGTGAG TA~ACACAGG AGGCTCCACG CGCTGCCACG
751 GTTCCTGACG CAGTCTCCTA AGGA~GGCCA CCTCGTAGCC A~ACCTGACC
801 CCTCTGCCAC CAAAGAACAG GTCCTTTCTG ACACCATGTC TGTGGA~AAC
20851 TCCAGAGAAG TCATTCTGAG ACAGGATTCA AATGGTGACA TATTAAGTGA
901 GCCAGCTGCT TTGTCTATTC TCAGTAACAT GAATAATTCT CCTTTTGACT
951 TATGTCATGT TTTGTTATCT CTATTGGA~A AAGTTTGTAA GTTTGACATT
1001 GCTTTGAATC ATAATTCTTC CCTAGCACTC AGTGTAGTAC CCACACTGAC
lC51 TGAGTTCCTA GCAGGCTTTG GGGACTGCTG TAACCAGAGT GACACTTTGG
251101 AGGGACAACT GGTTTCTGCA GGTTGGACAG AAGAGCCGGT AGCTTTGGTT
1151 CAACGGATGC TCTTTCGAAC CGTGCTGCAC CTTATGTCAG TAGACGTTAG
1201 CACTGCAGAG GCAATGCCAG A~AGTCTTAG GA~AAATTTG ACTGAATTGC
1251 TTAGGGCAGC TTTA~AAATT AGAGCTTGCT TGGA~AAGCA GCCTGAGCCT
1301 TTCTCCCCGA GACA~AAGAA AACACTACAG GAGGTCCAGG AGGGCTTTGT
301351 ATTTTCCAAG TATCGTCACC GAGCCCTTCT ACTACCTGAG CTTCTGGAAG
1401 GAGTTCTACA GCTCCTCATC TCTTGTCTTC AGAGTGCAGC TTCA~ATCCC
1451 TTTTACTTCA GTCAAGCCAT GGATTTAGTT C~AGAATTTA TCCAGCACCA
1501 AGGATTTAAT CTCTTTGGAA CAGCAGTTCT TCAGATGGAA TGGCTGCTTA
1551 CAAGGGACGG TGTTCCTTCA GAAGCTGCAG AACATTTGAA AGCTCTGATA
351601 AACAGTGTAA TA~AAATAAT GAGTACTGTG AAAAAGGTGA AATCAGAGCA
1651 ACTTCATCAT TCCATGTGCA CAAGGA~AAG ACACCGGCGT TGTGAGTATT
1701 CCCACTTCAT GCAGCACCAC CGCGATCTTT CAGGGCTCCT GGTTTCAGCT
1751 TTTA~AAATC AGCTTTCTAA AAGCCCCTTT GAAGAGACCG CAGAGGGAGA
1801 TGTGCAGTAT CCAGAGCGCT GCTGCTGCAT CGCCGTGTGC GCTCACCAGT
401851 GCTTGCGCTT GCTGCAGCAG GTTTCCCTGA GCACCACGTG TGTCCAGATC
1901 CTATCAGGTG TACACAGTGT TGGAATCTGT TGTTGTATGG ATCCTAAGTC
1951 TGTGATCGCC CCTTTACTGC ATGCTTTTAA GTTGCCAGCA CTGAAAGCTT
2001 TCCAGCAGCA TATACTGAAT GTCCTGAGCA AACTTCTTGT GGATCAGTTA
2051 GGAGGAGCAG AGCTATCACC GAGAATTA~A A~AGCAGCTT GCAACATCTG
452101 TACTGTGGAC TCTGACCAAC TGGCTAAGTT AGGAGAGACA CTGCAAGGCA
2151 CCTTGTGTGG TGCTGGTCCT ACCTCCGGCT TGCCCAGTCC TTCCTACCGA
2201 TTTCAGGGGA TCCTGCCCAG CAGCGGCTCT GAAGACTTGC TGTGGAAGTG
2251 GGATGCATTA GAGGCTTATC AGAGCTTTGT CTTTCAAGAA GACAGATTAC
-2301 ATAACATTCA GATTGCAAAT CACATTTGTA ATTTACTCCA GA~AGGCAAT
502351 GTAGTTGTTC AGTGGA~ATT GTATAATTAT ATCTTTAATC CTGTGCTCCA
2401 AAGAGGAGTT GAATTAGTAC ATCATTGTCA ACAGCTAAGC ATTCCTTCAG
2451 CTCAGACTCA CATGTGTAGC CAACTGAAAC AGTATTTGCC TCAGGAAGTG
2501 CTTCAGATTT ATTTAAAAAC TCTACCTGTC CTACTTA~AT CCAGGGTAAT
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2551 AAGAGATTTG TTTTTAAÇTT GTAATGGAGT A~ACCACATA ATTGAACTA~
2601 ATTACTTAGA TGGGATTCGA AGTCATTCCC TGA~AGCATT TGA~ACTCTG
2651 ATTGTCAGCC TAGGGGAACA ACAGA~AGAT GCTGCAGTTC TAGACGTCGA
2701 TGGGTTAGAC ATCCAACAGG AGTTGCCGTC CTTAAGTGTG GGTCCTTCTC
52751 TTCATAAGCA GCAAGCTTCT TCAGATTCTC CTTGCAGTCT CAGGAAGTTT
2801 TATGCCAGCC TCAGAGAGCC TGATCCA~AA A~ACGA~AGA CCATTCACCA
2851 GGATGTTCAC ATA~ACACCA TA~ACCTCTT CCTCTGTGTG GCTTTTCTAT
2901 GTGTCAGTAA AGAAGCAGAC TCTGATAGGG AGTCTGCCAA TGAGTCAGAA
2951 GATACTTCTG GCTATGACAG CCCTCCCAGT GAGCCATTAA GTCACATGCT
lO3001 ACCATGTCTG TCTCTTGAGG ACGTTGTCTT ACCTTCCCCT GAATGTTTGC
3051 ACCATGCAGC AGACATTTGG TCCATGTGTC GTTGGATCTA CATGTTGAAC
3101 TCAGTCTTCC AGA~ACAATT TCACAGGCTT GGTGGTTTCC AAGTGTGCCA
3151 TGAATTAATA TTTATGATAA TCCAGA~ACT ATTCAGAAGT CATACAGAGG
3201 ATCAAGGAAG AAGGCAGGGA GA~ATGAGTA GA~ATGA~AA CCAAGAGCTA
153251 ATCAGGATAT CTTACCCCGA GCTGACACTG A~GGGAGATG TATCATCTGC
3301 AACAGCACCA GACCTGGGAT TTCTGAGA~A GAGTGCTGAC AGCGTGCGTG
3351 GATTCCAGTC ACAGCCTGTG CTTCCCACAA GTGCAGAGCA GATTGTGGCT
3401 ACTGAATCTG TTCCTGGGGA ACGAAAGGCA TTTATGAGTC A~CA~AGTGA
3451 GACTTCTCTC CAGAGCATAC GACTTTTGGA GTCTCTCCTG GACATTTGTC
203501 TTCATAGTGC CAGAGCCTGT CAACAGAAGA TGGAATTGGA GCTACCGTCT
3551 CAGGGCTTGT CTGTGGA~AA TATATTGTGT GAACTGAGGG AACACCTTTC
3601 CCAGTCAAAG GTGGCAGAAA CAGAATTAGC A~AGCCTTTA TTTGATGCCC
3651 TGCTTCGAGT AGCCCTGGGG AATCATTCAG CAGATTTGGG CCCTGGTGAT
3701 GCTGTGACTG AGAAGAGTCA TCCCTCTGAG GAAGAGCTGT TGTCCCAGCC
253751 CGGAGATTTT TCAGAAGAAG CTGAGGATTC TCAGTGTTGT AGTTTGAAAC
3801 TTCTGGGTGA GGAAGAAGGC TATGA~GCGG ATAGTGA~AG CAATCCTGAG
3851 GATGTTGACA CCCAAGACGA TGGAGTAGAA TTA~ATCCTG AAGCAGAAGG
3901 TTTCAGTGGA TCGATTGTTT CAAACAACTT ACTTGA~AAC CTCACTCACG
3951 GGGA~ATAAT ATACCCTGAG ATTTGCATGC TGGGATTA~A TTTGCTTTCT
304001 GCTAGCA~AG CTAaACTTGA TGTGCTTGCT CATGTGTTTG AGAGCTTTCT
4051 GA~AATTGTC AGGCAGAAGG A~AAGAACAT TTCTCTCCTC ATACA~CAGG
4101 GAACTGTGAA AATCCTTCTA GGCGGGTTCT TGAATATTTT AACACAAACT
4151 AACTCTGATT TCCAAGCATG CCAGAGAGTA CTGGTGGATC TCTTGGTATC
4201 TTTGATGAGC TCAAGAACGT GTTCAGAAGA CTTAACTCTT CTTTGGAGAA
354251 TATTTCTGGA GA~ATCTCCT TGTACAGAAA TTCTTCTCCT TGGTATTCAC
4301 AAAATTGTTG A~AGTGATTT TACTATGAGC CCTTCACAGT GTCTGACCTT
4351 TCCTTTCCTG CATACCCCGA GTTTAAGCAA TGGTGTCTTA TCACAGA~AC
4401 CTCCTGGGAT TCTTAACAGT A~AGCCTTAG GCTTATTGAG AAGAGCACGG
4451 ATTTCCCGAG GCAAGA~AGA GGCTGATAGA GAGAGTTTTC CCTATAGGCT
404501 GCTTTCCTCT TGGCATATAG CCCCAATCCA CCTGCCGTTG CTGGGACAGA
4551 ACTGCTGGCC ACACCTGTCA GAAGGATTTA GTGTTTCTCT TGTGGGTTTA
4601 ATGTGGAATA CATCCAATGA ATCCGAGAGT GCTGCAGA~A GGGGA~AAAG
4651 AGTA~AGA~A AGA~ACAAAC CATCAGTTCT GGAAGACAGC AGTTTTGAAG
4701 GAGCAGAAGG TGATAGACCA GAAGTTACAG AATCCATCAA TCCTGGTGAC
454751 AGACTCATAG AAGACGGCTG TATTCATTTG ATTTCACTGG GGTCCA~AGC
4801 ATTGATGATC CAAGTGTGGG CTGATCCCCA CAGTGGCACT TTTATCTTTC
4851 GTGTGTGCAT GGACTCA~AT GATGACACGA A~GCTGTCTC ACTAGCACAG
4901 GTGGA~TCAC AGGAGAATAT TTTCTTTCCA AGCA~ATGGC AACACTTAGT
4951 ACTTACCTAT ATTCAGCATC CTCAAGGGAA A~AGAATGTC CATGGGGA~A
505001 TCTCCATATG GGTCTCTGGG CAGAGGAAGA CTGATGTCAT CTTGGATTTT
5051 GTGCTCCCAA GAAAAACAAG CTTATCATCA GACAGCAATA A~ACATTTTG
5101 CATGATTGGT CATTGCTTAA CATCCCAAGA AGAGTCTCTG CAATTAGCTG
5151 GAAAATGGGA CCTGGGGAAC TTGCTCCTCT TCAATGGAGC TA~AATTGGC
5201 TCACAAGAGG CCTTTTTCCT GTATGCTTGT GGACCCAACT ACACATCCAT
55~ 5251 CATGCCGTGT A~ATATGGAC AGCCAGTCAT TGACTACTCC AA~TACATTA
-
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5301 ATA~AGACAT TTTGAGATGT GATGA~ATCA GAGACCTTTT TATGACQAG
- 5351 A~AGAAGTGG ATGTTGGTCT CTTAATTGAA AGTCTTTCAG TTGTTTATAC
5401 AACTTGCTGT CCTGCTCAGT ACACCATCTA TGAACCAGTG ATTCGACTCA
5451= AGGGCCAAGT GA~AACTCAG CCCTCTCAAA GACCCTTCAG CTCA~AGGAA
5501 GCCCAGAGCA TCTTGCTAGA ACCTTCTCAA CTCA~AGGCC TCCAACCTAC
5551 GGAATGTAAA GCCATCCAGG GCATTCTGCA TGAGATTGGT GGGGCTGGCA
- 5601 CATTTGTTTT TCTCTTTGCT AGGGTTGTTG AACTTAGTAG CTGTGAAGAA
5651 ACTCAAGCAT TAGCACTGCG GGTTATACTG TCTTTAATTA AGTACAGCCA
5701 ACAGAGAACA CAGGAACTGG A~AATTGTAA TGGACTCTCT ATGATTCACC
5751 AAGTGTTGGT CA~ACAGA~A TGCATTGTTG GCTTTCACAT TTTGAAGACC
5801 CTTCTTGAAG GTTGCTGCGG TGAAGA~GTT ATCCACGTCA GTGAGCATGG
5851 AGAGTTCAAG CTGGATGTTG AGTCTCATGC TATAATCCAA GATGTTAAGC
5901 TGCTGCAGGA ACTGTTACTT GACTGGAAGA TATGGAATAA GGCAGAGCAA
5951 GGTGTGTGGG AGACTCTGCT AGCAGCTTTG GAAGTCCTCA TCCGGGTAGA
6001 GCACCACCAG CAGQAGTTTA ATATTAAGCA GTTGCTGAAC GCCCACGTGG
6051 TTCACCACTT CCTACTGACC TGTCAGGTTT TACAGGAACA CAGAGAGGGG
6101 CAGCTTACAT CTATGCCCCG AGA~GTTTGT AGATCATTTG TGA~AATCAT
6151 TGCAGAAGTC CTTGGTTCTC CTCCAGACTT GGAATTATTG ACAGTTATTT
6201 TCAATTTCCT GTTAGCTGTA CACCCTCCTA CTAATACTTA TGTTTGTCAC
6251 AATCCCACAA ACTTCTACTT CTCTTTGCAC ATAGATGGCA AGATCTTTCA
6301 GGAGA~AGTG CAGTCACTCG CGTACCTGAG GCATTCTAGC AGCGGAGGGC
6351 A~GCCTTTCC CAGCCCTGGA TTCCTGGTAA TAAGCCCATC TGCCTTTACT
6401 GCAGCTCCTC CTGAAGGAAC CAGTTCTTCC AATATTGTTC QACAGCGGAT
6451 GGCTGCTCAG ATGGTTCGAT CTAGAAGTCT ACCAGCATTT CCTACTTATT
6501 TACCACTAAT ACGAGCACAA AAACTGGCTG CAAGTTTGGG TTTTAGTGTT
6551 GACAAGTTAC A~AATATTGC AGATGCCAAC CCAGAGA~AC AGAATCTTTT
6601 AGGAAGACCC TACGCACTGA A~ACAAGCAA AGAGGAAGCA TTCATCAGCA
6651 GCTGTGAGTC TGCAAAGACT GTTTGTGAAA TGGAGGCTCT TCTTGGAGCC
6701 CACGCCTCTG CCAATGGGGT TTCCAGAGGA TCACCGAGGT TCCCCAGGGC
6751 CAGAGTAGAT CACAAAGATG TGGGAACAGA GCCCAGATCA GATGATGACA
6801 GTCCTGGGGA TGAGTCTTAC CCACGTCGGC CTGACAACCT CAAGGGACTG
6851 GCCT QATTCC AGCGAAGCCA AAGCACTGTC GCAAGCCTTG GGCTGGCGTT
6901 TCCCTCTCAG AATGGATCTG CAGTTGCTAG CAGGTGGCCA AGTCTTGTTG
6951 ATAGGAATGC TGATGACTGG GAGAACTTTA CCTTTTCTCC TGCTTATGAG
7001 GQ AGCTAQA ACCGAGCCAC AAGCACCCAC AGTGTCATTG AAGACTGTCT
7051 GATACCTATC TGCTGTGGAT TATATGAACT CTTAAGTGGG GTTCTTCTTG
7101 TCCTGCCTGA TGCTATGCTT GAAGATGTGA TGGACAGGAT TATTCAAGCA
7151 GATATTCTTC TAGTCCTTGT TAACCACCCA TCACCTGCTA TCCAGCAAGG
7201 AGTAATTA~A CTGTTACATG CATACATTAA TAGAGCATCA AAGGAGCAAA
7251 AGGACAAGTT TCTGAAGAAC CGTGGCTTTT CCTTATTAGC CAACCAGTTG
7301 TATCTTCATA GGGGAACTCA GGAGTTGTTG GAGTGCTTTG TTGA~ATGTT
7351 CTTTGGTCGA CCGATTGGCC TGGATGAAGA ATTTGATCTG GAGGAAGTGA
7401 AGCACATGGA ACTGTTCQAG A~GTGGTCTG TCATTCCCGT TCTCGGACTA
7451 ATAGAGACCT CTCTCTATGA CAATGTCCTC TTGQACAATG CTCTTTTACT
7501 TCTTCTGCAA GTTTTA~ACT CTTGTTCCAA GGTAGCAGAC ATGCTACTGG
7551 ACAATGGTCT ACTCTATGTA TTATGTAATA CAGTAGCAGC CCTGAATGGA
7601 TTAGA~AAGA ACATTCCTGT GAACGAATAC A~ATTGCTCG CATGTGATAT
7651 ACAGCAGCTT TTCATAGCAG TTACAATTCA TGCTTGCAGT TCCTCAGGCA
7701 CACAGTATTT TAGAGTGATT GAAGACCTTA TTGTACTTCT TGGATATCTT
7751 QATAATAGCA A~ACAAGAG GACACA~AAT ATGGCTTTGG CCCTGCAGCT
7801 TAGAGTTCTC CAGGCTGCTT TGGAATTTAT AAGGAGCACA GCCAATCATG
7851 ACTCTGA~AG TCCAGTGCAC TCGCCTTCTG CCCACCGCCA TTCAGTGCCT
7901 CCGAAGCGGA GAAGCATTGC TGGTTCTCGC A~ATTCCCTC TGGCTCAGAC
7951 AGAGTCTCTG CTGATGAAGA TGCGCTCAGT GGCCAGCGAT GAGCTACACT
8001 CTATGATGCA GAGGAGGATG AGCCAAGAGC ACCCCAGCCA GGCCTCGGAG
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8051 GCAGAGCTCG CTCAGAGÇCT GCAGAGGCTC ACCATCTTAG CTGTGAACAG
-8101 GATTATTTAC CAAGAGTTGA ATTCAGATAT TATTGACATT TTGAGAACTC
8151 CAGAAAATAC ATCCCA~AGC AAGACCTCAG TTTCTCAGAC TGA~ATTTCT
8201 GAAGAAGACA TGCATCATGA GCAACCTTCT GTATATAATC CATTTCA~AA
58251 AGA~ATGTTA ACCTATCTGT TGGATGGCTT CA~AGTGTGT ATTGGTTCAA
8301 GTA~AACTAG CGTTTCTAAG CAGCAGTGGA CTA~AATCCT GGGGTCTTGT
8351 A~AGA~ACCC TCCGAGACCA GCTTGGAAGA TTGCTAGCGC ATATTTTGTC
8401 TCCAACCCAC ACTGTACAAG AACGGAAGCA GATACTTGAG ATAGTTCATG
8451 AACCAGCTCA CCAGGATATA CTTCGTGACT GTCTTAGCCC CTCCCCACAA
108501 CATGGAGCCA AGTTGGTTTT GTATTTGTCA GAGTTGATAC ATAATCATCA
8551 GGATGAGTTA AGTGAAGAAG A~ATGGACAC AGCAGA~CTG CTTATGAATG
8601 CTCTA~AGTT ATGTGGCCAC AAGTGCATCC CGCCCAGTGC CCCTTCCA~A
8651 CCAGAGCTCA TTAAGATCAT CAGAGAGGAG CA~AAGAAGT ATGA~AGTGA
8701 AGAGAGTGTG AGCAAAGGCT CATGGCAGAA AACGGTGA~C AACAACCAGC
158751 A~AGTCTCTT CCAGAGGCTC GATTTCAAAT CCAAGGATAT ATCTA~AATC
8801 GCTGCAGACA TCACCCAGGC TGTATCACTC TCCCAAGGCA TTGA~AGGAA
8851 GAAGGTGATC CAGCACATCA GAGGGATGTA CAAAGTTGAC CTGAGTGCCA
8901 GCAGGCACTG GCAGGAATGC ATCCAGCAGC TGACACATGA CAGAGCAGTC
8951 TGGTATGACC CAATCTACTA TCCAACTTCA TGGCAGTTGG ATCCAACAGA
209001 AGGGCCA~AC CGAGAGAGGA GACGTTTGCA GA&ATGCTAT CTAACTATTC
9051 CCAATAAGTA CCTCCTGAGG GACAGACAGA AGTCAGAAGG TGTGCTCAGG
9101 CCCCCACTCT CTTACCTTTT TGA~GATA~A ACTCATTCTT CCTTCTCCTC
9151 TACTGTCAAA GACA~AGCTG CAAGTGAATC CATCAGAGTG AATCGAAGAT
9201 GTATCAGTGT TGCACCATCT AGAGAGACAG CTGGGGAATT GTTGTTAGGT
- 9251 A~ATGTGGGA TGTACTTTGT GGAAGACAAT GCCTCTGACG CAGTTGA~AG
9301 CTCGAGCCTC CAAGGGGAGT TAGAGCCGGC ATCATTTTCT TGGACATATG
9351 AGGA~ATTAA AGAAGTTCAC AGGCGCTGGT GGCAACTAAG AGATAATGCT
9401 GTAGA~ATCT TTTTAACAAA TGGCAGAACA CTCCTATTAG CATTTGACAA
9451 TAACAAGGTT CGTGATGACG TGTACCAGAG CATCCTCACA AATAACCTCC
309501 CAAATCTTCT GGAGTACGGC AACATCACCG CTCTGACAAA CCTGTGGTAT
9551 TCTGGACAAA TTACCAATTT TGAATATTTG ACTCATTTAA ACAAGCATGC
9601 GGGCCGGTCC TTCAATGATC TCATGCAGTA CCCGGTGTTC CCCTTCATCC
9651 TTTCTGACTA TGTTAGTGAG ACTCTTGACC TCAATGATCC ATCTATCTAC
9701 AGA~ACCTAT CTAAGCCTAT AGCTGTGCAG TATA~AGA~A AAGAAGACCG
- 9751 TTACGTTGAC ACATACAAGT ACTTGGAGGA GGAGTATCGC AAGGGAGCTC
9801 GAGAGGATGA CCCCATGCCT CCTGTGCAAC CCTACCACTA TGGCTCCCAC
9851 TACTCCAACA GCGGCACCGT GCTCCACTTC CTGGTCAGGA TGCCGCCTTT
9901 CACTA~AATG TTTCTAGCCT ATC~AGATCA GAGTTTCGAC ATTCCAGACC
9951 GAACATTTCA TTCTACAAAC ACAACTTGGC GCCTCTCCTC CTTTGAGTCC
4010001 ATGACTGATG TGAAGGAGCT GATTCCAGAG TTTTTCTATC TTCCTGAGTT
10051 CTTAGTGAAC CGTGAAGGCT TTGACTTCGG TGTTCGTCAG AATGGAGAGC
10101 GGGTTAACCA CGTCAATCTT CCTCCCTGGG CACGCAACGA TCCTCGGCTG
10151 TTCATCCTTA TTCACCGGCA AGCACTAGAG TCTGACCATG TGTCCCAGAA
10201 CATCTGTCAC TGGATCGACT TAGTGTTTGG CTACAAGCAA AAGGGGAAGG
45_ 10251 CGTCTGTTCA AGCCATCAAT GTCTTCCACC CTGCTACATA TTTTGGAATG
10301 GATGTCTCTG CAGTTGAAGA TCCAGTGCAG AGACGGGCTT TAGA~ACCAT
10351 GATA~AAACC TACGGGCAGA CCCCACGTCA GTTGTTCCAC ACAGCCCATG
10401 CCAGCCGACC TGGAGCCAAG CTTAACATCG AAGGAGAGCT TCCAGCAGCT
10451 GTTGGCTTGT TAGTCCAGTT CGCTTTCAGA GAGACCCGAG AACCAGTCAA
5010501 GGAAGTCACT CATCCGAGCC CTTTGTCATG GATAAAAGGC TTGAAGTGGG
10551 GGGAGTACGT AGGTTCCCCC AGTGCTCCAG TACCTGTGGT CTGCTTCAGC
10601 CAGCCCCATG GAGA~AGATT TGGTTCCCTG CAGGCACTGC CCACCAGAGC
10651 CATCTGTGGT TTATCACGAA ACTTCTGTCT TCTGATGACC TACAACAAGG
10701 AGCAAGGTGT GAGAAGCATG AACAACACCA ATATTCAGTG GTCTGCTATC
10751 CTAAGCTGGG GATATGCTGA CAACATCTTA CGGTTGA~AA GTAAGCAGAG
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- 10801 TGAGCCACCA ATCAACTTCA TTCAGAGTTC ACAGCAGCAC CAGGTAACCA
- 10851 GTTGTGCCTG GGTGCCTGAC AGTTGTCAGC TCTTCACTGG GAGCAAGTGT
10901 GGTGTCATCA CAGCCTATAC CAACAGGCTC ACCAGCAGCA CGCCCTCAGA
10951 AATTGA~ATG GAGAGTCAGA TGCATCTCTA TGGACACACA GAGGAGATCA
511001 CCGGCTTATG TGTCTGCAAG CCGTACAGCG TGATGATAAG CGTGAGCAGA
11051 GACGGGACCT GCATAGTATG GGACCTGAAC AGGCTGTGCT ATGTACAAAG
11101 TTTGGCTGGA CACA~AAGCC CTGTGACGGC TGTCTCTGCC AGTGA~ACGT
11151 CAGGTGACAT TGCTACTGTG TGTGACTCAG CTGGCGGGGG CAGTGACCTG
11201 AGACTCTGGA CCGTGAATGG GGACCTCGTT GGACATGTCC ACTGCAGAGA
1011251 GATCATTTGT TCTGTAGCTT TCTCCAACCA GCCTGAGGGA GTCTCCATCA
11301 ACGTCATTGC TGGGGGATTA GA~AATGGCA TTGTAAGGCT ATGGAGCACA
11351 TGGGACTTGA AGCCTGTGAG AGAGATTACA TTTCCCA~AT CA~ATAAGCC
11401 CATCATAAGC CTGACATTCT CCTGTGATGG CCACCATTTG TACACTGCCA
11451 ACAGTGAGGG GACAGTGATC GCATGGTGCC GGAAGGACCA GCAGCGTGTG
1511501 AAGCTGCCCA TGTTCTACTC TTTCCTCAGC AGCTACGCAG CTGGATGAAG
11551 AGAAGGAGTG TCCCCACAGG ACATAAGCAC CGCTCTGCGA GCCTGGCTCC
11601 ACCAACTGCA GAAGCAGATG ACTGA&CAGA TATCCAGGAA AGACAACACA
11651 CGTGCCTCTG TGCGCGCTTC CCCAGCCTCC GTGGGCCTGA GAGTA~AGCC
11701 CTGCCCTCAT TCCATAATGG CGTGGAAGGC TGGGTCTGCA CACACTAGCC
2011751 AATTA~AGTC AGAATCTTGA TGCTTTTTCC CA~AAGGTTA GGCTGAATCA
11801 AAGATCAGGC TCGTGCC
5.5.2 C~N~ SEQUENCE OF SHORTISOFORU~(SE Q nD N 0:5)
251 TGAGAGCTCA CGCTGGCCTG GCAGCCTTGG TGAGTCGGGA TTCTCCTGCA
51 CCGGCGGGCG AGAGCGCGCG GCGGACCACA GAGCGGAGGT GAAGCCTTAT
101 GCTGAGACAG TTTTATCTAG TTCATGAACC CA~ATTATAT ACAAGCTGAA
151 TGTIACAGAA GTGCTGAAAG ACTGCTCTGT CATGAGCACG GACAGCAACT
201 CATTGGCACG TGAGTTTCTG ATTGATGTCA ACCAGCTTTG CAATGCAGTG
3 02 51 GTCCAGAGGG CAGAAGCCAG GGAAGAAGAA GAAGAGGAGA CACACATGGC
301 A~CTCTTGGA CAGTACCTTG TCCATGGACG AGGATTTCTG TTACTTACCA
351 AACTA~ATTC TATCATTGAT CAGGCCCTGA CATGCAGAGA AGAACTCCTG
401 ACTCTTCTTC TGTCGCTCCT TCCCTTGGTG TGGAAGATAC CTGTCCAGGA
4 51 ACAGCAGGCA ACAGATTTTA ACCTGCCACT GTCATCTGAT ATAATCCTGA
355 01 CCAAAGA~AA GAACTCAAGT TTGCA~AAAT CAACTCAGGG A~AATTATAT
551 TTAGAAGGAA GTGCTCCATC TGGTCAGGTT TCTGCAi~AAG TA~ACCTTTT
601 TCGA~AAATC AGGCGACAGC GTA~AAGTAC CCATCGTTAT TCTGTAAGAG
651 ATGCAAGAAA GACACAGCTC TCCACCTCTG ACTCCGAAGG CA~CTCAGAT
701 GA~AAGAGTA CGGTTGTGAG TA~ACACAGG AGGCTCCACG CGCTGCCACG
40751 GTTCCTGACG CAGTCTCCTA AGGAAGGCCA CCTCGTAGCC A~ACCTGACC
801 CCTCTGCCAC CA~AGAACAG GTCCTTTCTG ACACCATGTC TGTGGA~AAC
851 TCCAGAGAAG TCATTCTGAG ACAGGATTCA AATGGTGACA TATTAAGTGA
901 GCCAGCTGCT TTGTCTATTC TCAGTAACAT GAATAATTCT CCTTTTGACT
951 TATGTCATGT TTTGTTATCT CTATTGGA~A AAGTTTGTAA GTTTGACATT
451001 GCTTTGAATC ATAATTCTTC CCTAGCACTC AGTGTAGTAC CCACACTGAC
1051 TGAGTTCCTA GCAGGCTTTG GGGACTGCTG TAACCAGAGT GACACTTTGG
1101 AGGGACAACT GGTTTCTGCA GGTTGGACAG AAGAGCCGGT AGCTTTGGTT
1151 CAACGGATGC TCTTTCGAAC CGTGCTGCAC CTTATGTCAG TAGACGTTAG
1201 CACTGCAGAG GCAATGCCAG A~AGTCTTAG GAAAAATTTG ACTGAATTGC
501251 TTAGGGCAGC TTTA ~ ATT AGAGCTTGCT TGGAP~AAGCA GCCTGAGCCT
1301 TTCTCCCCGA GACA~AAGAA A~CACTACAG GAGGTCCAGG AGGGCTTTGT
1351 ATTTTCCAAG TATCGTCACC GAGCCCTTCT ACTACCTGAG CTTCTGGAAG
CA 02244744 1998-07-29
WO 97/28262 PCTrUS97/01748
1401 GAGTTCTACA GCTCCTCATC TCTTGTCTTC AGAGTGCAGC TTCA~ATCCC
- 1451 TTTTACTTCA GTCAAGCCAT GGATTTAGTT CAAGAATTTA TCCAGCACCA
1501 AGGATTTAAT CTCTTTGGAA CAGCAGTTCT TCAGATGG~A TGGCTGCTTA
1551 CAAGGGACGG TGTTCCTTCA GAAGCTGCAG AACATTTGAA AGCTCTGATA
1601 AACAGTGTAA TA~AAATAAT GAGTACTGTG A~AAAGGTGA AATCAGAGCA
1651 ACTTCATCAT TCCATGTGCA CAAGGA~AAG ACACCGGCGT TGTGAGTATT
1701 CCCACTTCAT GCAGCACCAC CGCGATCTTT CAGGGCTCCT GGTTTCAGCT
1751 TTTA~AAATC AGCTTTCTAA AAGCCCCTTT GAAGAGACCG CAGAGGGAGA
1801 TGTGCAGTAT CCAGAGCGCT GCTGCTGCAT CGCCGTGTGC GCTCACCAGT
1851 GCTTGCGCTT GCTGCAGCAG GTTTCCCTGA GCACCACGTG TGTCCAGATC
l901 CTATCAGGTG TACACAGTGT TGGAATCTGT TGTTGTATGG ATCCTAAGTC
1951 TGTGATCGCC CCTTTACTGC ATGCTTTTAA GTTGCCAGCA CTGAAAGCTT
2001 TCCAGCAGCA TATACTGAAT GTCCTGAGCA AACTTCTTGT GGATCAGTTA
2051 GGAGGAGCAG AGCTATCACC GAGAATTAAA A~AGCAGCTT GCAACATCTG
2101 TACTGTGGAC TCTGACCAAC TGGCTAAGTT AGGAGAGACA CTGCAAGGCA
2151 CCTTGTGTGG TGCTGGTCCT ACCTCCGGCT TGCCCAGTCC TTCCTACCGA
2201 TTTCAGGGGA TCCTGCCCAG CAGCGGCTCT GAAGACTTGC TGTGGAAGTG
2251 GGATGCATTA GAGGCTTATC AGAGCTTTGT CTTTCAAGAA GACAGATTAC
2301 ATAACATTCA GATTGCA~AT CACATTTGTA ATTTACTCCA GA~AGGCAAT
2351 GTAGTTGTTC AGTGGA~ATT GTATAATTAT ATCTTTAATC CTGTGCTCCA
2401 AAGAGGAGTT GAATTAGTAC ATCATTGTCA ACAGCTAAGC ATTCCTTCAG
2451 CTCAGACTCA CATGTGTAGC CAACTGAAAC AGTATTTGCC TCAGGAAGTG
2501 CTTCAGATTT ATTTAAAAAC TCTACCTGTC CTACTTAAAT CCAGGGTAAT
2551 AAGAGATTTG TTTTTAAGTT GTAATGGAGT A~ACCACATA ATTGAACTAA
2601 ATTACTTAGA TGGGATTCGA AGTCATTCCC TGAAAGCATT TGA~ACTCTG
2651 ATTGTCAGCC TAGGGGAACA ACAGAAAGAT GCTGCAGTTC TAGACGTCGA
2701 TGGGTTAGAC ATCCAACAGG AGTTGCCGTC CTTAAGTGTG GGTCCTTCTC
2751 TTCATAAGCA GCAAGCTTCT TCAGATTCTC CTTGCAGTCT CAGGAAGTTT
2801 TATGCCAGCC TCAGAGAGCC TGATCCA~AA A~ACGA~AGA CCATTCACCA
2851 GGATGTTCAC ATA~ACACCA TAAACCTCTT CCTCTGTGTG GCTTTTCTAT
2901 GTGTCAGTAA AGAAGCAGAC TCTGATAGGG AGTCTGCCAA TGAGTCAGAA
2951 GATACTTCTG GCTATGACAG CCCTCCCAGT GAGCCATTAA GTCACATGCT
3001 ACCATGTCTG TCTCTTGAGG ACGTTGTCTT ACCTTCCCCT GAATGTTTGC
3051 ACCATGCAGC AGACATTTGG TCCATGTGTC GTTGGATCTA CATGTTGAAC
3101 TCAGTCTTCC AGAAACAATT TCACAGGCTT GGTGGTTTCC AAGTGTGCCA
3151 TGAATTAATA TTTATGATAA TCCAGAAACT ATTCAGAAGT CATACAGAGG
3201 ATCAAGGAAG AAGGCAGGGA GAAATGAGTA GAAATGA~AA CCAAGAGCTA
3251 ATCAGGATAT CTTACCCCGA GCTGACACTG AAGGGAGATG TATCATCTGC
3301 AACAGCACCA GACCTGGGAT TTCTGAGA~A GAGTGCTGAC AGCGTGCGTG
3351 GATTCCAGTC ACAGCCTGTG CTTCCCACAA GTGCAGAGCA GATTGTGGCT
3401 ACTGAATCTG TTCCTGGGGA ACGA~AGGCA TTTATGAGTC AACAAAGTGA
3451 GACTTCTCTC CAGAGCATAC GACTTTTGGA GTCTCTCCTG GACATTTGTC
3501 TTCATAGTGC CAGAGCCTGT CAACAGAAGA TGGAATTGGA GCTACCGTCT
3551 CAGGGCTTGT CTGTGGA~AA TATATTGTGT GAACTGAGGG AACACCTTTC
3601 CCAGTCAAAG GTGGCAGAAA CAGAATTAGC AAAGCCTTTA TTTGATGCCC
3651 TGCTTCGAGT AGCCCTGGGG AATCATTCAG CAGATTTGGG CCCTGGTGAT
3701 GCTGTGACTG AGAAGAGTCA TCCCTCTGAG GAAGAGCTGT TGTCCCAGCC
3751 CGGAGATTTT TCAGAAGAAG CTGAGGATTC TCAGTGTTGT AGTTTGA~AC
3801 TTCTGGGTGA GGAAGAAGGC TATGAAGCGG ATAGTGA~AG CAATCCTGAG
3851 GATGTTGACA CCCAAGACGA TGGAGTAGAA TTAAATCCTG AAGCAGAAGG
3901 TTTCAGTGGA TCGATTGTTT CA~ACAACTT ACTTGA~AAC CTCACTCACG
3951 GGGA~ATAAT ATACCCTGAG ATTTGCATGC TGGGATTA~A TTTGCTTTCT
4001 GCTAGCA~AG CTA~ACTTGA TGTGCTTGCT CATGTGTTTG AGAGCTTTCT
4051 GA~AATTGTC AGGCAGAAGG A~AAGA~CAT TTCTCTCCTC ATACAACAGG
4101 GAACTGTGAA AATCCTTCTA GGCGGGTTCT TGAATATTTT AACACAAACT
CA 02244744 1998-07-29
W O 97/28262 PCTrUS97/01748
Ivl
4151 AACTCTGATT TCCAAGCATG CCAGAGAGTA CTGGTGGATC TCTTGGTATC
4201 TTTGATGAGC TCAAGAA~GT GTTCAGAAGA CTTAACTCTT CTTTGGAGAA
4251 TATTTCTGGA GAAATCTCCT TGTACAGA~A TTCTTCTCCT TGGTATTCAC
4301 A~AATTGTTG A~AGTGATTT TACTATGAGC CCTTCACAGT GTCTGACCTT
4351 TCCTTTCCTG CATACCCCGA GTTTAAGCAA TGGTGTCTTA TCACAGA~AC
4401 CTCCTGGGAT TCTTAACAGT A~AGCCTTAG GCTTATTGAG AAGAGCACGG
4451 ATTTCCCGAG GCAAG~AAGA GGCTGATAGA GAGAGTTTTC CCTATAGGCT
4501 GCTTTCCTCT TGGCATATAG CCCCAATCCA CCTGCCGTTG CTGGGACAGA
4551 ACTGCTGGCC ACACCTGTCA GAAGGATTTA GTGTTTCTCT TGTGGGTTTA
4601 ATGTGGAATA CATCCAATGA ATCCGAGAGT GCTGCAGA~A GGGGA~AAAG
4651 AGTA~AGA~A AGA~ACA~AC CATCAGTTCT GGAAGACAGC AGTTTTGAAG
4701 GAGCAGGTAT GATGGCAGGG TCTGATCTAT ATACTAAGAT TCTTCA~ATA
4751 GCTGCTTGCC TGAGTTTTAA GCATATCTGG CAGTATTTtA ATGTATTCTT
4801 TA~ATGTTAT TCACCTTA~A GATCCTACTT CACTACTGAA TTACCA~AGC
4851 CTGAGTTTTC A~ACAGCCTT GA~ATCTTCA TTGTCTCTAA ACTTTAGATA
4901 GGGAAGTGGG GATGCTCTGT TTCTGCAACA GCTGTTGAAG TTAGCAGTCC
4951 CATGACTGTG TTAGTGTGGC TTCTGATACT AGATAGTTAT AAATA~AACC
5001 CTATGGCCAT TTTTATTTTA AGTTCTCCTT CTGTGTCTTA CACCAATGGC
5051 CCCTTCTAGT TACTGTCCCT GATCATTTAT ATGTAACAGT CCA~AGTTAG
5101 AACAGAGTTC ATCTGTAACT GAAGAACTGC TGTTAGGATG TACTGAAATT
5151 GAATTTTGTT TTTGTTCTCT TCTTTTTTAA GCAATCAACA GTTTCTTAAG
