CA2244744A1 - Lyst1 and lyst2 gene compositions and methods of use - Google Patents

Abstract

Disclosed are compositions comprising murine Lyst1 and Lyst2 genes and human LYST1 and LYST2 genes. Also disclosed are the Lyst1, Lyst2, LYST1, and LYST2 proteins encoded by these genes, respectively. Also disclosed are methods of using these genes in identifying patients with Chediak-Higashi Syndrome and detecting CHS-related nucleic acid and/or protein sequences. Also disclosed are methods for the recombinant expression of LYST1, Lyst1, LYST2, and Lyst2 polypeptides, antibodies raised against these polypeptides, and therapeutic approaches to treatment of autoimmune diseases and certain types of tumors. Assays for detection of the gene mutations resulting in CH Syndrome, as well as diagnostic probes for the detection of Lyst1, Lyst2, LYST1, and LYST2 genes are also provided.

Description

W O 97/28262 PC~rUS97/01748 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 W O 97/28262 PCT~US97/01748 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.

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.

WO 97/28262 PCT~US97/01748 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

W 097/28Z62 PCTnUS97/01748 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 W O 97J28262 PCTrUS97/01748 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, W O 97/28262 PCT~S97/0174 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 WO 97/28262 PC~AUS97/017~8 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.

W O 97/28262 PCTrUS97/01748 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 CA 02244744 l998-07-29 W O 97/2826~ PCTrUS97/01748 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 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 W O 97/28262 PCT~US97/01748 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 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 W O 97/28262 rcTrusg7/ol748 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 , CA 02244744 l998-07-29 WO 97/28262 PCT~US97/01748 - 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.

WO 97/28262 PCT~US97/01748 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 WO 97J28262 PCT~US97/01748 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 WO 97/28262 PCTrUS97/01748 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.

W O 97/28262 PCTA~S97101748 - 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 W O 97/28262 P~l-/u~7/01748 '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 W O 97/28262 PCT~US97/01748 - 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.

- 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 .

W O 97/28262 PCTrUS97/01748 ~3 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.

W O 97128262 PCT~US97/01748 Z~
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.

WO 97/28262 PCTrUS97/01748 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 w 097/28262 PCT~US97/01748 ;~
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 W O 97/28262 PCTrUS97/01748 '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.

WO 97/28262 PCTrUS97/01748 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-W 097128262 PCT~US97/01748 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.

W O 97/28262 ~CT~US97/01748 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 W O 97/28262 PCT~US97/01748 3 t 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.
-PCTnUS971~1748 ~Z
- 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 CA 02244744 l998-07-29 ~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 , -- -- === ===== = . = = = =

W O 97/28262 PCTnUS97/01748 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'.

PCT~US97/01748 - 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 w097/28262 PCTnUS97/01748 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 , W O 97/28262 PCTrUS97/01748 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 WO 97/28262 PCTrUS97/01748 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.

w 097/28262 PCT~US97~01748 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 W O 97/28262 PCT~US97/01748 - 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 - 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.

WO 97/28262 PCTnUS97r01748 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.

. ~ -- =

PCT~US97/01748 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 . ,.

W O 97/28262 PCTrUS97/01748 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 CA 02244744 l998-07-29 WO 97/28262 PCT~US97/01748 .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 WO 97/28262 PCTrUS97/01748 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.

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 W O 97/28262 PCT~US97/01748 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.

W O 97/28262 PCTrUS97/01748 ~) 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 W O 97/28262 PCTrUS97/01748 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 ~v097/28262 PCT~US97/01748 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.

WO 97/28262 PCT~US97/01748 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 W 097/28262 PCT~US97/0174B
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 W O 97/28262 PCTrUS97/01748 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 W O 97/28262 PCTrUS97/01748 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 W 097/28262 PCT~US97101748 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 W O 97/28262 PCTrUS97/01748 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.

CA 02244744 l998-07-29 W O 97/28262 PCT~US97/01748 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.

W O 97/28262 PCT~US97/01748 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.

w 097/28262 PCT~US97/01748 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 W 097128262 PCT~US97/01748 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 W O 97/28262 PCT~US97/Q1748 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

~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 WO 97/28262 PCTnUS97/01748 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 , CA 02244744 l998-07-29 W O 97/28262 PCTrUS97/01748 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 WO 97/28262 PCT~US97/01748 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 CA 02244744 l998-07-29 W O 97/28262 PCT~US97/01748 ~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.

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~o 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 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 W O 97/28262 PCTrUS97/01748 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 W O 97/28262 PCT~US97/01748 ~<~
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 W O 97/28262 PCT~US97/01748 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 -W O 97/28262 PCTrUS97/01748 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.

,.

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 W O 97128262 PCT~US97/01748 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 W 097/28262 PCT~US97/01748 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.

CA 02244744 l998-07-29 W O 97/28262 PCT~US97/01748 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 CA 02244744 l998-07-29 WO 97/28262 PCT~US97/01748 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 w 097/28262 PCTrUS97/01748 $1 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 W O 97/28262 PCTrUS97/01748 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.

W O 97/28262 PCT~US97/01748 ~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 W097/28262 PCT~US97/01748 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:

W 097/28262 PCTrUS97/01748 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 W O 97/28262 PCTrUS97/01748 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) w 097~28262 PCT~US97/01748 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 w 097/28262 PCTrUS97/01748 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.

W O 97/28262 PCT~US97~01748 ~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 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 VVO 97128262 PCT~US97/01748 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 W O 97/28262 PCTrUS97/01748 ~'~
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).

W O 97/28262 PCTrUS97/01748 ~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 CA 02244744 l998-07-29 WO 97/28262 PCT~US97/01748 9L~
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.

q~
5.5 EXAnIPLE5-- D NA SEQUENCES OF Mo~sE LYS~I

5.5.1CDNA SEQUENCE OF LONG ISOFORM (SE Q ID N 0:3) - 5101 GCTGAGACAG TTTTATCTAG TTCATGAACC CA~ATTATAT ACAAGCTGAA
151 TGTTACAGAA GTGCTGA~AG ACTGCTCTGT CATGAGCACG GACAGCAACT

10351 AACTA~ATTC TATCATTGAT CAGGCCCTGA CATGCAGAGA AGAACTCCTG

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

951 TATGTCATGT TTTGTTATCT CTATTGGA~A AAGTTTGTAA GTTTGACATT

lC51 TGAGTTCCTA GCAGGCTTTG GGGACTGCTG TAACCAGAGT GACACTTTGG

1201 CACTGCAGAG GCAATGCCAG A~AGTCTTAG GA~AAATTTG ACTGAATTGC
1251 TTAGGGCAGC TTTA~AAATT AGAGCTTGCT TGGA~AAGCA GCCTGAGCCT
1301 TTCTCCCCGA GACA~AAGAA AACACTACAG GAGGTCCAGG AGGGCTTTGT

1401 GAGTTCTACA GCTCCTCATC TCTTGTCTTC AGAGTGCAGC TTCA~ATCCC
1451 TTTTACTTCA GTCAAGCCAT GGATTTAGTT C~AGAATTTA TCCAGCACCA

351601 AACAGTGTAA TA~AAATAAT GAGTACTGTG AAAAAGGTGA AATCAGAGCA
1651 ACTTCATCAT TCCATGTGCA CAAGGA~AAG ACACCGGCGT TGTGAGTATT