5201 TCATATAGCA GCTAGAGGAA GTAGTCTTAA A~ACTGGCTG TGTATTTTTT
5251 TAACCTGTTA A~AATGGTGG CTAATATTTT TATACCCTAA TAATTGATAA
5301 TGTTCCTCTT TTTTA~AAGT CTGAGCTTTT GGACATGCAC TGTTTATGTT
5351 AGTACATCTT AGCTTAGTTT AACATA~AGT CACATCATAG TAACA~ATAG
5401 CTTATCACAC ATATTCCACC TGCCATTGCT GTCACAGATA ATGGGAATAT
5451 AGAGGCAACT CAAGATTTAA GTAGTAAGGT GCCATTGGGA GGGGTAAGCA
5501 GCTAGCTCAC AGCCATA~AC ACTTCTCTCA GCGGAGACAA ACTGTGATTC
5551 AGGGTTTGGC ATCACTTAGC ATGGTTATTT CAAGGTTGTT CACTACCTTA
5601 AATAATGATC ATTTGAGCAG TGCAGCTTTT CT~AGAAGAG TATTAATAAT
5651 ATTATAGATC GTGCCTTTGT AACAATTTTT TTAGTGCAAG GCATCTGTTG
5701 ATGGCATGTG CTCCCTGGGC CATGGTCAGT TGTGTTAGAG TGACCCAATC
5751 CAACAA~AGC AGAACCTTGG TATGGAGTGT GGCTGACGAT GGTCCTTTAG
5801 CACCCTCAGG CCTTGTAGTT TA~AGCATTT AATAACTTTT AAAACACTGG
5851 AGTCTTTAGT GAGGACCTGC CCGGGCGGCC GCCACCGCGG TGG
5.6 EXA~LE 6 -- DEDUCED AMINO ACID SEQU~NCES OF MOUSE LYST1 PROTEINS
.6.1 PEPT~DE ~EC2UENCE OF LONG ISOFORM (S1~Q ID NO:4)
1 MSTDSNSLAR EFLIDVNQLC NAW QRAEAR ~.F.F.F.~.F.THMA TLGQYLVHGR
51 GFLLLTKLNS IIDQALTCRE ELLTLLLSLL PLVWKIPVQE QQATDFNLPL
101 SSDIILTKEK NSSLQKSTQG KLYLEGSAPS GQVSAKVNLF RKIRRQRKST
151 HRYSVRDARK TQLSTSDSEG NSDEKSTW S KHRRLHALPR FLTQSPKEGH
201 LVAKPDPSAT KEQVLSDTMS VENSREVILR QDSNGDILSE PA~LSILSMM
~ 251 NNSPFDLCHV LLSLLEKVCK FDIALNHNSS LALS W PTLT EFLAGFGDCC
301 NQSDTLEGQL VSAGWTEEPV ALVQRMLFRT VLHLMSVDVS TAEAMPESLR
351 KNLTELLR~A LKIRACLEKQ PEPFSPRQKK TLQEVQEGFV FSKYRHRALL
401 LPELLEGVLQ LLISCLQSAA SNPFYFSQAM DLVQEFIQHQ GFNLFGTAVL
451 QMEWLLTRDG VPSEA~EHLK ALINSVIKIM STVKKVKSEQ LHHSMCTRKR
501 HRRCEYSHFM QHHRDLSGLL VSAFKNQLSK SPFEETAEGD VQYPERCCCI
551 AVCAHQCLRL LQQVSLSTTC VQILSGVHSV GICCCMDPKS VIAPLLHAFK
CA 02244744 1998-07-29
WO 97/28262 PCTAUS97/01748
601 LPALKAFQQH ILNVLSKLLV DQLGGAELSP RIKKAACNIC TVDSDQLAKL
651 GETLQGTLCG AGPTSGLPSP SYRFQGILPS SGSEDLLWKW DALEAYQSFV
701 FQEDRLHNIQ IANHICNLLQ KGNVVVQWKL YNYIFNPVLQ RGVELVHHCQ
751 QLSIPSAQTH MCSQLKQYLP QEVLQIYLKT LPVLLKSRVI RDLFLSCNGV
5801 NHIIELNYLD GIRSHSLKAF ETLIVSLGEQ QKDAAVLDVD GLDIQQELPS
851 LSVGPSLHKQ QASSDSPCSL RKFYASLREP DPKKRKTIHQ DVHINTINLF
901 LCVAFLCVSK EADSDRESAN ESEDTSGYDS PPSEPLSHML PCLSLEDW L
951 PSPECLHHAA DIWSMCRWIY MLNSVFQKQF HRLGGFQVCH ELIFMIIQKL
1001 FRSHTEDQGR RQGEMSRNEN QELIRISYPE LTLKGDVSSA TAPDLGFLRK
101051 SADSVRGFQS QPVLPTSAEQ IVATESVPGE RKAFMSQQSE TSLQSIRLLE
1101 SLLDICLHSA RACQQKMELE LPSQGLSVEN ILCELREHLS QSKVAETELA
1151 KPLFDALLRV ALGNHSADLG PGDAVTEKSH PS~F.~.T.T..~QP GDFSEEAEDS
1201 QCCSLKLLGE EEGYEADSES NPEDVDTQDD GVELNPEAEG FSGSIVSNNL
1251 LENLTHGEII YPEICMLGLN LLSASKAKLD VLAHVFESFL KIVRQKEKNI
15 ~1301 SLLIQQGTVK ILLGGFLNIL TQTNSDFQAC QRVLVDLLVS LMSSRTCSED
1351 LTLLWRIFLE KSPCTEILLL GIHKIVESDF TMSPSQCLTF PFLHTPSLSN
1401 GVLSQKPPGI LNSKALGLLR RARISRGKKE ADRESFPYRL LSSWHIAPIH
1451 LPLLGQNCWP HLSEGFSVSL VGLMWNTSNE SESAAERGKR VKKRNKPSVL
1501 EDSSFEGAEG DRPEVTESIN PGDRLIEDGC IHLISLGSKA LMIQVWADPH
201551 SGTFIFRVCM DSNDDTKAVS LAQVESQENI FFPSKWQHLV LTYIQHPQGK
1601 KNVHGEISIW VSGQRKTDVI LDFVLPRKTS LSSDSNKTFC MIGHCLTSQE
1651 ESLQLAGKWD LGNLLLFNGA KIGSQEAFFL YACGPNYTSI MPCKYGQPVI
1701 DYSKYINKDI LRCDEIRDLF MTKKEVDVGL LIESLSVVYT TCCPAQYTIY
1751 EPVIRLKGQV KTQPSQRPFS SKEAQSILLE PSQLKGLQPT ECKAIQGILH
251801 EIGGAGTFVF LFARVVELSS CEETQALALR VILSLIKYSQ QRTQELENCN
1851 GLSMIHQVLV KQKCIVGFHI LKTLLEGCCG EEVIHVSEHG EFKLDVESHA
1901 IIQDVKLLQE LLLDWKIWNK AEQGVWETLL AALEVLIRVE HHQQQFNIKQ
1951 LLNAHVVHHF LLTCQVLQEH REGQLTSMPR EVCRSFVKII AEVLGSPPDL
2001 ELLTVIFNFL LAVHPPTNTY VCHNPTNFYF SLHIDGKIFQ EKVQSLAYLR
302051 HSSSGGQAFP SPGFLVISPS AFTAAPPEGT SSSNIVPQRM AAQMVRSRSL
2101 PAFPTYLPLI RAQKLAASLG FSVDKLÇNIA DANPEKQNLL GRPYALKTSK
2151 EEAFISSCES AKTVCEMEAL LGAHASANGV SRGSPRFPRA RVDHKDVGTE
2201 PRSDDDSPGD ESYPRRPDNL KGLASFQRSQ STVASLGLAF PSQNGSAVAS
2251 RWPSLVDRNA DDWENFTFSP AYEASYNRAT STHSVIEDCL IPICCGLYEL
352301 LSGVLLVLPD AMLEDVMDRI IQADILLVLV NHPSPAIQQG VIKLLHAYIN
2351 RASKEQKDKF LKNRGFSLLA NQLYLHRGTQ ELLECFVEMF FGRPIGLDEE
2~01 FDLEEVKHME LFQKWSVIPV LGLIETSLYD NVLLHNALLL LLQVLNSCSK
2451 VADMLLDNGL LYVLCNTVAA LNGLEKNIPV NEYKLLACDI QQLFIAVTIH
2501 ACSSSGTQYF RVIEDLIVLL GYLHNSKNKR TQNMALALQL RVLQAALEFI
40~551 RSTANHDSES PVHSPSAHRH SVPPKRRSIA GSRKFPLAQT ESLLMKMRSV
2601 ASDELHSMMQ RRMSQEHPSQ ASEAELAQRL QRLTILAVNR IIYQELNSDI
2651 IDILRTPENT SQSKTSVSQT EISEEDMHHE QPSVYNPFQK EMLTYLLDGF
2701 KVCIGSSKTS VSKQQWTKIL GSCKETLRDQ LGRLLAHILS PTHTVQERKQ
2751 ILEIVHEPAH QDILRDCLSP SPQHGAKLVL YLSELIHNHQ DELSEEEMDT
452801 AELLMNALKL CGHKCIPPSA PSKPELIKII REEQKKYESE ESVSKGSWQK
2851 TVNNNQQSLF QRLDFKSKDI SKIAADITQA VSLSQGIERK KVIQHIRGMY
2901 KVDLSASRHW QECIQQLTHD RAVWYDPIYY PTSWQLDPTE GPNRERRRLQ
2951 RCYLTIPNKY LLRDRQKSEG VLRPPLSYLF EDKTHSSFSS TVKDK~ASES
3001 IRVNRRCISV APSRETAGEL LLGKCGMYFV EDNASDAVES SSLQGELEPA
503051 SFSWTYEEIK EVHRRWWQLR DNAVEIFLTN GRTLLLAFDN NKVRDDVYQS
3101 ILTNNLPNLL EYGNITALTN LWYSGQITNF EYLTHLNKHA GRSFNDLMQY
3151 PVFPFILSDY VSETLDLNDP SIYRNLSKPI AVQYKEKEDR YVDTYKYLEE
3201 EYRKGAREDD PMPPVQPYHY GSHYSNSGTV LHFLVRMPPF TKMFLAYQDQ
3251 SFDIPDRTFH STNTTWRLSS FESMTDVKEL IPEFFYLPEF LVNREGFDFG
553301 VRQNGERVNH VNLPPWARND PRLFILIHRQ ALESDHVSQN ICHWIDLVFG
CA 02244744 1998-07-29
WO 97/28262 PCTrUS97/01748
103
3351 YKQKGKASVQ AINVFHPATY FGMDVSAVED PVQRRALETM IKTYGQTPRQ
3401 LFHTAHASRP GAKLNIEGEL PA~VGLLVQF AFRETREPVK EVTHPSPLSW
- 3451 IKGLKWGEYV GSPSAPVPW CFSQPHGERF GSLQALPTRA ICGLSRNFCL
3501 LMTYNKEQGV RSMNNTNIQW SAILSWGYAD NILRLKSKQS EPPINFIQSS
3551 QQHQVTSCAW VPDSCQLFTG SKCGVITAYT NRLTSSTPSE IEMESQMHLY
3601 GHTEEITGLC VCKPYSVMIS VSRDGTCIVW DLNRLCYVQS LAGHKSPVTA
3651 VSASETSGDI ATVCDSAGGG SDLRLWTVNG DLVGHVHCRE IICSVAFSNQ
3701 PEGVSINVIA GGLENGIVRL WSTWDLKPVR EITFPKSNKP IISLTFSCDG
3751 HHLYTANSEG TVIAWCRKDQ QRVKLPMFYS FLSSYAAG
5.6.2 PEPTIDE SEQUENCE OF S~ORT ISOFORM (SEQ ID NO:6)
1 MSTDSNSLAR EFLIDVNQLC NA W QRAEAR ~.~F~EETHMA TLGQYLVHGR
51 GFLLLTKLNS IIDQALTCRE ELLTLLLSLL PLVWKIPVQE QQATDFNLPL
101 SSDIILTKEK NSSLQKSTQG KLYLEGSAPS GQVSAKVNLF RKIRRQRKST
151 HRYSVRDARK TQLSTSDSEG NSDEKST WS KHRRLHALPR FLTQSPKEGH
201 LVAKPDPSAT KEQVLSDTMS VENSREVILR QDSNGDILSE PAALSILSNM
251 NNSPFDLCHV LLSLLEKVCK FDIALNHNSS LALSWPTLT EFLAGFGDCC
301 NQSDTLEGQL VSAGWTEEPV ALVQRMLFRT VLHLMSVDVS TAEAMPESLR
351 KNLTELLR~A LKIRACLEKQ PEPFSPRQKK TLQEVQEGFV FSKYRHRALL
401 LPELLEGVLQ LLISCLQSAA SNPFYFSQAM DLVQEFIQHQ GFNLFGTAVL
451 QMEWLLTRDG VPSEAAEHLK ALINSVIKIM STVKKVKSEQ LHHSMCTRKR
501 HRRCEYSHFM QHHRDLSGLL VSAFKNQLSK SPFEETAEGD VQYPERCCCI
551 AVCAHQCLRL LQQVSLSTTC VQILSGVHSV GICCCMDPKS VIAPLLHAFK
601 LPALKAFQQH ILNVLSKLLV DQLGGAELSP RIKK~ACNIC TVDSDQLAKL
651 GETLQGTLCG AGPTSGLPSP SYRFQGILPS SGSEDLLWKW DALEAYQSFV
701 FQEDRLHNIQ IANHICNLLQ KGNV W QWKL YNYIFNPVLQ RGVELVHHCQ
751 QLSIPSAQTH MCSQLKQYLP QEVLQIYLKT LPVLLKSRVI RDLFLSCNGV
801 NHIIELNYLD GIRSHSLKAF ETLIVSLGEQ QKDA~VLDVD GLDIQQELPS
851 LSVGPSLHKQ QASSDSPCSL RKFYASLREP DPKKRKTIHQ DVHINTINLF
901 LCVAFLCVSK EADSDRESAN ESEDTSGYDS PPSEPLSHML PCLSLEDWL
951 PSPECLHHAA DIWSMCRWIY MLNSVFQKQF HRLGGFQVCH ELIFMIIQKL
1001 FRSHTEDQGR RQGEMSRNEN QELIRISYPE LTLKGDVSSA TAPDLGFLRK
1051 SADSVRGFQS QPVLPTSAEQ IVATESVPGE RKAFMSQQSE TSLQSIRLLE
1101 SLLDICLHSA RACQQKMELE LPSQGLSVEN ILCELREHLS QSKVAETELA
1151 KPLFDALLRV ALGNHSADLG PGDAVTEKSH PS~.F.F.T,T.SQP GDFSEEAEDS
1201 QCCSLKLLGE EEGYEADSES NPEDVDTQDD GVELNPEAEG FSGSIVSNNL
1251 LENLTHGEII YPEICMLGLN LLSASKAKLD VLAHVFESFL KIVRQKEKNI
1301 SLLIQQGTVK ILLGGFLNIL TQTNSDFQAC QRVLVDLLVS LMSSRTCSED
1351 LTLLWRIFLE KSPCTEILLL GIHKIVESDF TMSPSQCLTF PFLHTPSLSN
1401 GVLSQKPPGI LNSKALGLLR RARISRGKKE ADRESFPYRL LSSWHIAPIH
1451 LPLLGQNCWP HLSEGFSVSL VGLMWNTSNE SESAAERGKR VKKRNKPSVL
1501 EDSSFEGAGM MAGSDLYTKI LQIAACLSFK HIWQYFNVFF KCYSP
5.7 EXAMPLE7 -- ~NA SEQUENCESOFF~UMANLYST1 GENE
45 5.7.1 C:DNA SEQUENCEOFLONGISOFORM(SEQ ID NO:7)
1 CGCA~GGGCT TCTAAGAAGC CATCCCA~TG ACCTTTTGGC TTTGAGAAGA
51 GCAGTCCTCA TACCAGAGTG TTTGGGGTTT TGGCCTCTTT CAGTGTTTAT
CA 02244744 l998-07-29
WO 97/28262 PCT/US97/01748
101 TCATTCTTAC GTGGGA~AGT TGTATTCCGA GGTTTCTGTG GTGCATGAAG
151 CTTTTGCCTT CACCATCTGT TCCCGTGTCT TCCTCGGGTG ACATCAGAGT
- 201 ACAGCAGTAT TTTCCCTTGC CATCTAATGG GGTTTGGGCT GTTTGACTCA
251 ACCGTGTGTG TTCCTCAATG CCAGGGGAAT AATCCTACCC TAAGTCAGCT
301 GA~CAGAAGC CAGGATCTAA CTGCAAACAA GAGACCCAGC TTGCTTAACA
351 GCATGGAAGA GAACCAGTTT CCTTGCAGCT ACCTGGGAAG ACGGTTGCTA
401 ATTAGCCTGC AACA~AGAGT TCCTTGCTCA TCTA~AAGAG GCAATCACCG
451 TTCAGGTGAA GCTTTGTTCT AAGAATATTT GTTTCATCTA GTTTATGAGT
501 CCA~ATGATA TAGACTGTAA ATGTCACAGC AGTGGTGA~A GACTGCTCGG
551 TCATGAGCAC CGACAGTAAC TCACTGGCAC GTGAATTTCT GACCGATGTC
601 AACCGGCTTT GCAATGCAGT GGTCCAGAGG GTGGAGGCCA GGGAGGAAGA
651 AGAGGAGGAG ACGCACATGG CAACCCTTGG ACAGTACCTT GTCCATGGTC
701 GAGGATTTCT ATTACTTACC AAGCTA~ATT CTATAATTGA TCAGGCATTG
751 ACATGTAGAG AAGAACTCCT GACTCTTCTT CTGTCTCTCC TTCCACTGGT
801 ATGGAAGATA CCTGTCCAAG AAGAAAAGGC AACAGATTTT AACCTACCGC
851 TCTCAGCAGA TATAATCCTG ACCA~AGAAA AGAACTCAAG TTCACA~AGA
901 TCCACTCAGG A~AAATTACA TTTAGAAGGA AGTGCCCTGT CTAGTCAGGT
951 TTCTGCA~AA GTA~ATGTTT TTCGA~AAAG CAGACGACAG CGTA~AATTA
1001 CCCATCGCTA TTCTGTAAGA GATGCAAGAA AGACACAGCT CTCCACCTCA
1051 GATTCAGAAG CCAATTCAGA TGA~AAAGGC ATAGCAATGA ATAAGCATAG
1101 AAGGCCCCAT CTGCTGCATC ATTTTTTAAC ATCGTTTCCT A~ACAAGACC
1151 ACCCCAAAGC TA~ACTTGAC CGCTTAGCAA CCA~AGAACA GACTCCTCCA
1201 GATGCTATGG CTTTGGA~AA TTCCAGAGAG ATTATTCCAA GACAGGGGTC
1251 A~ACACTGAC ATTTTAAGTG AGCCAGCTGC CTTGTCTGTT ATCAGTAACA
1301 TGAACAATTC TCCATTTGAC TTATGTCATG ITTTGTTATC TTTATTAGAA
1351 A~AGTTTGTA AGTTTGACGT TACCTTGAAT CATAATTCTC CTTTAGCAGC
- 1401 CAGTGTAGTG CCCACACTAA CTGA~TTCCT A5CAGGCTTT GGGGACTGCT
1451 GCAGTCTGAG CGACAACTTG GAGAGTCGAG TAGTTTCTGC AGGTTGGACC
1501 GAAGAACCGG TGGCTTTGAT TCA;~AGGATG CTCTTTCGAA CAGTGTTGCA
1551 TCTTCTGTCA GTAGATGTTA GTACTGCAGA GATGATGCCA GA~AATCTTA
1601 GGA~AAATTT AACTGAATTG CTTAGAGCAG CTTTA~AAAT TAGAATATGC
16 51 CTAGAPAAGC AGCCTGACCC TTTTGCACCA AGACA~ GA A~ACACTGCA
1701 GGAGGTTCAG GAAGATTTTG TGTTTTCA~A GTATCGTCAT AGAGCCCTTC
1751 TTTTACCTGA GCTTTTGGAA GGAGTTCTTC AGATTCTGAT CTGTTGTCTT
1801 CAGAGTGCAG CTTCAAATCC CTTCTACTTC AGTCAAGCCA TGGATTTGGT
18 51 TCAAGAATTC ATTCAGCATC ATGGATTTAA TTTATTTGAA ACAGCAGTTC
1901 TTCA~ATGGA ATGGCTGGTT TTAAGAGATG GAGTTCCTCC CGAGGCCTCA
1951 GAGCATTTGA AAGCCCTAAT A~ATAGTGTG ATGA~AATAA TGAGCACTGT
2001 C~AAA~GTG A~ATCAGAGC AACTTCATCA TTCGATGTGT ACAAGAAAAA
2051 GGCACAGACG ATGTGAATAT TCTCATTTTA TGCATCATCA CCGAGATCTC
2101 TCAGGTCTTC TGGTTTCGGC TTTTA~AAAC CAGGTTTCCA A~AACCCATT
2151 TGAAGAGACT GCAGATGGAG ATGTTTATTA TCCTGAGCGG TGCTGTTGCA
2201 TTGCAGTGTG TGCCCATCAG TGCTTGCGCT TACTGCAGCA GGCTTCCTTG
2251 AGCAGCACTT GTGTCCAGAT CCTATCGGGT GTTCATAACA TTGGAATATG
~S 2301 CTGTTGTATG GATCCCA~AT CTGTAATCAT TCCTTTGCTC CATGCTTTTA
2351 AATTGCCAGC ACTGAAAAAT TTTCAGCAGC ATATATTGAA TATCCTTAAC
2401 A~ACTTATTT TGGATCAGTT AGGAGGAGCA GAGATATCAC CAAAAATTAA
2451 A~AAGCAGCT TGTAATATTT GTACTGTTGA CTCTGACCAA CTAGCCCAAT
2501 TAGAAGAGAC ACTGCAGGGA AACTTATGTG ATGCTGAACT CTCCTCAAGT
2551 TTATCCAGTC CTTCTTACAG ATTTCAAGGG ATCCTGCCCA GCAGTGGATC
2601 TGAAGATTTG TTGTGGA~AT GGGATGCTTT A~AGGCTTAT CAGAACTTTG
2651 TTTTTGGAGA AGACAGATTA CATAGTATAC AGATTGCAAA TCACATTTGC
2701 AATTTAATCC AGAAAGGCAA TATAGTTGTT CAGTGGAAAT TATATAATTA
2751 CATATTTAAT CCTGTGCTCC A~AGAGGAGT TGAATTAGCA CATCATTGTC
5 5 2 8 01 AACACCTAAG CGTTACTTCA GCTCA~AGTC ATGTATGTAG CCATCATAAC
CA 02244744 1998-07-29
W O 97/28262 PCTrUS97/01748
i ~C~
2851 CAGTGCTTGC CTCAGGACGT G TTCAGATT TATGTA~AAA CTCTGCCTAT
2901 CCTGCTTA~A TCCAGGGTAA TAAGAGATTT GTTTTTGAGT TGTAATGGAG
- 2951 TAAGTCA~AT AATCGAATTA AATTGCTTAA ATGGTATTCG AAGTCATTCT
3001 CTA~AAGCAT TTGA~ACTCT GATAATCAGC CTAGGGGAGC AACAGA~AGA
3051 TGCCTCAGTT CCAGATATTG ATGGGATAGA CATTGAACAG AAGGAGTTGT
3101 CCTCTGTACA TGTGGGTACT TCTTTTCATC ATCAGCAAGC TTATTCAGAT
3151 TCTCCTCAGA GTCTCAGCAA ATTTTATGCT GGCCTCA~AG AAGCTTATCC
- 3201 A~AGAGACGG AAGACTGTTA ACCAAGATGT TCATATCAAC ACAATA~ACC
3251 TATTCCTCTG TGTGGCTTTT TTATGCGTAA GTA~AGA~GC AGAGTCTGAC
3301 AGGGAGTCGG CCAATGACTC AGAAGATACT TCTGGCTATG ACAGCACAGC
3351 CAGCGAGCCT TTAAGTCATA TGCTGCCATG TATATCTCTC GAGAGCCTTG
3401 TCTTGCCTTC TCCTGAACAT ATGCACCAAG CAGCAGACAT TTGGTCTATG
3451 TGTCGTTGGA TCTACATGTT GAGTTCAGTG TTCCAGA~AC AGTTTTATAG
3501 GCTTGGTGGT TTCCGAGTAT GCCATAAGTT AATATTTATG ATAATACAGA
3551 AACTGTTCAG AAGTCACA~A GAGGAGCAAG GA~A~GGA GGGAGATACA
3601 AGTGTAAATG A~AACCAGGA TTTAAACAGA ATTTCTCAAC CTA~GAGAAC
3651 TATGAAGGAA GATTTATTAT CTTTGGCTAT AA~AAGTGAC CCCATACCAT
3701 CAGAACTAGG TAGTCTAAAA AAGAGTGCTG ACAGTTTAGG TA~ATTAGAG
3751 TTACAGCATA TTTCTTCCAT A~ATGTGGAA GAAGTTTCAG CTACTGAAGC
3801 CGCTCCCGAG GAAGCAAAGC TATTTACAAG TCAAGA~AGT GAGACCTCAC
3851 TTCAAAGTAT ACGACTTTTG GAAGCCCTTC TGGCCATTTG TCTTCATGGT
3901 GCCAGAACTA GTCAACAGAA GATGGAATTG GAGTTACCTA ATCAGAACTT
3951 GTCTGTGGAA AGTATATTAT TTGA~ATGAG GGACCATCTT TCCCAGTCAA
4001 AGGTGATTGA AACACAACTA GCA~AGCCTT TATTTGATGC CCTGCTTCGA
4051 GTTGCCCTCG GGAATTATTC AGCAGATTTT GAACATAATG ATGCTATGAC
4101 TGAGAAGAGT CATCAATCTG CAGAAGAATT GTCATCCCAG CCTGGTGATT
4151 TTTCAGAAGA AGCTGAGGAT TCTCAGTGTT GTAGTTTTAA ACTTTTAGTT
4201 GAAGAAGAAG GTTACGAAGC AGATAGTGAA AGCAATCCTG AAGATGGCGA
4251 AACCCAGGAT GATGGGGTAG ACTTA~AGTC TGA~ACAGAA GGTTTCAGTG
4301 CATCAAGCAG TCCAAATGAC TTACTCGAAA ACCTCACTCA AGGGGA~ATA
4351 ATTTATCCTG AGATTTGTAT GCTGGAATTA AATTTGCTTT CTGCTAGTAA
4401 AGCCAAACTT GATGTGCTTG CCCATGTATT TGAGAGTTTT TTGA~AATTA
4451 TTAGGCAGAA AGA~AAGAAT GTTTTTCTGC TCATGCAACA GGGAACTGTG
4501 AAAAATCTTT TAGGAGGGTT CTTGAGTATT TTAACACAGG ATGATTCTGA
4551 TTTTCAAGCA TGCCAGAGAG TATTGGTGGA TCTTTTGGTA TCTTTGATGA
4601 GTTCAAGAAC ATGTTCAGAA GAGCTAACCC TTCTTTTGAG AATATTTCTG
4651 GAGA~ATCTC CTTGTACAAA AATTCTTCTT CTGGGTATTC TGA~AATTAT
4701 TGAAAGTGAT ACTACTATGA GCCCTTCACA GTATCTAACC TTCCCTTTAC
4751 TGCACGCTCC A~ATTTAAGC AACGGTGTTT CATCACA~AA GTATCCTGGG
4801 ATTTTA~ACA GTAAGGCCAT GGGTTTATTG AGAAGAGCAC GAGTTTCACG
4851 GAGCAAGA~A GAGGCTGATA GAGAGAGTTT TCCCCATCGG CTGCTTTCAT
4901 CTTGGCACAT AGCCCCAGTC CACCTGCCGT TGCTGGGGCA A~ACTGCTGG
4951 CCACACCTAT CAGAAGGTTT CAGTGTTTCC.CTGTGGTTTA ATGTGGAGTG
5001 TATCCATGAA GCTGAGAGTA CTACAGA~AA AGGA~AGAAG ATA~AGA~AA
5051 GAAACAAATC ATTAATTTTA CCAGATAGCA GTTTTGATGG TACAGAGAGC
5101 GACAGACCAG AAGGTGCAGA GTACATA~AT CCTGGTGAAA GACTCATAGA
5151 AGAAGGATGT ATTCATATAA TTTCACTGGG ATCCAAAGCG TTGATGATCC
5201 AAGTGTGGGC TGATCCCCAC AATGCCACTC TTATCTTTCG TGTGTGCATG
5251 GATTCA~ATG ATGACATGAA AGCTGTTTTA CTAGCACAGG TTGAATCACA
5301 GGAGAATATT TTCCTCCCAA GCAAATGGCA ACATTTAGTA CTCACCTACT
5351 TACAGCAGCC CCAAGGGA~A AGGAGGATTC ATGGGA~AAT CTCCATATGG
5401 GTCTCTGGAC AGAGGAAGCC TGATGTTACT TTGGATTTTA TGCTTCCAAG
5451 A~AAACAAGT TTGTCATCTG ATAGCAATAA AACATTTTGC ATGATTGGCC
5501 ATTGTTTATC ATCCCAAGAA GAGTTTTTGC AGTTGGCTGG A~AATGGGAC
5551 CTGGGA~ATT TGCTTCTCTT CAACGGAGCT AAGGTTGGTT CACAAGAGGC
CA 02244744 1998-07-29
W O 97/28262 PCTrUS97/01748
5601 CTTTTATCTG TATGCTTGTG GACCCAACCA TACATCTGTA ATGCCATGTA
5651 AGTATGGCAA GCCAGTCAAT GACTACTCCA AATATATTAA TA~AGA~ATT
- 5701 TTGCGATGTG AACA~ATCAG AGAATTTTTT ATGACCAAGA AAGATGTGGA
5751 TATTGGTCTC TTAATTGGAG TCTTTCAGTT GTTTATACAA CTTACTGTCC
5801 TGCTCCAGTA TACCATCTAT GAACCAGTGA TTAGACTTA~ AGGTCA~ATG
5851 A~AACCCAAC TCTCTCA~AG ACCCTTCAGC TCA~AAGAAG TTCAGAGCAT
5901 CTTATTAGAA CCTCATCATC TAAAGAATCT CCAACCTACT GAATATA~AA
5951 CTATTCAAGG CATTCTGCAC GA~ATTGGTG G~ACTGGCAT ATTTGTTTTT
6001 CTCTTTGCCA GGGTTGTTGA ACTCAGTAGC TGTGAAGA~A CTCAAGCATT
6051 AGCACTGCGA GTTATACTCT CATTAATTAA ATACAACCAA CA~AGAGTAC
6101 ATGAATTAGA A~ATTGTAAT GGACTTTCTA TGATTCATCA GGTGTTGATC
6151 A~ACA~AAAT GCATTGTTGG GTTTTACATT TTGAAGACCC TTCTTGAAGG
6201 ATGCTGTGGT GAAGATATTA TTTATATGAA TGAGAATGGA GAGTTTAAGT
6251 TGGATGTAGA CTCTAATGCT ATAATCCAAG ATGTTAAGCT GTTAGAGGAA
6301 CTATTGCTTG ACTGGAAGAT ATGGAGTAAA GCAGAGCAAG GTGTTTGGGA
6351 AACTTTGCTA GCAGCTCTAG AAGTCCTCAT CAGAGCAGAT CACCACCAGC
6401 AGATGTTTAA TATTAAGCAG TTATTGAAAG CTCAAGTGGT TCATCACTTT
6451 CTACTGACTT GTCAGGTTTT GCAGGAATAC A~AGAGGGGC AACTCACACC
6501 CATGCCCCGA GAGATGGCAA GATCTTTCAG GAGA~AGTGC GGTCAATCAT
6551 GTACCTGAGG CATTCCAGCA GTGGAGGAAG GTCCCTTATG AGCCCTGGAT
6601 TTATGGTAAT AAGCCCATCT GGTTTTACTG CTTCACCATA TGAAGGAGAG
6651 AATTCCTCTA ATATTATTCC ACAACAGATG GCCGCCCATA TGCTGCGTTC
6701 TAGAAGCCTA CCAGCATTCC CTACTTCTTC ACTACTAACG CAATCACAAA
6751 AACTGACTGG AAGTTTGGGT TGTAGTATCG ACAGGTTACA A~ATATTGCA
6801 GATACTTATG TTGCCACCCA ATCAAAGA~A CA~AATTCTT TGGGGAGTTC
6851 CGACACACTG A~AAAAGGCA A~GAGGACGC ATTCATCAGT AGCTGTGAGT
6901 CTGCA~AAAC TGTTTGTGAA ATGGAAGCTG TCCTCTCAGC CCAGGTCTCT
6951 GTCAGTGATG TCCCAAAGGG AGTGCTGGGA TTTCCAGTGG TCAAAGCAGA
7001 TCATA~ACAG TTGGGAGCAG AACCCAGGTC AGAAGATGAC AGTCCTGGGG
7051 ATGAGTCCTG CCCANCGCCG AGCCCTATGC A
5.7.2 CDNA SEQUENCEOFSHORT ~OFORM(SEQID NO:9)
1 CGCAAGGGCT TCTAAGAAGC CATCCCAATG ACCTTTTGGC TTTGAGAAGA
51 GCAGTCCTCA TACCAGAGTG TTTGGGGTTT TGGCCTCTTT CAGTGTTTAT
101 TCATTCTTAC GTGGGA~AGT TGTATTCCGA GGTTTCTGTG GTGCATGAAG
151 CTTTTGCCTT CACCATCTGT TCCCGTGTCT TCCTCGGGTG ACATCAGAGT
201 ACAGCAGTAT TTTCCCTTGC CATCTAATGG GGTTTGGGCT GTTTGACTCA
251 ACCGTGTGTG TTCCTCAATG CCAGGGGAAT AATCCTACCC TAAGTCAGCT
301 GAACAGAAGC CAGGATCTAA CTGCA~ACAA GAGACCCAGC TTGCTTAACA
351 GCATGGAAGA GAACCAGTTT CCTTGCAGCT ACCTGGGA~G ACGGTTGCTA
401 ATTAGCCTGC AACA~AGAGT TCCT TGCTCA TC TA~AAGAG GCAATCACCG
451 TTCAGGTGAA GCTTTGTTCT AAGAATATTT GTTTCATCTA GTTTATGAGT
501 CCA~ATGATA TAGACTGTAA ATGTCACAGC AGTGGTGA~A GACTGCTCGG
551 TCATGAGCAC CGACAGTAAC TCACTGGCAC GTGAATTTCT GACCGATGTC
601 AACCGGCTTT GCAATGCAGT GGTCCAGAGG GTGGAGGCCA GGGAGGAAGA
651 AGAGGAGGAG ACGCACATGG CAACCCTTGG ACAGTACCTT GTCCATGGTC
701 GAGGATTTCT ATTACTTACC AAGCTA~ATT CTATAATTGA TCAGGCATTG
751 ACATGTAGAG AAGA~CTCCT GACTCTTCTT CTGTCTCTCC TTCCACTGGT
801 ATGGAAGATA CCTGTCCA~G AAGA~AAGGC AACAGATTTT AACCTACCGC
851 TCTCAGCAGA TATAATCCTG ACCAAAGAAA AGAACTCA~G TTCACAAAGA
901 TCCACTCAGG A~AAATTACA TTTAGAAGGA AGTGCCCTGT CTAGTCAGGT
951 TTCTGCA~AA GTA~ATGTTT TTCGA~AAAG CAGACGACAG CGTAAAATTA
CA 02244744 1998-07-29
WO 97/28262 PCT~US97/01748
1.001 CCCATCGCTA TTCTGTAAGA GATGCAAGAA AGACACAGCT CTCCACCTCA
1051 GATTCAGA~G CCAATTCAGA TGA~AAAGGC ATAGCAATGA ATAAGCATAG
4 - 1101 AAGGCCCCAT CTGCTGCATC ATTTTTTAAC ATCGTTTCCT A~ACAAGACC
1151 ACCCCA~AGC TAAACTTGAC CGCTTAGCAA CCA~AGAACA GACTCCTCCA
1201 GATGCTATGG CTTTGGA~AA TTCCAGAGAG ATTATTCCAA GACAGGGGTC
1251 AAACACTGAC ATTTTAAGTG AGCCAGCTGC CTTGTCTGTT ATCAGTAACA
' 1301 TGAACAATTC TCCATTTGAC TTATGTCATG TTTTGTTATC TTTATTAGAA
- 1351 AAAGTTTGTA AGTTTGACGT TACCTTGAAT CATAATTCTC CTTTAGCAGC
1401 CAGTGTAGTG CCCACACTAA CTGAATTCCT AGCAGGCTTT GGGGACTGCT
1451 GCAGTCTGAG CGACAACTTG GAGAGTCGAG TAGTTTCTGC AGGTTGGACC
1501 GAAGAACCGG TGGCTTTGAT TCAAAGGATG CTCTTTCGAA CAGTGTTGCA
1551 TCTTCTGTCA GTAGATGTTA GTACTGCAGA GATGATGCCA GA~AATCTTA
1601 GGAAAAATTT AACTGAATTG CTTAGAGCAG CTTTA~AAAT TAGAATATGC
1651 CTAGA~AAGC AGCCTGACCC TTTTGCACCA AGACA~AAGA A~ACACTGCA
1701 GGAGGTTCAG GAAGATTTTG TGTTTTCA~A GTATCGTCAT AGAGCCCTTC
1751 TTTTACCTGA GCTTTTGGAA GGAGTTCTTC AGATTCTGAT CTGTTGTCTT
1801 CAGAGTGCAG CTTCAAATCC CTTCTACTTC AGTCAAGCCA TGGATTTGGT
1851 TCAAGAATTC ATTCAGCATC ATGGATTTAA TTTATTTGAA ACAGCAGTTC
1901 TTCAAATGGA ATGGCTGGTT TTAAGAGATG GAGTTCCTCC CGAGGCCTCA
1951 GAGCATTTGA AAGCCCTAAT A~ATAGTGTG ATGA~AATAA TGAGCACTGT
2001 CLA~ALAGTG A~ATCAGAGC AACTTCATCA TTCGATGTGT ACAAGA~AAA
2051 GGCACAGACG ATGTGAATAT TCTCATTTTA TGCATCATCA CCGAGATCTC
2101 TCAGGTCTTC TGGTTTCGGC TTTTA~A~AC CAGGTTTCCA A~AACCCATT
2151 TGAAGAGACT GCAGATGGAG ATGTTTATTA TCCTGAGCGG TGCTGTTGCA
2201 TTGCAGTGTG TGCCCATCAG TGCTTGCGCT TACTGCAGCA GGCTTCCTTG
2251 AGCAGCACTT GTGTCCAGAT CCTATCGGGT GTTCATAACA TTGGAATATG
2301 CTGTTGTATG GATCCCAAAT CTGTAATCAT TCCTTTGCTC CATGCTTTTA
2351 AATTGCCAGC ACTGA~AAAT TTTCAGCAGC ATATATTGAA TATCCTTAAC
2401 A~ACTTATTT TGGATCAGTT AGGAGGAGCA GAGATATCAC CA~AAATTAA
2451 A~AAGCAGCT TGTAATATTT GTACTGTTGA CTCTGACCAA CTAGCCCAAT
2501 TAGAAGAGAC ACTGCAGGGA AACTTATGTG ATGCTGAACT CTCCTCAAGT
2551 TTATCCAGTC CTTCTTACAG ATTTCAAGGG ATCCTGCCCA GCAGTGGATC
2601 TGAAGATTTG TTGTGGAAAT GGGATGCTTT A~AGGCTTAT CAGAACTTTG
2651 TTTTTGGAGA AGACAGATTA CATAGTATAC AGATTGCA~A TCACATTTGC
2701 AATTTAATCC AGA~AGGCAA TATAGTTGTT CAGTGGA~AT TATATAATTA
2751 CATATTTAAT CCTGTGCTCC A~AGAGGAGT TGAATTAGCA CATCATTGTC
2801 AACACCTAAG CGTTACTTCA GCTCAAAGTC ATGTATGTAG CCATCATAAC
2851 CAGTGCTTGC CTCAGGACGT GCTTCAGATT TATGTA~AAA CTCTGCCTAT
2901 CCTGCTTA~A TCCAGGGTAA TAAGAGATTT GTTTTTGAGT TGTAATGGAG
2951 TAAGTCA~AT AATCGAATTA AATTGCTTAA ATGGTATTCG AAGTCATTCT
3001 CTAAAAGCAT TTGAAACTCT GATAATCAGC CTAGGGGAGC AACAGA~AGA