1751 TTTA~AAATC AGCTTTCTAA AAGCCCCTTT GAAGAGACCG CAGAGGGAGA

2051 GGAGGAGCAG AGCTATCACC GAGAATTA~A A~AGCAGCTT GCAACATCTG

-2301 ATAACATTCA GATTGCAAAT CACATTTGTA ATTTACTCCA GA~AGGCAAT
502351 GTAGTTGTTC AGTGGA~ATT GTATAATTAT ATCTTTAATC CTGTGCTCCA

2501 CTTCAGATTT ATTTAAAAAC TCTACCTGTC CTACTTA~AT CCAGGGTAAT

WO 97/28262 PCTnUS97/0~748 q~
2551 AAGAGATTTG TTTTTAAÇTT GTAATGGAGT A~ACCACATA ATTGAACTA~
2601 ATTACTTAGA TGGGATTCGA AGTCATTCCC TGA~AGCATT TGA~ACTCTG
2651 ATTGTCAGCC TAGGGGAACA ACAGA~AGAT GCTGCAGTTC TAGACGTCGA

2801 TATGCCAGCC TCAGAGAGCC TGATCCA~AA A~ACGA~AGA CCATTCACCA
2851 GGATGTTCAC ATA~ACACCA TA~ACCTCTT CCTCTGTGTG GCTTTTCTAT

lO3001 ACCATGTCTG TCTCTTGAGG ACGTTGTCTT ACCTTCCCCT GAATGTTTGC

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

3401 ACTGAATCTG TTCCTGGGGA ACGAAAGGCA TTTATGAGTC A~CA~AGTGA

3551 CAGGGCTTGT CTGTGGA~AA TATATTGTGT GAACTGAGGG AACACCTTTC
3601 CCAGTCAAAG GTGGCAGAAA CAGAATTAGC A~AGCCTTTA TTTGATGCCC

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

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

4601 ATGTGGAATA CATCCAATGA ATCCGAGAGT GCTGCAGA~A GGGGA~AAAG
4651 AGTA~AGA~A AGA~ACAAAC CATCAGTTCT GGAAGACAGC AGTTTTGAAG

454751 AGACTCATAG AAGACGGCTG TATTCATTTG ATTTCACTGG GGTCCA~AGC

4851 GTGTGTGCAT GGACTCA~AT GATGACACGA A~GCTGTCTC ACTAGCACAG
4901 GTGGA~TCAC AGGAGAATAT TTTCTTTCCA AGCA~ATGGC AACACTTAGT
4951 ACTTACCTAT ATTCAGCATC CTCAAGGGAA A~AGAATGTC CATGGGGA~A

5051 GTGCTCCCAA GAAAAACAAG CTTATCATCA GACAGCAATA A~ACATTTTG

5151 GAAAATGGGA CCTGGGGAAC TTGCTCCTCT TCAATGGAGC TA~AATTGGC

55~ 5251 CATGCCGTGT A~ATATGGAC AGCCAGTCAT TGACTACTCC AA~TACATTA
-W O 97/28262 PCTrUS97/01748 qr1 5301 ATA~AGACAT TTTGAGATGT GATGA~ATCA GAGACCTTTT TATGACQAG
- 5351 A~AGAAGTGG ATGTTGGTCT CTTAATTGAA AGTCTTTCAG TTGTTTATAC

5451= AGGGCCAAGT GA~AACTCAG CCCTCTCAAA GACCCTTCAG CTCA~AGGAA
5501 GCCCAGAGCA TCTTGCTAGA ACCTTCTCAA CTCA~AGGCC TCCAACCTAC

5701 ACAGAGAACA CAGGAACTGG A~AATTGTAA TGGACTCTCT ATGATTCACC
5751 AAGTGTTGGT CA~ACAGA~A TGCATTGTTG GCTTTCACAT TTTGAAGACC
5801 CTTCTTGAAG GTTGCTGCGG TGAAGA~GTT ATCCACGTCA GTGAGCATGG

6101 CAGCTTACAT CTATGCCCCG AGA~GTTTGT AGATCATTTG TGA~AATCAT

6301 GGAGA~AGTG CAGTCACTCG CGTACCTGAG GCATTCTAGC AGCGGAGGGC
6351 A~GCCTTTCC CAGCCCTGGA TTCCTGGTAA TAAGCCCATC TGCCTTTACT

6551 GACAAGTTAC A~AATATTGC AGATGCCAAC CCAGAGA~AC AGAATCTTTT
6601 AGGAAGACCC TACGCACTGA A~ACAAGCAA AGAGGAAGCA TTCATCAGCA

7201 AGTAATTA~A CTGTTACATG CATACATTAA TAGAGCATCA AAGGAGCAAA

7301 TATCTTCATA GGGGAACTCA GGAGTTGTTG GAGTGCTTTG TTGA~ATGTT

7401 AGCACATGGA ACTGTTCQAG A~GTGGTCTG TCATTCCCGT TCTCGGACTA

7501 TCTTCTGCAA GTTTTA~ACT CTTGTTCCAA GGTAGCAGAC ATGCTACTGG

7601 TTAGA~AAGA ACATTCCTGT GAACGAATAC A~ATTGCTCG CATGTGATAT

7751 QATAATAGCA A~ACAAGAG GACACA~AAT ATGGCTTTGG CCCTGCAGCT

7851 ACTCTGA~AG TCCAGTGCAC TCGCCTTCTG CCCACCGCCA TTCAGTGCCT
7901 CCGAAGCGGA GAAGCATTGC TGGTTCTCGC A~ATTCCCTC TGGCTCAGAC

WO 97~8262 PCTAUS97/01748 8051 GCAGAGCTCG CTCAGAGÇCT GCAGAGGCTC ACCATCTTAG CTGTGAACAG

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

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

209001 AGGGCCA~AC CGAGAGAGGA GACGTTTGCA GA&ATGCTAT CTAACTATTC

9101 CCCCCACTCT CTTACCTTTT TGA~GATA~A ACTCATTCTT CCTTCTCCTC
9151 TACTGTCAAA GACA~AGCTG CAAGTGAATC CATCAGAGTG AATCGAAGAT

- 9251 A~ATGTGGGA TGTACTTTGT GGAAGACAAT GCCTCTGACG CAGTTGA~AG

9351 AGGA~ATTAA AGAAGTTCAC AGGCGCTGGT GGCAACTAAG AGATAATGCT
9401 GTAGA~ATCT TTTTAACAAA TGGCAGAACA CTCCTATTAG CATTTGACAA

9701 AGA~ACCTAT CTAAGCCTAT AGCTGTGCAG TATA~AGA~A AAGAAGACCG

9901 CACTA~AATG TTTCTAGCCT ATC~AGATCA GAGTTTCGAC ATTCCAGACC

45_ 10251 CGTCTGTTCA AGCCATCAAT GTCTTCCACC CTGCTACATA TTTTGGAATG
10301 GATGTCTCTG CAGTTGAAGA TCCAGTGCAG AGACGGGCTT TAGA~ACCAT
10351 GATA~AAACC TACGGGCAGA CCCCACGTCA GTTGTTCCAC ACAGCCCATG