3051 TGCCTCAGTT CCAGATATTG ATGGGATAGA CATTGAACAG AAGGAGTTGT
3101 CCTCTGTACA TGTGGGTACT TCTTTTCATC ATCAGCAAGC TTATTCAGAT
3151 TCTCCTCAGA GTCTCAGCAA ATTTTATGCT GGCCTCAAAG AAGCTTATCC
3201 AAAGAGACGG AAGACTGTTA ACCAAGATGT TCATATCAAC ACAATA~ACC
3251 TATTCCTCTG TGTGGCTTTT TTATGCGTAA GTA~AGAAGC AGAGTCTGAC
3301 AGGGAGTCGG CCAATGACTC AGAAGATACT TCTGGCTATG ACAGCACAGC
3351 CAGCGAGCCT TTAAGTCATA TGCTGCCATG TATATCTCTC GAGAGCCTTG
3401 TCTTGCCTTC TCCTGAACAT ATGCACCAAG CAGCAGACAT TTGGTCTATG
3451 TGTCGTTGGA TCTACATGTT GAGTTCAGTG TTCCAGA~AC AGTTTTATAG
3501 GCTTGGTGGT TTCCGAGTAT GCCATAAGTT AATATTTATG ATAATACAGA
3551 AACTGTTCAG AAGTCACAAA GAGGAGCAAG GAAAAAAGGA GGGAGATACA
3601 AGTGTA~ATG A~AACCAGGA TTTA~ACAGA ATTTCTCA~C CTAAGAGAAC
3651 TATGAAGGAA GATTTATTAT CTTTGGCTAT A~AAAGTGAC CCCATACCAT
3701 CAGAACTAGG TAGTCTA~AA AAGAGTGCTG ACAGTTTAGG TA~ATTAGAG
CA 02244744 1998-07-29
W097/28262 PCTrUS97/01748
l~g
3751 TTACAGCATA TTTCTTCCAT A~ATGTGGAA GAAGTTTCAG CTACTGAAGC
3801 CGCTCCCGAG GAAGCA~AGC TATTTACAAG TCAAGA~AGT GAGACCTCAC
3851 TTCA~AGTAT ACGACTTTTG GAAGCCCTTC TGGCCATTTG TCTTCATGGT
3901 GCCAGAACTA GTCAACAGAA GATGGAATTG GAGTTACCTA ATCAGAACTT
53951 GTCTGTGGAA AGTATATTAT TTGA~ATGAG GGACCATCTT TCCCAGTCAA
4001 AGGTGATTGA AACACAACTA GCA~AGCCTT TATTTGATGC CCTGCTTCGA
4051 GTTGCCCTCG GGAATTATTC AGCAGATTTT GAACATAATG ATGCTATGAC
4101 TGAGAAGAGT CATCAATCTG CAGAAGAATT GTCATCCCAG CCTGGTGATT
4151 TTTCAGAAGA AGCTGAGGAT TCTCAGTGTT GTAGTTTTAA ACTTTTAGTT
= 4201 GAAGAAGAAG GTTACGAAGC AGATAGTGAA AGCAATCCTG AAGATGGCGA
4251 AACCCAGGAT GATGGGGTAG ACTTA~AGTC TGA~ACAGAA GGTTTCAGTG
4301 CATCAAGCAG TCCAAATGAC TTACTCGA~A ACCTCACTCA AGGGGA~ATA
4351 ATTTATCCTG AGATTTGTAT GCTGGAATTA AATTTGCTTT CTGCTAGTAA
4401 AGCCA~ACTT GATGTGCTTG CCCATGTATT TGAGAGTTTT TTGAAAATTA
. 4451 TTAGGCAGAA AGAAAAGAAT GTTTTTCTGC TCATGCAACA GGGAACTGTG
4501 A~A~ATCTTT TAGGAGGGTT CTTGAGTATT TTAACACAGG ATGATTCTGA
4551 TTTTCAAGCA TGCCAGAGAG TATTGGTGGA TCTTTTGGTA TCTTTGATGA
4601 GTTCAAGAAC ATGTTCAGAA GAGCTAACCC TTCTTTTGAG AATATTTCTG
4651 GAGA~ATCTC CTTGTACAAA AATTCTTCTT CTGGGTATTC TGA~AATTAT
204701 TGA~AGTGAT ACTACTATGA GCCCTTCACA GTATCTAACC TTCCCTTTAC
4751 TGCACGCTCC A~ATTTAAGC AACGGTGTTT CATCACA~AA GTATCCTGGG
4801 ATTTTA~ACA GTAAGGCCAT GGGTTTATTG AGAAGAGCAC GAGTTTCACG
4851 GAGCAAGA~A GAGGCTGATA GAGAGAGTTT TCCCCATCGG CTGCTTTCAT
4901 CTTGGCACAT AGCCCCAGTC CACCTGCCGT TGCTGGGGCA A~ACTGCTGG
254951 CCACACCTAT CAGAAGGTTT CAGTGTITCC CTGTGGTTTA ATGTGGAGTG
5001 TATCCATG~A GCTGAGAGTA CTACAGAAAA AGGA~AGAAG ATA~AGAAAA
5051 GA~ACA~ATC ATTAATTTTA CCAGATAGCA GTTTTGATGG TACAGGTATG
5101 ATGACAGGAT TATCTGATTT GTACACAAAG ATTGTTTTCA GACTATAATT
5151 TTCCTTGAGC CGTA~AAATG TGGTAGTGTT CTTAACACTC TTAACATGTT
305201 ATTCACCTTA AAGATCCTAC T
5.8EXAMPLE 8 -- DEDUC~D AMINO ACID SEQUENCES OF HUMAN LYST1 PROTEIN
5.8.1 PEPTIDE SEQUENCE OF LONG ISOFORM (SEQ ID NO:8~
1 MSTDSNSLAR EFLTDVNRLC NAW QRVEAR F.~.~.F.~THMA TLGQYLVHGR
3551 GFLLLTKLNS IIDQALTCRE ELLTLLLSLL PLVWKIPVQE EKATDFNLPL
101 SADIILTKEK NSSSQRSTQE KLHLEGSALS SQVSAKVNVF RKSRRQRKIT
151 HRYSVRDARK TQLSTSDSEA NSDEKGIAMN KHRRPHLLHH FLTSFPKQDH
201 PKAKLDRLAT KEQTPPDAMA LENSREIIPR QGSNTDILSE PA~LSVISNM
251 NNSPFDLCHV LLSLLEKVCK FDVTLNHNSP LAAS W PTLT EFLAGFGDCC
40301 SLSDNLESRV VSAGWTEEPV ALIQRMLFRT VLHLLSVDVS TAEMMPENLR
351 KNLTELLRAA LKIRICLEKQ PDPFAPRQKK TLQEVQEDFV FSKYRHRALL
401 LPELLEGVLQ ILICCLQSAA SNPFYFSQAM DLVQEFIQHH GFNLFETAVL
451 QMEWLVLRDG ~PPEASEHLK ALINSVMKIM STVKKVKSEQ LHHSMCTRKR
501 HRRCEYSHFM HHHRDLSGLL VSAFKNQVSK NPFEETADGD VYYPERCCCI
45551 AVCAHQCLRL LQQASLSSTC VQILSGVHNI GICCCMDPKS VIIPLLHAFK
601 LPALKNFQQH ILNILNKLIL DQLGGAEISP KIKKAACNIC TVDSDQLAQL
651 EETLQGNLCD AELSSSLSSP SYRFQGILPS SGSEDLLWKW DALKAYQNFV
701 FGEDRLHSIQ IANHICNLIQ KGNI W QWKL YNYIFNPVLQ RGVELAHHCQ
751 HLSVTSAQSH VCSHHNQCLP QDVLQIYVKT LPILLKSRVI RDLFLSCNGV
50801 SQIIELNCLN GIRSHSLKAF ETLIISLGEQ QKDASVPDID GIDIEQKELS
CA 02244744 1998-07-29
W O 97/28262 PCTAUS97/01748
~ ~OCl
851 SVHVGTSFHH QQAYSDSPQS LSKFYAGLKE AYPKRRKTVN QDVHINTINL
901 FLCVAFLCVS KEAESDRESA NDSEDTSGYD STASEPLSHM LPCISLESLV
951 LPSPEHMHQA ADIWSMCRWI YMLSSVFQKQ FYRLGGFRVC HKLIFMIIQK
1001 LFRSHKEEQG KKEGDTSVNE NQDLNRISQP KRTMKEDLLS LAIKSDPIPS
51051 ELGSLKKSAD SLGKLELQHI SSINVEEVSA TEAAPEEAKL FTSQESETSL
1101 QSIRLLEALL AICLHGARTS QQKMELELPN QNLSVESILF EMRDHLSQSK
1151 VIETQLAKPL FDALLRVALG NYSADFEHND AMTEKSHQSA EELSSQPGDF
1201 SEEAEDSQCC SFKLLVEEEG YEADSESNPE DGETQDDGVD LKSETEGFSA
1251 SSSPNDLLEN LTQGEIIYPE ICMLELNLLS ASKAKLDVLA HVFESFLKII
101301 RQKEKNVFLL MQQGTVKNLL GGFLSILTQD DSDFQACQRV LVDLLVSLMS
1351 SRTCSEELTL LLRIFLEKSP CTKILLLGIL KIIESDTTMS PSQYLTFPLL
1401 HAPNLSNGVS SQKYPGILNS KAMGLLRRAR VSRSKKEADR ESFPHRLLSS
1451 WHIAPVHLPL LGQNC~PHLS EGFSVSLWFN VECIHEAEST TEKGKKIKKR
1501 NKSLILPDSS FDGTESDRPE GAEYINPGER LIEEGCIHII SLGSKALMIQ
151551 VWADPHNATL IFRVCMDSND DMKAVLLAQV ESQENIFLPS KWQHLVLTYL
1601 QQPQGKRRIH GKISIWVSGQ RKPDVTLDFM LPRKTSLSSD SNKTFCMIGH
1651 CLSSQEEFLQ LAGKWDLGNL LLFNGAKVGS QEAFYLYACG PNHTSVMPCK
1701 YGKPVNDYSK YINKEILRCE QIREFFMTKK DVDIGLLIGV FQLFIQLTVL
1751 LQYTIYEPVI RLKGQMKTQL SQRPFSSKEV QSILLEPHHL KNLQPTEYKT
201801 IQGILHEIGG TGIFVFLFAR WELSSCEET QALALRVILS LIKYNQQRVH
1851 ELENCNGLSM IHQVLIKQKC IVGFYILKTL LEGCCGEDII YMNENGEFKL
1901 DVDSNAIIQD VKLLEELLLD WKIWSKAEQG VWETLLAALE VLIRADHHQQ
1951 MFNIKQLLKA QVV~LTC QVLQEYKEGQ LTPMPREMAR SFRRKCGQSC
2001 T
5.8.2PEPTIDE SEQUENCE OF ~HORT ISOFORM (SEQ ID NO:10)
1 MSTDSNSLAR EFLTDVNRLC NA W QRVEAR EEEEEETHMA TLGQYLVHGR
51 GFLLLTKLNS IIDQALTCRE ELLTLLLSLL PLVWKIPVQE EKATDFNLPL
101 SADIILTKEK NSSSQRSTQE KLHLEGSALS SQVSAKVNVF RKSRRQRKIT
30151 HRYSVRDARK TQLSTSDSEA NSDEKGIAMN KHRRP~T.T.~H FLTSFPKQDH
201 PKAKLDRLAT KEQTPPDAMA LENSREIIPR QGSNTDILSE PAALSVISNM
251 NNSPFDLCHV LLSLLEKVCK FDVTLNHNSP LAAS W PTLT EFLAGFGDCC
301 SLSDNLESRV VSAGWTEEPV ALIQRMLFRT VLHLLSVDVS TAEMMPENLR
351 KNLTELLR~A LKIRICLEKQ PDPFAPRQKK TLQEVQEDFV FSKYRHRALL
35401 LPELLEGVLQ ILICCLQSAA SNPFYFSQAM DLVQEFIQHH GFNLFETAVL
451 QMEWLVLRDG VPPEASEHLK ALINSVMKIM STVKKVKSEQ LHHSMCTRKR
501 HRRCEYSHFM HHHRDLSGLL VSAFKNQVSK NPFEETADGD VYYPERCCCI
551 AVCAHQCLRL LQQASLSSTC VQILSGVHNI GICCCMDPKS VIIPLLHAFK
601 LPALKNFQQH ILNILNKLIL DQLGGAEISP KIKK~ACNIC TVDSDQLAQL
40651 EETLQGNLCD AELSSSLSSP SYRFQGILPS SGSEDLLWKW DALKAYQNFV
701 FGEDRLHSIQ IANHICNLIQ KGNIVVQWKL YNYIFNPVLQ RGVELAHHCQ
751 HLSVTSAQSH VCSHHNQCLP QDVLQIYVKT LPILLKSRVI RDLFLSCNGV
801 SQIIELNCLN GIRSHSLKAF ETLIISLGEQ QKDASVPDID GIDIEQKELS
851 SVHVGTSFHH QQAYSDSPQS LSKFYAGLKE AYPKRRKTVN QDVHINTINL
45901 FLCVAFLCVS KEAESDRESA NDSEDTSGYD STASEPLSHM LPCISLESLV
951 LPSPEHMHQA ADIWSMCRWI YMLSSVFQKQ FYRLGGFRVC HKLIFMIIQK
1001 LFRSHKEEQG KKEGDTSVNE NQDLNRISQP KRTMKEDLLS LAIKSDPIPS
1051 ELGSLKKSAD SLGKLELQHI SSINVEEVSA TEAAPEEAKL FTSQESETSL
1101 QSIRLLEALL AICLHGARTS QQKMELELPN QNLSVESILF EMRDHLSQSK
501151 VIETQLAKPL FDALLRVALG NYSADFEHND AMTEKSHQSA EELSSQPGDF
1201 SEEAEDSQCC SFKLLVEEEG YEADSESNPE DGETQDDGVD LKSETEGFSA
1251 SSSPNDLLEN LTQGEIIYPE ICMLELNLLS ASKAKLDVLA HVFESFLKII
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1301 RQKEKNVFLL MQQGTVKNLL GGFLSI~TQD DSDFQACQRV LVDLLVSLMS
1351 SRTCSEELTL LLRIFLEKSP CTKILLLGIL KIIESDTTMS PSQYLTFPL~
1401 HAPNLSNGVS SQKYPGILNS KAMGL~RRAR VSRSKKEADR ESFPHRLLSS
1451 WHIAPVHLPL LGQNCWPHLS EGFSVSLWFN VECIHEAEST TEKGKKIKKR
1501 NKSLILPDSS FDGTGMMTGL SDLYTKIVFR L
5.9 EXAMPLE 9 -- IDENTIFICATION OF A DNA SEGMENT ENCODINC~ LYST2
Lyst2 was identified in a search for human genes similar in sequence to Lystl (the C~I
gene). Mouse Lystl cDNA sequence was compared with Genbank sequences, and significant
similarity (52%) was noted between residues 3275 to 3413 of Lystl ~Genbank Accession number
U7001~) and R17955. R17955 is an uncharacterized human expressed sequence tag 292 bp in
length. The corresponding partial length cDNA clone (#32273) was obtained from Image
consortium This cDNA clone was derived from a cDNA libra~y of human infant brain, and is
1979-bp in length. The clone was de~ign~ted human LYST2.
5.10 ~XA~rPLE 10 -- DN A SEQUENCE OFTH~ HUMAN LYST2 GENE
The LYST2 clone was sequenced using standard methodologies. The DNA sequence is
given below (SEQ ID NO: 11):
1 ATACTTCTGA TGTA~AGGAA CTAATTCCAG AGTTCTACTA CCTACCAGAG
51 ATGTTTGTCA ACAGTAATGG ATATAATCTT GGAGTCAGAG AAGATGAAGT
101 AGTGGTA~AT GATGTTGATC TTCCCCCTTG GGCAA~APaA CCTGAAGACT
151 TTGTGCGGAT CA~CAGGATG GCCCTAGA~A GTGAATTTGT TTCTTGCQA
201 CTTCATCAGT GGATCGACCT TATATTTGGC TATAAGCAGC GAGGACCAGA
251 AGCAGTTCGT GCTCTGAATG TTTTTCACTA CTTGACTTAT GAAGGCTCTG
301 TGAACCTGGA TAGTATCACT GATCCTGTGC TCAGGGAGGC CATGGAGGCA
351 CAGATACAGA ACTTTGGACA GACGCCATCT CAGTTGCTTA TTGAGCCACA
401 TCCGCCTCGG AACTCTGCCA TGCACCTGTG TTTCCTTCCA CAGAGTCCGC
451 TCATGTTTAA AGATCAGATG CAACAGGATG TGATAATGGT GCTGAAGTTT
501 CCTTCAAATT CTCCAGTAAC CCATGTGGCA GCCAACACTC TGCCCCACTT
551 GACCATCCCC GCAGTGGTGA CAGTGACTTG CAGCCGACTC TTTGCAGTGA
601 ATAGATGGCA CAACACAGTA GGCCTCAGAG GAGCTCCAGG ATACTCCTTG
651 GATCAAGCCC ACCATCTTCC CATTGA~ATG GATCCATTAA TAGCCAATAA
701 TTCAGGTGTA AACA~ACGGC AGATCACAGA CCTCGTTGAC CAGAGTATAC
751 AAATCAATGC ACATTGTTTT GTGGTAACAG CAGATAATCG CTATATTCTT
801 ATCTGTGGAT TCTGGGATAA GAGCTTCAGA GTTTATACTA CAGA~ACAGG
851 GA~ATTGACT CAGATTGTAT TTGGCCATTG GGATGTGGTC ACTTGCTTGG
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901 CCAGGTCCGA GTCATACATT GGTGGGGACT GCTACATCGT GTCCGGATCT
- 951 CGAGATGCCA CCCTGCTGCT CTGGTACTGG AGTGGGCGGC ACCATATCAT
1001 AGGAGACAAC CCTAACAGCA GTGACTATCC GGCACCAAGA GCCGTCCTCA
1051 CAGGCCATGA CCATGAAGTT GTCTGTGTTT CTGTCTGTGC AGAACTTGGG
1101 CTTGTTATCA GTGGTGCTAA AGAGGGCCCT TGCCTTGTCC ACACCATCAC
1151 TGGAGATTTG CTGAGAGCCC TTGAAGGACC AGAAAACTGC TTATTCCCAC
1201 GCTTGATATC TGTCTCCAGC GAAGGCCACT GTATCATATA CTATGAACGA
1251 GGGCGATTCA GTAATTTCAG CATTAATGGG A~ACTTTTGG CTCA~ATGGA
1301 GATCAATGAT TCAACACGGG CCATTCTCCT GAGCAGTGAC GGCCAGAACC
1351 TGGTCACCGG AGGGGACAAT GGGGTAGTAG AGGTCTGGCA GGCCTGTGAC
1401 TTCAAGCAAC TGTACATTTA ACCCTGGATG TGATGCTGGC ATTAGAGCAA
1451 TGGACTTGTC CCATGACCAG AGGACTCTGA TCACTGGCAT GGCTTCTGGT
1501 AGCATTGTAG CTTTTAATAT AGATTTTAAT CGGTGGCATT ATGAGCATCA
1551 GAACAGATAC TGAAGATA~A GGAAGA~CCA A~AGCCAAGT TAAAGCTGAG
1601 GGCACAAGTG CTGCATGGAA AGGCAATATC TCTGGTGGAA A~AATTCGTC
1651 TACATCGACC TCCGTTTGTA CATTCCATCA CACCCAGCAA TAGCTGTACA
1701 TTGTAGTCAG CA~CCATTTT ACTTTGTGTG TTTTTTCACG ACTGAACACC
1751 AGCTGCTATC AAGCAAGCTT ATATCATGTA AATTATATGA ATTAGGAGAT
1801 GTTTTGGTA~ TTATTTCATA TATTGTTGTT TATTGAGAAA AGGTTGTAGG
1851 ATGTGTCACA AGAGACTTTT GACAATTCTG AGGAACCTTG TGTCCAGTTG
1901 TTACAAAGTT TAAGCTTTGA ACCTA~CCTG CATCCCATTT CCAGCCTCTT
1951 TTCAAGCTGA GAA~AAAAAA AAAAAAAAA (SEQ ID NO:ll)
This DNA sequence corresponds to the 3' end of the coding domain of human LYS~2 and
the 3' untr~n~l~ted region.
5.11 EXA~IPLE 11 -- AM~NO ACID SEQUENCE oFTHElIuMAN LYST2 PROTEIN
Translation of the DNA of SEQ ID NO:l 1 provided the deduced amino acid sequence of
the LYST2 protein ~S:~Q ID NO:12) which is shown below:
1 TSDVKELIPE FYYLPEMFVN SNGYNLGVRE DE W VNDVDL PPWAKKPEDF
51 VRINRMALES EFVSCQLHQW IDLIFGYKQR GPEAVR~LNV FHYLTYEGSV
101 NLDSITDPVL REAMEAQIQN FGQTPSQLLI EPHPPRNSAM HLCFLPQSPL
151 MFKDQMQQDV IMVLKFPSNS PVTHVA~NTL PHLTIPAW T VTCSRLFAVN
201 RWHNTVGLRG APGYSLDQAH HLPIEMDPLI ANNSGVNKRQ ITDLVDQSIQ
251 INAHCFVVTA DNRYILICGF WDKSFRVYTT ETGKLTQIVF GHWD W TCLA
301 RSESYIGGDC YIVSGSRDAT LLLWYWSGRH HIIGDNPNSS DYPAPRAVLT
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351 GHDHE W CVS VCAELGLVIS GAKEGPCLVH TITGDLLRAL EGPENCLFP~
- 401 LISVSSEGHC IIYYERGRFS NFSINGKLLA QMEINDSTRA IL~SSDGQNL
451 VTGGDNGVVE VWQACDFKQL YI (SEQ ID NO:2)
Amino acids 2 to 140 of the predicted human LYST2 protein share only a 51.8% amino
acid identity with amino acids 3275 to 3413 of mouse and human Lystl . The C-terminal residues
of LYST2 are not similar to LYST1, but do have a similar predicted secondary structure: This
region of LYSTl contains WD repeats and is predicted to assume a propellor-like secondary
structure, similar to the beta subunit of heterotrimeric G proteins. The corresponding region of
1 0 LYST2 also contains WD repeats and is also similar in sequence to the beta subunit of
heterotrimeric G proteins (30.4% identity from LYST2 amino acid 285 to 418 to the guanine
nucleotide-binding protein beta subunit-like protein P49027). Furthermore, the stop codons of
mouse Lystl and human LYST2 occur approximately the same distance from the matching region.
~.12 EXAMPLE 12 -- GENETIC MAPPING OF THE LYS~2 GENE
By hybridization to Southern blots of human-rodent somatic cell hybrids, LYST2 was
sho~n to map on human Chromosome 13. This is in contrast to ~ YSTl, which maps on human
Chromosome 1. Using an MspI restriction fragment length polymorphism, Lyst2 was mapped by
cros-hybridization in the mouse. Linkage analysis using DNA from 93 intersubspecific backcross
~(~57BL/6J-bgJX (C57BL/6~-bgJx CAST/EiJ)Fl] mice revealedLyst2 to map to mouse
2 o Chromosome3 between ~3Mit21 and D3Mit22. This contrasts with Lyst, which maps on mouse
Chromosome 13. Pulsed field gel electrophoresis blots of mouse DNA hybridized with a ~.yst2
probe showed a single band, indicating that Lyst2 is a single genetic locus.
.13 EXAMPLE 13 -- EXPRESSION ANALYSIS OF THE LYST2 GENE
Hybridization of northern blots of human and mouse tissues with LYST2 revealed the
2 5 following pattern of expression: Lyst2 is abundantly expressed in mouse brain, and moderately
expressed in mouse kidney, and weakly expressed in mouse heart, lung, skeletal muscle, and
testis. Lysf2 is not expressed in mouse spleen or liver. The largest (and most prominent) band
observed on northern blots was 13kb in size (very sirnilar to the largest Lyst rr~A). Additional
transcripts on 6kb and 5kb were evident in mouse brain RNA.
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In selected human tissues, LYST2 was expressed as follows: Moderate expression was
~ observed in melanoma cells, weak expression in HeLa cells, colorectal carcinoma cells, and in
spleen, Iymph node, thymus, and appendix. No expression was detected in peripheral blood
leucocyte, bone marrow, fetal liver, lung carcinoma, or }ellkemi~ cell lines (K562, MOLT4, Raji,
5 HL60).
The major transcript was 1 3-kb in size in human RNA.
In sl-m m~ry, LYST2 appears to be similar in size to the largest LYSTl mRNA, but has a
very dirre~ L tissue distribution of expression, being abundantly expressed only in brain. LYST2
appears to be a brain-specific homologue of LYSTl, and may function to regulate protein
0 trafficking to the Iysosome and late endosome within the brain.
The relative abundance of LYST2 mRNA isoforms in human tissues at di~elell~
developmental stages was examined by sequential hybridization of a poly(A)+ RNA dot blot with
a LYST2 cDNA probe The quantity of poly(A~+ RNA loaded on the blot was norm~li7ed to
eight housekeeping genes ~phospholipase, ribosomal protein S9, tubulin, a highly basic 23-kDa
5 protein, glyceraldehyde-3-phosphate dehydrogenase, hypo-~nthin~ guanine phosphoribosil
transferase, 13-actin, and ubiquitin) to allow estimation of the relative abundance of LYST2 mRNA
isoforms in di~erent tissues.
Abundant LYST2 transcripts were detected in all brain regions and in kidney. LYST~
transcripts were detected in those regions at all developmental stages.
2 o 5.14 EXAMPLE 14 -- IDENTIFICATION OF MOUSE L YsT2 CDNA CLONES
A mouse embryo (day 14.5 post-coitum) cDNA library was hybridized with a probe
corresponding to human LYST2. Two clones were isolated and sequenced. They contained
overlapping sequences that were assembled by alignment with human LYST2 and represent 2543
bp of cDNA sequence.
" 2 5 5.15 EXAMPI.E 15 -- DNA SEQUENCE OF THE ~OUSE LYST2 GENE
-
1 GCAGCAGGGC GAACCGGACC TCTGTGATGT TTAATTTTCC TGACCAAGCA
51 ACAGTTA~AA AAGTTGTCTA CAGCTTGCCT CGGGTTGGAG TGGGGACCAG
101 CTATGGTTTG CCACAAGCCA GGAGGATATC ACTGGCCACT CCTCGACAGC
30151 TGTATA~GTC TTCCAATATG ACTCAGCGCT GGCA~AGAAG GGAAATCTCC
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201 AACTTTGAGT ATTTGATGTT TCTCAACACG ATAGCAGGTC GGACGTATAA
251 TGATCTGAAC CAGTATCCTG TGTTTCCATG GGTGTTAACA AACTATGAAT
301 CAGAGGAGTT GGACCTGACT CTCCCAGGAA ACTTCAGGCA TCTGTCAAAG
351 CCA~AAGGTG CTTTGAACCC GAAGAGAGCA GTGTTTTACG CAGAGCGCTA
5401 TGAGACATGG GAGGAGGATC A~AGCCCACC CTTCCACTAC AACACACATT
451 ACTCAACGGC GACTTCCCCC CTTTCATGGC TTGTTCGGAT TGAGCCATTC
501 ACAACCTTCT TCCTCAATGC A~ATGATGGG A~ATTTGACC ATCCAGACCG
551 AACCTTCTCA TCCATTGCAA GGTCATGGAG AACCAGTCAG AGAGATACAT
601 CCGATGTCAA GGAACTAATT CCAGAGTTCT ATTACGTACC AGAGATGTTT
10651 GTCAACAGCA ATGGGTACCA TCTTGGAGTG AGGGAGGACG AAGTGGTGGT
701 TAATGATGTG GACCTGCCCC CCTGGGCCAA GAAGCCAGAA GACTTTGTGC
751 GGATCAACAG GATGGCCCTG GA~AGTGAAT TTGTTTCTTG CCAACTCCAT
801 CAATGGATTG ACCTTATATT TGGCTACA~A CAGCGAGGGC CAGAGGCAGT
851 CCGTGCTCTC AATGTTTTCC ACTACTTGAC CTACGAAGGC TCTGTA~ACC
15 ~901 TGGACAGCAT CACAGACCCT GTGCTCCGGG AGGCCATGGT TGCACAGATA
951 CAGAACTTTG CCCAGACGCC ATCTCAGTTG CTCATTGAGC CGCATCCGCC
lO01 TAGGACTTCA GCCATGCATC TGTGTTCCCT TCCACAGAGC CCACTCATGT
1051 TCA~AGATCA GATGCAGCAG GATGTGATCA TGGTGCTGAA GTTTCCATCC
1101 AATTCTCCTG TGACTCATGT GGCTGCCAAC ACCCTGCCCC ACCTGACCAT
201151 CCCTGCAGTG GTGACAGTGA CCTGCAGCCG ACTGTTTGCA GTGAACAGAT
1201 GGCAC~ACAC AGTCGGCCTC AGAGGAGCCC CCGGATACTC CTTGGATCAA
1251 GCACACCATC TTCCCATTGA GATGGACCCA TTAATCGCAA ATAACTCTGG
1301 TGTGAACAAG CGGCAGATCA CAGACCTTGT AGACCAGAGC ATCCAGATCA
1351 ATGCCCACTG CTTCGTGGTC ACAGCTGATA ATCGCTACAT CCTCATCTGT
251401 GGGTTTTGGG ATA~AAGTTT CAGAGTTTAC TCGACAGA~A CAGGGA~ACT
1451 GACACAGATT GTATTTGGCC ACTGGGATGT TGTCACATGC CTGGCCAGGT
1501 CGGAGTCCTA CATTGGTGGA GACTGCTACA TAGTGTCTGG ATCTCGGGAC
1551 GCCACCTTGC TTCTCTGGTA CTGGAGTGGG CGTCACCACA TCATCGGAGA
1601 CAACCCCAAT AGCAGTGACT ATCCTGCGCC CAGAGCTGTC CTCACAGGCC
301651 ATGACCATGA AGTTGTCTGT GTCTCCGTCT GTGCAGAACT CGGACTCGTT
1701 ATCAGTGGTG CTA~AGAGGG CCCTTGCCTC GTTCATACCA TCACTGGA~A
1751 TCTGCTGAAG GCCCTGGAAG GACCAGA~AA CTGCTTATTT CCACGCCTAA
1801 TTTCGGTATC CAGTGAAGGC CACTGCATCA TATATTATGA GCGAGGACGG
1851 TTTAGCAACT TCAGCATCAA TGGGA~ACTT TTGGCTCAAA TGGAGATCAA
:~ l901 TGATTCCACT AGGGCTATTC TCCTGAGCAG CGATGGACAG AACCTGGTGA
1951 CTGGAGGGGA CAATGGTGTG GTGGAGGTCT GGCAGGCCTG TGACTTTA~G
2001 CAGCTGTACA TTTACCCAGG ATGTGATGCT GGCATTAGAG CGATGGATTT
2051 ATCCCATGAC CA~AGGACTC TGATCACTGG CATGGCTTCC GGCAGCATTG
2101 TACTTTTAAT ATAGATTTTA ATCGGTGGCA TTATGAGCAT CAGAACAGTA
2151 CTGAAGAGAA GCAGCAGAAG CCACATTCA~ GTGAGAGCAC AAGTGCTTCT
2201 GTGGA~AGGC AGTATCTCTG GTGGGACGCT GGTCCACATC GGCCTCTGCT
2251 TGTACATCCA TCCCACCCAG CAGTCGCCGA ACATCATAGT CGGGAGCCAT
2301 TTCACCCTGT TTTTCCAGGA CTGAACACCA GCTGCTGTCA AGCAAGCTTA
2351 TATCATGTAA ATTATCTGA~ TTAGGAGCCG TTTTGGTAAT TATTTCATAT
2401 ATCGCCGTTT ATTGAGA~AA GGTTGTAGGA AGCCTCACAA GAGACTTTTG
2451 ACAATTCTGA GGAACCTTGT GCCCAGTTGT TACA~AGTTT AAGCTTTGA~
2501 CCTAACTTGC ATCCCATTTC CAGCCTCGGG CTTCACTCGT GCC
(SEQ ID NO:13)
50 5.16 EXAMPLE 16 -- DEDUCED AMINO ACID SEQUENCE OF MOUSE LYST2 PROTEIN
1 SRANRTSVMF NFPDQATVKK W YSLPRVGV GTSYGLPQAR RISLATPRQL
51 YKSSNMTQRW QRREISNFEY LMFLNTIAGR TYNDLNQYPV FPWVLTNYES
101 EELDLTLPGN FRHLSKPKGA LNPKRAVFYA ERYETWEEDQ SPPFHYNTHY
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151 STATSPLSWL VRIEPFTTFF LNANDGKFDH PDRTFSSIAR SWRTSQRDTS
201 DVKELIPEFY YVPEMF~NSN GYHLGVREDE VVVNDVDLPP WAKKPEDFVR
251 INRMALESEF VSCQLHQWID LIFGYKQRGP EAVRALNVFH YLTYEGSVNL
301 DSITDPVLRE AMVAQIQNFA QTPSQLLIEP HPPRTSAMHL CSLPQSPLMF
351 KDQMQQDVIM VLKFPSNSPV THVA~NTLPH LTIPAWTVT CSRLFAVNRW
401 HNTVGLRGAP GYSLDQAHHL PIEMDPLIAN NSGVNKRQIT DLVDQSIQIN
451 AHCFVVTADN RYILICGFWD KSFRVYSTET GKLTQIVFGH WDWTCLARS
501 ESYIGGDCYI VSGSRDATLL LWYWSGRHHI IGDNPNSSDY PAPRAVLTGH
551 DHE WCVSVC AELGLVISGA KEGPCLVHTI TGNLLKALEG PENCLFPRLI
601 SVSSEGHCII YYERGRFSNF SINGKLLAQM EINDSTRAIL LSSDGQNLVT
651 GGDNGVVEVW QACDFKQLYI YPGCDAGIRA MDLSHDQRTL ITGMASGSIV
701 LLI (SEQ ID NO:14)
Mouse Lyst2 shares 98% amino acid identity with human LYST
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6. References
The following literature citations as well as those cited above are incorporated in pertinent
part by reference herein for the reasons cited in the above text:
United States Patent 3,791,932.
United States Patent 3,949,064.
United States Patent 4,174,384.
United States Patent 4,196,265
United States Patent 4,271,147.