10601 CAGCCCCATG GAGA~AGATT TGGTTCCCTG CAGGCACTGC CCACCAGAGC

10751 CTAAGCTGGG GATATGCTGA CAACATCTTA CGGTTGA~AA GTAAGCAGAG

W O 97/28262 PCT~US97/01748 ~' 10951 AATTGA~ATG GAGAGTCAGA TGCATCTCTA TGGACACACA GAGGAGATCA

11101 TTTGGCTGGA CACA~AAGCC CTGTGACGGC TGTCTCTGCC AGTGA~ACGT

11301 ACGTCATTGC TGGGGGATTA GA~AATGGCA TTGTAAGGCT ATGGAGCACA
11351 TGGGACTTGA AGCCTGTGAG AGAGATTACA TTTCCCA~AT CA~ATAAGCC

11601 ACCAACTGCA GAAGCAGATG ACTGA&CAGA TATCCAGGAA AGACAACACA
11651 CGTGCCTCTG TGCGCGCTTC CCCAGCCTCC GTGGGCCTGA GAGTA~AGCC

2011751 AATTA~AGTC AGAATCTTGA TGCTTTTTCC CA~AAGGTTA GGCTGAATCA

5.5.2 C~N~ SEQUENCE OF SHORTISOFORU~(SE Q nD N 0:5) 101 GCTGAGACAG TTTTATCTAG TTCATGAACC CA~ATTATAT ACAAGCTGAA

301 A~CTCTTGGA CAGTACCTTG TCCATGGACG AGGATTTCTG TTACTTACCA
351 AACTA~ATTC TATCATTGAT CAGGCCCTGA CATGCAGAGA AGAACTCCTG

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

951 TATGTCATGT TTTGTTATCT CTATTGGA~A AAGTTTGTAA GTTTGACATT

1201 CACTGCAGAG GCAATGCCAG A~AGTCTTAG GAAAAATTTG ACTGAATTGC
501251 TTAGGGCAGC TTTA ~ ATT AGAGCTTGCT TGGAP~AAGCA GCCTGAGCCT
1301 TTCTCCCCGA GACA~AAGAA A~CACTACAG GAGGTCCAGG AGGGCTTTGT

WO 97/28262 PCTrUS97/01748 1401 GAGTTCTACA GCTCCTCATC TCTTGTCTTC AGAGTGCAGC TTCA~ATCCC

1501 AGGATTTAAT CTCTTTGGAA CAGCAGTTCT TCAGATGG~A TGGCTGCTTA

1601 AACAGTGTAA TA~AAATAAT GAGTACTGTG A~AAAGGTGA AATCAGAGCA
1651 ACTTCATCAT TCCATGTGCA CAAGGA~AAG ACACCGGCGT TGTGAGTATT

1751 TTTA~AAATC AGCTTTCTAA AAGCCCCTTT GAAGAGACCG CAGAGGGAGA

l901 CTATCAGGTG TACACAGTGT TGGAATCTGT TGTTGTATGG ATCCTAAGTC

2051 GGAGGAGCAG AGCTATCACC GAGAATTAAA A~AGCAGCTT GCAACATCTG

2301 ATAACATTCA GATTGCA~AT CACATTTGTA ATTTACTCCA GA~AGGCAAT
2351 GTAGTTGTTC AGTGGA~ATT GTATAATTAT ATCTTTAATC CTGTGCTCCA

2551 AAGAGATTTG TTTTTAAGTT GTAATGGAGT A~ACCACATA ATTGAACTAA
2601 ATTACTTAGA TGGGATTCGA AGTCATTCCC TGAAAGCATT TGA~ACTCTG

2801 TATGCCAGCC TCAGAGAGCC TGATCCA~AA A~ACGA~AGA CCATTCACCA
2851 GGATGTTCAC ATA~ACACCA TAAACCTCTT CCTCTGTGTG GCTTTTCTAT

3201 ATCAAGGAAG AAGGCAGGGA GAAATGAGTA GAAATGA~AA CCAAGAGCTA

3301 AACAGCACCA GACCTGGGAT TTCTGAGA~A GAGTGCTGAC AGCGTGCGTG

3401 ACTGAATCTG TTCCTGGGGA ACGA~AGGCA TTTATGAGTC AACAAAGTGA

3551 CAGGGCTTGT CTGTGGA~AA TATATTGTGT GAACTGAGGG AACACCTTTC

3751 CGGAGATTTT TCAGAAGAAG CTGAGGATTC TCAGTGTTGT AGTTTGA~AC
3801 TTCTGGGTGA GGAAGAAGGC TATGAAGCGG ATAGTGA~AG CAATCCTGAG

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

W O 97/28262 PCTrUS97/01748 Ivl 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

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

4951 CATGACTGTG TTAGTGTGGC TTCTGATACT AGATAGTTAT AAATA~AACC

5051 CCCTTCTAGT TACTGTCCCT GATCATTTAT ATGTAACAGT CCA~AGTTAG

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

5501 GCTAGCTCAC AGCCATA~AC ACTTCTCTCA GCGGAGACAA ACTGTGATTC

5601 AATAATGATC ATTTGAGCAG TGCAGCTTTT CT~AGAAGAG TATTAATAAT

5751 CAACAA~AGC AGAACCTTGG TATGGAGTGT GGCTGACGAT GGTCCTTTAG
5801 CACCCTCAGG CCTTGTAGTT TA~AGCATTT AATAACTTTT AAAACACTGG

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

201 LVAKPDPSAT KEQVLSDTMS VENSREVILR QDSNGDILSE PA~LSILSMM
~ 251 NNSPFDLCHV LLSLLEKVCK FDIALNHNSS LALS W PTLT EFLAGFGDCC

351 KNLTELLR~A LKIRACLEKQ PEPFSPRQKK TLQEVQEGFV FSKYRHRALL

451 QMEWLLTRDG VPSEA~EHLK ALINSVIKIM STVKKVKSEQ LHHSMCTRKR

1151 KPLFDALLRV ALGNHSADLG PGDAVTEKSH PS~F.~.T.T..~QP GDFSEEAEDS

15 ~1301 SLLIQQGTVK ILLGGFLNIL TQTNSDFQAC QRVLVDLLVS LMSSRTCSED

2101 PAFPTYLPLI RAQKLAASLG FSVDKLÇNIA DANPEKQNLL GRPYALKTSK

2~01 FDLEEVKHME LFQKWSVIPV LGLIETSLYD NVLLHNALLL LLQVLNSCSK

40~551 RSTANHDSES PVHSPSAHRH SVPPKRRSIA GSRKFPLAQT ESLLMKMRSV

2951 RCYLTIPNKY LLRDRQKSEG VLRPPLSYLF EDKTHSSFSS TVKDK~ASES

WO 97/28262 PCTrUS97/01748 3401 LFHTAHASRP GAKLNIEGEL PA~VGLLVQF AFRETREPVK EVTHPSPLSW

5.6.2 PEPTIDE SEQUENCE OF S~ORT ISOFORM (SEQ ID NO:6) 1 MSTDSNSLAR EFLIDVNQLC NA W QRAEAR ~.~F~EETHMA TLGQYLVHGR

351 KNLTELLR~A LKIRACLEKQ PEPFSPRQKK TLQEVQEGFV FSKYRHRALL

601 LPALKAFQQH ILNVLSKLLV DQLGGAELSP RIKK~ACNIC TVDSDQLAKL

801 NHIIELNYLD GIRSHSLKAF ETLIVSLGEQ QKDA~VLDVD GLDIQQELPS

1151 KPLFDALLRV ALGNHSADLG PGDAVTEKSH PS~.F.F.T,T.SQP GDFSEEAEDS

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

CA 02244744 l998-07-29 101 TCATTCTTAC GTGGGA~AGT TGTATTCCGA GGTTTCTGTG GTGCATGAAG

301 GA~CAGAAGC CAGGATCTAA CTGCAAACAA GAGACCCAGC TTGCTTAACA

401 ATTAGCCTGC AACA~AGAGT TCCTTGCTCA TCTA~AAGAG GCAATCACCG

501 CCA~ATGATA TAGACTGTAA ATGTCACAGC AGTGGTGA~A GACTGCTCGG

701 GAGGATTTCT ATTACTTACC AAGCTA~ATT CTATAATTGA TCAGGCATTG

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

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

1351 A~AGTTTGTA AGTTTGACGT TACCTTGAAT CATAATTCTC CTTTAGCAGC
- 1401 CAGTGTAGTG CCCACACTAA CTGA~TTCCT A5CAGGCTTT GGGGACTGCT

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

1901 TTCA~ATGGA ATGGCTGGTT TTAAGAGATG GAGTTCCTCC CGAGGCCTCA
1951 GAGCATTTGA AAGCCCTAAT A~ATAGTGTG ATGA~AATAA TGAGCACTGT
2001 C~AAA~GTG A~ATCAGAGC AACTTCATCA TTCGATGTGT ACAAGAAAAA

2101 TCAGGTCTTC TGGTTTCGGC TTTTA~AAAC CAGGTTTCCA A~AACCCATT

~S 2301 CTGTTGTATG GATCCCA~AT CTGTAATCAT TCCTTTGCTC CATGCTTTTA

2401 A~ACTTATTT TGGATCAGTT AGGAGGAGCA GAGATATCAC CAAAAATTAA
2451 A~AAGCAGCT TGTAATATTT GTACTGTTGA CTCTGACCAA CTAGCCCAAT

2601 TGAAGATTTG TTGTGGA~AT GGGATGCTTT A~AGGCTTAT CAGAACTTTG

2751 CATATTTAAT CCTGTGCTCC A~AGAGGAGT TGAATTAGCA CATCATTGTC
5 5 2 8 01 AACACCTAAG CGTTACTTCA GCTCA~AGTC ATGTATGTAG CCATCATAAC

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

3151 TCTCCTCAGA GTCTCAGCAA ATTTTATGCT GGCCTCA~AG AAGCTTATCC
- 3201 A~AGAGACGG AAGACTGTTA ACCAAGATGT TCATATCAAC ACAATA~ACC
3251 TATTCCTCTG TGTGGCTTTT TTATGCGTAA GTA~AGA~GC AGAGTCTGAC

3451 TGTCGTTGGA TCTACATGTT GAGTTCAGTG TTCCAGA~AC AGTTTTATAG

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

3951 GTCTGTGGAA AGTATATTAT TTGA~ATGAG GGACCATCTT TCCCAGTCAA
4001 AGGTGATTGA AACACAACTA GCA~AGCCTT TATTTGATGC CCTGCTTCGA

4251 AACCCAGGAT GATGGGGTAG ACTTA~AGTC TGA~ACAGAA GGTTTCAGTG
4301 CATCAAGCAG TCCAAATGAC TTACTCGAAA ACCTCACTCA AGGGGA~ATA

4401 AGCCAAACTT GATGTGCTTG CCCATGTATT TGAGAGTTTT TTGA~AATTA
4451 TTAGGCAGAA AGA~AAGAAT GTTTTTCTGC TCATGCAACA GGGAACTGTG

4651 GAGA~ATCTC CTTGTACAAA AATTCTTCTT CTGGGTATTC TGA~AATTAT

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

5101 GACAGACCAG AAGGTGCAGA GTACATA~AT CCTGGTGAAA GACTCATAGA

5251 GATTCA~ATG ATGACATGAA AGCTGTTTTA CTAGCACAGG TTGAATCACA

5351 TACAGCAGCC CCAAGGGA~A AGGAGGATTC ATGGGA~AAT CTCCATATGG

5451 A~AAACAAGT TTGTCATCTG ATAGCAATAA AACATTTTGC ATGATTGGCC
5501 ATTGTTTATC ATCCCAAGAA GAGTTTTTGC AGTTGGCTGG A~AATGGGAC
5551 CTGGGA~ATT TGCTTCTCTT CAACGGAGCT AAGGTTGGTT CACAAGAGGC

W O 97/28262 PCTrUS97/01748 5651 AGTATGGCAA GCCAGTCAAT GACTACTCCA AATATATTAA TA~AGA~ATT
- 5701 TTGCGATGTG AACA~ATCAG AGAATTTTTT ATGACCAAGA AAGATGTGGA

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

6451 CTACTGACTT GTCAGGTTTT GCAGGAATAC A~AGAGGGGC AACTCACACC
6501 CATGCCCCGA GAGATGGCAA GATCTTTCAG GAGA~AGTGC GGTCAATCAT

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

7001 TCATA~ACAG TTGGGAGCAG AACCCAGGTC AGAAGATGAC AGTCCTGGGG

5.7.2 CDNA SEQUENCEOFSHORT ~OFORM(SEQID NO:9) 101 TCATTCTTAC GTGGGA~AGT TGTATTCCGA GGTTTCTGTG GTGCATGAAG

301 GAACAGAAGC CAGGATCTAA CTGCA~ACAA GAGACCCAGC TTGCTTAACA
351 GCATGGAAGA GAACCAGTTT CCTTGCAGCT ACCTGGGA~G ACGGTTGCTA
401 ATTAGCCTGC AACA~AGAGT TCCT TGCTCA TC TA~AAGAG GCAATCACCG

501 CCA~ATGATA TAGACTGTAA ATGTCACAGC AGTGGTGA~A GACTGCTCGG

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

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

' 1301 TGAACAATTC TCCATTTGAC TTATGTCATG TTTTGTTATC TTTATTAGAA

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

1951 GAGCATTTGA AAGCCCTAAT A~ATAGTGTG ATGA~AATAA TGAGCACTGT
2001 CLA~ALAGTG A~ATCAGAGC AACTTCATCA TTCGATGTGT ACAAGA~AAA

2101 TCAGGTCTTC TGGTTTCGGC TTTTA~A~AC CAGGTTTCCA A~AACCCATT

2351 AATTGCCAGC ACTGA~AAAT TTTCAGCAGC ATATATTGAA TATCCTTAAC
2401 A~ACTTATTT TGGATCAGTT AGGAGGAGCA GAGATATCAC CA~AAATTAA
2451 A~AAGCAGCT TGTAATATTT GTACTGTTGA CTCTGACCAA CTAGCCCAAT