United States Patent 4,554,101.
o United States Patent 4,578,770.
United States Patent 4,596,792.
United States Patent 4,599,230.
United States Patent 4,599,231.
United States Patent 4,601,903.
United States Patent 4,608,251.
United States Patent 4,683,195.
United States Patent 4,683,202.
United States Patent 4,952,496.
United States Patent 5,168,050.
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CA 02244744 1998-07-29
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S~QUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: University o~ Florida
(B) STREET: 223 Grinter Hall
(C) CITY: Gainesville
(D) STATE: Florida
(E) COUNTRY: USA
(F) POSTA1 CODE (ZIP): 32611
(ii) TITLE OF INVENTION: Lystl and Lyst2 Gene Compositions and Methods
o~ Use
(iii) NUMBER OF SEQUENCES: 78
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/011,146
(B) FILING DATE: 01-FEB-1996
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US UNKNOWN
(B) FILING DATE: 23-DEC-1996
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/033,599
(B) FILING DATE: 20-DEC-1996
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3514 ~ase pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPO~OGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
TTTAAAAATT AGAGCTTGCT TGGA~AAGCA GCCTGAGCCT TTCTCCCCGA GACAAAAGAA 60
AACACTACAG GAGGTCCAGG AGGGCTTTGT ATTTTCCAAG TATCGTCACC GAGCCCTTCT 120
ACTACCTGAG CTTCTGGAAG GAGTTCTACA GCTCCTCATC TCTTGTCTTC AGAGTGCAGC 180
TTCA~ATCCC TTTTACTTCA GTCAAGCCAT GGATTTAGTT CAAGAATTTA TC Q GCACCA 240
AGGATTTAAT CTCTTTGGAA CAGCAGTTCT TCAGATGGAA TGGCTGCTTA CAAGGGACGG 300
TGTTCCTTCA GAAGCTGCAG AACATTTGAA AGCTCTGATA AACAGTGTAA TA~AAATAAT 360
GAGTACTGTG AAAAAGGTGA AATCAGAGCA ACTTCATCAT TCCATGTGCA CAAGGAAAAG 420
_ ACACCGGCGT TGTGAGTATT CCCACTTCAT GCAGCACCAC CGCGATCTTT CAGGGCTCCT 480
GGTTTCAGCT TTTAAAAATC AGCTTTCTAA AAGCCCCTTT GAAGAGACCG CAGAGGGAGA 540
CA 02244744 1998-07-29
W097/28262 PCTrUS97/01748
f'~g
TGTGCAGTAT CCAGAGCGCT GCTGCTGCAT CGCCGTGTGC GCTCACCAGT GCTTGCGCTT 600
GCTGCAGCAG GTTTCCCTGA GCACCACGTG TGTCCAGATC CTATCAGGTG TACACAGTGT 660
TGGAATCTGT TGTTGTATGG ATCCTAAGTC TGTGATCGCC CCTTTACTGC ATGCTTTTAA 720
GTTGCCAGCA CTGAAAGCTT TCCAGCAGCA TATACTGAAT GTCCTGAGCA AACTTCTTGT 780
GGATCAGTTA GGAGGAGCAG AGCTATCACC GAGAATTAAA ARAGCAGCTT GCAACATCTG 840
TACTGTGGAC TCTGACCAAC TGGCTAAGTT AGGAGAGACA CTGCAAGGCA CCTTGTGTGG 900
TGCTGGTCCT ACCTCCGGCT TGCCCAGTCC TTCCTACCGA TTTCAGGGGA TCCTGCCCAG g60
CAGCGGCTCT GAAGACTTGC TGTGGAAGTG GGATGCATTA GAGGCTTATC AGAGCTTTGT 1020
CTTTCAAGAA GACAGATTAC ATAACATTCA GATTGCAAAT CACATTTGTA ATTTACTCCA 1080
GAAAGGCAAT GTAGTTGTTC AGTGGAAATT GTATAATTAT ATCTTTAATC CTGTGCTCCA 1140
AAGAGGAGTT GAATTAGTAC ATCATTGTCA ACAGCTAAGC ATTCCTTCAG CTCAGACTCA 1200
CATGTGTAGC CAACTGAAAC AGTATTTGCC TCAGGAAGTG CTTCAGATTT ATTTAAAAAC 1260
TCTACCTGTC CTACTTAAAT CCAGGGTAAT AAGAGATTTG TTTTTAAGTT GTAATGGAGT 1320
AAACCACATA ATTGAACTAA ATTACTTAGA TGGGATTCGA AGTCATTCCC TGAAAGCATT 1380
TGPAACTCTG ATTGTCAGCC TAGGGGAACA ACAGAAAGAT GCTGCAGTTC TAGACGTCGA 1440
TGGGTTAGAC ATCCAACAGG AGTTGCCGTC CTTAAGTGTG GGTCCTTCTC TTCATAAGCA 1500
GCAAGCTTCT TCAGATTCTC CTTGCAGTCT CAGGAAGTTT TATGCCAGCC TCAGAGAGCC 1560
TGATCCAPAA AAACGAAAGA CCATTCACCA GG~TGTTCAC ATAAACACCA TAAACCTCTT 1620
CCTCTGTGTG GCTTTTCTAT GTGTCAGTAA AGAAGCAGAC TCTGATAGGG AGTCTGCCAA 1680
TGAGTCAGAA GATACTTCTG GCTATGACAG CCCTCCCAGT GAGCCATTAA GTCACATGCT 1740
ACCATGTCTG TCTCTTGAGG ACGTTGTCTT ACCTTCCCCT GAATGTTTGC ACCATGCAGC 1800
AGACATTTGG TCCATGTGTC GTTGGATCTA CATGTTGAAC TCAGTCTTCC AGAAACAATT 1860
TCACAGGCTT GGTGGTTTCC AAGTGTGCCA TGAATTAATA TTTATGATAA TCCAGAAACT 1920
ATTCAGAAGT CATACAGAGG ATCAAGGAAG AAGGCAGGGA GAAATGAGTA GAAATGAAAA 1980
CCAAGAGCTA ATCAGGATAT CTTACCCCGA GCTGACACTG AAGGGAGATG TATCATCTGC 2040
AACAGCACCA GACCTGGGAT TTCTGAGAAA GAGTGCTGAC AGCGTGCGTG GATTCCAGTC 2100
ACAGCCTGTG CTTCCCACAA GTGCAGAGCA GATTGTGGCT ACTGAATCTG TTCCTGGGGA 2160
ACGAAAGGCA TTTATGAGTC AACAAAGTGA GACTTCTCTC CAGAGCATAC GACTTTTGGA 2220
GTCTCTCCTG GACATTTGTC TTCATAGTGC CAGAGCCTGT CAACAGAAGA TGGAATTGGA 2280
GCTACCGTCT CAGGGCTTGT CTGTGGAAAA TATATTGTGT GAACTGAGGG AACACCTTTC 2340
CCAGTCAAAG GTGGCAGAAA CAGAATTAGC AAAGCCTTTA TTTGATGCCC TGCTTCGAGT 2400
AGCCCTGGGG AATCATTCAG CAGATTTGGG CCCTGGTGAT GCTGTGACTG AGAAGAGTCA 2460
TCCCTCTGAG GAAGAGCTGT TGTCCCAGCC CGGAGATTTT TcAGAAGAAG CTGAGGATTc 2520
CA 02244744 l998-07-29
W 097/28262 PCTrUS97/01748
1~
TCAGTGTTGT AGTTTGAAAC TTCTGGGTGA GGAAGAAGGC TATGAAGCGG ATAGTGAAAG 2580
CAATCCTGAG GATGTTGACA CCCAAGACGA TGGAGTAGAA TTAAATCCTG AAGCAGAAGG 2640
TTTCAGTGGA TCGATTGTTT CAAACAACTT ACTTGAAAAC CTCACTCACG GGGAAATAAT 2700
ATACCCTGAG ATTTGCATGC TGGGATTAAA TTTGCTTTCT GCTAGCAAAG CTAAACTTGA 2760
TGTGCTTGCT CATGTGTTTG AGAGCTTTCT GAAAATTGTC AGGCAGAAGG AAAAGAACAT 2820
TTCTCTCCTC ATACAACAGG GAACTGTGAA AATCCTTCTA GGCGGGTTCT TGAATATTTT 2880
AACACAAACT AACTCTGATT TCCAAGCATG CCAGAGAGTA CTGGTGGATC TCTTGGTATC 2940
TTTGATGAGC TCAAGAACGT GTTCAGAAGA CTTAACTCTT CTTTGGAGAA TATTTCTGGA 3000
GAAATCTCCT TGTACAGAAA TTCTTCTCCT TGGTATTCAC AAAATTGTTG AAAGTGATTT 3060
TACTATGAGC CCTTCACAGT GTCTGACCTT TCCTTTCCTG CATACCCCGA GTTTAAGCAA 3120
TGGTGTCTTA TCACAGAAAC CTCCTGGGAT TCTTAACAGT A~AGCCTTAG GCTTATTGAG 3180
AAGAGCACGG ATTTCCCGAG GCAAGAAAGA GGCTGATAGA GAGAGTTTTC CCTATAGGCT 3240
GCTTTCCTCT TGGCATATAG CCCCAATCCA CCTGCCGTTG CTGGGACAGA ACTGCTGGCC 3300
ACACCTGTCA GAAGGATTTA GTGTTTCTCT TGTGGGTTTA ATGTGGAATA CATCCAATGA 3360
ATCCGAGAGT GCTGCAGAAA GGGGAAAAGG AGTAAGGA~A AGAACAAACC ATCAGTTCTG 3420
GAAGACAGCA GTTTTGAAGG AGCAGAAGGT GATAGACCAG AAGTTACAGA ATCCATCAAT 3480
- CCTGGTGACA GCTCGTGCCG AATTCGGCAA CGAG 3514
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1185 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Leu Lys Ile Arg Ala Cys Leu Glu Lys Gln Pro Glu Pro Phe Ser Pro
1 5 10 15
Arg Gln Lys Lys Thr Leu Gln Glu Val Gln Glu Gly Phe Val Phe Ser
Lys Tyr Arg His Arg Ala Leu Leu Leu Pro Glu Leu Leu Glu Gly Val
Leu Gln Leu Leu Ile Ser Cys Leu Gln Ser Ala Ala Ser Asn Pro Phe
Tyr Phe Ser Gln Ala Met Asp Leu Val Gln Glu Phe Ile Gln His Gln
_ Gly Phe Asn Leu Phe Gly Thr Ala Val Leu Gln Met Glu Trp Leu Leu
Thr Arg Asp Gly Val Pro Ser Glu Ala Ala Glu His Leu Lys Ala Leu
CA 02244744 1998-07-29
W 097/28262 PCT~US97/01748J3~
100 105 110
Ile Asn Ser Val Ile Lys Ile Met Ser Thr Val Lys Lys Val Lys Ser
115 120 125
Glu Gln Leu His His Ser Met Cys Thr Arg Lys Arg His Arg Arg Cys
130 135 140
Glu Tyr Ser His Phe Met Gln His His Arg Asp Leu Ser Gly Leu Leu
145 150 155 160
~al Ser Ala Phe Lys Asn Gln Leu Ser Lys Ser Pro Phe Glu Glu Thr
165 170 175
~la Glu Gly Asp Val Gln Tyr Pro Glu Arg Cys Cys Cys Ile Ala Val
180 185 190
Cys Ala His Gln Cys Leu Arg Leu Leu Gln Gln Val Ser Leu Ser Thr
195 200 205
Thr Cys Val Gln Ile Leu Ser Gly Val His Ser Val Gly Ile Cys Cys
210 215 220
Cys Met Asp Pro Lys Ser Val Ile Ala Pro Leu Leu His Ala Phe Lys
225 230 235 240
~eu Pro Ala Leu Lys Ala Phe Gln Gln His lle Leu Asn Val Leu Ser
245 250 255
~ys Leu Leu Val Asp Gln Leu Gly Gly Ala Glu Leu Ser Pro Arg Ile
260 265 270
Lys Lys Ala Ala Cys Asn Ile Cys Thr Val Asp Ser Asp Gln Leu Ala
275 280 285
Lys Leu Gly Glu Thr Leu Gln Gly Thr Leu Cys Gly Ala Gly Pro Thr
290 295 300
Ser Gly Leu Pro Ser Pro Ser Tyr Arg Phe Gln Gly Ile Leu Pro Ser
305 310 315 320
~er Gly Ser Glu Asp Leu Leu Trp Lys Trp Asp Ala Leu Glu Ala Tyr
325 330 335
~ln Ser Phe Val Phe Gln Glu Asp Arg Leu His Asn Ile Gln Ile Ala
340 345 350
Asn His Ile Cys Asn Leu Leu Gln Lys Gly Asn Val Val Val Gln Trp
355 360 365
Lys Leu Tyr Asn Tyr Ile Phe Asn Pro Val Leu Gln Arg Gly Val Glu
370 375 380
Leu Val His His Cys Gln Gln Leu Ser Ile Pro Ser Ala Gln Thr His
385 390 395 400
~et Cys Ser Gln Leu Lys Gln Tyr Leu Pro Gln Glu Val Leu Gln Ile
405 410 415
~yr Leu Lys Thr Leu Pro Val Leu Leu Lys Ser Arg Val Ile Arg Asp
420 425 430
~eu Phe Leu Ser Cys Asn Gly Val Asn His Ile Ile Glu Leu Asn Tyr
435 440 445
CA 02244744 1998-07-29
W 097/28262 PCTrUS97/Q1748
1~1
Leu Asp Gly Ile Arg Ser His Ser Leu Lys Ala Phe Glu Thr Leu Ile
450 455, 460
Val Ser Leu Gly Glu Gln Gln Lys Asp Ala Ala Val Leu Asp Val Asp
465 470 475 480
Gly Leu Asp Ile Gln Gln Glu Leu Pro Ser Leu Ser Val Gly Pro Ser
485 490 495
Leu His Lys Gln Gln Ala Ser Ser Asp Ser Pro Cys Ser Leu Arg Lys
500 505 510
Phe Tyr Ala Ser Leu Arg Glu Pro Asp Pro Lys Lys Arg Lys Thr Ile
515 520 525
His Gln Asp Val His Ile Asn Thr Ile Asn Leu Phe Leu Cys Val Ala
530 535 540
Phe Leu Cys Val Ser Lys Glu Ala Asp Ser Asp Arg Glu Ser Ala Asn
545 550 555 560
Glu Ser Glu Asp Thr Ser Gly Tyr Asp Ser Pro Pro Ser Glu Pro Leu
565 570 575
Ser His Met Leu Pro Cys Leu Ser Leu Glu Asp Val Val Leu Pro Ser
580 585 590
Pro Glu Cys Leu His His Ala Ala Asp Ile Trp Ser Met Cys Arg Trp
595 600 605
Ile Tyr Met Leu Asn Ser Val Phe Gln Lys Gln Phe His Arg Leu Gly
610 615 620
Gly Phe Gln Val Cys His Glu Leu Ile Phe Met Ile Ile Gln Lys Leu
625 630 635 640
Phe Arg Ser His Thr Glu Asp Gln Gly Arg Arg Gln Gly Glu Met Ser
645 650 655
Arg Asn Glu Asn Gln Glu Leu Ile Arg Ile Ser Tyr Pro Glu Leu Thr
660 665 670
Leu Lys Gly Asp Val Ser Ser Ala Thr Ala Pro Asp Leu Gly Phe Leu
675 680 685
Arg Lys Ser Ala Asp Ser Val Arg Gly Phe Gln Ser Gln Pro Val Leu
690 695 700
Pro Thr Ser Ala Glu Gln Ile Val Ala Thr Glu Ser Val Pro Gly Glu
705 710 715 720
Arg Lys Ala Phe Met Ser Gln Gln Ser Glu Thr Ser Leu Gln Ser Ile
725 730 735
Arg Leu Leu Glu Ser Leu Leu Asp Ile Cys Leu His Ser Ala Arg Ala
740 745 750
Cys Gln Gln Lys Met Glu Leu Glu Leu Pro Ser Gln Gly Leu Ser Val
~ 755 760 765
Glu Asn Ile Leu Cys Glu Leu Arg Glu His Leu Ser Gln Ser Lys Val
770 775 780
Ala Glu Thr Glu Leu Ala Lys Pro Leu Phe Asp Ala Leu Leu Arg Val
785 790 795 800
CA 02244744 l998-07-29
W097/28262 PCTAUS97/01748
132,
Ala Leu Gly Asn His Ser Ala ~Asp Leu Gly Pro Gly Asp Ala Val Thr
- 805 810 815
Glu Lys Ser His Pro Ser Glu Glu Glu Leu Leu Ser Gln Pro Gly Asp
820 825 830
Phe Ser Glu Glu Ala Glu Asp Ser Gln Cys Cys Ser Leu Lys Leu Leu
835 840 845
Gly Glu Glu Glu Gly Tyr Glu Ala Asp Ser Glu Ser Asn Pro Glu Asp
850 855 860
Val Asp Thr Gln Asp Asp Gly Val Glu Leu Asn Pro Glu Ala Glu Gly
865 870 875 880
Phe Ser Gly Ser Ile Val Ser Asn Asn Leu Leu Glu Asn Leu Thr His
885 890 895
Gly Glu Ile Ile Tyr Pro Glu Ile Cys Met Leu Gly Leu Asn Leu Leu
900 905 910
Ser Ala Ser Lys Ala Lys Leu Asp Val Leu Ala His Val Phe Glu Ser
915 920 925
Phe Leu Lys Ile Val Arg Gln Lys Glu Lys Asn Ile Ser Leu Leu Ile
930 935 940
Gln Gln Gly Thr Val Lys Ile Leu Leu Gly Gly Phe Leu Asn Ile Leu
945 950 955 960
Thr Gln Thr Asn Ser Asp Phe Gln Ala Cys Gln Arg Val Leu Val Asp
965 970 975
Leu Leu Val Ser Leu Met Ser Ser Arg Thr Cys Ser Glu Asp Leu Thr
980 985 990
Leu Leu Trp Arg Ile Phe Leu Glu Lys Ser Pro Cys Thr Glu Ile Leu
995 1000 1005
Leu Leu Gly ~le His Lys Ile Val Glu Ser Asp Phe Thr Met Ser Pro
1010 1015 1020
Ser Gln Cys Leu Thr Phe Pro Phe Leu His Thr Pro Ser Leu Ser Asn
1025 1030 1035 1040
Gly Val Leu Ser Gln Lys Pro Pro Gly Ile Leu Asn Ser Lys Ala Leu
1045 1050 1055
Gly Leu Leu Arg Arg Ala Arg Ile Ser Arg Gly Lys Lys Glu Ala Asp
1060 1065 1070
Arg Glu Ser Phe Pro Tyr Arg Leu Leu Ser Ser Trp His Ile Ala Pro
1075 1080 1085
Ile His Leu Pro Leu Leu Gly Gln Asn Cys Trp Pro His Leu Ser Glu
1090 1095 1100
Gly Phe Ser Val Ser Leu Val Gly Leu Met Trp Asn Thr Ser Asn Glu
1105 1110 1115 1120
Ser Glu Ser Ala Ala Glu Arg Gly Lys Arg Val Lys Lys Arg Asn Lys
1125 1130 1135
Pro Ser Val Leu Glu Asp Ser Ser Phe Glu Gly Ala Gly Met Met Ala
-
CA 02244744 1998-07-29
W O 97/28262 PCTrUS97/01748
1~3
1140 1145 1150
Gly Ser Asp Leu Tyr Thr Lys Ile Leu Gln Ile Ala Ala Cys Leu Ser
1155 1160 1165
Phe Lys His Ile Trp Gln Tyr Phe Asn Val Phe Phe Lys Cys Tyr Ser
1170 1175 1180
Pro
1185
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11817 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
TGAGAGCTCA CGCTGGCCTG GCAGCCTTGG TGAGTCGGGA TTCTCCTGCA CCGGCGGGCG 60
AGAGCGCGCG GCGGACCACA GAGCGGAGGT GAAGCCTTAT GCTGAGACAG TTTTATCTAG 120
TTCATGAACC CAAATTATAT ACAAGCTGAA TGTTACAGAA GTGCTGAAAG ACTGCTCTGT 180
CATGAGCACG GACAGCAACT CATTGGCACG TGAGTTTCTG ATTGATGTCA ACCAGCTTTG 240
CAATGCAGTG GTCCAGAGGG CAGAAGCCAG GGAAGAAGAA GAAGAGGAGA CACACATGGC 300
A~CTCTTGGA CAGTACCTTG TCCATGGACG AGGATTTCTG TTACTTACCA AACTAAATTC 360
TATCATTGAT CAGGCCCTGA CATGCAGAGA AGAACTCCTG ACTCTTCTTC TGTCGCTCCT 420
TCCCTTGGTG TGGAAGATAC CTGTCCAGGA ACAGCAGGCA ACAGATTTTA ACCTGCCACT 480
GTCATCTGAT ATAATCCTGA CCAAAGAAAA GAACTCAAGT TTGCAAAAAT CAACTCAGGG 540
AAAATTATAT TTAGAAGGAA GTGCTCCATC TGGTCAGGTT TCTGCAAAAG TAAACCTTTT 600
TCGAAAAATC AGGCGACAGC GTAAAAGTAC CCATCGTTAT TCTGTAAGAG ATGCAAGAAA 660
GACACAGCTC TCCACCTCTG ACTCCGAAGG CAACTCAGAT GAAAAGAGTA CGGTTGTGAG 720
TAAACACAGG AGGCTCCACG CGCTGCCACG GTTCCTGACG CAGTCTCCTA AGGAAGGCCA 780
CCTCGTAGCC AAACCTGACC CCTCTGCCAC CAAAGAACAG GTCCTTTCTG ACACCATGTC 840
TGTGGAAAAC TCCAGAGAAG TCATTCTGAG ACAGGATTCA AATGGTGACA TATTAAGTGA 900
GCCAGCTGCT TTGTCTATTC TCAGTAACAT GAATAATTCT CCTTTTGACT TATGTCATGT 960
TTTGTTATCT CTATTGGAAA AAGTTTGTAA GTTTGACATT GCTTTGAATC ATAATTCTTC 1020
CCTAGCACTC AGTGTAGTAC CCA Q CTGAC TGAGTTCCTA GCAGGCTTTG GGGACTGCTG 1080
TAACCAGAGT GA QCTTTGG AGGGA QACT GGTTTCTGCA GGTTGGACAG AAGAGCCGGT 1140
AGCTTTGGTT CAACGGATGC TCTTTCGAAC CGTGCTGCAC CTTATGTCAG TAGACGTTAG lZ00
CACTGCAGAG GCAATGC Q G AAAGTCTTAG GAAAAATTTG ACTGAATTGC TTAGGG QGC 1260
TTTAAAAATT AGAGCTTGCT TGGAAAAG Q GCCTGAGCCT TTCTCCCCGA GACAAAAGAA 13Z0
CA 02244744 l998-07-29
W O 97/28262 PCTnUS97/01748
AACACTACAG GAGGTCCAGG AGGGCTTTGT ~ATTTTCCAAG TATCGTCACC GAGCCCTTCT 1380
ACTACCTGAG CTTCTGGAAG GAGTTCTACA GCTCCTCATC TCTTGTCTTC AGAGTGCAGC 1440
TTCAAATCCC TTTTACTTCA GTCAAGCCAT GGATTTAGTT CAAGAATTTA TCCAGCACCA 1500
AGGATTTAAT CTCTTTGGAA CAGCAGTTCT TCAGATGGAA TGGCTGCTTA CAAGGGACGG 1560
TGTTCCTTCA GAAGCTGCAG AACATTTGAA AGCTCTGATA AACAGTGTAA TAAAAATAAT 1620
GAGTACTGTG AAAAAGGTGA AATCAGAGCA ACTTCATCAT TCCATGTGCA CAAGGAAAAG 1680
ACACCGGCGT TGTGAGTATT CCCACTTCAT GCAGCACCAC CGCGATCTTT CAGGGCTCCT 1740
GGTTTCAGCT TTTAAAAATC AGCTTTCTAA AAGCCCCTTT GAAGAGACCG CAGAGGGAGA 1800
TGTGCAGTAT CCAGAGCGCT GCTGCTGCAT CGCCGTGTGC GCTCACCAGT GCTTGCGCTT 1860
GCTGCAGCAG GTTTCCCTGA GCACCACGTG TGTCCAGATC CTATCAGGTG TACACAGTGT 1920
TGGAATCTGT TGTTGTATGG ATCCTAAGTC TGTGATCGCC CCTTTACTGC ATGCTTTTAA 1980
GTTGCCAGCA CTGAAAGCTT TCCAGCAGCA TATACTGAAT GTCCTGAGCA AACTTCTTGT 2040
GGATCAGTTA GGAGGAGCAG AGCTATCACC GAGAATTAAA AAAGCAGCTT GCAACATCTG 2100
TACTGTGGAC TCTGACCAAC TGGCTAAGTT AGGAGAGACA CTGCAAGGCA CCTTGTGTGG 2160
TGCTGGTCCT ACCTCCGGCT TGCCCAGTCC TTCCTACCGA TTTCAGGGGA TCCTGCCCAG 2220
CAGCGGCTCT GAAGACTTGC TGTGGAAGTG GGATGCATTA GAGGCTTATC AGAGCTTTGT 2280
CTTTCAAGAA GACAGATTAC ATAACATTCA GATTGCAAAT CAC~TTTGTA ATTTACTCCA 2340
GAAAGGCAAT GTAGTTGTTC AGTGGAAATT GTATAATTAT ATCTTTAATC CTGTGCTCCA 2400
AAGAGGAGTT GAATTAGTAC ATCATTGTCA ACAGCTAAGC ATTCCTTCAG CTCAGACTCA 2460
CATGTGTAGC CAACTGAAAC AGTATTTGCC TCAGGAAGTG CTTCAGATTT ATTTAAAAAC 2520
TCTACCTGTC CTACTTAAAT CCAGGGTAAT AAGAGATTTG TTTTTAAGTT GTAATGGAGT 2580
AAACCACATA ATTGAACTAA ATTACTTAGA TGGGATTCGA AGTCATTCCC TGAAAGCATT 2640
TGAAACTCTG ATTGTCAGCC TAGGGGAACA ACAGAAAGAT GCTGCAGTTC TAGACGTCGA 2700
TGGGTTAGAC ATCCAACAGG AGTTGCCGTC CTTAAGTGTG GGTCCTTCTC TTCATAAGCA 2760
GCAAGCTTCT TCAGATTCTC CTTGCAGTCT CAGGAAGTTT TATGCCAGCC TCAGAGAGCC 2820
TGATCCAAAA AAACGAAAGA CCATTCACCA GGATGTTCAC ATAAACACCA TAAACCTCTT 2880
CCTCTGTGTG GCTTTTCTAT GTGTCAGTAA AGAAGCAGAC TCTGATAGGG AGTCTGCCAA 2940
TGAGTCAGAA GATACTTCTG GCTATGACAG CCCTCCCAGT GAGCCATTAA GTCACATGCT 3000
ACCATGTCTG TCTCTTGAGG ACGTTGTCTT ACCTTCCCCT GAATGTTTGC ACCATGCAGC 3060
AGACATTTGG TCCATGTGTC GTTGGATCTA CATGTTGAAC TCAGTCTTCC AGAAACAATT 3120
TCACAGGCTT GGTGGTTTCC AAGTGTGCCA TGAATTAATA TTTATGATAA TCCAGAAACT 3180
ATTCAGAAGT CATACAGAGG ATCAAGGAAG AAGGCAGGGA GAAATGAGTA GAAATGAAAA 3240
CA 02244744 1998-07-29
W 097/28262 PCT~US97/01748
1~
CCAAGAGCTA ATCAGGATAT CTTACCCCGA GCTGACACTG AAGGGAGATG TATCATCTGC 3300
AACAGCACCA GACCTGGGAT TTCTGAGAAA GAGTGCTGAC AGCGTGCGTG GATTCCAGTC 3360
ACAGCCTGTG CTTCCCACAA GTGCAGAGCA GATTGTGGCT ACTGAATCTG TTCCTGGGGA 3420
ACGAAAGGCA TTTATGAGTC AACAAAGTGA GACTTCTCTC CAGAGCATAC GACTTTTGGA 3480
GTCTCTCCTG GACATTTGTC TTCATAGTGC CAGAGCCTGT CAACAGAAGA TGGAATTGGA 3540
GCTACCGTCT CAGGGCTTGT CTGTGGAAAA TATATTGTGT GAACTGAGGG AACACCTTTC 3600
CCAGTCAAAG GTGGCAGAAA CAGAATTAGC AAAGCCTTTA TTTGATGCCC TGCTTCGAGT 3660
AGCCCTGGGG AATCATTCAG CAGATTTGGG CCCTGGTGAT GCTGTGACTG AGAAGAGTCA 3720
TCCCTCTGAG GAAGAGCTGT TGTCCCAGCC CGGAGATTTT TCAGAAGAAG CTGAGGATTC 3780
TCAGTGTTGT AGTTTGAAAC TTCTGGGTGA GGAAGAAGGC TATGAAGCGG ATAGTGAAAG 3840
CAATCCTGAG GATGTTGACA CCCAAGACGA TGGAGTAGAA TTAAATCCTG AAGCAGAAGG 3900
TTTCAGTGGA TCGATTGTTT CAAACAACTT ACTTGAAAAC CTCACTCACG GGGAAATAAT 3960
ATACCCTGAG ATTTGCATGC TGGGATTAAA TTTGCTTTCT GCTAGCAAAG CTA~ACTTGA 4020
TGTGCTTGCT CATGTGTTTG AGAGCTTTCT GAAAATTGTC AGGCAGAAGG AAAAGAACAT 4080
TTCTCTCCTC ATACAACAGG GAACTGTGAA AATCCTTCTA GGCGGGTTCT TGAATATTTT 4140
AACACAAACT AACTCTGATT TCCAAGCATG CCAGAGAGTA CTGGTGGATC TCTTGGTATC 4200
TTTGATGAGC TCAAGAACGT GTTCAGAAGA CTTAACTCTT CTTTGGAGAA TATTTCTGGA 4260
GAAATCTCCT TGTACAGAAA TTCTTCTCCT TGGTATTCAC AAAATTGTTG AAAGTGATTT 4320
TACTATGAGC CCTTCACAGT GTCTGACCTT TCCTTTCCTG CATACCCCGA GTTTAAGCAA 4380
TGGTGTCTTA TCACAGAAAC CTCCTGGGAT TCTTAACAGT AAAGCCTTAG GCTTATTGAG 4440
AAGAGCACGG ATTTCCCGAG GCAAGAAAGA GGCTGATAGA GAGAGTTTTC CCTATAGGCT 4500
GCTTTCCTCT TGGCATATAG CCCCAATCCA CCTGCCGTTG CTGGGACAGA ACTGCTGGCC 4560
ACACCTGTCA GAAGGATTTA GTGTTTCTCT TGTGGGTTTA ATGTGGAATA CATCCAATGA 4620
ATCCGAGAGT GCTGCAGAAA GGGGAAAAAG AGTAAAGAAA AGAAACAAAC CATCAGTTCT 4680
GGAAGACAGC AGTTTTGAAG GAGCAGAAGG TGATAGACCA GAAGTTACAG AATCCATCAA 4740
TCCTGGTGAC AGACTCATAG AAGACGGCTG TATTCATTTG ATTTCACTGG GGTCCAAAGC 4800
ATTGATGATC CAAGTGTGGG CTGATCCCCA CAGTGGCACT TTTATCTTTC GTGTGTGCAT 4860
GGACTCAAAT GATGACACGA AAGCTGTCTC ACTAGCACAG GTGGAATCAC AGGAGAATAT 4g20
TTTCTTTCCA AGCAAATGGC AACACTTAGT ACTTACCTAT ATTCAGCATC CTCAAGGGAA 4980
AAAGAATGTC CATGGGGAAA TCTCCATATG GGTCTCTGGG CAGAGGAAGA CTGATGTCAT 5040
CTTGGATTTT GTGCTCCCAA GAAAAACAAG CTTATCATCA GACAGCAATA A~ACATTTTG 5100
CATGATTGGT CATTGCTTAA CATCCCAAGA AGAGTCTCTG CAATTAGCTG GAAAATGGGA 5160
CCTGGGGAAC TTGCTCCTCT TCAATGGAGC TAAAATTGGC TCACAAGAGG CcTTTTTccT 5220
CA 02244744 l998-07-29
W 097/28262 PCTnUS97/01748
1~
GTATGCTTGT GGACCCAACT ACACATCCAT ,CATGCCGTGT AAATATGGAC AGCCAGTCAT 5280
TGACTACTCC AAATACATTA ATAAAGACAT TTTGAGATGT GATGAAATCA GAGACCTTTT 5340
TATGACCAAG AAAGAAGTGG ATGTTGGTCT CTTAATTGAA AGTCTTTCAG TTGTTTATAC 5400
AACTTGCTGT CCTGCTCAGT ACACCATCTA TGAACCAGTG ATTCGACTCA AGGGCCAAGT 5460
GAAAACTCAG CCCTCTCAAA GACCCTTCAG CTCAPAGGAA GCCCAGAGCA TCTTGCTAGA 5520
ACCTTCTCAA CTCAAAGGCC TCCAACCTAC GGAATGTAAA GCCATCCAGG GCATTCTGCA 5580
TGAGATTGGT GGGGCTGGCA CATTTGTTTT TCTCTTTGCT AGGGTTGTTG AACTTAGTAG 5640
CTGTGAAGAA ACTCAAGCAT TAGCACTGCG GGTTATACTG TCTTTAATTA AGTACAGCCA 5700
ACAGAGAACA CAGGAACTGG AAAATTGTAA TGGACTCTCT ATGATTCACC AAGTGTTGGT 5760
CAAACAGAAA TGCATTGTTG GCTTTCACAT TTTGAAGACC CTTCTTGAAG GTTGCTGCGG 5820
TGAAGAAGTT ATCCACGTCA GTGAGCATGG AGAGTTCAAG CTGGATGTTG AGTCTCATGC 5880
TATAATCCAA GATGTTAAGC TGCTGCAGGA ACTGTTACTT GACTGGAAGA TATGGAATAA 5940
GGCAGAGCAA GGTGTGTGGG AGACTCTGCT AGCAGCTTTG GAAGTCCTCA TCCGGGTAGA 6000
GCACCACCAG CAGCAGTTTA ATATTAAGCA GTTGCTGAAC GCCCACGTGG TTCACCACTT 6060
CCTACTGACC TGTCAGGTTT TACAGGAACA CAGAGAGGGG CAGCTTACAT CTATGCCCCG 6120
AGAAGTTTGT AGATCATTTG TGAAAATCAT TGCAGAAGTC CTTGGTTCTC CTCCAGACTT 6180
GGAATTATTG ACAGTTATTT TCAATTTCCT GTTAGCTGTA CACCCTCCTA CTAATACTTA 6240
TGTTTGTCAC AATCCCACAA ACTTCTACTT CTCTTTGCAC ATAGATGGCA AGATCTTTCA 6300
GGAGAAAGTG CAGTCACTCG CGTACCTGAG GCATTCTAGC AGCGGAGGGC AAGCCTTTCC 6360
CAGCCCTGGA TTCCTGGTAA TAAGCCCATC TGCCTTTACT GCAGCTCCTC CTGAAGGAAC 6420
CAGTTCTTCC AATATTGTTC CACAGCGGAT GGCTGCTCAG ATGGTTCGAT CTAGAAGTCT 6480
ACCAGCATTT CCTACTTATT TACCACTAAT ACGAGCACAA AAACTGGCTG CAAGTTTGGG 6540
TTTTAGTGTT GACAAGTTAC AAAATATTGC AGATGCCAAC CCAGAGAAAC AGAATCTTTT 6600
AGGAAGACCC TACGCACTGA AAACAAGCAA AGAGGAAGCA TTCATCAGCA GCTGTGAGTC 6660
TGCAAAGACT GTTTGTGAAA TGGAGGCTCT TCTTGGAGCC CACGCCTCTG CCAATGGGGT 6720
TTCCAGAGGA TCACCGAGGT TCCCCAGGGC CAGAGTAGAT CACAAAGATG TGGGAACAGA 6780
GCCCAGATCA GATGATGACA GTCCTGGGGA TGAGTCTTAC CCACGTCGGC CTGACAACCT 6840
CAAGGGACTG GCCTCATTCC AGCGAAGCCA AAGCACTGTC GCAAGCCTTG GGCTGGCGTT 6900
TCCCTCTCAG AATGGATCTG CAGTTGCTAG CAGGTGGCCA AGTCTTGTTG ATAGGAATGC 6960
TGATGACTGG GAGAACTTTA CCTTTTCTCC TGCTTATGAG GCAAGCTACA ACCGAGCCAC 7020
AAGCACCCAC AGTGTCATTG AAGACTGTCT GATACCTATC TGCTGTGGAT TATATGAACT 7080
CTTAAGTGGG GTTCTTCTTG TCCTGCCTGA TGCTATGCTT GAAGATGTGA TGGACAGGAT 7140
CA 02244744 l998-07-29
W 0 97/28262 PCTrUS97/01748
13'~
TATTCAAGCA GATATTCTTC TAGTCCTTGT TAACCACCCA TCACCTGCTA TCCAGCAAGG 7200
AGTAATTAAA CTGTTACATG CATACATTAA TAGAGCATCA AAGGAGCAAA AGGACAAGTT 7260
TCTGAAGAAC CGTGGCTTTT CCTTATTAGC CAACCAGTTG TATCTTCATA GGGGAACTCA 7320
GGAGTTGTTG GAGTGCTTTG TTGAAATGTT CTTTGGTCGA CCGATTGGCC TGGATGAAGA 7380
ATTTGATCTG GAGGAAGTGA AGCACATGGA ACTGTTCCAG AAGTGGTCTG TCATTCCCGT 7440
TCTCGGACTA ATAGAGACCT CTCTCTATGA CAATGTCCTC TTGCACAATG CTCTTTTACT 7500
TCTTCTGCAA GTTTTAAACT CTTGTTCCAA GGTAGCAGAC ATGCTACTGG ACAATGGTCT 7560
ACTCTATGTA TTATGTAATA CAGTAGCAGC CCTGAATGGA TTAGAAAAGA ACATTCCTGT 7620
GAACGAATAC AAATTGCTCG CATGTGATAT ACAGCAGCTT TTCATAGCAG TTACAATTCA 7680
TGCTTGCAGT TCCTCAGGCA CACAGTATTT TAGAGTGATT GAAGACCTTA TTGTACTTCT 7740
TGGATATCTT CATAATAGCA AAAACAAGAG GACACAAAAT ATGGCTTTGG CCCTGCAGCT 7800
TAGAGTTCTC CAGGCTGCTT TGGAATTTAT AAGGAGCACA GCCAATCATG ACTCTGAAAG 7860
TCCAGTGCAC TCGCCTTCTG CCCACCGCCA TTCAGTGCCT CCGAAGCGGA GAAGCATTGC 7920
TGGTTCTCGC AAATTCCCTC TGGCTCAGAC AGAGTCTCTG CTGATGAAGA TGCGCTCAGT 7980
GGCCAGCGAT GAGCTACACT CTATGATGCA GAGGAGGATG AGCCAAGAGC ACCCCAGCCA 8040
GGCCTCGGAG GCAGAGCTCG CTCAGAGGCT GCAGAGGCTC ACCATCTTAG CTGTGAACAG 8100
GATTATTTAC CAAGAGTTGA ATTCAGATAT TATTGACATT TTGAGAACTC CAGAAAATAC 8160
ATCCCAAAGC AAGACCTCAG TTTCTCAGAC TGAAATTTCT GAAGAAGACA TGCATCATGA 8220
GCAACCTTCT GTATATAATC CATTTCAAAA AGAAATGTTA ACCTATCTGT TGGATGGCTT 8280
CAAAGTGTGT ATTGGTTCAA GTAAAACTAG CGTTTCTAAG CAGCAGTGGA CTAAAATCCT 8340
GGGGTCTTGT AAAGAAACCC TCCGAGACCA GCTTGGAAGA TTGCTAGCGC ATATTTTGTC 8400
TCCAACCCAC ACTGTACAAG AACGGAAGCA GATACTTGAG ATAGT'rCATG AACCAGCTCA 8460
CCAGGATATA CTTCGTGACT GTCTTAGCCC CTCCCCACAA CATGGAGCCA AGTTGGTTTT 8520
GTATTTGTCA GAGTTGATAC ATAATCATCA GGATGAGTTA AGTGAAGAAG AAATGGACAC 8580
AGCAGAACTG CTTATGAATG CTCTAAAGTT ATGTGGCCAC AAGTGCATCC CGCCCAGTGC 8640
CCCTTCCAAA CCAGAGCTCA TTAAGATCAT CAGAGAGGAG CAAAAGAAGT ATGAAAGTGA 8700
AGAGAGTGTG AGCAAAGGCT CATGGCAGAA AACGGTGAAC AACAACCAGC AAAGTCTCTT 8760
C Q GAGGCTC GATTTCAAAT CCAAGGATAT ATCTAAAATC GCTGCAGACA TCACCCAGGC 8820
TGTATCACTC TCCCAAGGCA TTGAAAGGAA GAAGGTGATC CAGCACATCA GAGGGATGTA 8880
CAAAGTTGAC CTGAGTGCCA GCAGGCACTG GCAGGAATGC ATCCAGCAGC TGACACATGA 8940
CAGAGCAGTC TGGTATGACC CAATCTACTA TCCAACTTCA TGGCAGTTGG ATCCAACAGA 9000
AGGGCCAAAC CGAGAGAGGA GACGTTTGCA GAGATGCTAT CTAACTATTC CCAATAAGTA 9060
CCTCCTGAGG GACAGACAGA AGTCAGAAGG TGTGCTCAGG CCCCCACTCT CTTACCTTTT 9120
CA 02244744 1998-07-29
WO 97/28262 PCTtUS97tO1748
13~
TGAAGATAAA ACTCATTCTT CCTTCTCCTC TACTGTCAAA GACAAAGCTG CA~GTGAATC 9180
CATCAGAGTG AATCGAAGAT GTATCAGTGT TGCACCATCT AGAGAGACAG CTGGGGAATT 9240
GTTGTTAGGT AAATGTGGGA TGTACTTTGT GGAAGACAAT GCCTCTGACG CAGTTGAAAG 9300
CTCGAGCCTC CAAGGGGAGT TAGAGCCGGC ATCATTTTCT TGGACATATG AGGAAATTAA 9360
AGAAGTTCAC AGGCGCTGGT GGCAACTAAG AGATAATGCT GTAGAAATCT TTTTAACAAA 9420
TGGCAGAACA CTCCTATTAG CATTTGACAA TAACAAGGTT CGTGATGACG TGTACCAGAG 9480
CATCCTCACA AATAACCTCC CAAATCTTCT GGAGTACGGC AACATCACCG CTCTGACAAA 9540
CCTGTGGTAT TCTGGACAAA TTACCAATTT TGAATATTTG ACTCATTTAA ACAAGCATGC 9600
GGGCCGGTCC TTCAATGATC TCATGCAGTA CCCGGTGTTC CCCTTCATCC TTTCTGACTA 9660
TGTTAGTGAG ACTCTTGACC TCAATGATCC ATCTATCTAC AGAAACCTAT CTA~GCCTAT 9720
AGCTGTGCAG TATAAAGAAA AAGAAGACCG TTACGTTGAC ACATACA~GT ACTTGGAGGA 9780
GGAGTATCGC AAGGGAGCTC GAGAGGATGA CCCCATGCCT CCTGTGCAAC CCTACCACTA 9840
TGGCTCCCAC TACTCCAACA GCGGCACCGT GCTCCACTTC CTGGTCAGGA TGCCGCCTTT 9900
CACTAAAATG TTTCTAGCCT ATCAAGATCA GAGTTTCGAC ATTCCAGACC GAACATTTCA 9960
TTCTACAAAC ACAACTTGGC GCCTCTCCTC CTTTGAGTCC ATGACTGATG TGAAGGAGCT 10020
GATTCCAGAG TTTTTCTATC TTCCTGAGTT CTTAGTGAAC CGTGAAGGCT TTGACTTCGG 10080
TGTTCGTCAG AATGGAGAGC GGGTTAACCA CGTCAATCTT CCTCCCTGGG CACGCAACGA 10140
TCCTCGGCTG TTCATCCTTA TTCACCGGCA AGCACTAGAG TCTGACCATG TGTCCCAGAA 10200
CATCTGTCAC TGGATCGACT TAGTGTTTGG CTACAAGCAA AAGGGGAAGG CGTCTGTTCA 10260
AGCCATCAAT GTCTTCCACC CTGCTACATA TTTTGGAATG GATGTCTCTG CAGTTGAAGA 10320
TCCAGTGCAG AGACGGGCTT TAGAAACCAT GATAAAAACC TACGGGCAGA CCCCACGTCA 10380
GTTGTTCCAC ACAGCCCATG C QGCCGACC TGGAGCCAAG CTTAACATCG AAGGAGAGCT 10440
TCCAGCAGCT GTTGGCTTGT TAGTCCAGTT CGCTTTCAGA GAGACCCGAG AACCAGTCAA 10500
GGAAGTCACT CATCCGAGCC CTTTGTCATG GATAAAAGGC TTGAAGTGGG GGGAGTACGT 10560
AGGTTCCCCC AGTGCTCCAG TACCTGTGGT CTGCTTCAGC CAGCCCCATG GAGAAAGATT 10620
TGGTTCCCTG CAGGCACTGC CCACCAGAGC CATCTGTGGT TTATCACGAA ACTTCTGTCT 10680
TCTGATGACC TACAACAAGG AGCAAGGTGT GAGAAGCATG AACAACACCA ATATTCAGTG 107gO
GTCTGCTATC CTAAGCTGGG GATATGCTGA CAACATCTTA CGGTTGAAAA GTAAGCAGAG 10800
TGAGCCACCA ATCAACTTCA TTCAGAGTTC ACAGCAGCAC CAGGTAACCA GTTGTGCCTG 10860
GGTGCCTGAC AGTTGTCAGC TCTTCACTGG GAGCAAGTGT GGTGTCATCA CAGCCTATAC 10920
CAACAGGCTC ACCAGCAGCA CGCCCTCAGA AATTGAAATG GAGAGTCAGA TGCATCTCTA 10980
TGGACACACA GAGGAGATCA CCGGCTTATG TGTCTGCAAG CCGTACAGCG TGATGATAAG 11040
-
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CGTGAGCAGA GACGGGACCT GCATAGTATG GGACCTGAAC AGGCTGTGCT ATGTACAAAG 11100
- TTTGGCTGGA CACAAAAGCC CTGTGACGGC TGTCTCTGCC AGTGAAACGT CAGGTGACAT 11160
TGCTACTGTG TGTGACTCAG CTGGCGGGGG CAGTGACCTG AGACTCTGGA CCGTGAATGG 11220
GGACCTCGTT GGACATGTCC ACTGCAGAGA GATCATTTGT TCTGTAGCTT TCTCCAACCA 11280
GCCTGAGGGA GTCTC QTCA ACGTCATTGC TGGGGGATTA GAAAATGGCA TTGTAAGGCT 11340
ATGGAGCACA TGGGACTTGA AGCCTGTGAG AGAGATTACA TTTCCCAAAT CAAATAAGCC 11400
CATCATAAGC CTGACATTCT CCTGTGATGG CCACCATTTG TACACTGCCA ACAGTGAGGG 11460
GACAGTGATC GCATGGTGCC GGAAGGACCA GCAGCGTGTG AAGCTGCCCA TGTTCTACTC 11520
TTTCCTCAGC AGCTACGCAG CTGGATGAAG AGAAGGAGTG TCCCCACAGG ACATAAGCAC 11580
CGCTCTGCGA GCCTGGCTCC ACCAACTGCA GAAGCAGATG ACTGAGCAGA TATCCAGGAA 11640
AGACAACACA CGTGCCTCTG TGCGCGCTTC CCCAGCCTCC GTGGGCCTGA GAGTAAAGCC 11700
CTGCCCTCAT TCCATAATGG CGTGGAAGGC TGGGTCTGCA CACACTAGCC AATTAAAGTC 11760
AGAATCTTGA TGCTTTTTCC CAAAAGGTTA GGCTGAATCA AAGATCAGGC TCGTGCC 11817
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3788 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D! TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Met Ser Thr Asp Ser Asn Ser Leu Ala Arg Glu Phe Leu Ile Asp Val
1 5 10 15
Asn Gln Leu Cys Asn Ala Val Val Gln Arg Ala Glu Ala Arg Glu Glu
Glu Glu Glu Glu Thr His Met Ala Thr Leu Gly Gln Tyr Leu Val His
Gly Arg Gly Phe Leu Leu Leu Thr Lys Leu Asn Ser Ile Ile Asp Gln
Ala Leu Thr Cys Arg Glu Glu Leu Leu Thr Leu Leu Leu Ser Leu Leu
Pro Leu Val Trp Lys Ile Pro Val Gln Glu Gln Gln Ala Thr Asp Phe
Asn Leu Pro Leu Ser Ser Asp Ile Ile Leu Thr Lys Glu Lys Asn Ser
100 105 110
Ser Leu Gln Lys Ser Thr Gln Gly Lys Leu Tyr Leu Glu Gly Ser Ala
115 120 125
_ Pro Ser Gly Gln Val Ser Ala Lys Val Asn Leu Phe Arg Lys Ile Arg 130 135 140
Arg Gln Arg Lys Ser Thr His Arg Tyr Ser Val Arg Asp Ala Arg Lys
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145 150 155 160
Thr Gln Leu Ser Thr Ser Asp Ser Glu Gly Asn Ser Asp Glu Lys Ser
165 170 175
~hr Val Val Ser Lys His Arg Arg Leu His Ala Leu Pro Arg Phe Leu
180 185 190
Thr Gln Ser Pro Lys Glu Gly His Leu Val Ala Lys Pro Asp Pro Ser
195 200 205
Ala Thr Lys Glu Gln Val Leu Ser Asp Thr Met Ser Val Glu Asn Ser
210 215 220
Arg Glu Val Ile Leu Arg Gln Asp Ser Asn Gly Asp Ile Leu Ser Glu
225 230 235 240
~ro Ala Ala Leu Ser Ile Leu Ser Asn Met Asn Asn Ser Pro Phe Asp
245 250 255
~eu Cys His Val Leu Leu Ser Leu Leu Glu Lys Val Cys Lys Phe Asp
260 265 270
Ile Ala Leu Asn His Asn Ser Ser Leu Ala Leu Ser Val Val Pro Thr
275 Z80 285
Leu Thr Glu Phe Leu Ala Gly Phe Gly Asp Cys Cys Asn Gln Ser Asp
290 295 300
Thr Leu Glu Gly Gln Leu Val Ser Ala Gly Trp Thr Glu Glu Pro Val
305 310 315 .. 320
~la Leu Val Gln Arg Met Leu Phe Arg Thr Val Leu His Leu Met Ser
325 330 335
~al Asp Val Ser Thr Ala Glu Ala Met Pro Glu Ser Leu Arg Lys Asn
340 345 350
Leu Thr Glu Leu Leu Arg Ala Ala Leu Lys Ile Arg Ala Cys Leu Glu
355 360 365
Lys Gln Pro Glu Pro Phe Ser Pro Arg Gln Lys Lys Thr Leu Gln Glu
370 375 380
Val Gln Glu Gly Phe Val Phe Ser Lys Tyr Arg His Arg Ala Leu Leu
385 390 395 400
~eu Pro Glu Leu Leu Glu Gly Val Leu Gln Leu Leu Ile Ser Cys Leu
405 410 415
~ln Ser Ala Ala Ser Asn Pro Phe Tyr Phe Ser Gln Ala Met Asp Leu
420 425 430
Val Gln Glu Phe Ile Gln His Gln Gly Phe Asn Leu Phe Gly Thr Ala
435 440 445
Val Leu Gln Met Glu Trp Leu Leu Thr Arg Asp Gly Val Pro Ser Glu
450 455 460
Ala Ala Glu Xis Leu Lys Ala Leu Ile Asn Ser Val Ile Lys Ile Met
465 470 475 480
Ser Thr Val Lys Lys Val Lys Ser Glu Gln Leu His Xis Ser Met Cys
485 490 495
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Thr Arg Lys Arg His Arg Arg Cys Glu Tyr Ser His Phe Met Gln His
500 ~ 505 510
His Arg Asp Leu Ser Gly Leu Leu Val Ser Ala Phe Lys Asn Gln Leu
515 520 525
Ser Lys Ser Pro Phe Glu Glu Thr Ala Glu Gly Asp Val Gln Tyr Pro
530 535 540
Glu Arg Cys Cys Cys Ile ALa Val Cys ALa His Gln Cys Leu Arg Leu
545 550 555 560
Leu Gln Gln Val Ser Leu Ser Thr Thr Cys Val Gln Ile Leu Ser Gly
565 570 575
Val His Ser Val Gly Ile Cys Cys Cys Met Asp Pro Lys Ser Val Ile
580 585 590
Ala Pro Leu Leu His ALa Phe Lys Leu Pro ALa Leu Lys Ala Phe Gln
595 600 605
Gln His Ile Leu Asn Val Leu Ser Lys Leu Leu Val Asp Gln Leu Gly
610 615 620
Gly Ala Glu Leu Ser Pro Arg Ile Lys Lys Ala Ala Cys Asn Ile Cys
625 630 635 640
Thr Val Asp Ser Asp Gln Leu ALa Lys Leu Gly Glu Thr Leu Gln Gly
645 650 655
Thr Leu Cys Gly Ala Gly Pro Thr Ser Gly Leu Pro Ser Pro Ser Tyr
660 665 670
Arg Phe Gln GLy Ile T eu Pro Ser Ser GLy Ser Glu Asp Leu Leu Trp
675 680 685
Lys Trp Asp Ala Leu Glu ALa Tyr Gln Ser Phe Val Phe Gln Glu Asp
690 695 700
Arg Leu Hls Asn Ile Gln Ile ALa Asn His Ile Cys Asn Leu Leu Gln
705 710 715 720
Lys Gly Asn Val Val Val Gln Trp Lys Leu Tyr Asn Tyr Ile Phe Asn
725 730 735
Pro Val Leu Gln Arg Gly Val Glu Leu Val His His Cys Gln Gln Leu
740 745 750
Ser Ile Pro Ser ALa Gln Thr His Met Cys Ser Gln Leu Lys Gln Tyr
755 760 765
Leu Pro Gln Glu Val Leu Gln Ile Tyr Leu Lys Thr Leu Pro Val Leu
770 775 780
Leu Lys Ser Arg Val Ile Arg Asp Leu Phe Leu Ser Cys Asn Gly Val
785 790 795 800
Asn His Ile Ile Glu Leu Asn Tyr Leu Asp Gly Ile Arg Ser His Ser
805 810 815
Leu Lys Ala Phe Glu Thr Leu Ile Val Ser Leu Gly Glu Gln Gln Lys
820 825 830
Asp Ala Ala Val Leu Asp Val Asp Gly Leu Asp Ile Gln Gln Glu Leu
835 840 845
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Pro Ser Leu Ser Val Gly Pro Ser Leu Hi s Lys Gln Gln Ala Ser Ser
- 850 855 860
Asp Ser Pro Cys Ser Leu Arg Lys Phe Tyr Ala Ser Leu Arg Glu Pro
865 870 875 880
Asp Pro Lys Lys Arg Lys Thr ILe His Gln Asp Val His Ile Asn Thr
885 890 895
Ile Asn Leu Phe Leu Cys Val Ala Phe Leu Cys Val Ser Lys Glu Ala
900 905 910
Asp Ser Asp Arg Glu Ser Ala Asn Glu Ser Glu Asp Thr Ser Gly Tyr
915 920 925
Asp Ser Pro Pro Ser Glu Pro Leu Ser His Met Leu Pro Cys Leu Ser
930 935 940
Leu Glu Asp Val Val Leu Pro Ser Pro Glu Cys Leu His His Ala Ala
945 950 955 960
Asp Ile Trp Ser Met Cys Arg Trp Ile Tyr Met Leu Asn Ser Val Phe
965 970 975
Gln Lys Gln Phe His Arg Leu Gly Gly Phe Gln Val Cys His Glu Leu
980 985 990
Ile Phe Met Ile Ile Gln Lys Leu Phe Arg Ser His Thr Glu Asp Gln
995 1000 1005
Gly Arg Arg Gln Gly Glu Met Ser Arg Asn Glu Asn Gln Glu Leu Ile
1010 1015 1020
Arg Ile Ser Tyr Pro Glu Leu Thr Leu Lys Gly Asp Val Ser Ser Ala
1025 1030 1035 1040
Thr Ala Pro Asp Leu Gly Phe Leu Arg Lys Ser Ala Asp Ser Val Arg
1045 1050 1055
Gly Phe Gln Ser Gln Pro Val Leu Pro Thr Ser Ala Glu Gln Ile Val
1060 1065 1070
Ala Thr Glu Ser Val Pro Gly Glu Arg Lys Ala Phe Met Ser Gln Gln
1075 1080 1085
Ser Glu Thr Ser Leu Gln Ser Ile Arg Leu Leu Glu Ser Leu Leu Asp
1090 1095 llO0
Ile Cys Leu His Ser Ala Arg Ala Cys Gln Gln Lys Met Glu Leu Glu
1105 lllO 1115 1120
Leu Pro Ser Gln Gly Leu Ser Val Glu Asn Ile Leu Cys Glu Leu Arg
1125 1130 1135
Glu His Leu Ser Gln Ser Lys Val Ala Glu Thr Glu Leu Ala Lys Pro
1140 1145 1150
Leu Phe ~sp Ala Leu Leu Arg Val Ala Leu Gly Asn His Ser Ala Asp
1155 1160 1165
Leu Gly Pro Gly Asp Ala Val Thr Glu Lys Ser His Pro Ser Glu Glu
1170 1175 1180
Glu Leu Leu Ser Gln Pro Gly Asp Phe Ser Glu Glu Ala Glu Asp Ser
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.1185 1190 1195 1200
Gln Cys Cys Ser Leu Lys Leu Leu Gly Glu Glu Glu Gly Tyr Glu Ala
1205 1210 1215
~sp Ser Glu Ser Asn Pro Glu Asp Val Asp Thr Gln Asp Asp Gly Val
1220 1225 1230
Glu Leu Asn Pro Glu Ala Glu Gly Phe Ser Gly Ser Ile Val Ser Asn
1235 1240 1245
Asn Leu Leu Glu Asn Leu Thr His Gly Glu Ile Ile Tyr Pro Glu Ile
1250 1255 1260
Cys Met Leu Gly Leu Asn Leu Leu Ser Ala Ser Lys Ala Lys Leu Asp
1265 1270 1275 1280
~al Leu Ala His Val Phe Glu Ser Phe Leu Lys Ile Val Arg Gln Lys
1285 1290 1295
~lu Lys Asn Ile Ser Leu Leu Ile Gln Gln Gly Thr Val Lys Ile Leu
1300 1305 1310
Leu Gly Gly Phe Leu Asn Ile Leu Thr Gln Thr Asn Ser Asp Phe Gln
1315 1320 1325
Ala Cys Gln Arg Val Leu Val Asp Leu Leu Val Ser Leu Met Ser Ser
1330 1335 1340
Arg Thr Cys Ser Glu Asp Leu Thr Leu Leu Trp Arg Ile Phe Leu Glu
1345 1350 1355 136~)
~ys Ser Pro Cys Thr Glu Ile Leu Leu Leu Gly Ile His Lys Ile Val
1365 1370 1375
~lu Ser Asp Phe Thr Met Ser Pro Ser Gln Cys Leu Thr Phe Pro Phe
1380 1385 1390
Leu His Thr Pro Ser Leu Ser Asn Gly Val Leu Ser Gln Lys Pro Pro
1395 1400 1405
Gly Ile Leu Asn Ser Lys Ala Leu Gly Leu Leu Arg Arg Ala Arg Ile
1410 1415 1420
Ser Arg Gly Lys Lys Glu Ala Asp Arg Glu Ser Phe Pro Tyr Arg Leu
1425 1430 1435 1440
~eu Ser Ser Trp His Ile Ala Pro Ile His Leu Pro Leu Leu Gly Gln
1445 1450 1455
~sn Cys Trp Pro His Leu Ser Glu Gly Phe Ser Val Ser Leu Val Gly
1460 1465 1470
Leu Met Trp Asn Thr Ser Asn Glu Ser Glu Ser Ala Ala Glu Arg Gly
1475 1480 1485
Lys Arg Val Lys Lys Arg Asn Lys Pro Ser Val Leu Glu Asp Ser Ser
1490 1495 1500
Phe Glu Gly Ala Glu Gly Asp Arg Pro Glu Val Thr Glu Ser Ile Asn
1505 1510 1515 1520
Pro Gly Asp Arg Leu Ile Glu Asp Gly Cys Ile His Leu Ile Ser Leu
1525 1530 1535
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Gly Ser Lys Ala Leu Met Ile Gln Val Trp Ala Asp Pro His Ser Gly
1540 , 1545 1550
Thr Phe Ile Phe Arg Val Cys Met Asp Ser Asn Asp Asp Thr Lys Ala
1555 1560 1565
Val Ser Leu Ala Gln Val Glu Ser Gln Glu Asn Ile Phe Phe Pro Ser
1570 1575 1580
Lys Trp Gln His Leu Val Leu Thr Tyr Ile Gln His Pro Gln Gly Lys
1585 1590 1595 1600
~ys Asn Val His Gly Glu Ile Ser Ile Trp Val Ser Gly Gln Arg Lys
1605 1610 1615
~hr Asp Val Ile Leu Asp Phe Val Leu Pro Arg Lys Thr Ser Leu Ser
1620 1625 1630
Ser Asp Ser Asn Lys Thr Phe Cys Met Ile Gly His Cys Leu Thr Ser
1635 1640 1645
Gln Glu Glu Ser Leu Gln Leu Ala Gly Lys Trp Asp Leu Gly Asn Leu
1650 1655 1660
Leu Leu Phe Asn Gly Ala Lys Ile Gly Ser Gln Glu Ala Phe Phe Leu
1665 1670 1675 1680
~yr Ala Cys Gly Pro Asn Tyr Thr Ser Ile Met Pro Cys Lys Tyr Gly
1685 1690 1695
~ln Pro Val Ile Asp Tyr Ser Lys Tyr Ile Asn Lys Asp Ile Leu Arg
1700 1705 1710
Cys Asp Glu Ile Arg Asp Leu Phe Met Thr Lys Lys Glu Val Asp Val
1715 1720 1725
Gly Leu Leu Ile Glu Ser Leu Ser Val Val Tyr Thr Thr Cys Cys Pro
1730 1735 1740
Ala Gln Tyr Thr Ile Tyr Glu Pro Val Ile Arg Leu Lys Gly Gln Val
1745 1750 1755 1760
~ys Thr Gln Pro Ser Gln Arg Pro Phe Ser Ser Lys Glu Ala Gln Ser
1765 1770 1775
~le Leu Leu Glu Pro Ser Gln Leu Lys Gly Leu Gln Pro Thr Glu Cys
1780 1785 1790
Lys Ala Ile Gln Gly Ile Leu His Glu Ile Gly Gly Ala Gly Thr Phe
1795 1800 1805
Val Phe Leu Phe Ala Arg Val Val Glu Leu Ser Ser Cys Glu Glu Thr
1810 1815 1820
Gln Ala Leu Ala Leu Arg Val Ile Leu Ser Leu Ile Lys Tyr Ser Gln
1825 1830 1835 1840
~ln Arg Thr Gln Glu Leu Glu Asn Cys Asn Gly Leu Ser Met Ile His
1845 1850 1855
~ln Val Leu Val Lys Gln Lys Cys Ile Val Gly Phe His Ile Leu Lys
1860 1865 1870
~hr Leu Leu Glu Gly Cys Cys Gly Glu Glu Val Ile His Val Ser Glu
1875 1880 1885
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~ His Gly Glu Phe Lys Leu Asp Val Glu Ser His Ala Ile Ile Gln Asp
_ 1890 1895 1900
Val Lys Leu Leu Gln Glu Leu Leu Leu Asp Trp Lys Ile Trp Asn Lys
1905 1910 1915 = 1920
Ala Glu Gln Gly Val Trp Glu Thr Leu Leu ALa Ala Leu Glu Val Leu
1925 1930 1935
Ile Arg Val Glu His His Gln Gln Gln Phe Asn Ile Lys Gln Leu Leu
1940 1945 1950
Asn ALa His Val Val His His Phe Leu Leu Thr Cys Gln Val Leu Gln
1955 1960 1965
Glu His Arg Glu Gly Gln Leu Thr Ser Met Pro Arg Glu Val Cys Arg
1970 1975 1980
Ser Phe Val Lys Ile Ile ALa Glu Val Leu Gly Ser Pro Pro Asp Leu
1985 1990 1995 2000
Glu Leu Leu Thr Val Ile Phe Asn Phe Leu Leu Ala Val His Pro Pro
2005 2010 2015
Thr Asn Thr Tyr Val Cys His Asn Pro Thr Asn Phe Tyr Phe Ser Leu
2020 2025 2030
His ILe Asp Gly Lys Ile Phe Gln Glu Lys VaL Gln Ser Leu Ala Tyr
2035 2040 2045
Leu Arg His Ser Ser Ser Gly Gly Gln Ala Phe Pro Ser Pro Gly Phe
2050 2055 2060
Leu Val Ile Ser Pro Ser Ala Phe Thr Ala Ala Pro Pro Glu Gly Thr
2065 2070 2075 2080
Ser Ser Ser Asn Ile Val Pro Gln Arg Met Ala ALa GLn Met Val Arg
2085 2090 2095
Ser Arg Ser Leu Pro Ala Phe Pro Thr Tyr Leu Pro Leu Ile Arg ALa
2100 2105 2110
Gln Lys Leu Ala ALa Ser Leu Gly Phe Ser Val Asp Lys Leu Gln Asn
2115 2120 2125
Ile ALa Asp Ala Asn Pro Glu Lys Gln Asn Leu Leu Gly Arg Pro Tyr
2130 2135 2140
ALa Leu Lys Thr Ser Lys Glu Glu Ala Phe Ile Ser Ser Cys Glu Ser
2145 2150 2155 2160
Ala Lys Thr Val Cys Glu Met Glu Ala Leu Leu Gly Ala His Ala Ser
2165 2170 2175
Ala Asn Gly Val Ser Arg Gly Ser Pro Arg Phe Pro Arg ALa Arg Val
2180 2185 2190
~ Asp His Lys Asp Val Gly Thr Glu Pro Arg Ser Asp Asp Asp Ser Pro
2195 2200 2205
_ Gly Asp Glu Ser Tyr Pro Arg Arg Pro Asp Asn Leu Lys Gly Leu Ala 2210 2215 2220
Ser Phe Gln Arg Ser Gln Ser Thr Val Ala Ser Leu Gly Leu Ala Phe
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222~ 2230 2235 2240
Pro Ser Gln Asn Gly Ser Ala Val Ala Ser Arg Trp Pro Ser Leu Val
2245 2250 2255
Asp Arg A5n Ala Asp Asp Trp Glu Asn Phe Thr Phe Ser Pro Ala Tyr
2260 2265 22~0
Glu Ala Ser Tyr Asn Arg Ala Thr Ser Thr His Ser Val Ile Glu Asp
2275 2280 2285
Cys Leu Ile Pro Ile Cys Cys Gly Leu Tyr Glu Leu Leu Ser Gly Val
2290 2295 2300
Leu Leu Val Leu Pro Asp Ala Met Leu Glu Asp Val Met Asp Arg Ile
2305 2310 2315 2320
Ile Gln Ala Asp Ile Leu Leu Val Leu Val Asn His Pro Ser Pro Ala
2325 2330 2335
Ile Gln Gln Gly Val Ile Lys Leu Leu His Ala Tyr Ile Asn Arg Ala
2340 2345 2350
Ser Lys Glu Gln Lys Asp Lys Phe Leu Lys Asn Arg Gly Phe Ser Leu
2355 2360 2365
Leu Ala Asn Gln Leu Tyr Leu His Arg Gly Thr Gln Glu Leu Leu Glu
2370 2375 2380
Cys Phe Val Glu Met Phe Phe Gly Arg Pro Ile Gly Leu Asp Glu Glu
2385 2390 2~95 2400
Phe Asp Leu Glu Glu Val Lys His Met Glu Leu Phe Gln Lys Trp Ser
'7405 241~) 2415
Val Il~ Pro Val Leu Gly Leu Ile Glu Thr Ser Leu Tyr Asp Asn Val
242;~ 2425 2430
Leu Leu His Asn Ala Leu Leu Leu Leu Leu Gln Val Leu Asn Ser Cys
2435 2440 2445
Ser Lys Val Ala Asp Met Leu Leu Asp Asn Gly Leu Leu Tyr Val Leu
2450 2455 2460
Cys Asn Thr Val Ala Ala Leu Asn Gly Leu Glu Lys Asn Ile Pro Val
2465 2470 2475 2480
Asn Glu Tyr Lys Leu Leu Ala Cys Asp Ile Gln Gln Leu Phe Ile Ala
2485 2490 2495
Val Thr Ile His Ala Cys Ser Ser Ser Gly Thr Gln Tyr Phe Arg Val
2500 2505 2510
Ile Glu Asp Leu Ile Val Leu Leu Gly Tyr Leu ~is Asn Ser Lys Asn
2515 2520 2525
Lys Arg Thr Gln Asn Met Ala Leu Ala Leu Gln Leu Arg Val Leu Gin
2530 2535 2540
Ala Ala Leu Glu Phe Ile Arg Ser Thr Ala Asn His Asp Ser Glu Ser
2545 2550 2555 2560
Pro Val His Ser Pro Ser Ala His Arg His Ser Val Pro Pro Lys Arg
2565 2570 2575
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Arg Ser Ile Ala Gly Ser Arg Lys Phe Pro Leu Ala Gln Thr Glu Ser
2580 2585 2590
Leu Leu Met Lys Met Arg Ser Val Ala Ser Asp Glu Leu His Ser Met
2595 2600 2605
Met Gln Arg Arg Met Ser Gln Glu His Pro Ser Gln Ala Ser Glu Ala
2610 2615 2620
Glu Leu Ala Gln Arg Leu Gln Arg Leu Thr Ile Leu Ala Val Asn Arg
2625 2630 2635 2640
Ile Ile Tyr Gln Glu Leu Asn Ser Asp Ile Ile Asp Ile Leu Arg Thr
2645 2650 2655
Pro Glu Asn Thr Ser Gln Ser Lys Thr Ser Val Ser Gln Thr Glu Ile
2660 2665 2670
Ser Glu Glu Asp Met His His Glu Gln Pro Ser Val Tyr Asn Pro Phe
2675 2680 2685
Gln Lys Glu Met Leu Thr Tyr Leu Leu Asp Gly Phe Lys Val Cys Ile
2690 2695 2700
Gly Ser Ser Lys Thr Ser Val Ser Lys Gln Gln Trp Thr Lys Ile Leu
2705 2710 2715 2720
Gly Ser Cys Lys Glu Thr Leu Arg Asp Gln Leu Gly Arg Leu Leu Ala
2725 2730 2735
His Ile Leu Ser Pro Thr His Thr Val Gln Glu Arg Lys Gln Ile Leu
2740 2745 2750
Glu Ile Val His Glu Pro Ala His Glrl Asp Ile Leu Arg Asp Cys Leu
2755 2760 2765
Ser Pro Ser Pro Gln His Gly Ala Lys Leu Val Leu Tyr Leu Ser Glu
2770 2775 2780
Leu Ile His Asn His Gln Asp Glu Leu Ser Glu Glu Glu Met Asp Thr
2785 2790 2795 2800
Ala Glu Leu Leu Met Asn Ala Leu Lys Leu Cys Gly His Lys Cys Ile
2805 2810 2815
Pro Pro Ser Ala Pro Ser Lys Pro Glu Leu Ile Lys Ile Ile Arg Glu
2820 2825 2830
Glu Gln Lys Lys Tyr Glu Ser Glu Glu Ser Val Ser Lys Gly Ser Trp
2835 2840 2845
Gln Lys Thr Val Asn Asn Asn Gln Gln Ser Leu Phe Gln Arg Leu Asp
2850 2855 2860
Phe Lys Ser Lys Asp Ile Ser Lys Ile Ala Ala Asp Ile Thr Gln Ala
2865 2870 2875 2880
Val Ser Leu Ser Gln Gly Ile Glu Arg Lys Lys Val Ile Gln His Ile
2885 2890 2895
Arg Gly Met Tyr Lys Val Asp Leu Ser Ala Ser Arg His Trp Gln Glu
2900 2905 2910
Cys Ile Gln Gln Leu Thr His Asp Arg Ala Val Trp Tyr Asp Pro Ile
2915 2920 2925
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Tyr Tyr Pro Thr Ser Trp Gln Leu Asp Pro Thr Glu Gly Pro Asn Arg
- 2930 2935 2940
Glu Arg Arg Arg Leu Gln Arg Cys Tyr Leu Thr Ile Pro Asn Lys Tyr
2945 2950 2955 2960
Leu Leu Arg Asp Arg Gln Lys Ser Glu Gly Val Leu Arg Pro Pro Leu
2965 2970 2975
Ser Tyr Leu Phe Glu Asp Lys Thr His Ser Ser Phe Ser Ser Thr Val
2980 2985 2990
Lys Asp Lys Ala Ala Ser Glu Ser Ile Arg Val Asn Arg Arg Cys Ile
2995 3000 3005
Ser Val Ala Pro Ser Arg Glu Thr Ala Gly Glu Leu Leu Leu Gly Lys
3010 3015 30Z0
Cys Gly Met Tyr Phe Val Glu Asp Asn Ala Ser Asp Ala Val Glu Ser
3025 3030 3035 3040
Ser Ser Leu Gln Gly Glu Leu Glu Pro Ala Ser Phe Ser Trp Thr Tyr
3045 3050 3055
Glu Glu Ile Lys Glu Val His Arg Arg Trp Trp Gln Leu Arg Asp Asn
3060 3065 3070
Ala Val Glu Ile Phe Leu Thr Asn Gly Arg Thr Leu Leu Leu Ala Phe
3075 3080 3085
Asp Asn Asn Lys Val Arg Asp Asp Val Tyr Gln Ser Ile Leu Thr Asn
3090 3095 3100
Asn Leu Pro Asn Leu Leu Glu Tyr Gly Asn Ile Thr Ala Leu Thr Asn
3105 3110 3115 3120
Leu Trp Tyr Ser Gly Gln Ile Thr Asn Phe Glu Tyr Leu Thr His Leu
3125 3130 3135
Asn Lys His Ala Gly Arg Ser Phe Asn Asp Leu Met Gln Tyr Pro Val
3140 3145 - 3150
Phe Pro Phe Ile Leu Ser Asp Tyr Val Ser Glu Thr Leu Asp Leu Asn
3155 3160 3165
Asp Pro Ser Ile Tyr Arg Asn Leu Ser Lys Pro Ile Ala Val Gln Tyr
3170 3175 3180
Lys Glu Lys Glu Asp Arg Tyr Val Asp Thr Tyr Lys Tyr Leu Glu Glu
3185 3190 3195 3200
Glu Tyr Arg Lys Gly Ala Arg Glu Asp Asp Pro Met Pro Pro Val Gln
3205 3210 3215
Pro Tyr His Tyr Gly Ser His Tyr Ser Asn Ser Gly Thr Val Leu His
3220 3225 3230
Phe Leu Val Arg Met Pro Pro Phe Thr Lys Met Phe Leu Ala Tyr Gln
3235 3240 3245
Asp Gln Ser Phe Asp Ile Pro Asp Arg Thr Phe His Ser Thr Asn Thr
3250 3255 3260
Thr Trp Arg Leu Ser Ser Phe Glu Ser Met Thr Asp Val Lys Glu Leu
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3265 3270 3275 3280
Ile Pro Glu Phe Phe Tyr Leu Pro Glu Phe Leu Val Asn Arg Glu Gly
3285 3290 3295
Phe Asp Phe Gly Val Arg Gln Asn Gly Glu Arg Val Asn His Val Asn
3300 3305 3310
Leu Pro Pro Trp Ala Arg Asn Asp Pro Arg Leu Phe Ile Leu Ile His
3315 3320 3325
Arg Gln Ala Leu Glu Ser Asp His Val Ser Gln Asn Ile Cys His Trp
3330 3335 3340
Ile Asp Leu Val Phe Gly Tyr Lys Gln Lys Gly Lys Ala Ser Val Gln
3345 3350 3355 3360
Ala Ile Asn Val Phe His Pro Ala Thr Tyr Phe Gly Met Asp Val Ser
3365 3370 3375
Ala Val Glu Asp Pro Val Gln Arg Arg Ala Leu Glu Thr Met Ile Lys
3380 3385 3390
Thr Tyr Gly Gln Thr Pro Arg Gln Leu Phe His Thr Ala His Ala Ser
3395 3400 3405
Arg Pro Gly Ala Lys Leu Asn Ile Glu Gly Glu Leu Pro Ala Ala Val
3410 3415 3420
Gly Leu Leu Val Gln Phe Ala Phe Arg Glu Thr Arg Glu Pro Val Lys
3425 3430 3435 3440
Glu Val Thr His Pro Ser Pro Leu Ser Trp Ile Lys Gly Leu Lys Trp
3445 3450 3455
Gly Glu Tyr Val Gly Ser Pro Ser Ala Pro Val Pro Val Val Cys Phe
3460 3465 3470
Ser Gln Pro His Gly Glu Arg Phe Gly Ser Leu Gln Ala Leu Pro Thr
3475 3480 3485
Arg Ala Ile Cys Gly Leu Ser Arg Asn Phe Cys Leu Leu Met Thr Tyr
3490 3495 3500
Asn Lys Glu Gln Gly Val Arg Ser Met Asn Asn Thr Asn Ile Gln Trp
3505 3510 3515 3520
Ser Ala Ile Leu Ser Trp Gly Tyr Ala Asp Asn Ile Leu Arg Leu Lys
3525 3530 3535
Ser Lys Gln Ser Glu Pro Pro Ile Asn Phe Ile Gln Ser Ser Gln Gln
3540 3545 3550
His Gln Val Thr Ser Cys Ala Trp Val Pro Asp Ser Cys Gln Leu Phe
3555 3560 3565
Thr Gly Ser Lys Cys Gly Val Ile Thr Ala Tyr Thr Asn Arg Leu Thr
3570 3575 3580
Ser Ser Thr Pro Ser Glu Ile Glu Met Glu Ser Gln Met His Leu Tyr
3585 3590 3595 3600
Gly His Thr Glu Glu Ile Thr Gly Leu Cys Val Cys Lys Pro Tyr Ser
3605 3610 3615
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Val Met Ile Ser Val Ser Arg Asp Gly Thr Cys Ile Val Trp Asp Leu
3620 - 3625 3630
Asn Arg Leu Cys Tyr Val Gln Ser Leu Ala Gly His Lys Ser Pro Val
3635 3640 3645
Thr Ala Val Ser Ala Ser Glu Thr Ser Gly Asp Ile Ala Thr Val Cys
3650 3655 3660
Asp Ser Ala Gly Gly Gly Ser Asp Leu Arg Leu Trp Thr Val Asn Gly
3665 3670 3675 3680
Asp Leu Val Gly His Val His Cys Arg Glu Ile Ile Cys Ser Val Ala
3685 3690 3695
Phe Ser Asn Gln Pro Glu Gly Val Ser Ile Asn Val Ile Ala Gly Gly
3700 3705 3710
Leu Glu Asn Gly Ile Val Arg Leu Trp Ser Thr Trp Asp Leu Lys Pro
3715 3720 3725
Val Arg Glu Ile Thr Phe Pro Lys Ser Asn Lys Pro Ile Ile Ser Leu
3730 3735 3740
Thr Phe Ser Cys Asp Gly His His Leu Tyr Thr Ala Asn Ser Glu Gly
3745 3750 3755 3760