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

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

3201 AAAGAGACGG AAGACTGTTA ACCAAGATGT TCATATCAAC ACAATA~ACC
3251 TATTCCTCTG TGTGGCTTTT TTATGCGTAA GTA~AGAAGC AGAGTCTGAC

3451 TGTCGTTGGA TCTACATGTT GAGTTCAGTG TTCCAGA~AC AGTTTTATAG

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

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

53951 GTCTGTGGAA AGTATATTAT TTGA~ATGAG GGACCATCTT TCCCAGTCAA
4001 AGGTGATTGA AACACAACTA GCA~AGCCTT TATTTGATGC CCTGCTTCGA

= 4201 GAAGAAGAAG GTTACGAAGC AGATAGTGAA AGCAATCCTG AAGATGGCGA
4251 AACCCAGGAT GATGGGGTAG ACTTA~AGTC TGA~ACAGAA GGTTTCAGTG
4301 CATCAAGCAG TCCAAATGAC TTACTCGA~A ACCTCACTCA AGGGGA~ATA

4401 AGCCA~ACTT GATGTGCTTG CCCATGTATT TGAGAGTTTT TTGAAAATTA
. 4451 TTAGGCAGAA AGAAAAGAAT GTTTTTCTGC TCATGCAACA GGGAACTGTG
4501 A~A~ATCTTT TAGGAGGGTT CTTGAGTATT TTAACACAGG ATGATTCTGA

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

5001 TATCCATG~A GCTGAGAGTA CTACAGAAAA AGGA~AGAAG ATA~AGAAAA
5051 GA~ACA~ATC ATTAATTTTA CCAGATAGCA GTTTTGATGG TACAGGTATG

5151 TTCCTTGAGC CGTA~AAATG TGGTAGTGTT CTTAACACTC TTAACATGTT

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

201 PKAKLDRLAT KEQTPPDAMA LENSREIIPR QGSNTDILSE PA~LSVISNM

451 QMEWLVLRDG ~PPEASEHLK ALINSVMKIM STVKKVKSEQ LHHSMCTRKR

~ ~OCl 1451 WHIAPVHLPL LGQNC~PHLS EGFSVSLWFN VECIHEAEST TEKGKKIKKR

1951 MFNIKQLLKA QVV~LTC QVLQEYKEGQ LTPMPREMAR SFRRKCGQSC

5.8.2PEPTIDE SEQUENCE OF ~HORT ISOFORM (SEQ ID NO:10) 30151 HRYSVRDARK TQLSTSDSEA NSDEKGIAMN KHRRP~T.T.~H FLTSFPKQDH

351 KNLTELLR~A LKIRICLEKQ PDPFAPRQKK TLQEVQEDFV FSKYRHRALL

601 LPALKNFQQH ILNILNKLIL DQLGGAEISP KIKK~ACNIC TVDSDQLAQL

W O 97/28262 PCTrUS97/01748 1301 RQKEKNVFLL MQQGTVKNLL GGFLSI~TQD DSDFQACQRV LVDLLVSLMS
1351 SRTCSEELTL LLRIFLEKSP CTKILLLGIL KIIESDTTMS PSQYLTFPL~
1401 HAPNLSNGVS SQKYPGILNS KAMGL~RRAR VSRSKKEADR ESFPHRLLSS

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

101 AGTGGTA~AT GATGTTGATC TTCCCCCTTG GGCAA~APaA CCTGAAGACT
151 TTGTGCGGAT CA~CAGGATG GCCCTAGA~A GTGAATTTGT TTCTTGCQA

651 GATCAAGCCC ACCATCTTCC CATTGA~ATG GATCCATTAA TAGCCAATAA
701 TTCAGGTGTA AACA~ACGGC AGATCACAGA CCTCGTTGAC CAGAGTATAC

801 ATCTGTGGAT TCTGGGATAA GAGCTTCAGA GTTTATACTA CAGA~ACAGG
851 GA~ATTGACT CAGATTGTAT TTGGCCATTG GGATGTGGTC ACTTGCTTGG

1251 GGGCGATTCA GTAATTTCAG CATTAATGGG A~ACTTTTGG CTCA~ATGGA

1551 GAACAGATAC TGAAGATA~A GGAAGA~CCA A~AGCCAAGT TAAAGCTGAG
1601 GGCACAAGTG CTGCATGGAA AGGCAATATC TCTGGTGGAA A~AATTCGTC

1701 TTGTAGTCAG CA~CCATTTT ACTTTGTGTG TTTTTTCACG ACTGAACACC

1801 GTTTTGGTA~ TTATTTCATA TATTGTTGTT TATTGAGAAA AGGTTGTAGG

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:

51 VRINRMALES EFVSCQLHQW IDLIFGYKQR GPEAVR~LNV FHYLTYEGSV

151 MFKDQMQQDV IMVLKFPSNS PVTHVA~NTL PHLTIPAW T VTCSRLFAVN

W O 97/28262 PCTrUS97/01748 I~Z, 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.

W O 97/28262 PCTrUS97101748 1l3 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
-51 ACAGTTA~AA AAGTTGTCTA CAGCTTGCCT CGGGTTGGAG TGGGGACCAG

30151 TGTATA~GTC TTCCAATATG ACTCAGCGCT GGCA~AGAAG GGAAATCTCC

CA 02244744 l998-07-29 W O 97/28262 PCT~US97/01748 i~

351 CCA~AAGGTG CTTTGAACCC GAAGAGAGCA GTGTTTTACG CAGAGCGCTA
5401 TGAGACATGG GAGGAGGATC A~AGCCCACC CTTCCACTAC AACACACATT

501 ACAACCTTCT TCCTCAATGC A~ATGATGGG A~ATTTGACC ATCCAGACCG

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

lO01 TAGGACTTCA GCCATGCATC TGTGTTCCCT TCCACAGAGC CCACTCATGT
1051 TCA~AGATCA GATGCAGCAG GATGTGATCA TGGTGCTGAA GTTTCCATCC

1201 GGCAC~ACAC AGTCGGCCTC AGAGGAGCCC CCGGATACTC CTTGGATCAA

251401 GGGTTTTGGG ATA~AAGTTT CAGAGTTTAC TCGACAGA~A CAGGGA~ACT

1701 ATCAGTGGTG CTA~AGAGGG CCCTTGCCTC GTTCATACCA TCACTGGA~A
1751 TCTGCTGAAG GCCCTGGAAG GACCAGA~AA CTGCTTATTT CCACGCCTAA

1851 TTTAGCAACT TCAGCATCAA TGGGA~ACTT TTGGCTCAAA TGGAGATCAA
:~ l901 TGATTCCACT AGGGCTATTC TCCTGAGCAG CGATGGACAG AACCTGGTGA
1951 CTGGAGGGGA CAATGGTGTG GTGGAGGTCT GGCAGGCCTG TGACTTTA~G