Thr Val Ile Ala Trp Cys Arg Lys Asp Gln Gln Arg Val Lys Leu Pro
3765 3770 3775
Met Phe Tyr Ser Phe Leu Ser Ser Tyr Ala Ala Gly
3780 3785
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHA~ACTERISTICS:
(A) LENGTH: 5893 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
TGAGAGCTCA CGCTGGCCTG GCAGCCTTGG TGAGTCGGGA TTCTCCTGCA CCGGCGGGCG 60
AGAGCGCGCG GCGGACCACA GAGCGGAGGT GAAGCCTTAT GCTGAGACAG TTTTATCTAG 120
TTCATGAACC CAAATTATAT ACAAGCTGAA TGTTACAGAA GTGCTGAAAG ACTGCTCTGT 180
CATGAGCACG GACAGCAACT CATTGG Q CG TGAGTTTCTG ATTGATGTCA ACCAGCTTTG 240
CAATGCAGTG GTCCAGAGGG CAGAAGCCAG GGAAGAAGAA GAAGAGGAGA CACACATGGC 300
AACTCTTGGA CAGTACCTTG TCCATGGACG AGGATTTCTG TTACTTACCA AACTA~ATTC 360
TATCATTGAT CAGGCCCTGA CATGCAGAGA AGAACTCCTG ACTCTTCTTC TGTCGCTCCT 420
TCCCTTGGTG TGGAAGATAC CTGTCCAGGA ACAGCAGGCA ACAGATTTTA ACCTGCCACT 480
GTCATCTGAT ATAATCCTGA CCAAAGAAAA GAACTCAAGT TTGCAAAAAT CAACTCAGGG 540
AAAATTATAT TTAGAAGGAA GTGCTCCATC TGGTCAGGTT TCTGCAAAAG TAAACCTTTT 600
TCGAAAAATC AGGCGACAGC GTAAAAGTAC CCATCGTTAT TcTGTAAGAG ATGCAAGAAA 660
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GACACAGCTC TCCACCTCTG ACTCCGAAGG CAACTCAGAT GAAAAGAGTA CGGTTGTGAG 720
TAAACACAGG AGGCTCCACG CGCTGCCACG GTTCCTGACG CAGTCTCCTA AGGAAGGCCA 780
CCTCGTAGCC AAACCTGACC CCTCTGCCAC CAAAGAACAG GTCCTTTCTG ACACCATGTC 840
TGTGGAAAAC TCCAGAGAAG TCATTCTGAG ACAGGATTCA AATGGTGACA TATTAAGTGA 900
GCCAGCTGCT TTGTCTATTC TCAGTAACAT GAATAATTCT CCTTTTGACT TATGTCATGT 960
TTTGTTATCT CTATTGGAAA AAGTTTGTAA GTTTGACATT GCTTTGAATC ATAATTCTTC 1020
CCTAGCACTC AGTGTAGTAC CCACACTGAC TGAGTTCCTA GCAGGCTTTG GGGACTGCTG 1080
TAACCAGAGT GACACTTTGG AGGGACAACT GGTTTCTGCA GGTTGGACAG AAGAGCCGGT 1140
AGCTTTGGTT CAACGGATGC TCTTTCGAAC CGTGCTGCAC CTTATGTCAG TAGACGTTAG 1200
CACTGCAGAG GCAATGCCAG AAAGTCTTAG GAAAAATTTG ACTGAATTGC TTAGGGCAGC 1260
TTTAAAAATT AGAGCTTGCT TGGAAAAGCA GCCTGAGCCT TTCTCCCCGA GACAAAAGAA 1320
AACACTACAG GAGGTCCAGG AGGGCTTTGT ATTTTCCAAG TATCGTCACC GAGCCCTTCT 1380
ACTACCTGAG CTTCTGGAAG GAGTTCTACA GCTCCTCATC TCTTGTCTTC AGAGTGCAGC 1440
TTCAAATCCC TTTTACTTCA GTCAAGCCAT GGATTTAGTT CAAGAATTTA TCCAGCACCA 1500
AGGATTTAAT CTCTTTGGAA CAGCAGTTCT TCAGATGGAA TGGCTGCTTA CAAGGGACGG 1560
TGTTCCTTCA GAAGCTGCAG AACATTTGAA AGCTCTGATA AACAGTGTAA TAAAAATAAT 1620
GAGTACTGTG AAAAAGGTGA AATCAGAGCA ACTTCATCAT TCCATGTGCA CAAGGAAAAG 1680
ACACCGGCGT TGTGAGTATT CCCACTTCAT GCAGCACCAC CGCGATCTTT CAGGGCTCCT 1740
GGTTTCAGCT TTTAAAAATC AG~lll~lAA AAGCCCCTTT GAAGAGACCG CAGAGGGAGA 1800
TGTGCAGTAT CCAGAGCGCT GCTGCTGCAT CGCCGTGTGC GCTCACCAGT GCTTGCGCTT 1860
GCTGCAGCAG GTTTCCCTGA GCACCACGTG TGTCCAGATC CTATCAGGTG TACACAGTGT 1920
TGGAATCTGT TGTTGTATGG ATCCTAAGTC TGTGATCGCC CCTTTACTGC ATGCTTTTAA 1980
GTTGCCAGCA CTGAAAGCTT TCCAGCAGCA TATACTGAAT GTCCTGAGCA AACTTCTTGT 2040
GGATCAGTTA GGAGGAGCAG AGCTATCACC GAGAATTAAA AAAGCAGCTT GCAACATCTG 2100
TACTGTGGAC TCTGACCAAC TGGCTAAGTT AGGAGAGACA CTGCAAGGCA CCTTGTGTGG 2160
TGCTGGTCCT ACCTCCGGCT TGCCCAGTCC TTCCTACCGA TTTCAGGGGA TCCTGCCCAG 2220
CAGCGGCTCT GAAGACTTGC TGTGGAAGTG GGATGCATTA GAGGCTTATC AGAGCTTTGT 2280
CTTTCAAGAA GACAGATTAC ATAACATTCA GATTGCAAAT CACATTTGTA ATTTACTCCA 2340
GAAAGGCAAT GTAGTTGTTC AGTGGAAATT GTATAATTAT ATCTTTAATC CTGTGCTCCA 2400
AAGAGGAGTT GAATTAGTAC ATCATTGTCA ACAGCTAAGC ATTCCTTCAG CTCAGACTCA 2460
CATGTGTAGC CAACTGAAAC AGTATTTGCC TCAGGAAGTG CTTCAGATTT ATTTAAAAAC 2520
TCTACCTGTC CTACTTAAAT CCAGGGTA~T AAGAGATTTG TTTTTAAGTT GTAATGGAGT 2580
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AAACCACATA ATTGAACTAA ATTACTTAGA TGGGATTCGA AGTCATTCCC TGAAAGCATT 2640
TGAAACTCTG ATTGTCAGCC TAGGGGAACA ACAGAAAGAT GCTGCAGTTC TAGACGTCGA 2700 r
TGGGTTAGAC ATCCAACAGG AGTTGCCGTC CTTAAGTGTG GGTCCTTCTC TTCATAAGCA 2760
GCAAGCTTCT TCAGATTCTC CTTGCAGTCT CAGGAAGTTT TATGCCAGCC TCAGAGAGCC 2820
TGATCCAAAA AAACGAAAGA CCATTCACCA GGATGTTCAC ATAAACACCA TAAACCTCTT 2880
CCTCTGTGTG GCTTTTCTAT GTGTCAGTAA AGAAGCAGAC TCTGATAGGG AGTCTGCCAA 2940
TGAGTCAGAA GATACTTCTG GCTATGACAG CCCTCCCAGT GAGCCATTAA GTCACATGCT 3000
ACCATGTCTG TCTCTTGAGG ACGTTGTCTT ACCTTCCCCT GAATGTTTGC AC QTGCAGC 3060
AGACATTTGG TCCATGTGTC GTTGGATCTA CATGTTGAAC TCAGTCTTCC AGAAACAATT 3120
TCACAGGCTT GGTGGTTTCC AAGTGTGCCA TGAATTAATA TTTATGATAA TCCAGAAACT 3180
ATTCAGAAGT CATACAGAGG ATCAAGGAAG AAGGCAGGGA GAAATGAGTA GAAATGAAAA 3240
CCAAGAGCTA ATCAGGATAT CTTACCCCGA GCTGACACTG AAGGGAGATG TATCATCTGC 3300
AACAGCACCA GACCTGGGAT TTCTGAGAAA GAGTGCTGAC AGCGTGCGTG GATTCCAGTC 3360
ACAGCCTGTG CTTCCCACAA GTGCAGAGCA GATTGTGGCT ACTGAATCTG TTCCTGGGGA 3420
ACGAAAGGCA TTTATGAGTC AACAAAGTGA GACTTCTCTC CAGAGCATAC GACTTTTGGA 3480
GTCTCTCCTG GACATTTGTC TTCATAGTGC CAGAGCCTGT CAACAGAAGA TGGAATTGGA 3540
GCTACCGTCT CAGGGCTTGT CTGTGGAAAA TATATTGTGT GAACTGAGGG AACACCTTTC 3600
CCAGTCAAAG GTGGCAGAAA CAGAATTAGC AAAGCCTTTA TTTGATGCCC TGCTTCGAGT 3660
AGCCCTGGGG AATCATTCAG CAGATTTGGG CCCTGGTGAT GCTGTGACTG AGAAGAGTCA 3720
TCCCTCTGAG GAAGAGCTGT TGTCCCAGCC CGGAGATTTT TCAGAAGAAG CTGAGGATTC 3780
TCAGTGTTGT AGTTTGAAAC TTCTGGGTGA GGAAGAAGGC TATGAAGCGG ATAGTGAAAG 3840
CAATCCTGAG GATGTTGACA CCCAAGACGA TGGAGTAGAA TTAAATCCTG AAGCAGAAGG 3900
TTTCAGTGGA TCGATTGTTT CAAACAACTT ACTTGAAAAC CTCACTCACG GGGAAATAAT 3960
ATACCCTGAG ATTTGCATGC TGGGATTAAA TTTGCTTTCT GCTAGCAAAG CTAAACTTGA 4020
TGTGCTTGCT CATGTGTTTG AGAGCTTTCT GAAAATTGTC AGGCAGAAGG AAAAGAACAT 4080
TTCTCTCCTC ATACAACAGG GAACTGTGAA AATCCTTCTA GGCGGGTTCT TGAATATTTT 4140
AACACAAACT AACTCTGATT TCCAAGCATG CCAGAGAGTA CTGGTGGATC TCTTGGTATC 4200
TTTGATGAGC TCAAGAACGT GTTCAGAAGA CTTAACTCTT CTTTGGAGAA TATTTCTGGA 4260
GAAATCTCCT TGTACAGAAA TTCTTCTCCT TGGTATTCAC AAAATTGTTG AAAGTGATTT 4320
TACTATGAGC CCTTCACAGT GTCTGACCTT TCCTTTCCTG CATACCCCGA GTTTAAGCAA 4380
TGGTGTCTTA TCACAGAAAC CTCCTGGGAT TCTTAACAGT AAAGCCTTAG GCTTATTGAG 4440
AAGAGCACGG ATTTCCCGAG GCAAGAAAGA GGCTGATAGA GAGAGTTTTC CCTATAGGCT 4500
GCTTTCCTCT TGGCATATAG CCCCAATCCA CCTGCCGTTG CTGGGACAGA ACTGCTGGCC 4560
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ACACCTGTCA GAAGGATTTA GTGTTTCTCT ~GTGGGTTTA ATGTGGAATA CATCCAATGA 4620
ATCCGAGAGT GCTGCAGAAA GGGGAAAAAG AGTAAAGAAA AGAAACAAAC CATCAGTTCT 4680
GGAAGACAGC AGTTTTGAAG GAGCAGGTAT GATGGCAGGG TCTGATCTAT ATACTAAGAT 4740
TCTTCAAATA GCTGCTTGCC TGAGTTTTAA GCATATCTGG CAGTATTTTA ATGTATTCTT 4800
TAAATGTTAT TCACCTTAAA GATCCTACTT CACTACTGAA TTACCAAAGC CTGAGTTTTC 4860
AAACAGCCTT GAAATCTTCA TTGTCTCTAA ACTTTAGATA GGGAAGTGGG GATGCTCTGT 4920
TTCTGCAACA GCTGTTGAAG TTAGCAGTCC CATGACTGTG TTAGTGTGGC TTCTGATACT 4980
AGATAGTTAT AAATAAAACC CTATGGC QT TTTTATTTTA AGTTCTCCTT CTGTGTCTTA 5040
CACCAATGGC CCCTTCTAGT TACTGTCCCT GATCATTTAT ATGTA~CAGT CCAAAGTTAG 5100
AACAGAGTTC ATCTGTAACT GAAGAACTGC TGTTAGGATG TACTGAAATT GAATTTTGTT 5160
TTTGTTCTCT T~ll~ AA GCAATCAACA ~l"l"l~l"l~AAG TCATATAGCA GCTAGAGGAA 5220
GTAGTCTTAA AAACTGGCTG TGTATTTTTT TAACCTGTTA AAAATGGTGG CTAATATTTT 5280
TATACCCTAA TAATTGATAA TGTTCCTCTT TTTTAAAAGT CTGAGCTTTT GGACATGCAC 5340
TGTTTATGTT AGTACATCTT AGCTTAGTTT AACATAAAGT CACATCATAG TAACAAATAG 5400
CTTATCACAC ATATTCCACC TGCCATTGCT GTCACAGATA ATGGGAATAT AGAGGCAACT 5460
CAAGATTTAA GTAGTAAGGT GCCATTGGGA GGGGTAAGCA GCTAGCTCAC AGCCATAAAC 5520
ACTTCTCTCA GCGGAGACAA ACTGTGATTC AGGGTTTGGC ATCACTTAGC ATGGTTATTT 5580
CAAGGTTGTT CACTACCTTA AATAATGATC ATTTGAGCAG TGCAGCTTTT CTAAGAAGAG 5640
TATTAATAAT ATTATAGATC GTGCCTTTGT AACAATTTTT TTAGTGCAAG GCATCTGTTG 5700
ATGGCATGTG CTCCCTGGGC CATGGTCAGT TGTGTTAGAG TGACCCAATC CAACA~AAGC 5760
AGAACCTTGG TATGGAGTGT GGCTGACGAT GGTCCTTTAG CACCCTCAGG CCTTGTAGTT 5820
TAAAGCATTT AATAACTTTT AAAACACTGG AGTCTTTAGT GAGGACCTGC CCGGGCGGCC 5880
GCCACCGCGG TGG 5893
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1545 amino acids
(B) TYPE: amino acid
(C) ST~ANDEDNESS:
(D) TOPOLOGY: linear
8 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Met Ser Thr Asp Ser Asn Ser Leu Ala Arg Glu Phe Leu Ile Asp Val
1 5 10 15
_ Asn Gln Leu Cys Asn Ala Val Val Gln Arg Ala Glu Ala Arg Glu Glu
20 25 30
Glu Glu Glu Glu Thr His Met Ala Thr Leu Gly Gln Tyr Leu Val His
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- Gly Arg Gly Phe Leu Leu Leu Thr Lys Leu Asn Ser Ile Ile Asp Gln
Ala Leu Thr Cys Arg Glu Glu Leu Leu Thr Leu Leu Leu Ser Leu Leu
Pro Leu Val Trp Lys Ile Pro Val Gln Glu Gln Gln Ala Thr Asp Phe
Asn Leu Pro Leu Ser Ser Asp Ile Ile Leu Thr Lys Glu Lys Asn Ser
100 105 110
Ser Leu Gln Lys Ser Thr Gln Gly Lys Leu Tyr Leu Glu Gly Ser Ala
115 lZ0 125
Pro Ser Gly Gln Val Ser Ala Lys Val Asn Leu Phe Arg Lys Ile Arg
130 135 140
Arg Gln Arg Lys Ser Thr His Arg Tyr Ser Val Arg Asp Ala Arg Lys
145 150 155 160
Thr Gln Leu Ser Thr Ser Asp Ser Glu Gly Asn Ser Asp Glu Lys Ser
165 170 175
Thr Val Val Ser Lys His Arg Arg Leu His Ala Leu Pro Arg Phe Leu
180 185 190
Thr Gln Ser Pro Lys Glu Gly His Leu Val Ala Lys Pro Asp Pro Ser
195 200 205
Ala Thr Lys Glu Gln Val Leu Ser Asp Thr Met Ser Val Glu Asn Ser
210 215 220
Arg Glu Val Ile Leu Arg Gln Asp Ser Asn Gly Asp Ile Leu Ser Glu
225 230 235 . 240
Pro Ala Ala Leu Ser Ile Leu Ser Asn Met Asn Asn Ser Pro Phe Asp
245 250 255
Leu Cys His Val Leu Leu Ser Leu Leu Glu Lys Val Cys Lys Phe Asp
260 265 270
Ile Ala Leu Asn His Asn Ser Ser Leu Ala Leu Ser Val Val Pro Thr
275 280 285
Leu Thr Glu Phe Leu Ala Gly Phe Gly Asp Cys Cys Asn Gln Ser Asp
290 295 300
Thr Leu Glu Gly Gln Leu Val Ser Ala Gly Trp Thr Glu Glu Pro Val
305 310 315 320
Ala Leu Val Gln Arg Met Leu Phe Arg Thr Val Leu His Leu Met Ser
325 330 335
Val Asp Val Ser Thr Ala Glu Ala Met Pro Glu Ser Leu Arg Lys Asn
340 345 350
Leu Thr Glu Leu Leu Arg Ala Ala Leu Lys Ile Arg~la Cys Leu Glu
355 360 365
Lys Gln Pro Glu Pro Phe Ser Pro Arg Gln Lys Lys Thr Leu Gln Glu
370 375 380
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Val Gln Glu Gly Phe Val Phe Ser Lys Tyr Arg His Arg Ala Leu Leu
385 390 395 400
Leu Pro Glu Leu Leu Glu Gly Val Leu Gln Leu Leu Ile Ser Cys Leu
~' 405 410 415
Gln Ser Ala Ala Ser Asn Pro Phe Tyr Phe Ser Gln Ala Met Asp Leu
420 425 430
Val Gln Glu Phe Ile Gln His Gln Gly Phe Asn Leu Phe Gly Thr Ala
435 440 445
Val Leu Gln Met Glu Trp Leu Leu Thr Arg Asp Gly Val Pro Ser Glu
450 455 460
Ala Ala Glu His Leu Lys Ala Leu Ile Asn Ser Val Ile Lys Ile Met
465 470 475 480
Ser Thr Val Lys Lys Val Lys Ser Glu Gln Leu His His Ser Met Cys
485 490 495
Thr Arg Lys Arg His Arg Arg Cys Glu Tyr Ser His Phe Met Gln His
500 505 510
His Arg Asp Leu Ser Gly Leu Leu Val Ser Ala Phe Lys Asn Gln Leu
515 520 525
Ser Lys Ser Pro Phe Glu Glu Thr Ala Glu Gly Asp Val Gln Tyr Pro
530 535 540
Glu Arg Cys Cys Cys Ile Ala Val Cys Ala His Gln Cys Leu Arg Leu
545 550 555 560
Leu Gln Gln Val Ser Leu Ser Thr Thr Cys Val Gln Ile Leu Ser Gly
565 570 575
Val His Ser Val Gly Ile Cys Cys Cys Met Asp Pro Lys Ser Val Ile
580 585 590
Ala Pro Leu Leu His Ala Phe Lys Leu Pro Ala Leu Lys Ala Phe Gln
595 600 605
Gln His Ile Leu Asn Val Leu Ser Lys Leu Leu Val Asp Gln Leu Gly
610 615 620
Gly Ala Glu Leu Ser Pro Arg Ile Lys Lys Ala Ala Cys Asn Ile Cys
625 630 635 640
Thr Val Asp Ser Asp Gln Leu Ala Lys Leu Gly Glu Thr Leu Gln Gly
645 650 655
Thr Leu Cys Gly Ala Gly Pro Thr Ser Gly Leu Pro Ser Pro Ser Tyr
660 665 670
Arg Phe Gln Gly Ile Leu Pro Ser Ser Gly Ser Glu Asp Leu Leu Trp
675 680 685
Lys Trp Asp Ala Leu Glu Ala Tyr Gln Ser Phe Val Phe Gln Glu Asp
690 695 700
Arg Leu His Asn Ile Gln Ile Ala Asn His Ile Cys Asn Leu Leu Gln
705 710 715 720
Lys Gly Asn Val Val Val Gln Trp Lys Leu Tyr Asn Tyr Ile Phe Asn
725 730 735
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f~
Pro Val Leu Gln Arg Gly VaL Glu Leu Val His His Cys Gln Gln Leu
740 745 750
Ser Ile Pro Ser Ala Gln Thr His Met Cys Ser Gln Leu Lys Gln Tyr
755 760 765
Leu Pro Gln Glu Val Leu Gln Ile Tyr Leu Lys Thr Leu Pro Val Leu
770 775 780
Leu Lys Ser Arg Val Ile Arg Asp Leu Phe Leu Ser Cys Asn Gly Val
785 790 795 800
~sn His Ile Ile Glu Leu Asn Tyr Leu Asp Gly Ile Arg Ser His Ser
805 810 815
~eu Lys Ala Phe Glu Thr Leu Ile Val Ser Leu Gly Glu Gln Gln Lys
820 825 830
Asp Ala Ala Val Leu Asp Val Asp Gly Leu Asp Ile Gln Gln Glu Leu
835 840 845
Pro Ser Leu Ser Val Gly Pro Ser Leu His Lys Gln Gln Ala Ser Ser
850 855 860
Asp Ser Pro Cys Ser Leu Arg Lys Phe Tyr Ala Ser Leu Arg Glu Pro
865 870 875 880
~sp Pro Lys Lys Arg Lys Thr Ile His Gln Asp Val His Ile Asn Thr
885 890 895
~le Asn Leu Phe Leu Cys Val Ala Phe Leu Cys Val Ser Lys Glu Ala
900 905 910
Asp Ser Asp Arg Glu Ser Ala Asn Glu Ser Glu Asp Thr Ser Gly Tyr
915 920 925
Asp Ser Pro Pro Ser Glu Pro Leu Ser His Met Leu Pro Cys Leu Ser
930 935 940
Leu Glu Asp Val Val Leu Pro Ser Pro Glu Cys Leu His His Ala Ala
945 950 955 960
~sp Ile Trp Ser Met Cys Arg Trp Ile Tyr Met Leu Asn Ser Val Phe
965 970 975
~ln Lys Gln Phe His Arg Leu Gly Gly Phe Gln Val Cys His Glu Leu
980 985 990
Ile Phe Met Ile Ile Gln Lys Leu Phe Arg Ser His Thr Glu Asp Gln
995 1000 1005
Gly Arg Arg Gln Gly Glu Met Ser Arg Asn Glu Asn Gln Glu Leu Ile
1010 1015 1020
Arg Ile Ser Tyr Pro Glu Leu Thr Leu Lys Gly Asp Val Ser Ser Ala
1025 1030 1035 1040
~hr Ala Pro Asp Leu Gly Phe Leu Arg Lys Ser Ala Asp Ser Val Arg
1045 1050 1055
~ly Phe Gln Ser Gln Pro Val Leu Pro Thr Ser Ala Glu Gln Ile Val
1060 1065 1070
~la Thr Glu Ser Val Pro Gly Glu Arg Lys Ala Phe Met Ser Gln Gln
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15~
1075 1080 1085
Ser Glu Thr Ser Leu Gln Ser Ile Arg Leu Leu Glu Ser Leu Leu Asp
1090 1095 1100
Ile Cys Leu His Ser Ala Arg Ala Cys Gln Gln Lys Met Glu Leu Glu
1105 1110 1115 1120
Leu Pro Ser Gln Gly Leu Ser Val Glu Asn Ile Leu Cys Glu Leu Arg
1125 1130 1135
Glu His Leu Ser Gln Ser Lys Val Ala Glu Thr Glu Leu Ala Lys Pro
1140 1145 1150
Leu Phe Asp Ala Leu Leu Arg Val Ala Leu Gly Asn His Ser Ala Asp
1155 1160 1165
Leu Gly Pro Gly Asp Ala Val Thr Glu Lys Ser His Pro Ser Glu Glu
1170 1175 1180
Glu Leu Leu Ser Gln Pro Gly Asp Phe Ser Glu Glu Ala Glu Asp Ser
1185 1190 1195 1200
Gln Cys Cys Ser Leu Lys Leu Leu Gly Glu Glu Glu Gly Tyr Glu Ala
1205 1210 1215
Asp Ser Glu Ser Asn Pro Glu Asp Val Asp Thr Gln Asp Asp Gly Val
1220 1225 1230
Glu Leu Asn Pro Glu Ala Glu Gly Phe Ser Gly Ser Ile Val Ser Asn
1235 1240 1245
Asn Leu Leu Glu Asn Leu Thr His Gly Glu Ile Ile Tyr Pro Glu Ile
1250 1255 1260
Cys Met Leu Gly Leu Asn Leu Leu Ser Ala Ser Lys Ala Lys Leu Asp
1265 1270 1275 . 1280
Val Leu Ala His Val Phe Glu Ser Phe Leu Lys Ile Val Arg Gln Lys
1285 1290 1295
Glu Lys Asn Ile Ser Leu Leu Ile Gln Gln Gly Thr Val Lys Ile Leu
1300 1305 1310
Leu Gly Gly Phe Leu Asn Ile Leu Thr Gln Thr Asn Ser Asp Phe Gln
1315 1320 1325
Ala Cys Gln Arg Val Leu Val Asp Leu Leu Val Ser Leu Met Ser Ser
1330 1335 1340
Arg Thr Cys Ser &lu Asp Leu Thr Leu Leu Trp Arg Ile Phe Leu Glu
1345 1350 1355 1360
Lys Ser Pro Cys Thr Glu Ile Leu Leu Leu Gly Ile His Lys Ile Val
1365 1370 1375
Glu Ser Asp Phe Thr Met Ser Pro Ser Gln Cys Leu Thr Phe Pro Phe
1380 1385 1390
Leu His Thr Pro Ser Leu Ser Asn Gly Val Leu Ser Gln Lys Pro Pro
1395 1400 1405
Gly Ile Leu Asn Ser Lys Ala Leu Gly Leu Leu Arg Arg Ala Arg Ile
1410 1415 1420
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~Ser Arg Gly Lys Lys Glu Ala Asp Arg Glu Ser Phe Pro Tyr Arg Leu
1425 1430 ' 1435 1440
Leu Ser Ser Trp His Ile Ala Pro Ile ~is Leu Pro Leu Leu Gly Gln
1445 1450 1455
Asn Cys Trp Pro His Leu Ser Glu Gly Phe Ser Val Ser Leu Val Gly
1460 1465 1470
Leu Met Trp Asn Thr Ser Asn Glu Ser Glu Ser Ala Ala Glu Arg Gly
1475 1480 1485
Lys Arg Val Lys Lys Arg Asn Lys Pro Ser Val Leu Glu Asp Ser Ser
1490 1495 1500
Phe Glu Gly Ala Gly Met Met Ala Gly Ser Asp Leu Tyr Thr Lys Ile
1505 1510 1515 1520
Leu Gln Ile Ala Ala Cys Leu Ser Phe Lys His Ile Trp Gln Tyr Phe
1525 1530 1535
Asn Val Phe Phe Lys Cys Tyr Ser Pro
1540 1545
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7080 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
CGCAAGGGCT TCTAAGAAGC CATCCCAATG ACCTTTTGGC TTTGAGAAGA GCAGTCCTCA 60
TACCAGAGTG TTTGGGGTTT TGGCCTCTTT CAGTGTTTAT TCATTCTTAC GTGGGAAAGT 120
TGTATTCCGA GGTTTCTGTG GTGCATGAAG CTTTTGCCTT CACCATCTGT TCCCGTGTCT 180
TCCTCGGGTG ACATCAGAGT ACAGCAGTAT TTTCCCTTGC CATCTAATGG GGTTTGGGCT 240
GTTTGACTCA ACCGTGTGTG TTCCTCAATG CCAGGGGAAT AATCCTACCC TAAGTCAGCT 300
GAACAGAAGC CAGGATCTAA CTGCAAACAA GAGACCCAGC TTGCTTAACA GCATGGAAGA 360
GAACCAGTTT CCTTGCAGCT ACCTGGGAAG ACGGTTGCTA ATTAGCCTGC AACAAAGAGT 420
TCCTTGCTCA TCTAAAAGAG GCAATCACCG TTCAGGTGAA GCTTTGTTCT AAGAATATTT 480
GTTTCATCTA GTTTATGAGT CCAAATGATA TAGACTGTAA ATGTCACAGC AGTGGTGAAA 540
GACTGCTCGG TCATGAGCAC CGACAGTAAC TCACTGGCAC GTGAATTTCT GACCGATGTC 600
AACCGGCTTT GCAATGCAGT GGTCCAGAGG GTGGAGGCCA GGGAGGAAGA AGAGGAGGAG 660
ACGCACATGG CAACCCTTGG ACAGTACCTT GTCCATGGTC GAGGATTTCT ATTACTTACC 720
AAGCTA~ATT CTATAATTGA TCAGGCATTG ACATGTAGAG AAGAACTCCT GACTCTTCTT 780
CTGTCTCTCC TTCCACTGGT ATGGA~GATA CCTGTCCAAG AAGAAAAGGC AACAGATTTT 840
AACCTACCGC TCTCAGCAGA TATAATCCTG ACCAAAGAAA AGAACTCAAG TTCACAAAGA 900
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TCCACTCAGG AAAAATTACA TTTAGAAGGA AGTGCCCTGT CTAGTCAGGT TTCTGCAAAA 960
GTAAATGTTT TTCGAAAAAG CAGACGACAG CGTAAAATTA CCCATCGCTA TTCTGTAAGA 1020
GATGCAAGAA AGACACAGCT CTCCACCTCA GATTCAGAAG CCAATTCAGA TGAAAAAGGC 1080
ATAGCAATGA ATAAGCATAG AAGGCCCCAT CTGCTGCATC ATTTTTTAAC ATCGTTTCCT 1140
AAACAAGACC ACCCCAAAGC TAAACTTGAC CGCTTAGCAA CCAAAGAACA GACTCCTCCA 1200
GATGCTATGG CTTTGGAAAA TTCCAGAGAG ATTATTCCAA GACAGGGGTC AAACACTGAC 1260
ATTTTAAGTG AGCCAGCTGC CTTGTCTGTT ATCAGTAACA TGAACAATTC TCCATTTGAC 1320
TTATGTCATG TTTTGTTATC TTTATTAGAA AAAGTTTGTA AGTTTGACGT TACCTTGAAT 1380
CATAATTCTC CTTTAGCAGC CAGTGTAGTG CCCACACTAA CTGAATTCCT AGCAGGCTTT 1440
GGGGACTGCT GCAGTCTGAG CGACAACTTG GAGAGTCGAG TAGTTTCTGC AGGTTGGACC 1500
GAAGAACCGG TGGCTTTGAT TCAAAGGATG CTCTTTCGAA CAGTGTTGCA TCTTCTGTCA 1560
GTAGATGTTA GTACTGCAGA GATGATGCCA GAAAATCTTA GGAAAAATTT AACTGAATTG 1620
CTTAGAGCAG CTTTAAAAAT TAGAATATGC CTAGAAAAGC AGCCTGACCC TTTTGCACCA 1680
AGACAAAAGA AAACACTGCA GGAGGTTCAG GAAGATTTTG TGTTTTCAAA GTATCGTCAT 1740
AGAGCCCTTC TTTTACCTGA GCTTTTGGAA GGAGTTCTTC AGATTCTGAT CTGTTGTCTT 1800
CAGAGTGCAG CTTCAAATCC CTTCTACTTC AGTCAAGCCA TGGATTTGGT TCAAGAATTC 1860
ATTCAGCATC ATGGATTTAA TTTATTTGAA ACAGCAGTTC TTCAAATGGA ATGGCTGGTT 1920
TTAAGAGATG GAGTTCCTCC CGAGGCCTCA GAGCATTTGA AAGCCCTAAT AAATAGTGTG 1980
ATGAAAATAA TGAGCACTGT CAAAAAAGTG AAATCAGAGC AACTTCATCA TTCGATGTGT 2040
ACAAGAAAAA GGCACAGACG ATGTGAATAT TCTCATTTTA TGCATCATCA CCGAGATCTC 2100
TCAGGTCTTC TGGTTTCGGC TTTTAAAAAC CAGGTTTCCA AAAACCCATT TGAAGAGACT 2160
GCAGATGGAG ATGTTTATTA TCCTGAGCGG TGCTGTTGCA TTGGAGTGTG TGCCCATCAG 2220
TGCTTGCGCT TACTGCAGCA GGCTTGCTTG AGCAGCACTT GTGTCCAGAT CCTATCGGGT 2280
GTTCATAACA TTGGAATATG CTGTTGTATG GATCCCAAAT CTGTAATCAT TCCTTTGCTC 2340
CATGCTTTTA AATTGCCAGC ACTGAAAAAT TTTCAGCAGC ATATATTGAA TATCCTTAAC 2400
AAACTTATTT TGGATCAGTT AGGAGGAGCA GAGATATCAC CAAAAATTAA AAAAGCAGCT 2460
TGTAATATTT GTACTGTTGA CTCTGACCAA CTAGCCCAAT TAGAAGAGAC ACTGCAGGGA 2520
AACTTATGTG ATGCTGAACT CTCCTCAAGT TTATCCAGTC CTTCTTACAG ATTTCAAGGG 2580
ATCCTGCCCA GCAGTGGATC TGAAGATTTG TTGTGGAAAT GGGATGCTTT AAAGGCTTAT 2640
CAGAACTTTG TTTTTGGAGA AGACAGATTA CATAGTATAC AGATTGCAAA TCACATTTGC 2700
AATTTAATCC AGA~AGGCAA TATAGTTGTT CAGTGGAAAT TATATAATTA CATATTTAAT 2760
CCTGTGCTCC AAAGAGGAGT TGAATTAGCA CATCATTGTC AACACCTAAG CGTTACTTCA 2820
GCTCAAAGTC ATGTATGTAG CCATCATAAC CAGTGCTTGC CTCAGGACGT GCTTCAGATT 2880
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TATGTAAAAA CTCTGCCTAT CCTGCTTAAA TCCAGGGTAA TAAGAGATTT GTTTTTGAGT 2940
TGTAATGGAG TAAGTCAAAT AATCGAATTA AATTGCTTAA ATGGTATTCG AAGTCATTCT 3000
CTAAAAGCAT TTGAAACTCT GATAATCAGC CTAGGGGAGC AACAGAAAGA TGCCTCAGTT 3060
CCAGATATTG ATGGGATAGA CATTGAACAG AAGGAGTTGT CCTCTGTACA TGTGGGTACT 3120
T~l~lll~ATC ATCAGCAAGC TTATTCAGAT TCTCCTCAGA GTCTCAGCAA ATTTTATGCT 3180
GGCCTCAAAG AAGCTTATCC AAAGAGACGG AAGACTGTTA ACCAAGATGT TCATATCAAC 3240
ACAATAAACC TATTCCTCTG TGTGGCTTTT TTATGCGTAA GTA~AGAAGC AGAGTCTGAC 3300
AGGGAGTCGG CCAATGACTC AGAAGATACT TCTGGCTATG ACAGCACAGC CAGCGAGCCT 3360
TTAAGTCATA TGCTGCCATG TATATCTCTC GAGAGCCTTG TCTTGCCTTC TCCTGAACAT 3420
ATGCACCAAG CAGCAGACAT TTGGTCTATG TGTCGTTGGA TCTACATGTT GAGTTCAGTG 3480
TTCCAGAAAC AGTTTTATAG GCTTGGTGGT TTCCGAGTAT GCCATAAGTT AATATTTATG 3540
ATAATACAGA AACTGTTCAG AAGTCACAAA GAGGAGCAAG GAAAAAAGGA GGGAGATACA 3600
AGTGTAAATG AAAACCAGGA TTTAAACAGA ATTTCTCAAC CTAAGAGAAC TATGAAGGAA 3660
GATTTATTAT CTTTGGCTAT AAAAAGTGAC CCCATACCAT CAGAACTAGG TAGTCTAAAA 3720
AAGAGTGCTG ACAGTTTAGG TAAATTAGAG TTACAGCATA TTTCTTCCAT AAATGTGGAA 3780
GAAGTTTCAG CTACTGAAGC CGCTCCCGAG GAAGCAAAGC TATTTACAAG TCAAGAAAGT 3840
GAGACCTCAC TTCAAAGTAT ACGACTTTTG GAAGCCCTTC TGGCCATTTG TCTTCATGGT 3900
GCCAGAACTA GTCAACAGAA GATGGAATTG GAGTTACCTA ATCAGAACTT GTCTGTGGAA 3960
AGTATATTAT TTGAAATGAG GGACCATCTT TCCCAGTCAA AGGTGATTGA AACACAACTA 4020
GCAAAGCCTT TATTTGATGC CCTGCTTCGA GTTGCCCTCG GGAATTATTC AGCAGATTTT 4080
GAACATAATG ATGCTATGAC TGAGAAGAGT CATCAATCTG CAGAAGAATT GTCATCCCAG 4140
CCTGGTGATT TTTCAGAAGA AGCTGAGGAT TCTCAGTGTT GTAGTTTTAA ACTTTTAGTT 4200
GAAGAAGAAG GTTACGAAGC AGATAGTGAA AGCAATCCTG AAGATGGCGA AACCCAGGAT 4260
GATGGGGTAG ACTTAAAGTC TGAAACAGAA GGTTTCAGTG CATCAAGCAG TCCAAATGAC 4320
TTACTCGAAA ACCTCACTCA AGGGGAAATA ATTTATCCTG AGATTTGTAT GCTGGAATTA 4380
AATTTGCTTT CTGCTAGTAA AGCCAAACTT GATGTGCTTG CCCATGTATT TGAGAGTTTT 4440
TTGAAAATTA TTAGGCAGAA AGAAAAGAAT GTTTTTCTGC TCATGCAACA GGGAACTGTG 4500
AAAAATCTTT TAGGAGGGTT CTTGAGTATT TTAACACAGG ATGATTCTGA TTTTCAAGCA 4560
TGCCAGAGAG TATTGGTGGA TCTTTTGGTA TCTTTGATGA GTTCAAGAAC ATGTTCAGAA 4620
GAGCTAACCC TTCTTTTGAG AATATTTCTG GAGAAATCTC CTTGTACAAA AATTCTTCTT 4680
CTGGGTATTC TGAAAATTAT TGAAAGTGAT ACTACTATGA GCCCTTCACA GTATCTAACC 4740
TTCCCTTTAC TGCACGCTCC AAATTTAAGC AACGGTGTTT CATCACAAAA GTATCCTGGG 4800
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ATTTTAAACA GTAAGGCCAT GGGTTTATTG AGAAGAGCAC GAGTTTCACG GAGCAAGA~A 4860
- GAGGCTGATA GAGAGAGTTT TCCCCATCGG CTGCTTTCAT CTTGGCACAT AGCCCCAGTC 4920
CACCTGCCGT TGCTGGGGCA AAACTGCTGG CCACACCTAT CAGAAGGTTT CAGTGTTTCC 4980
CTGTGGTTTA ATGTGGAGTG TATCCATGAA GCTGAGAGTA CTACAGAAAA AGGAAAGAAG 5040
~ ATAAAGAAAA GAAACAAATC ATTAATTTTA CQGATAGCA GTTTTGATGG TACAGAGAGC S100
GACAGACCAG AAGGTGCAGA GTACATAAAT CCTGGTGAAA GACTCATAGA AGAAGGATGT 5160
ATTCATATAA TTTCACTGGG ATCCAAAGCG TTGATGATCC AAGTGTGGGC TGATCCCCAC 5Z20
AATGCCACTC TTATCTTTCG TGTGTGCATG GATTCAAATG ATGACATGAA AGCTGTTTTA 5280
CTAGCACAGG TTGAATCACA GGAGAATATT TTCCTCCCAA GCAAATGGCA ACATTTAGTA 5340
CTCACCTACT TACAGCAGCC CCAAGGGAAA AGGAGGATTC ATGGGAAAAT CTCCATATGG 5400
GTCTCTGGAC AGAGGAAGCC TGATGTTACT TTGGATTTTA TGCTTCCAAG AAAAACAAGT 5460
TTGTCATCTG ATAGCAATAA AACATTTTGC ATGATTGGCC ATTGTTTATC ATCCCAAGAA 5520
GAGTTTTTGC AGTTGGCTGG AAAATGGGAC CTGGGAAATT TGCTTCTCTT CAACGGAGCT 5580
AAGGTTGGTT CACAAGAGGC CTTTTATCTG TATGCTTGTG GACCCAACCA TACATCTGTA 5640
ATGCCATGTA AGTATGGCAA GCCAGTCAAT GACTACTCCA AATATATTAA TAAAGAAATT 5700
TTGCGATGTG AACAAATCAG AGAA'l~ ATGACCAAGA AAGATGTGGA TATTGGTCTC 5760
TTAATTGGAG TCTTTCAGTT GTTTATACAA CTTACTGTCC TGCTCCAGTA TACCATCTAT 5820
GAACCAGTGA TTAGACTTAA AGGTCAAATG AAAACCCAAC TCTCTCAAAG ACCCTTCAGC 5880
TCAAAAGAAG TTCAGAGCAT CTTATTAGAA CCTCATCATC TAAAGAATCT CCAACCTACT 5940
GAATATAAAA CTATTCAAGG CATTCTGCAC GAAATTGGTG GAACTGGCAT ATTTGTTTTT 6000
CTCTTTGCCA GGGTTGTTGA ACTCAGTAGC TGTGAAGAAA CTCAAGCATT AGCACTGCGA 6060
GTTATACTCT CATTAATTAA ATACAACCAA CAAAGAGTAC ATGAATTAGA AAATTGTAAT 6120
GGACTTTCTA TGATTCATCA GGTGTTGATC AAACAAA~AT GCATTGTTGG GTTTTACATT 6180
TTGAAGACCC TTCTTGAAGG ATGCTGTGGT GAAGATATTA TTTATATGAA TGAGAATGGA 6240
GAGTTTAAGT TGGATGTAGA CTCTAATGCT ATAATCCAAG ATGTTAAGCT GTTAGAGGAA 6300
CTATTGCTTG ACTGGAAGAT ATGGAGTAAA GCAGAGCAAG GTGTTTGGGA AACTTTGCTA 6360
GCAGCTCTAG AAGTCCTCAT CAGAGCAGAT CACCACCAGC AGATGTTTAA TATTAAGCAG 6420
TTATTGAAAG CTCAAGTGGT TCATCACTTT CTACTGACTT GTCAGGTTTT GCAGGAATAC 6480
AAAGAGGGGC AACTCA QCC CATGCCCCGA GAGATGGCAA GATCTTTCAG GAGAAAGTGC 6540
GGTCAATCAT GTACCTGAGG CATTCCAGCA GTGGAGGAAG GTCCCTTATG AGCCCTGGAT 6600
TTATGGTAAT AAGCCCATCT GGTTTTACTG CTTCACCATA TGAAGGAGAG AATTCCTCTA 6660
ATATTATTCC ACAACAGATG GCCGCCCATA TGCTGCGTTC TAGAAGCCTA CCAGCATTCC 6720
CTACTTCTTC ACTACTAACG CAATCACAAA AACTGACTGG AAGTTTGGGT TGTAGTATcG 6780
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ACAGGTTACA AAATATTGCA GATACTTATG TTGCCACCCA ATCAAAGAAA CAAAATTCTT 6840
TGGGGAGTTC CGACACACTG AAAAAAGGCA AAGAGGACGC ATTCATCAGT AGCTGTGAGT 6900
CTGCAAAAAC TGTTTGTGAA ATGGAAGCTG TCCTCTCAGC CCAGGTCTCT GTCAGTGATG 6960
TCCCAAAGGG AGTGCTGGGA TTTCCAGTGG TCAAAGCAGA TCATAAACAG TTGGGAGCAG 7020
AACCCAGGTC AGAAGATGAC AGTCCTGGGG ATGAGTCCTG CCCACGCCGA GCCCTATGCA 7080
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2001 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Met Ser Thr Asp Ser Asn Ser Leu Ala Arg Glu Phe Leu Thr Asp Val
1 5 10 15
Asn Arg Leu Cys Asn Ala Val Val Gln Arg Val Glu Ala Arg Glu Glu
Glu Glu Glu Glu Thr His Met Ala Thr Leu Gly Gln Tyr Leu Val His
Gly Arg Gly Phe Leu Leu Leu Thr Lys Leu Asn Ser Ile Ile Asp Gln
Ala Leu Thr Cys Arg Glu Glu Leu Leu Thr Leu Leu Leu Ser Leu Leu
Pro Leu Val Trp Lys Ile Pro Val Gln Glu Glu Lys Ala Thr Asp Phe
Asn Leu Pro Leu Ser Ala Asp Ile IIe Leu Thr Lys Glu Lys Asn Ser
100 105 110
Ser Ser Gln Arg Ser Thr Gln Glu Lys Leu His Leu Glu Gly Ser Ala
115 . 120 125
Leu Ser Ser Gln Val Ser Ala Lys Val Asn Val Phe Arg Lys Ser Arg
130 135 140
Arg Gln Arg Lys Ile Thr His Arg Tyr Ser Val Arg Asp Ala Arg Lys
145 150 155 160
Thr Gln Leu Ser Thr Ser Asp Ser Glu Ala Asn Ser Asp Glu Lys Gly
165 170 175
Ile Ala Met Asn Lys His Arg Arg Pro His Leu Leu ~is His Phe Leu
~80 185 190
Thr Ser Phe Pro Lys Gln Asp His Pro Lys Ala Lys Leu Asp Arg Leu
195 200 205
A~a Thr Lys Glu Gln Thr Pro Pro Asp Ala Met Ala Leu Glu Asn Ser
210 215 220
Arg Glu Ile Ile P~o Arg Gln Gly Ser Asn Thr Asp Ile Leu Ser Glu
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225 230 235 240
- Pro Ala Ala Leu Ser Val Ile Ser Asn Met Asn Asn Ser Pro Phe Asp
245 250 255
Leu Cys His Val Leu Leu Ser Leu Leu Glu Lys Val Cys Lys Phe Asp
260 26S 270
Val Thr Leu Asn His Asn Ser Pro Leu Ala Ala Ser Val Val Pro Thr
275 280 285
Leu Thr Glu Phe Leu Ala Gly Phe Gly Asp Cys Cys Ser Leu Ser Asp
290 295 300
Asn Leu Glu Ser Arg Val Val Ser Ala Gly Trp Thr Glu Glu Pro Val
305 310 315 320
Ala Leu Ile Gln Arg Met Leu Phe Arg Thr Val Leu His Leu Leu Ser
325 330 335
Val Asp Val Ser Thr Ala Glu Met Met Pro Glu Asn Leu Arg Lys Asn
340 345 350
Leu Thr Glu Leu Leu Arg Ala Ala Leu Lys Ile Arg Ile Cys Leu Glu
355 360 365
Lys Gln Pro Asp Pro Phe Ala Pro Arg Gln Lys Lys Thr Leu Gln Glu
370 375 380
Val Gln Glu Asp Phe Val Phe Ser Lys Tyr Arg His Arg Ala Leu Leu
385 390 395 400
Leu Pro Glu Leu Leu Glu Gly Val Leu Gln Ile Leu Ile Cys Cys Leu
405 410 415
Gln Ser Ala Ala Ser Asn Pro Phe Tyr Phe Ser Gln Ala Met Asp Leu
420 425 430
Val Gln Glu Phe Ile Gln His His Gly Phe Asn Leu Phe Glu Thr Ala
435 440 445
Val Leu Gln Met Glu Trp Leu Val Leu Arg Asp Gly Val Pro Pro Glu
450 455 460
Ala Ser Glu His Leu Lys Ala Leu Ile Asn Ser Val Met Lys Ile Met
465 470 475 480
Ser Thr Val Lys Lys Val Lys Ser Glu Gln Leu His His Ser Met Cys
485 490 495
Thr Arg Lys Arg His Arg Arg Cys Glu Tyr Ser His Phe Met His His
500 505 510
His Arg Asp Leu Ser Gly Leu Leu Val Ser Ala Phe Lys Asn Gln Val
515 520 525
.. Ser Lys Asn Pro Phe Glu Glu Thr Ala Asp Gly Asp Val Tyr Tyr Pro
530 535 540
Glu Arg Cys Cys Cys Ile Ala Val Cys Ala His Gln Cys Leu Arg Leu
545 550 555 560
Leu Gln Gln Ala Ser Leu Ser Ser Thr Cys Val Gln Ile Leu Ser Gly
565 570 575
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Val His Asn Ile Gly Ile Cys Cys Cys Met Asp Pro Lys Ser Val Ile
580 585 590
.
Ile Pro Leu Leu His Ala Phe Lys Leu Pro Ala Leu Lys Asn Phe Gln
595 600 605
Gln His Ile Leu Asn Ile Leu Asn Lys Leu Ile Leu Asp Gln Leu Gly
610 615 620
Gly Ala Glu Ile Ser Pro Lys Ile Lys Lys Ala Ala Cys Asn Ile Cys
625 630 635 640
~hr Val Asp Ser Asp Gln Leu Ala Gln Leu Glu Glu Thr Leu Gln Gly
645 650 655
~sn Leu Cys Asp Ala Glu Leu Ser Ser Ser Leu Ser Ser Pro Ser Tyr
660 665 670
Arg Phe Gln Gly Ile Leu Pro Ser Ser Gly Ser Glu Asp Leu Leu Trp
675 680 685
Lys Trp Asp Ala Leu Lys Ala Tyr Gln Asn Phe Val Phe Gly Glu Asp
690 695 700
Arg Leu His Ser Ile Gln Ile Ala Asn His Ile Cys Asn Leu Ile Gln
705 710 715 720
~ys Gly Asn Ile Val Val Gln Trp Lys Leu Tyr Asn Tyr Ile Phe Asn
725 730 735
~ro Val Leu Gln Arg Gly Val Glu Leu Ala His His Cys Gln His Leu
740 745 750
Ser Val Thr Ser Ala Gln Ser His Val Cys Ser His His Asn Gln Cys
755 760 765
Leu Pro Gln Asp Val Leu Gln Ile Tyr Val Lys Thr Leu Pro Ile Leu
770 775 780
Leu Lys Ser Arg Val Ile Arg Asp Leu Phe Leu Ser Cys Asn Gly Val
785 790 795 800
~er Gln Ile Ile Glu Leu Asn Cys Leu Asn Gly Ile Arg Ser His Ser
805 810 815
~eu Lys Ala Phe Glu Thr Leu Ile Ile Ser Leu Gly Glu Gln Gln Lys
820 825 830
Asp Ala Ser Val Pro Asp Ile Asp Gly Ile Asp Ile Glu Gln Lys Glu
835 840 845
Leu Ser Ser Val His Val Gly Thr Ser Phe His His Gln Gln Ala Tyr
850 855 860
Ser Asp Ser Pro Gln Ser Leu Ser Lys Phe Tyr Ala Gly Leu Lys Glu
865 870 875 880
~la Tyr Pro Lys Arg Arg Lys Thr Val Asn Gln Asp Val His Ile Asn
885 890 895
~hr Ile Asn Leu Phe Leu Cys Val Ala Phe Leu Cys Val Ser Lys Glu
900 905 910
~la Glu Ser Asp Arg Glu Ser Ala Asn Asp Ser Glu Asp Thr Ser Gly
915 920 925
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Tyr Asp Ser Thr Ala Ser Glu Pro Leu Ser His Met Leu Pro Cys Ile
- 930 935 940
Ser Leu Glu Ser Leu Val Leu Pro Ser Pro Glu His Met His Gln Ala
945 950 955 960
Ala Asp Ile Trp Ser Met Cys Arg Trp Ile Tyr Met Leu Ser Ser Val
965 970 975
Phe Gln Lys Gln Phe Tyr Arg Leu Gly Gly Phe Arg Val Cys His Lys
980 985 990
Leu Ile Phe Met Ile Ile Gln Lys Leu Phe Arg Ser His Lys Glu Glu
995 1000 1005
Gln Gly Lys Lys Glu Gly Asp Thr Ser Val Asn Glu Asn Gln Asp Leu
1010 1015 1020
Asn Arg Ile Ser Gln Pro Lys Arg Thr Met Lys Glu Asp Leu Leu Ser
1025 1030 1035 1040
Leu Ala Ile Lys Ser Asp Pro Ile Pro Ser Glu Leu Gly Ser Leu Lys
1045 1050 1055
Lys Ser Ala Asp Ser Leu Gly Lys Leu Glu Leu Gln His Ile Ser Ser
1060 1065 1070
Ile Asn Val Glu Glu Val Ser Ala Thr Glu Ala Ala Pro Glu Glu Ala
1075 1080 1085
Lys Leu Phe Thr Ser Gln Glu Ser Glu Thr Ser Leu Gln Ser Ile Arg
1090 1095 1100
Leu Leu Glu Ala Leu Leu Ala Ile Cys Leu His Gly Ala Arg Thr Ser
1105 1110 1115 1120
Gln Gln Lys Met Glu Leu Glu Leu Pro Asn Gln Asn Leu Ser Val Glu
1125 1130 1135
Ser Ile Leu Phe Glu Met Arg Asp His Leu Ser Gln Ser Lys Val Ile
1140 1145 1150
Glu Thr Gln Leu Ala Lys Pro Leu Phe Asp Ala Leu Leu Arg Val Ala
1155 1160 1165
Leu Gly Asn Tyr Ser Ala Asp Phe Glu His Asn Asp Ala Met Thr Glu
1170 1175 1180
Lys Ser Hls Gln Ser Ala Glu Glu Leu Ser Ser Gln Pro Gly Asp Phe
1185 1190 1195 1200
Ser Glu Glu Ala Glu Asp Ser Gln Cys Cys Ser Phe Lys Leu Leu Val
1205 1210 1215
Glu Glu Glu Gly Tyr Glu Ala Asp Ser Glu Ser Asn Pro Glu Asp Gly
.~ 1220 1225 1230
Glu Thr Gln Asp Asp Gly Val Asp Leu Lys Ser Glu Thr Glu Gly Phe
,. 1235 1240 1245
,~ Ser Ala Ser Ser Ser Pro Asn Asp Leu Leu Glu Asn Leu Thr Gln Gly
1250 1255 1260
Glu Ile Ile Tyr Pro Glu Ile Cys Met Leu Glu Leu Asn Leu Leu Ser
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1265 1270 1275 1280
Ala Ser Lys Ala Lys Leu Asp Val Leu Ala His Val Phe Glu Ser Phe "
1285 1290 1295
Leu Lys Ile Ile Arg Gln Lys Glu Lys Asn Val Phe Leu Leu Met Gln
1300 1305 1310
Gln Gly Thr Val Lys Asn Leu Leu Gly Gly Phe Leu Ser Ile Leu Thr
1315 1320 1325
Gln Asp Asp Ser Asp Phe Gln Ala Cys Gln Arg Val Leu Val Asp Leu
1330 - 1335 1340
Leu Val Ser Leu Met Ser Ser Arg Thr Cys Ser Glu Glu Leu Thr Leu
1345 1350 1355 1360
Leu Leu Arg Ile Phe Leu Glu Lys Ser Pro Cys Thr Lys Ile Leu Leu
1365 1370 1375
Leu Gly Ile Leu Lys Ile Ile Glu Ser Asp Thr Thr Met Ser Pro Ser
1380 1385 1390
G- n Tyr Leu Thr Phe Pro Leu Leu His Ala Pro Asn Leu Ser Asn Gly
1395 1400 1405
Val Ser Ser Gln Lys Tyr Pro Gly Ile Leu Asn Ser Lys Ala Met Gly
1410 1415 1420
Leu Leu Arg Arg Ala Arg Val Ser Arg Ser Lys Lys Glu Ala Asp Arg
1425 1430 1435 1440
Glu Ser Phe Pro His Arg Leu Leu Ser Ser Trp His Ile Ala Pro Val
1445 1450 1455
His Leu Pro Leu Leu Gly Gln Asn Cys Trp Pro His Leu Ser Glu Gly
1460 1465 1470
Phe Ser Val Ser Leu Trp Phe Asn Val Glu Cys Ile His Glu Ala Glu
1475 1480 1485
Ser Thr Thr Glu Lys Gly Lys Lys Ile Lys Lys Arg Asn Lys Ser Leu
1490 1495 1500
Ile Leu Pro Asp Ser Ser Phe Asp Gly Thr Glu Ser Asp Arg Pro Glu
1505 1510 1515 1520
Gly Ala Glu Tyr Ile Asn Pro Gly Glu Arg Leu Ile Glu Glu Gly Cys
1525 1530 1535
Ile His Ile Ile Ser Leu Gly Ser Lys Ala Leu Met Ile Gln Val Trp
1540 1545 1550
Ala Asp Pro His Asn Ala Thr Leu Ile Phe Arg Val Cys Met Asp Ser
1555 1560 1565
Asn Asp Asp Met Lys Ala Val Leu Leu Ala Gln Val Glu Ser Gln Glu
1570 1575 1580
Asn Ile Phe Leu Pro Ser Lys Trp Gln His Leu Val Leu Thr Tyr Leu ~.
1585 1590 1595 1600
Gln Gln Pro Gln Gly Lys Arg Arg Ile His Gly Lys Ile Ser Ile Trp
1605 1610 1615
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Val Ser Gly Gln Arg Lys Pro Asp Val Thr Leu Asp Phe Met Leu Pro
1620 1625 1630
Arg Lys Thr Ser Leu Ser Ser Asp Ser Asn Lys Thr Phe Cys Met Ile
1635 1640 1645
Gly His Cys Leu Ser Ser Gln Glu Glu Phe Leu Gln Leu Ala Gly Lys
1650 1655 1660
Trp Asp Leu Gly Asn Leu Leu Leu Phe Asn Gly Ala Lys Val Gly Ser
1665 1670 1675 1680
Gln Glu Ala Phe Tyr Leu Tyr Ala Cys Gly Pro Asn His Thr Ser Val
1685 1690 1695
Met Pro Cys Lys Tyr Gly Lys Pro Val Asn Asp Tyr Ser Lys Tyr Ile
1700 1705 1710
Asn Lys Glu Ile Leu Arg Cys Glu Gln Ile Arg Glu Phe Phe Met Thr
1715 1720 1725
Lys Lys Asp Val Asp Ile Gly Leu Leu Ile Gly Val Phe Gln Leu Phe
1730 1735 1740
Ile Gln Leu Thr Val Leu Leu Gln Tyr Thr Ile Tyr Glu Pro Val Ile
1745 1750 1755 1760
Arg Leu Lys Gly Gln Met Lys Thr Gln Leu Ser Gln Arg Pro Phe Ser
1765 1770 1775
Ser Lys Glu Val Gln Ser Ile Leu Leu Glu Pro His His Leu Lys Asn
1780 1785 1790
Leu Gln Pro Thr Glu Tyr Lys Thr Ile Gln Gly Ile Leu His Glu Ile
1795 1800 1805
Gly Gly Thr Gly Ile Phe Val Phe Leu Phe Ala Arg Val Val Glu Leu
1810 1815 1820
Ser Ser Cys Glu Glu Thr Gln Ala Leu Ala Leu Arg Val Ile Leu Ser
1825 1830 1835 1840
Leu Ile Lys Tyr Asn Gln Gln Arg Val His Glu Leu Glu Asn Cys Asn
1845 1850 1855
Gly Leu Ser Met Ile His Gln Val Leu Ile Lys Gln Lys Cys Ile Val
1860 1865 1870
Gly Phe Tyr Ile Leu Lys Thr Leu Leu Glu Gly Cys Cys Gly Glu Asp
1875 1880 1885
Ile Ile Tyr Met Asn Glu Asn Gly Glu Phe Lys Leu Asp Val Asp Ser
1890 1895 l900
~Asn Ala Ile Ile Gln Asp Val Lys Leu Leu Glu Glu Leu Leu Leu Asp
1905 1910 l 915 1920
Trp Lys Ile Trp Ser Lys Ala Glu Gln Gly Val Trp Glu Thr Leu Leu
1925 1930 1935
Ala Ala Leu Glu Val Leu Ile Arg Ala Asp His His Gln Gln Met Phe
-~ 1940 1945 1950
Asn Ile Lys Gln Leu Leu Lys Ala Gln Val Val His His Phe Leu Leu
1955 1960 1965
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Thr Cys Gln Val Leu Gln Glu Tyr Lys Glu Gly Gln Leu Thr Pro Met
- 1970 1975 1980
Pro Arg Glu Met Ala Arg Ser Phe Arg Arg Lys Cys Gly Gln Ser Cys
1985 1990 1995 2000
Thr
(2) INFORMATION FOR SEQ ID NO: 9:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5221 base pairs
(B) TYPE: nucleic acid
~C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
CGCAAGGGCT TCTAAGAAGC CATCCCAATG ACCTTTTGGC TTTGAGAAGA GCAGTCCTCA 60
TACCAGAGTG TTTGGGGTTT TGGCCTCTTT CAGTGTTTAT TCATTCTTAC GTGGGAAAGT 120
TGTATTCCGA GGTTTCTGTG GTGCATGAAG CTTTTGCCTT CACCATCTGT TCCCGTGTCT 180
TCCTCGGGTG ACATCAGAGT ACAGCAGTAT TTTCCCTTGC CATCTAATGG GGTTTGGGCT 240
GTTTGACTCA ACCGTGTGTG TTCCTCAATG CCAGGGGAAT AATCCTACCC TAAGTCAGCT 300
GAACAGAAGC CAGGATCTAA CTGCAAACAA GAGACCCAGC TTGCTTAACA GCATGGAAGA 360
GAACCAGTTT CCTTG QGCT ACCTGGGAAG ACGGTTGCTA ATTAGCCTGC AACAAAGAGT 420
TCCTTGCTCA TCTAAAAGAG GCAATCACCG TTCAGGTGAA GCTTTGTTCT AAGAATATTT 480
GTTTCATCTA GTTTATGAGT CCAAATGATA TAGACTGTAA ATGTCACAGC AGTGGTGA~A 540
GACTGCTCGG TCATGAGCAC CGACAGTAAC TCACTGGCAC GTGAATTTCT GACCGATGTC 600
AACCGGCTTT GCAATGCAGT GGTCCAGAGG GTGGAGGCCA GGGAGGAAGA AGAGGAGGAG 660
ACGCACATGG CAACCCTTGG ACAGTACCTT GTCCATGGTC GAGGATTTCT ATTACTTACC 720
AAGCTAAATT CTATAATTGA TCAGGCATTG ACATGTAGAG AAGAACTCCT GACTCTTCTT 780
CTGTCTCTCC TTCCACTGGT ATGGAAGATA CCTGTCCAAG AAGAAAAGGC AACAGATTTT 840
AACCTACCGC TCTCAGCAGA TATAATCCTG ACCAAAGAAA AGAACTCAAG TTCACAAAGA 900
TCCACTCAGG A~AAATTACA TTTAGAAGGA AGTGCCCTGT CTAGTCAGGT TTCTGCAAAA 960
GTAAATGTTT TTCGAAAAAG CAGACGACAG CGTA~AATTA CCCATCGCTA TTCTGTAAGA 1020
GATGCAAGAA AGACACAGCT CTCCACCTCA GATTCAGAAG CCAATTCAGA TGAAAAAGGC 1080
ATAGCAATGA ATAAGCATAG AAGGCCCCAT CTGCTGCATC A~L~ "l"l'AAC ATCGTTTCCT 1140
AAACAAGACC ACCCCA~AGC TAAACTTGAC CGCTTAGCAA CCAAAGAACA GACTCCTCCA 1200
GATGCTATGG CTTTGGAAAA TTCCAGAGAG ATTATTCCAA GACAGGGGTC A~ACACTGAC 1260
ATTTTAAGTG AGCCAGCTGC CTTGTCTGTT ATCAGTAACA TGAACAATTC TCCATTTGAC 1320
CA 02244744 l998-07-29
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TTATGTCATG TTTTGTTATC TTTATTAGAA AAAGTTTGTA AGTTTGACGT TACCTTGAAT 1380
CATAATTCTC CTTTAGCAGC CAGTGTAGTG CCCACACTAA CTGAATTCCT AGCAGGCTTT 1440
GGGGACTGCT GCAGTCTGAG CGACAACTTG GAGAGTCGAG TAGTTTCTGC AGGTTGGACC 1500
GAAGAACCGG TGGCTTTGAT TCAAAGGATG CTCTTTCGAA CAGTGTTGCA TCTTCTGTCA 1560
GTAGATGTTA GTACTGCAGA GATGATGCCA GAAAATCTTA GGA~AAATTT AACTGAATTG 1620
CTTAGAGCAG CTTTAAAAAT TAGAATATGC CTAGAAAAGC AGCCTGACCC TTTTGCACCA 1680
AGACAAAAGA AAACACTGCA GGAGGTTCAG GAAGATTTTG TGTTTTCAAA GTATCGTCAT 1740
AGAGCCCTTC TTTTACCTGA GCTTTTGGAA GGAGTTCTTC AGATTCTGAT CTGTTGTCTT 1800
CAGAGTGCAG CTTCAAATCC CTTCTACTTC AGTCAAGCCA TGGATTTGGT TCAAGAATTC 1860
ATTCAGCATC ATGGATTTAA TTTATTTGAA ACAGCAGTTC TTCAAATGGA ATGGCTGGTT 1920
TTAAGAGATG GAGTTCCTCC CGAGGCCTCA GAGCATTTGA AAGCCCTAAT AAATAGTGTG 1980
ATGAAAATAA TGAGCACTGT CAAAAAAGTG AAATCAGAGC AACTTCATCA TTCGATGTGT 2040
ACAAGAAAAA GGCACAGACG ATGTGAATAT TCTCATTTTA TGCATCATCA CCGAGATCTC 210Q
TCAGGTCTTC TGGTTTCGGC TTTTAAAAAC CAGGTTTCCA AAAACCCATT TGAAGAGACT 2160
GCAGATGGAG ATGTTTATTA TCCTGAGCGG TGCTGTTGCA TTGCAGTGTG TGCCCATCAG 2220
TGCTTGCGCT TACTGCAGCA GGCTTCCTTG AGCAGCACTT GTGTCCAGAT CCTATCGGGT ~280
GTTCATAACA TTGGAATATG CTGTTGTATG GATCCCAAAT CTGTAATCAT TCCTTTGCTC 2340
CATGCTTTTA AATTGCCAGC ACTGAAAAAT TTTCAGCAGC ATATATTGAA TATCCTTAAC 2400
AAACTTATTT TGGATCAGTT AGGAGGAGCA GAGATATCAC CAAAAATTAA AAAAGCAGCT 2460
TGTAATATTT GTACTGTTGA CTCTGACCAA CTAGCCCAAT TAGAAGAGAC ACTGCAGGGA 2520
AACTTATGTG ATGCTGAACT CTCCTCAAGT TTATCCAGTC CTTCTTACAG ATTTCAAGGG 2580
ATCCTGCCCA GCAGTGGATC TGAAGATTTG TTGTGGAAAT GGGATGCTTT AAAGGCTTAT 2640
CAGAACTTTG TTTTTGGAGA AGACAGATTA CATAGTATAC AGATTGCAAA TCACATTTGC 2700
AATTTAATCC AGAAAGGCAA TATAGTTGTT CAGTGGAAAT TATATAATTA CATATTTAAT 2760
CCTGTGCTCC AAAGAGGAGT TGAATTAGCA CATCATTGTC AACACCTAAG CGTTACTTCA 2820
GCTCAAAGTC ATGTATGTAG CCATCATAAC CAGTGCTTGC CTCAGGACGT GCTTCAGATT 2880
TATGTAAAAA CTCTGCCTAT CCTGCTTAAA TCCAGGGTAA TAAGAGATTT GTTTTTGAGT 2940
TGTAATGGAG TAAGTCAAAT AATCGAATTA AATTGCTTAA ATGGTATTCG AAGTCATTCT 3000
CTAAAAGCAT TTGAAACTCT GATAATCAGC CTAGGGGAGC AACAGAAAGA TGCCTCAGTT 3060
CCAGATATTG ATGGGATAGA CATTGAACAG AAGGAGTTGT CCTCTGTACA TGTGGGTACT 3120
TCTTTTCATC ATCAGCAAGC TTATTCAGAT TCTCCTCAGA GTCTCAGCAA ATTTTATGCT 3180
GGCCTCAAAG AAGCTTATCC AAAGAGACGG AAGACTGTTA ACCAAGATGT TCATATCAAC 3240
ACAATAAACC TATTCCTCTG TGTGGCTTTT TTATGCGTAA GTAAAGAAGC AGAGTCTGAC 3300
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AGGGAGTCGG CCAATGACTC AGAAGATACT TCTGGCTATG ACAGCACAGC CAGCGAGCCT 3360
TTAAGTCATA TGCTGCCATG TATATCTCTC GAGAGCCTTG TCTTGCCTTC TCCTGAACAT 3420
ATGCACCAAG CAGCAGACAT TTGGTCTATG TGTCGTTGGA TCTACATGTT GAGTTCAGTG 3480
TTCCAGAAAC AGTTTTATAG GCTTGGTGGT TTCCGAGTAT GCCATAAGTT AATATTTATG 3540
ATAATACAGA AACTGTTCAG AAGTCACAAA GAGGAGCAAG GAAAAAAGGA GGGAGATACA 3600
AGTGTAAATG AAAACCAGGA TTTAAACAGA ATTTCTCAAC CTAAGAGAAC TATGAAGGAA 3660
GATTTATTAT CTTTGGCTAT AAAAAGTGAC CCCATACCAT CAGAACTAGG TAGTCTAAAA 3720
AAGAGTGCTG ACAGTTTAGG TAAATTAGAG TTACAGCATA TTTCTTCCAT AAATGTGGAA 3780
GAAGTTTCAG CTACTGAAGC'CGCTCCCGAG GAAGCAAAGC TATTTACAAG TCAAGAAAGT 3840
GAGACCTCAC TTCAAAGTAT ACGACTTTTG GAAGCCCTTC TGGCCATTTG TCTTCATGGT 3900
GCCAGAACTA GTCAACAGAA GATGGAATTG GAGTTACCTA ATCAGAACTT GTCTGTGGAA 3960
AGTATATTAT TTGAAATGAG GGACCATCTT TCCCAGTCAA AGGTGATTGA AACACAACTA 4020
GCAAAGCCTT TATTTGATGC CCTGCTTCGA GTTGCCCTCG GGAATTATTC AGCAGATTTT 4080
GAACATAATG ATGCTATGAC TGAGAAGAGT CATCAATCTG CAGAAGAATT GTCATCCCAG 4140
CCTGGTGATT TTTCAGAAGA AGCTGAGGAT TCTCAGTGTT GTAGTTTTAA ACTTTTAGTT 4200
GAAGAAGAAG GTTACGAAGC AGATAGTGAA AGCAATCCTG AAGATGGCGA AACCCAGGAT 4260
GATGGGGTAG ACTTAAAGTC TGAAA Q GAA GGTTTCAGTG CATCAAGCAG TCCAAATGAC 4320
TTACTCGAAA ACCT Q CTCA AGGGGAAATA ATTTATCCTG AGATTTGTAT GCTGGAATTA 4380
AATTTGCTTT CTGCTAGTAA AGCCAAACTT GATGTGCTTG CC QTGTATT TGAGAGTTTT 4440
TTGAAAATTA TTAGGCAGAA AGAAAAGAAT GTTTTTCTGC T Q TGCAACA GGGAACTGTG 4500
AAAAATCTTT TAGGAGGGTT CTTGAGTATT TTAA QCAGG ATGATTCTGA TTTTCAAGCA 4560
TGCCAGAGAG TATTGGTGGA TCTTTTGGTA TCTTTGATGA GTT QAGAAC ATGTTCAGAA 4620
GAGCTAACCC 'l"l'~ GAG AATATTTCTG GAGAAATCTC CTTGTACAAA AATTCTTCTT 4680
CTGGGTATTC TGAAAATTAT TGAAAGTGAT ACTACTATGA GCCCTTCACA GTATCTAACC 4740
TTCCCTTTAC TG QCGCTCC AAATTTAAGC AACGGTGTTT CATCACAAAA GTATCCTGGG 4800
ATTTTAAACA GTAAGGC QT GGGTTTATTG AGAAGAGCAC GAGTTTCACG GAGCAAGAAA 4860
GAGGCTGATA GAGAGAGTTT TCCC QTCGG CTGCTTTCAT CTTGGCACAT AGCCCCAGTC 4920
CACCTGCCGT TGCTGGGG Q AAACTGCTGG CCA QCCTAT CAGAAGGTTT CAGTGTTTCC 4980
CTGTGGTTTA ATGTGGAGTG TATCCATGAA GCTGAGAGTA CTACAGAAAA AGGAAAGAAG 5040
ATAAAGAAAA GAAACAAATC ATTAATTTTA CCAGATAGCA GTTTTGATGG TACAGGTATG 5100
ATGACAGGAT TATCTGATTT GTACACAAAG ATTGTTTTCA GACTATAATT TTCCTTGAGC 5160
CGTAAAAATG TGGTAGTGTT CTTAACACTC TTAACATGTT ATT QCCTTA AAGATCCTAC 5220
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T 1~ 5221
(2) INFORMATION FOR SEO ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1531 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Met Ser Thr Asp Ser Asn Ser Leu Ala Arg Glu Phe Leu Thr Asp Val
1 5 10 15
Asn Arg Leu Cys Asn Ala Val Val Gln Arg Val Glu Ala Arg Glu Glu
Glu Glu Glu Glu Thr His Met Ala Thr Leu Gly Gln Tyr Leu Val His
Gly Arg Gly Phe Leu Leu Leu Thr Lys Leu Asn Ser Ile Ile Asp Gln
Ala Leu Thr Cys Arg Glu Glu Leu Leu Thr Leu Leu Leu Ser Leu Leu
Pro Leu Val Trp Lys Ile Pro Val Gln Glu Glu Lys Ala Thr Asp Phe
Asn Leu Pro Leu Ser Ala Asp Ile Ile Leu Thr Lys Glu Lys Asn Ser
100 105 110
Ser Ser Gln Arg Ser Thr Gln Glu Lys Leu His Leu Glu Gly Ser Ala
115 120 125
Leu Ser Ser Gln Val Ser Ala Lys Val Asn Val Phe Arg Lys Ser Arg
130 135 140
Arg Gln Arg Lys Ile Thr His Arg Tyr Ser Val Arg Asp Ala Arg Lys
145 150 155 160
Thr Gln Leu Ser Thr Ser Asp Ser Glu Ala Asn Ser Asp Glu Lys Gly
165 170 175
Ile Ala Met Asn Lys His Arg Arg Pro His Leu Leu His His Phe Leu
180 185 190
Thr Ser Phe Pro Lys Gln Asp His Pro Lys Ala Lys Leu Asp Arg Leu
195 200 205
Ala Thr Lys Glu Gln Thr Pro Pro Asp Ala Met Ala Leu Glu Asn Ser
210 215 220
Arg Glu Ile Ile Pro Arg Gln Gly Ser Asn Thr Asp Ile Leu Ser Glu
.~ 225 230 235 240
Pro Ala Ala Leu Ser Val Ile Ser Asn Met Asn Asn Ser Pro Phe Asp
245 250 255
-~ Leu Cys His Val Leu Leu Ser Leu Leu Glu Lys Val Cys Lys Phe Asp
260 265 270
Val Thr Leu Asn His Asn Ser Pro Leu Ala Ala Ser Val Val Pro Thr
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275 280 285
Leu Thr Glu Phe Leu Ala Gly Phe Gly Asp Cys Cys Ser Leu Ser Asp
290 295 300
Asn Leu Glu Ser Arg Val Val Ser Ala Gly Trp Thr Glu Glu Pro Val
305 310 315 320
Ala Leu Ile Gln Arg Met Leu Phe Arg Thr Val Leu His Leu Leu Ser
325 330 335
Val Asp Val Ser Thr Ala Glu Met Met Pro Glu Asn Leu Arg Lys Asn
340 345 350
Leu Thr Glu Leu Leu Arg Ala Ala Leu Lys Ile Arg Ile Cys Leu Glu
355 360 365
Lys Gln Pro Asp Pro Phe Ala Pro Arg Gln Lys Lys Thr Leu Gln Glu
370 375 380
Val Gln Glu Asp Phe Val Phe Ser Lys Tyr Arg His Arg Ala Leu Leu
385 390 395 400
Leu Pro Glu Leu Leu Glu Gly Val Leu Gln Ile Leu Ile Cys Cys Leu
405 410 415
Gln Ser Ala Ala Ser Asn Pro Phe Tyr Phe Ser Gln Ala Met Asp Leu
420 425 430
Val Gln Glu Phe Ile Gln His His Gly Phe Asn Leu Phe Glu Thr Ala
435 440 445
Val Leu Gln Met Glu Trp Leu Val Leu Arg Asp Gly Val Pro Pro Glu
450 455 460
Ala Ser Glu His Leu Lys Ala Leu Ile Asn Ser Val Met Lys Ile Met
465 470 475 480
Ser Thr Val Lys Lys Val Lys Ser Glu Gln Leu His His Ser Met Cys
485 490 495
Thr Arg Lys Arg His Arg Arg Cys Glu Tyr Ser His Phe Met His His
500 505 510
His Arg Asp Leu Ser Gly Leu Leu Val Ser Ala Phe Lys Asn Gln Val
515 520 525
Ser Lys Asn Pro Phe Glu Glu Thr Ala Asp Gly Asp Val Tyr Tyr Pro
530 535 540
Glu Arg Cys Cys Cys Ile Ala Val Cys Ala His Gln Cys Leu Arg Leu
545 550 555 560
Leu Gln Gln Ala Ser Leu Ser Ser Thr Cys Val Gln Ile Leu Ser Gly
565 570 575
Val His Asn Ile Gly Ile Cys Cys Cys Met Asp Pro Lys Ser Val Ile
580 585 590
Ile Pro Leu Leu His Ala Phe Lys Leu Pro Ala Leu Lys Asn Phe Gln
595 600 605
Gln His Ile Leu Asn Ile Leu Asn Lys Leu Ile Leu Asp Gln Leu Gly
610 615 620
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Gly Ala Glu Ile Ser Pro Lys Ile Lys Lys Ala Ala Cys Asn Ile Cys
625 630 635 640
~hr Val Asp Ser Asp Gln Leu Ala Gln Leu Glu Glu Thr Leu Gln Gly
645 650 655
Asn Leu Cys Asp Ala Glu Leu Ser Ser Ser Leu Ser Ser Pro Ser Tyr
660 665 670
Arg Phe Gln Gly Ile Leu Pro Ser Ser Gly Ser Glu Asp Leu Leu Trp
675 680 685
Lys Trp Asp Ala Leu Lys Ala Tyr Gln Asn Phe Val Phe Gly Glu Asp
690 695 700
Arg Leu His Ser Ile Gln Ile Ala Asn His Ile Cys Asn Leu Ile Gln
705 710 715 720
Lys Gly Asn Ile Val Val Gln Trp Lys Leu Tyr Asn Tyr Ile Phe Asn
725 730 735
Pro Val Leu Gln Arg Gly Val Glu Leu Ala His His Cys Gln His Leu
740 745 750
Ser Val Thr Ser Ala Gln Ser His Val Cys Ser His His Asn Gln Cys
755 760 - 765
Leu Pro Gln Asp Val Leu Gln Ile Tyr Val Lys Thr Leu Pro Ile Leu
770 775 780
Leu Lys Ser Arg Val Ile Arg Asp Leu Phe Leu Ser Cys Asn Gly Val
785 790 = 795 800
Ser Gln Ile Ile Glu Leu Asn Cys Leu Asn Gly Ile Arg Ser His Ser
805 810 815
Leu Lys Ala Phe Glu Thr Leu Ile Ile Ser Leu Gly Glu Gln Gln Lys
820 825 830
Asp Ala Ser Val Pro Asp Ile Asp Gly Ile Asp Ile Glu Gln Lys Glu
835 840 845
Leu Ser Ser Val His Val Gly Thr Ser Phe His His Gln Gln Ala Tyr
850 855 860
Ser Asp Ser Pro Gln Ser Leu Ser Lys Phe Tyr Ala Gly Leu Lys Glu
865 870 875 880
Ala Tyr Pro Lys Arg Arg Lys Thr Val Asn Gln Asp Val His Ile Asn
885 890 895
Thr Ile Asn Leu Phe Leu Cys Val Ala Phe Leu Cys Val Ser Lys Glu