2051 ATCCCATGAC CA~AGGACTC TGATCACTGG CATGGCTTCC GGCAGCATTG

2151 CTGAAGAGAA GCAGCAGAAG CCACATTCA~ GTGAGAGCAC AAGTGCTTCT
2201 GTGGA~AGGC AGTATCTCTG GTGGGACGCT GGTCCACATC GGCCTCTGCT

2351 TATCATGTAA ATTATCTGA~ TTAGGAGCCG TTTTGGTAAT TATTTCATAT
2401 ATCGCCGTTT ATTGAGA~AA GGTTGTAGGA AGCCTCACAA GAGACTTTTG
2451 ACAATTCTGA GGAACCTTGT GCCCAGTTGT TACA~AGTTT AAGCTTTGA~

(SEQ ID NO:13) 50 5.16 EXAMPLE 16 -- DEDUCED AMINO ACID SEQUENCE OF MOUSE LYST2 PROTEIN

W O 97/28262 PCT~US97/01748 201 DVKELIPEFY YVPEMF~NSN GYHLGVREDE VVVNDVDLPP WAKKPEDFVR

351 KDQMQQDVIM VLKFPSNSPV THVA~NTLPH LTIPAWTVT CSRLFAVNRW

701 LLI (SEQ ID NO:14) Mouse Lyst2 shares 98% amino acid identity with human LYST

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W O 97/28262 PCT~US97/01748 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 TTCA~ATCCC TTTTACTTCA GTCAAGCCAT GGATTTAGTT CAAGAATTTA TC Q GCACCA 240 TGTTCCTTCA GAAGCTGCAG AACATTTGAA AGCTCTGATA AACAGTGTAA TA~AAATAAT 360 _ ACACCGGCGT TGTGAGTATT CCCACTTCAT GCAGCACCAC CGCGATCTTT CAGGGCTCCT 480 W097/28262 PCTrUS97/01748 f'~g TGCTGGTCCT ACCTCCGGCT TGCCCAGTCC TTCCTACCGA TTTCAGGGGA TCCTGCCCAG g60 TGATCCAPAA AAACGAAAGA CCATTCACCA GG~TGTTCAC ATAAACACCA TAAACCTCTT 1620 TCCCTCTGAG GAAGAGCTGT TGTCCCAGCC CGGAGATTTT TcAGAAGAAG CTGAGGATTc 2520 CA 02244744 l998-07-29 W 097/28262 PCTrUS97/01748 1~

TGGTGTCTTA TCACAGAAAC CTCCTGGGAT TCTTAACAGT A~AGCCTTAG GCTTATTGAG 3180 ATCCGAGAGT GCTGCAGAAA GGGGAAAAGG AGTAAGGA~A AGAACAAACC ATCAGTTCTG 3420 (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 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 W 097/28262 PCT~US97/01748J3~

Ile Asn Ser Val Ile Lys Ile Met Ser Thr Val Lys Lys Val Lys Ser Glu Gln Leu His His Ser Met Cys Thr Arg Lys Arg His Arg Arg Cys Glu Tyr Ser His Phe Met Gln His His Arg Asp Leu Ser Gly Leu Leu ~al Ser Ala Phe Lys Asn Gln Leu Ser Lys Ser Pro Phe Glu Glu Thr ~la Glu Gly Asp Val Gln Tyr Pro Glu Arg Cys Cys Cys Ile Ala Val Cys Ala His Gln Cys Leu Arg Leu Leu Gln Gln Val Ser Leu Ser Thr Thr Cys Val Gln Ile Leu Ser Gly Val His Ser Val Gly Ile Cys Cys Cys Met Asp Pro Lys Ser Val Ile Ala Pro Leu Leu His Ala Phe Lys ~eu Pro Ala Leu Lys Ala Phe Gln Gln His lle Leu Asn Val Leu Ser ~ys Leu Leu Val Asp Gln Leu Gly Gly Ala Glu Leu Ser Pro Arg Ile Lys Lys Ala Ala Cys Asn Ile Cys Thr Val Asp Ser Asp Gln Leu Ala Lys Leu Gly Glu Thr Leu Gln Gly Thr Leu Cys Gly Ala Gly Pro Thr Ser Gly Leu Pro Ser Pro Ser Tyr Arg Phe Gln Gly Ile Leu Pro Ser ~er Gly Ser Glu Asp Leu Leu Trp Lys Trp Asp Ala Leu Glu Ala Tyr ~ln Ser Phe Val Phe Gln Glu Asp Arg Leu His Asn Ile Gln Ile Ala Asn His Ile Cys Asn Leu Leu Gln Lys Gly Asn Val Val Val Gln Trp Lys Leu Tyr Asn Tyr Ile Phe Asn Pro Val Leu Gln Arg Gly Val Glu Leu Val His His Cys Gln Gln Leu Ser Ile Pro Ser Ala Gln Thr His ~et Cys Ser Gln Leu Lys Gln Tyr Leu Pro Gln Glu Val Leu Gln Ile ~yr Leu Lys Thr Leu Pro Val Leu Leu Lys Ser Arg Val Ile Arg Asp ~eu Phe Leu Ser Cys Asn Gly Val Asn His Ile Ile Glu Leu Asn Tyr 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 Gly Leu Asp Ile Gln Gln Glu Leu Pro Ser Leu Ser Val Gly Pro Ser Leu His Lys Gln Gln Ala Ser Ser Asp Ser Pro Cys Ser Leu Arg Lys Phe Tyr Ala Ser Leu Arg Glu Pro Asp Pro Lys Lys Arg Lys Thr Ile His Gln Asp Val His Ile Asn Thr Ile Asn Leu Phe Leu Cys Val Ala Phe Leu Cys Val Ser Lys Glu Ala Asp Ser Asp Arg Glu Ser Ala Asn Glu Ser Glu Asp Thr Ser Gly Tyr Asp Ser Pro Pro Ser Glu Pro Leu Ser His Met Leu Pro Cys Leu Ser Leu Glu Asp Val Val Leu Pro Ser Pro Glu Cys Leu His His Ala Ala Asp Ile Trp Ser Met Cys Arg Trp Ile Tyr Met Leu Asn Ser Val Phe Gln Lys Gln Phe His Arg Leu Gly Gly Phe Gln Val Cys His Glu Leu Ile Phe Met Ile Ile Gln Lys Leu Phe Arg Ser His Thr Glu Asp Gln Gly Arg Arg Gln Gly Glu Met Ser Arg Asn Glu Asn Gln Glu Leu Ile Arg Ile Ser Tyr Pro Glu Leu Thr Leu Lys Gly Asp Val Ser Ser Ala Thr Ala Pro Asp Leu Gly Phe Leu Arg Lys Ser Ala Asp Ser Val Arg Gly Phe Gln Ser Gln Pro Val Leu Pro Thr Ser Ala Glu Gln Ile Val Ala Thr Glu Ser Val Pro Gly Glu Arg Lys Ala Phe Met Ser Gln Gln Ser Glu Thr Ser Leu Gln Ser Ile Arg Leu Leu Glu Ser Leu Leu Asp Ile Cys Leu His Ser Ala Arg Ala 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 Ala Glu Thr Glu Leu Ala Lys Pro Leu Phe Asp Ala Leu Leu Arg Val CA 02244744 l998-07-29 132, Ala Leu Gly Asn His Ser Ala ~Asp Leu Gly Pro Gly Asp Ala Val Thr Glu Lys Ser His Pro Ser Glu Glu Glu Leu Leu Ser Gln Pro Gly Asp Phe Ser Glu Glu Ala Glu Asp Ser Gln Cys Cys Ser Leu Lys Leu Leu Gly Glu Glu Glu Gly Tyr Glu Ala Asp Ser Glu Ser Asn Pro Glu Asp Val Asp Thr Gln Asp Asp Gly Val Glu Leu Asn Pro Glu Ala Glu Gly Phe Ser Gly Ser Ile Val Ser Asn Asn Leu Leu Glu Asn Leu Thr His Gly Glu Ile Ile Tyr Pro Glu Ile Cys Met Leu Gly Leu Asn Leu Leu Ser Ala Ser Lys Ala Lys Leu Asp Val Leu Ala His Val Phe Glu Ser Phe Leu Lys Ile Val Arg Gln Lys Glu Lys Asn Ile Ser Leu Leu Ile Gln Gln Gly Thr Val Lys Ile Leu Leu Gly Gly Phe Leu Asn Ile Leu Thr Gln Thr Asn Ser Asp Phe Gln Ala Cys Gln Arg Val Leu Val Asp Leu Leu Val Ser Leu Met Ser Ser Arg Thr Cys Ser Glu Asp Leu Thr Leu Leu Trp Arg Ile Phe Leu Glu Lys Ser Pro Cys Thr Glu Ile Leu Leu Leu Gly ~le His Lys Ile Val Glu Ser Asp Phe Thr Met Ser Pro Ser Gln Cys Leu Thr Phe Pro Phe Leu His Thr Pro Ser Leu Ser Asn Gly Val Leu Ser Gln Lys Pro Pro Gly Ile Leu Asn Ser Lys Ala Leu Gly Leu Leu Arg Arg Ala Arg Ile Ser Arg Gly Lys Lys Glu Ala Asp Arg Glu Ser Phe Pro Tyr Arg Leu Leu Ser Ser Trp His Ile Ala Pro Ile His Leu Pro Leu Leu Gly Gln Asn Cys Trp Pro His Leu Ser Glu Gly Phe Ser Val Ser Leu Val Gly Leu Met Trp Asn Thr Ser Asn Glu Ser Glu Ser Ala Ala Glu Arg Gly Lys Arg Val Lys Lys Arg Asn Lys Pro Ser Val Leu Glu Asp Ser Ser Phe Glu Gly Ala Gly Met Met Ala -W O 97/28262 PCTrUS97/01748 1~3 Gly Ser Asp Leu Tyr Thr Lys Ile Leu Gln Ile Ala Ala Cys Leu Ser Phe Lys His Ile Trp Gln Tyr Phe Asn Val Phe Phe Lys Cys Tyr Ser Pro (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:

A~CTCTTGGA CAGTACCTTG TCCATGGACG AGGATTTCTG TTACTTACCA AACTAAATTC 360 AGCTTTGGTT CAACGGATGC TCTTTCGAAC CGTGCTGCAC CTTATGTCAG TAGACGTTAG lZ00 CA 02244744 l998-07-29 W O 97/28262 PCTnUS97/01748 AACACTACAG GAGGTCCAGG AGGGCTTTGT ~ATTTTCCAAG TATCGTCACC GAGCCCTTCT 1380 CTTTCAAGAA GACAGATTAC ATAACATTCA GATTGCAAAT CAC~TTTGTA ATTTACTCCA 2340 W 097/28262 PCT~US97/01748 1~

ATACCCTGAG ATTTGCATGC TGGGATTAAA TTTGCTTTCT GCTAGCAAAG CTA~ACTTGA 4020 GGACTCAAAT GATGACACGA AAGCTGTCTC ACTAGCACAG GTGGAATCAC AGGAGAATAT 4g20 CTTGGATTTT GTGCTCCCAA GAAAAACAAG CTTATCATCA GACAGCAATA A~ACATTTTG 5100 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 CA 02244744 l998-07-29 W 0 97/28262 PCTrUS97/01748 13'~

TCCAACCCAC ACTGTACAAG AACGGAAGCA GATACTTGAG ATAGT'rCATG AACCAGCTCA 8460 WO 97/28262 PCTtUS97tO1748 13~
TGAAGATAAA ACTCATTCTT CCTTCTCCTC TACTGTCAAA GACAAAGCTG CA~GTGAATC 9180 TGTTAGTGAG ACTCTTGACC TCAATGATCC ATCTATCTAC AGAAACCTAT CTA~GCCTAT 9720 AGCTGTGCAG TATAAAGAAA AAGAAGACCG TTACGTTGAC ACATACA~GT ACTTGGAGGA 9780 TCTGATGACC TACAACAAGG AGCAAGGTGT GAGAAGCATG AACAACACCA ATATTCAGTG 107gO

-WO 97/28262 PCTrUS97/0l748 1~

(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 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 Ser Leu Gln Lys Ser Thr Gln Gly Lys Leu Tyr Leu Glu Gly Ser Ala _ 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 CA 02244744 l998-07-29 1~