900 905 910
Ala Glu Ser Asp Arg Glu Ser Ala Asn Asp Ser Glu Asp Thr Ser Gly
915 920 925
Tyr Asp Ser Thr Ala Ser Glu Pro Leu Ser His Met Leu Pro Cys Ile
930 935 9~0
Ser Leu Glu Ser Leu Val Leu Pro Ser Pro Glu His Met His Gln Ala
~, 945 950 955 960
Ala Asp Ile Trp Ser Met Cys Arg Trp Ile Tyr Met Leu Ser Ser Val
965 970 975
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Phe Gln Lys Gln Phe Tyr Arg Leu Gly Gly Phe Arg Val Cys His Lys
- 980 985 990
Leu Ile Phe Met Ile Ile Gln Lys Leu Phe Arg Ser His Lys Glu Glu
995 1000 1005
Gln Gly Lys Lys Glu Gly Asp Thr Ser Val Asn Glu Asn Gln Asp Leu
1010 1015 1020
Asn Arg Ile Ser Gln Pro Lys Arg Thr Met Lys Glu Asp Leu Leu Ser
1025 1030 1035 1040
Leu Ala Ile Lys Ser Asp Pro Ile Pro Ser Glu Leu Gly Ser Leu Lys
1045 1050 1055
Lys Ser Ala Asp Ser Leu Gly Lys Leu Glu Leu Gln His Ile Ser Ser
1060 1065 1070
Ile Asn Val Glu Glu Val Ser Ala Thr Glu Ala Ala Pro Glu Glu Ala
1075 1080 1085
Lys Leu Phe Thr Ser Gln Glu Ser Glu Thr Ser Leu Gln Ser Ile Arg
1090 1095 1100
Leu Leu Glu Ala Leu Leu Ala Ile Cys Leu His Gly Ala Arg Thr Ser
1105 1110 1115 I120
Gln Gln Lys Met Glu Leu Glu Leu Pro Asn Gln Asn Leu .Ser Val Glu
1125 1130 1135
Ser Ile Leu Phe Glu Met Arg Asp His Leu Ser Gln Ser Lys Val Ile
1140 1145 1150
Glu Thr Gln Leu Ala Lys Pro Leu Phe Asp Ala Leu Leu Arg Val Ala
1155 1160 1165
Leu Gly Asn Tyr Ser Ala Asp Phe Glu His Asn Asp Ala Met Thr Glu
1170 1175 1180
Lys Ser His Gln Ser A~a Glu Glu Leu Ser Ser Gln Pro Gly Asp Phe
1185 1190 1195 . 1200
Ser Glu Glu Ala Glu Asp Ser Gln Cys Cys Ser Phe Lys Leu Leu Val
1205 . 1210 1215
Glu Glu Glu Gly Tyr Glu Ala Asp Ser Glu Ser Asn Pro Glu Asp Gly
1220 1225 1230
Glu Thr Gln Asp Asp Gly Val Asp Leu Lys Ser Glu Thr Glu Gly Phe
1235 1240 1245
Ser Ala Ser Ser Ser Pro Asn Asp Leu Leu Glu Asn Leu Thr Gln Gly
1250 1255 1260
Glu Ile Ile Tyr Pro Glu Ile Cys Met Leu Glu Leu Asn Leu Leu Ser
1265 1270 1275 1280
Ala Ser Lys Ala Lys Leu Asp Val Leu Ala His Val Phe Glu Ser Phe
1285 1290 1295
Leu Lys Ile Ile Arg Gln Lys Glu Lys Asn Val Phe Leu Leu Met Gln
1300 1305 1310
Gln Gly Thr Val Lys Asn Leu Leu Gly Gly Phe Leu Ser Ile Leu Thr
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1'7~
1315 1320 1325
- Gln Asp Asp Ser Asp Phe Gln Ala Cys Gln Arg Val Leu Val Asp Leu
1330 1335 1340
Leu Val Ser Leu Met Ser Ser Arg Thr Cys Ser Glu Glu Leu Thr Leu
1345 1350 1355 1360
Leu Leu Arg Ile Phe Leu Glu Lys Ser Pro Cys Thr Lys Ile Leu Leu
1365 1370 1375
Leu Gly Ile Leu Lys Ile Ile Glu Ser Asp Thr Thr Met Ser Pro Ser
1380 1385 1390
Gln Tyr Leu Thr Phe Pro Leu Leu His Ala Pro Asn Leu Ser Asn Gly
1395 1400 1405
Val Ser Ser Gln Lys Tyr Pro Gly Ile Leu Asn Ser Lys Ala Met Gly
1410 1415 1420
Leu Leu Arg Arg Ala Arg Val Ser Arg Ser Lys Lys Glu Ala Asp Arg
1425 1430 1435 1440
Glu Ser Phe Pro His Arg Leu Leu Ser Ser Trp His Ile Ala Pro Val
1445 1450 1455
His Leu Pro Leu Leu Gly Gln Asn Cys Trp Pro His Leu Ser Glu Gly
1460 1465 1470
Phe Ser Val Ser Leu Trp Phe Asn Val Glu Cys Ile His Glu Ala Glu
1475 1480 1485
Ser Thr Thr Glu Lys Gly Lys Lys Ile Lys Lys Arg Asn Lys Ser Leu
1490 1495 1500
Ile Leu Pro Asp Ser Ser Phe Asp Gly Thr Gly Met Met Thr Gly Leu
1505 1510 1515 1520
Ser Asp Leu Tyr Thr Lys Ile Val Phe Arg Leu
1525 1530
(2) INFORMATION FOR SEQ ID NO~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1979 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
ATACTTCTGA TGTA~AGGAA CTAATTCCAG AGTTCTACTA CCTACCAGAG ATGTTTGTCA 60
ACAGTAATGG ATATAATCTT GGAGTCAGAG AAGATGAAGT AGTGGTAAAT GATGTTGATC 120
TTCCCCCTTG GGC~AAAA CCTGAAGACT TTGTGCGGAT CAACAGGATG GCCCTAGAAA 180
GTGAATTTGT TTCTTGCCAA CTTCATCAGT GGATCGACCT TATATTTGGC TATAAGCAGC 240
GAGGACCAGA AGCAGTTCGT GCTCTGAATG TTTTTCACTA CTTGACTTAT GAAGGCTCTG 300
TGAACCTGGA TAGTATCACT GATCCTGTGC TCAGGGAGGC CATGGAGGCA CAGATACAGA 360
ACTTTGGACA GACGCCATCT CAGTTGCTTA TTGAGCCACA TCCGCCTCGG AACTCTGCCA 420
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TGCACCTGTG TTTCCTTCCA CAGAGTCCGC TCATGTTTAA AGATCAGATG CAACAGGATG 480
TGATAATGGT GCTGAAGTTT CCTTCAAATT CTCCAGTAAC CCATGTGGCA GCCAACACTC 540
TGCCCCACTT GACCATCCCC GCAGTGGTGA CAGTGACTTG CAGCCGACTC TTTGCAGTGA 600
ATAGATGGCA CAACACAGTA GGCCTCAGAG GAGCTCCAGG ATACTCCTTG GATCAAGCCC 660
ACCATCTTCC CATTGAAATG GATCCATTAA TAGCCAATAA TTCAGGTGTA AACAAACGGC 720
AGAT Q CAGA CCTCGTTGAC CAGAGTATAC A~ATCAATGC ACATTGTTTT GTGGTAACAG 780
CAGATAATCG CTATATTCTT ATCTGTGGAT TCTGGGATAA GAGCTTCAGA GTTTATACTA 840
CAGAAACAGG GAAATTGACT CAGATTGTAT TTGGCCATTG GGATGTGGTC ACTTGCTTGG 900
CCAGGTCCGA GTCATACATT GGTGGGGACT GCTACATCGT GTCCGGATCT CGAGATGCCA 960
CCCTGCTGCT CTGGTACTGG AGTGGGCGGC ACCATATCAT AGGAGACAAC CCTAACAGCA 1020
GTGACTATCC GGCACCAAGA GCCGTCCTCA CAGGCCATGA CCATGAAGTT GTCTGTGTTT 1080
CTGTCTGTGC AGAACTTGGG CTTGTTATCA GTGGTGCTAA AGAGGGCCCT TGCCTTGTCC 1140
ACACCATCAC TGGAGATTTG CTGAGAGCCC TTGAAGGACC AGAAAACTGC TTATTCCCAC 1200
GCTTGATATC TGTCTCCAGC GAAGGCCACT GTATCATATA CTATGAACGA GGGCGATTCA 1260
GTAATITCAG CATTAATGGG AAACTTTTGG CTCAAATGGA GATCAATGAT TCAACACGGG 1320
CCATTCTCCT GAGCAGTGAC GGCCAGAACC TGGTCACCGG AGGGGACAAT GGGGTAGTAG 1380
AGGTCTGGCA GGCCTGTGAC TTCAAGCAAC TGTACATTTA ACCCTGGATG TGATGCTGGC 1440
ATTAGAGCAA TGGACTTGTC CCATGACCAG AGGACTCTGA TCACTGGCAT GGCTTCTGGT 1500
AGCATTGTAG CTTTTAATAT AGATTTTAAT CGGTGGCATT ATGAGCATCA GAACAGATAC 1560
TGAAGATAAA GGAAGAAC Q AAAGCCAAGT TA~AGCTGAG GGCACAAGTG CTGCATGGAA 1620
AGGCAATATC TCTGGTGGAA AAAATTCGTC TA QTCGACC TCCGTTTGTA QTTCCATCA 1680
CACCCAGCAA TAGCTGTACA TTGTAGT QG CAAC QTTTT ACTTTGTGTG TTTTTTCACG 1740
ACTGAACACC AGCTGCTATC AAGCAAGCTT ATATCATGTA AATTATATGA ATTAGGAGAT 1800
GTTTTGGTAA TTATTTCATA TATTGTTGTT TATTGAGAAA AGGTTGTAGG ATGTGTCACA 1860
AGAGACTTTT GACAATTCTG AGGAACCTTG TGTCCAGTTG TTACAAAGTT TAAGCTTTGA 1920
ACCTAACCTG CATCCCATTT CCAGCCTCTT TTCAAGCTGA GA~2~UhUVAA APP~AA 1979
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 472 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Thr Ser Asp Val Lys Glu Leu Ile Pro Glu Phe Tyr Tyr Leu Pro Glu
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- Met Phe Val Asn Ser Asn Gly Tyr Asn Leu Gly Val Arg Glu Asp Glu
Val Val Val Asn Asp Val Asp Leu Pro Pro Trp Ala Lys Lys Pro Glu
Asp Phe Val Arg Ile Asn Arg Met Ala Leu Glu Ser Glu Phe Val Ser
Cys Gln Leu His Gln Trp Ile Asp Leu Ile Phe Gly Tyr Lys Gln Arg
Gly Pro Glu Ala Val Arg Ala Leu Asn Val Phe His Tyr Leu Thr Tyr
Glu Gly Ser Val Asn Leu Asp Ser Ile Thr Asp Pro Val Leu Arg Glu
100 105 110
Ala Met Glu Ala Gln Ile Gln Asn Phe Gly Gln Thr Pro Ser Gln Leu
115 120 125
Leu Ile Glu Pro His Pro Pro Arg Asn Ser Ala Met His Leu Cys Phe
130 135 140
Leu Pro Gln Ser Pro Leu Met Phe Lys Asp Gln Met Gln Gln Asp Val
145 150 lS5 160
Ile Met Val Leu Lys Phe Pro Ser Asn Ser Pro Val Thr His Val Ala
165 170 175
Ala Asn Thr Leu Pro His Leu Thr Ile Pro Ala Val Val Thr Val Thr
180 185 190
Cys Ser Arg Leu Phe Ala Val Asn Arg Trp His Asn Thr Val Gly Leu
195 200 205
Arg Gly Ala Pro Gly Tyr Ser Leu Asp Gln Ala His His Leu Pro Ile
210 215 220
Glu Met Asp Pro Leu Ile Ala Asn Asn Ser Gly Val Asn Lys Arg Gln
225 230 235 240
Ile Thr Asp Leu Val Asp Gln Ser Ile Gln Ile Asn Ala His Cys Phe
245 250 255
Val Val Thr Ala Asp Asn Arg Tyr Ile Leu Ile Cys Gly Phe Trp Asp
260 265 270
Lys Ser Phe Arg Val Tyr Thr Thr Glu Thr Gly Lys Leu Thr Gln Ile
275 280 285
Val Phe Gly His Trp Asp Val Val Thr Cys Leu Ala Arg Ser Glu Ser
290 295 300
Tyr Ile Gly Gly Asp Cys Tyr Ile Val Ser Gly Ser Arg Asp Ala Thr
305 310 315 320
Leu Leu Leu Trp Tyr Trp Ser Gly Arg His His Ile Ile Gly Asp Asn
325 330 335
Pro Asn Ser Ser Asp Tyr Pro Ala Pro Arg Ala Val Leu Thr Gly His
340 345 350
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Asp His Glu Val Val Cys Val Ser Val Cys Ala Glu Leu Gly Leu Val
355 .360 365
Ile Ser Gly Ala Lys Glu Gly Pro Cys Leu Val His Thr Ile Thr Gly
370 375 380
Asp Leu Leu Arg Ala Leu Glu Gly Pro Glu Asn Cys Leu Phe Pro Arg
385 390 395 400
Leu Ile Ser Val Ser Ser Glu Gly His Cys Ile Ile Tyr Tyr Glu Arg
405 410 415
Gly Arg Phe Ser Asn Phe Ser Ile Asn Gly Lys Leu Leu Ala Gln Met
420 425 430
Glu Ile Asn Asp Ser Thr Arg Ala Ile Leu Leu Ser Ser Asp Gly Gln
435 440 445
Asn Leu Val Thr Gly Gly Asp Asn Gly Val Val Glu Val Trp Gln Ala
450 455 460
Cys Asp Phe Lys Gln Leu Tyr Ile
465 470
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2543 hase pairs
(B) TYPE: nucleic ac~d
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
GCAGCAGGGC GAACCGGACC TCTGTGATGT TTAATTTTCC TGACCAAGCA ACAGTTAAAA 60
AAGTTGTCTA CAGCTTGCCT CGGGTTGGAG TGGGGACCAG CTATGGTTTG CCACAAGCCA 120
GGAGGATATC ACTGGCCACT CCTCGACAGC TGTATAAGTC TTCCAATATG ACTCAGCGCT 180
GGCAAAGAAG GGAAATCTCC AACTTTGAGT ATTTGATGTT TCTCAACACG ATAGCAGGTC 240
GGACGTATAA TGATCTGAAC CAGTATCCTG TGTTTCCATG GGTGTTAACA AACTATGAAT 300
CAGAGGAGTT GGACCTGACT CTCCCAGGAA ACTTCAGGCA TCTGTCAAAG CCAAAAGGTG 360
CTTTGAACCC GAAGAGAGCA GTGTTTTACG CAGAGCGCTA TGAGACATGG GAGGAGGATC 420
AAAGCCCACC CTTCCACTAC AACACACATT ACTCAACGGC GACTTCCCCC CTTTCATGGC 480
TTGTTCGGAT TGAGCCATTC ACAACCTTCT TCCTCAATGC AAATGATGGG AAATTTGACC 540
ATCCAGACCG AACCTTCTCA TCCATTGCAA GGTCATGGAG AACCAGT QG AGAGATACAT 600
CCGATGTCAA GGAACTAATT CCAGAGTTCT ATTACGTACC AGAGATGTTT GTCAACAGCA 660
ATGGGTACCA TCTTGGAGTG AGGGAGGACG AAGTGGTGGT TAATGATGTG GACCTGCCCC 720
CCTGGGCCAA GAAGCCAGAA GACTTTGTGC GGATCAACAG GATGGCCCTG GAAAGTGAAT 780
TTGTTTCTTG CCAACTCCAT CAATGGATTG ACCTTATATT TGGCTACAAA CAGCGAGGGC 840
CAGAGGCAGT CCGTGCTCTC AATGTTTTCC ACTACTTGAC CTACGAAGGC TCTGTAAACC 900
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Iflq
TGGACAGCAT CACAGACCCT GTGCTCCGGG AGGCCATGGT TGCACAGATA CAGAACTTTG 960
CCCAGACGCC ATCTCAGTTG CTCATTGAGC CGCATCCGCC TAGGACTTCA GCCATGCATC 1020
TGTGTTCCCT TCCACAGAGC CCACTCATGT TCA~AGATCA GATGCAGCAG GATGTGATCA 1080
TGGTGCTGAA GTTTCCATCC AATTCTCCTG TGACTCATGT GGCTGCCAAC ACCCTGCCCC 1140
ACCTGACCAT CCCTGCAGTG GTGACAGTGA CCTGCAGCCG ACTGTTTGCA GTGAACAGAT 1200
GGCACAACAC AGTCGGCCTC AGAGGAGCCC CCGGATACTC CTTGGATCAA GCACACCATC 1260
TTCCCATTGA GATGGACCCA TTAATCGCAA ATAACTCTGG TGTGAACAAG CGGCAGATCA 1320
CAGACCTTGT AGACCAGAGC ATCCAGATCA ATGCCCACTG CTTCGTGGTC ACAGCTGATA 1380
ATCGCTACAT CCTCATCTGT GGGTTTTGGG ATAAAAGTTT CAGAGTTTAC TCGACAGAAA 1440
CAGGGAAACT GACACAGATT GTATTTGGCC ACTGGGATGT TGTCACATGC CTGGCCAGGT 1500
CGGAGTCCTA CATTGGTGGA GACTGCTACA TAGTGTCTGG ATCTCGGGAC GCCACCTTGC lS60
TTCTCTGGTA CTGGAGTGGG CGTCACCACA TCATCGGAGA CAACCCCAAT AGCAGTGACT 1620
ATCCTGCGCC CAGAGCTGTC CTCACAGGCC ATGACCATGA AGTTGTCTGT GTCTCCGTCT 1680
GTGCAGAACT CGGACTCGTT ATCAGTGGTG CTAAAGAGGG CCCTTGCCTC GTTCATACCA 1740
TCACTGGAAA TCTGCTGAAG GCCCTGGAAG GACCAGAAAA CTGCTTATTT CCACGCCTAA 1800
TTTCGGTATC CAGTGAAGGC CACTGCATCA TATATTATGA GCGAGGACGG TTTAGCAACT 1860
TCAGCATCAA TGGGAAACTT TTGGCTCAAA TGGAGATCAA TGATTCCACT AGGGCTATTC 1920
TCCTGAGCAG CGATGGACAG AACCTGGTGA CTGGAGGGGA CAATGGTGTG GTGGAGGTCT 1980
GGCAGGCCTG TGACTTTAAG CAGCTGTACA TTTACCCAGG ATGTGATGCT GGCATTAGAG 2040
CGATGGATTT ATCCCATGAC CA~AGGACTC TGATCACTGG CATGGCTTCC GGCAGCATTG 2100
TACTTTTAAT ATAGATTTTA ATCGGTGGCA TTATGAGCAT CAGAACAGTA CTGAAGAGAA 2160
GCAGCAGAAG.CCACATTCAA GTGAGAGCAC AAGTGCTTCT GTGGAAAGGC AGTATCTCTG 2220
GTGGGACGCT GGTCCACATC GGCCTCTGCT TGTACATCCA TCCCACCCAG CAGTCGCCGA 2280
ACATCATAGT CGGGAGCCAT TTCACCCTGT TTTTCCAGGA CTGAACACCA GCTGCTGTCA 2340
AGCAAGCTTA TATCATGTAA ATTATCTGAA TTAGGAGCCG TTTTGGTAAT TATTTCATAT 2400
ATCGCCGTTT ATTGAGAAAA GGTTGTAGGA AGCCTCACAA GAGACTTTTG ACAATTCTGA 2460
GGAACCTTGT GCCCAGTTGT TACAAAGTTT AAGCTTTGAA CCTAACTTGC ATCCCATTTC 2520
CAGCCTCGGG CTTCACTCGT GCC 2543
-
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 703 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
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1~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
- Ser Arg Ala Asn Arg Thr Ser Val Met Phe Asn Phe Pro Asp Gln Ala
1 5 10 15
Thr Val Lys Lys Val Val Tyr Ser Leu Pro Arg Val Gly Val Gly Thr
Ser Tyr Gly Leu Pro Gln Ala Arg Arg Ile Ser Leu Ala Thr Pro Arg
Gln Leu Tyr Lys Ser Ser Asn Met Thr Gln Arg Trp Gln Arg Arg Glu
Ile Ser Asn Phe Glu Tyr Leu Met ~he Leu Asn Thr Ile Ala Gly Arg
Thr Tyr Asn Asp Leu Asn Gln Tyr Pro Val Phe Pro Trp Val Leu Thr
Asn Tyr Glu Ser Glu Glu Leu Asp Leu Thr Leu Pro Gly Asn Phe Arg
100 105 110
His Leu Ser Lys Pro Lys Gly Ala Leu Asn Pro Lys Arg Ala Val Phe
115 120 125
Tyr Ala Glu Arg Tyr Glu Thr Trp Glu Glu Asp Gln Ser Pro Pro Phe
130 135 140
His Tyr Asn Thr His Tyr Ser Thr Ala Thr Ser Pro Leu Ser Trp Leu
145 150 155 160
Val Arg Ile Glu Pro Phe Thr Thr Phe Phe Leu Asn Ala Asn Asp Gly
165 170 175
Lys Phe Asp His Pro Asp Arg Thr Phe Ser Ser Ile Ala Arg Ser Trp
180 185 190
Arg Thr Ser Gln Arg Asp Thr Ser Asp Val Lys Glu Leu Ile Pro Glu
195 200 205
Phe Tyr Tyr Val Pro Glu Met Phe Val Asn Ser Asn Gly Tyr His Leu
210 215 220
Gly Val Arg Glu Asp Glu Val Val Val Asn Asp Val Asp Leu Pro Pro
225 230 235 240
Trp Ala Lys Lys Pro Glu Asp Phe Val Arg Ile Asn Arg Met Ala Leu
245 250 255
Glu Ser Glu Phe Val Ser Cys Gln Leu His Gln Trp Ile Asp Leu Ile
260 265 270
Phe Gly Tyr Lys Gln Arg Gly Pro Glu Ala Val Arg Ala Leu Asn Val
275 280 285
Phe His Tyr Leu Thr Tyr Glu Gly Ser Val Asn Leu Asp Ser Ile Thr
290 295 300
Asp Pro Val Leu Arg Glu Ala Met Val Ala Gln Ile Gln Asn Phe Ala
305 310 315 320
Gln Thr Pro Ser Gln Leu Leu Ile Glu Pro His Pro Pro Arg Thr Ser
325 330 335
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1~\
Ala Met His Leu Cys Ser Leu Pro Gln Ser Pro Leu Met Phe Lys Asp
340 345 350
Gln Met Gln Gln Asp Val Ile Met Val Leu Lys Phe Pro Ser Asn Ser
355 360 365
Pro Val Thr His Val Ala Ala Asn Thr Leu Pro His Leu Thr Ile Pro
370 375 380
Ala Val Val Thr Val Thr Cys Ser Arg Leu Phe Ala Val Asn Arg Trp
385 390 395 400
His Asn Thr Val Gly Leu Arg Gly Ala Pro Gly Tyr Ser Leu Asp Gln
405 410 415
Ala His His Leu Pro Ile Glu Met Asp Pro Leu Ile Ala Asn Asn Ser
420 425 430
Gly Val Asn Lys Arg Gln Ile Thr Asp Leu Val Asp Gln Ser Ile Gln
435 440 445
Ile Asn Ala His Cys Phe Val Val Thr Ala Asp Asn Arg Tyr Ile Leu
450 455 46Q
Ile Cys Gly Phe Trp Asp Lys Ser Phe Arg Val Tyr Ser Thr Glu Thr
465 470 475 480
Gly Lys Leu Thr Gln Ile Val Phe Gly His Trp Asp Val Val Thr Cys
485 490 495
Leu Ala Arg Ser Glu Ser Tyr Ile Gly Gly Asp Cys Tyr Ile Val Ser
500 505 510
Gly Ser Arg Asp Ala Thr Leu Leu Leu Trp Tyr Trp Ser Gly Arg His
515 520 525
His Ile Ile Gly Asp Asn Pro Asn Ser Ser Asp Tyr Pro Ala Pro Arg
530 535 540
Ala Val Leu Thr Gly His Asp His Glu Val Val Cys Val Ser Val Cys
545 550 555 560
Ala Glu Leu Gly Leu Val Ile Ser Gly Ala Lys Glu Gly Pro Cys Leu
565 570 575
Val His Thr Ile Thr Gly Asn Leu Leu Lys Ala Leu Glu Gly Pro Glu
580 585 590
Asn Cys Leu Phe Pro Arg Leu Ile Ser Val Ser Ser Glu Gly His Cys
595 600 = 605 .
Ile Ile Tyr Tyr Glu Arg Gly Arg Phe Ser Asn Phe Ser Ile Asn Gly
610 615 620
Lys Leu Leu Ala Gln Met Glu Ile Asn Asp Ser Thr Arg Ala Ile Leu
625 630 635 640
Leu Ser Ser Asp Gly Gln Asn Leu Val Thr Gly Gly Asp Asn Gly Val
645 650 655
Val Glu Val Trp Gln Ala Cys Asp Phe Lys Gln Leu Tyr Ile Tyr Pro
660 665 670
Gly Cys Asp Ala Gly Ile Arg Ala Met Asp Leu Ser His Asp Gln Arg
675 680 685
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Thr Leu Ile Thr Gly Met Ala Ser Gly Ser Ile Val Leu Leu Ile
- 690 695 700
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
CATTCTTTAT TGACAGTGTT 20
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GTGAACCCTA CCATATCT 18
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
ATGCCATTCT TTATTGACAG 20
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
CATTGGCACA GGAAACAAC 19
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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~3
(xi) SEQUENCE DESC~IPTION: SEQ ID NO: 19:
- AAACCCTGTC TCGAAACAA 19
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
GAATTCC QA GGACAGGT 18
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
TAGGAGGTGT GGCCTTG 17
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
AGAGACGGCG GA Q CTTA 18
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
TAAATATGAG GCGGG Q G 18
~r
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTICN: SEQ ID NO: 24:
- ATACTCTAAG TAAGATACAC 20
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
TGTTTTAACT GTTTGCTAA 19
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
ACGCAGTGGG CATGCTG 17
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
CACAGGCTGT GACTGGAA 18
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
AGTAGCCACA GGCCCTA 17
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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lg~'
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
~ GAGATTACCC CAATAGTA 18
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
AGTGGAAGGA GGCTGTC 17
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 31:
CCATGGCGAT GAAGCGG 17
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
ATGATGCA~A GAACCCAG 18
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
~D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
TTCAACTACA TAGTGAATT 19
c (2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
- TCCCTAACAC ATCCCTAA 18
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
CCTCATGTTA GGGTAGAG 18
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
CCGTTAGTGT GTAGTCTC 18
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
CACATTGGCA CAGGAAAC 18
(2) INFORMATION FOR SEQ ID NO: 38:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
TTACGAATGT GCCTGGTG 18
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
~ TCCAAACACA CTAAACCTG 19
~.
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
CATGCCATTC TTTATTGACA 20
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
CTCAGTAGAC TATACGAG 18
(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
GAGTTCAAGG TCATCCTC 18
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
GCCTAAGCCC ATTATCG 17
-
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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i~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
- TAAATGCTGC CATA~ACTCC 20
(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
AGAAGTGACT TCAGGTAATA 20
(2) INFORMATION FOR SEQ ID NO: 46:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
AGTCTCTCAC ACTTACAC 18
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: n~cleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
GACCCAAGTC AGCTTTC 17
(2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TY~E: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
CCAGTGTGTC ACTTAAGC 18
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
- AGGGAGATGT ATCATCTGC 19
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
AGGGATCACC ATGCTTTG 18
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
ACTTGGTCTT GGGGTCC 17
(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
GAAAGCAGTG TAATGAGG 18
(2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
TGCCTCTACA TGGGAGC 17
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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~q~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
- GCAAGCATTT AGTTAAACG
(2) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
CTTGTTCTTG TATATCTG
(2) INFORMATION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
ACAATGA~AT CCTCCACC
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
GTGACTTGAT CCAGACTG
(2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
CTTGCTCTCA CTGTTCTC
(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: ~8 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
~ CAGGTGGAGA TGCTGTTC 18
(2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
GAGATGCCTT CAGGCAGT 18
(2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
CCGTTAGTGT GTAGTCTC 18
(2) INFORMATION FOR SEQ ID NO: 62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:
CTTGCTCTCA CTGTTCTC 18
(2) INFORMATION FOR SEQ ID NO: 63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:
TGGATGGGCT GTCTGAACGC 20
(2) INFORMATION FOR SEQ ID NO: 64:
(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 NO: 64:
- TGCTGGCAGA TGCTGGCATA 20
(2) INFORMATION FOR SEQ ID NO: 65:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
CCAAGATGAA AGCAGCCGAT GGGGAAAACT 30
(2) INFORMATION FOR SEQ ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
TCAGCCTCTT TCTTGCTCCG TGA~ACTGCT 30
(2) INFORMATION FOR SEQ ID NO: 67:
(ij SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:
AGTTTATGAG TCCAAATGAT 20
(2) INFORMATION FOR SEQ ID NO: 68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:
GAATGATGAA GTTGCTCTGA 20
(2) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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W097/28262 PCTAUS97/0~748
Iq'~
(~i) SEQUENCE DESCRIPTION: SEQ ID NO: 69:
- CAGCAGTTCT TCAGATGGA 19
r
(2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic aci~
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:
ATCTTTCTGT TGTTCCCCTA 20
(2) INFORMATION FOR SEQ ID NO: 71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:
TAGGGGAGCA ACAGAAAGAT 20
(2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:
GCTCATAGTA GTATCACTTT 20
(2) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHA~ACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:
CGCACATGGC AACCCTT 17
(2) INFORMATION FOR SEQ ID NO: 74:
(i) SEQUENCE CHA~ACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: si ngle
(D) TOPOLOGY: linear
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W097/28262 PCTAUS97/01748
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
- GCACATGGGC AACCCTT 17
(2) INFORMATION FOR SEQ ID NO: 75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
CCAGGACACC AGGGCTACAG AG 22
(2) INFORMATION FOR SEQ ID NO: 76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
CCCGAGTGCT GGGATTAAAG 20
(2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:
GTTGTAAAAC GACGGCCAGT GGCAAGTTCA GCCTGGTTAA G 41
(2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:
CACAGGAAAC AGCTATGACC AGAGTATTTC TTCCAGGGTA 40
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W O 97/28262 PCTrUS97/01748
19~
All of the compositions and methods disclosed and claimed herein can be made and" ~ executed without undue experimentation in light of the present disclosure. While the
compositions and methods of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that variations may be applied to the
- 5 composition, methods and in the steps or in the sequence of steps of the method described herein
without departing from the concept, spirit and scope of the invention. More specifically, it will be
~ppal c;lll that certain agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or similar results would be achieved.
All such similar substitutes and modifications apparent to those skilled in the art are deemed to be
0 within the spirit, scope and concept of the invention as defined by the appended claims.
Accordingly, the exclusive rights sought to be patented are as described in the claims below.