Thr Gln Leu Ser Thr Ser Asp Ser Glu Gly Asn Ser Asp Glu Lys Ser ~hr Val Val Ser Lys His Arg Arg Leu His Ala Leu Pro Arg Phe Leu Thr Gln Ser Pro Lys Glu Gly His Leu Val Ala Lys Pro Asp Pro Ser Ala Thr Lys Glu Gln Val Leu Ser Asp Thr Met Ser Val Glu Asn Ser Arg Glu Val Ile Leu Arg Gln Asp Ser Asn Gly Asp Ile Leu Ser Glu ~ro Ala Ala Leu Ser Ile Leu Ser Asn Met Asn Asn Ser Pro Phe Asp ~eu Cys His Val Leu Leu Ser Leu Leu Glu Lys Val Cys Lys Phe Asp Ile Ala Leu Asn His Asn Ser Ser Leu Ala Leu Ser Val Val Pro Thr Leu Thr Glu Phe Leu Ala Gly Phe Gly Asp Cys Cys Asn Gln Ser Asp 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 ~al Asp Val Ser Thr Ala Glu Ala Met Pro Glu Ser Leu Arg Lys Asn Leu Thr Glu Leu Leu Arg Ala Ala Leu Lys Ile Arg Ala Cys Leu Glu Lys Gln Pro Glu Pro Phe Ser Pro Arg Gln Lys Lys Thr Leu Gln Glu Val Gln Glu Gly Phe Val Phe Ser Lys Tyr Arg His Arg Ala Leu Leu ~eu Pro Glu Leu Leu Glu Gly Val Leu Gln Leu Leu Ile Ser Cys Leu ~ln 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 Xis Leu Lys Ala Leu Ile Asn Ser Val Ile Lys Ile Met Ser Thr Val Lys Lys Val Lys Ser Glu Gln Leu His Xis Ser Met Cys W O 97/28262 PCTrUS97/01748 1~
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 Ser Lys Ser Pro Phe Glu Glu Thr Ala Glu Gly Asp Val Gln Tyr Pro Glu Arg Cys Cys Cys Ile ALa Val Cys ALa His Gln Cys Leu Arg Leu Leu Gln Gln Val Ser Leu Ser Thr Thr Cys Val Gln Ile Leu Ser Gly Val His Ser Val Gly Ile Cys Cys Cys Met Asp Pro Lys Ser Val Ile Ala Pro Leu Leu His ALa Phe Lys Leu Pro ALa Leu Lys Ala Phe Gln Gln His Ile Leu Asn Val Leu Ser Lys Leu Leu Val Asp Gln Leu Gly Gly Ala Glu Leu Ser Pro Arg Ile Lys Lys Ala Ala Cys Asn Ile Cys Thr Val Asp Ser Asp Gln Leu ALa Lys Leu Gly Glu Thr Leu Gln Gly Thr Leu Cys Gly Ala Gly Pro Thr Ser Gly Leu Pro Ser Pro Ser Tyr Arg Phe Gln GLy Ile T eu Pro Ser Ser GLy Ser Glu Asp Leu Leu Trp Lys Trp Asp Ala Leu Glu ALa Tyr Gln Ser Phe Val Phe Gln Glu Asp Arg Leu Hls Asn Ile Gln Ile ALa Asn His Ile Cys Asn Leu Leu Gln Lys Gly Asn Val Val Val Gln Trp Lys Leu Tyr Asn Tyr Ile Phe Asn Pro Val Leu Gln Arg Gly Val Glu Leu Val His His Cys Gln Gln Leu Ser Ile Pro Ser ALa Gln Thr His Met Cys Ser Gln Leu Lys Gln Tyr Leu Pro Gln Glu Val Leu Gln Ile Tyr Leu Lys Thr Leu Pro Val Leu Leu Lys Ser Arg Val Ile Arg Asp Leu Phe Leu Ser Cys Asn Gly Val Asn His Ile Ile Glu Leu Asn Tyr Leu Asp Gly Ile Arg Ser His Ser Leu Lys Ala Phe Glu Thr Leu Ile Val Ser Leu Gly Glu Gln Gln Lys Asp Ala Ala Val Leu Asp Val Asp Gly Leu Asp Ile Gln Gln Glu Leu W O 97/28262 PCT~US97/01748 Pro Ser Leu Ser Val Gly Pro Ser Leu Hi s Lys Gln Gln Ala Ser Ser Asp Ser Pro Cys Ser Leu Arg Lys Phe Tyr Ala Ser Leu Arg Glu Pro Asp Pro Lys Lys Arg Lys Thr ILe His Gln Asp Val His Ile Asn Thr Ile Asn Leu Phe Leu Cys Val Ala Phe Leu Cys Val Ser Lys Glu Ala Asp Ser Asp Arg Glu Ser Ala Asn Glu Ser Glu Asp Thr Ser Gly Tyr Asp Ser Pro Pro Ser Glu Pro Leu Ser His Met Leu Pro Cys Leu Ser Leu Glu Asp Val Val Leu Pro Ser Pro Glu Cys Leu His His Ala Ala Asp Ile Trp Ser Met Cys Arg Trp Ile Tyr Met Leu Asn Ser Val Phe Gln Lys Gln Phe His Arg Leu Gly Gly Phe Gln Val Cys His Glu Leu Ile Phe Met Ile Ile Gln Lys Leu Phe Arg Ser His Thr Glu Asp Gln Gly Arg Arg Gln Gly Glu Met Ser Arg Asn Glu Asn Gln Glu Leu Ile Arg Ile Ser Tyr Pro Glu Leu Thr Leu Lys Gly Asp Val Ser Ser Ala Thr Ala Pro Asp Leu Gly Phe Leu Arg Lys Ser Ala Asp Ser Val Arg Gly Phe Gln Ser Gln Pro Val Leu Pro Thr Ser Ala Glu Gln Ile Val Ala Thr Glu Ser Val Pro Gly Glu Arg Lys Ala Phe Met Ser Gln Gln 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 Glu His Leu Ser Gln Ser Lys Val Ala Glu Thr Glu Leu Ala Lys Pro Leu Phe ~sp Ala Leu Leu Arg Val Ala Leu Gly Asn His Ser Ala Asp Leu Gly Pro Gly Asp Ala Val Thr Glu Lys Ser His Pro Ser Glu Glu Glu Leu Leu Ser Gln Pro Gly Asp Phe Ser Glu Glu Ala Glu Asp Ser W 097/28262 PCT~US97/01748 ~3 .1185 1190 1195 1200 Gln Cys Cys Ser Leu Lys Leu Leu Gly Glu Glu Glu Gly Tyr Glu Ala ~sp Ser Glu Ser Asn Pro Glu Asp Val Asp Thr Gln Asp Asp Gly Val Glu Leu Asn Pro Glu Ala Glu Gly Phe Ser Gly Ser Ile Val Ser Asn Asn Leu Leu Glu Asn Leu Thr His Gly Glu Ile Ile Tyr Pro Glu Ile Cys Met Leu Gly Leu Asn Leu Leu Ser Ala Ser Lys Ala Lys Leu Asp ~al Leu Ala His Val Phe Glu Ser Phe Leu Lys Ile Val Arg Gln Lys ~lu Lys Asn Ile Ser Leu Leu Ile Gln Gln Gly Thr Val Lys Ile Leu Leu Gly Gly Phe Leu Asn Ile Leu Thr Gln Thr Asn Ser Asp Phe Gln Ala Cys Gln Arg Val Leu Val Asp Leu Leu Val Ser Leu Met Ser Ser 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 ~lu Ser Asp Phe Thr Met Ser Pro Ser Gln Cys Leu Thr Phe Pro Phe Leu His Thr Pro Ser Leu Ser Asn Gly Val Leu Ser Gln Lys Pro Pro Gly Ile Leu Asn Ser Lys Ala Leu Gly Leu Leu Arg Arg Ala Arg Ile Ser Arg Gly Lys Lys Glu Ala Asp Arg Glu Ser Phe Pro Tyr Arg Leu ~eu Ser Ser Trp His Ile Ala Pro Ile His Leu Pro Leu Leu Gly Gln ~sn Cys Trp Pro His Leu Ser Glu Gly Phe Ser Val Ser Leu Val Gly Leu Met Trp Asn Thr Ser Asn Glu Ser Glu Ser Ala Ala Glu Arg Gly Lys Arg Val Lys Lys Arg Asn Lys Pro Ser Val Leu Glu Asp Ser Ser Phe Glu Gly Ala Glu Gly Asp Arg Pro Glu Val Thr Glu Ser Ile Asn Pro Gly Asp Arg Leu Ile Glu Asp Gly Cys Ile His Leu Ile Ser Leu CA 02244744 l998-07-29 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 Val Ser Leu Ala Gln Val Glu Ser Gln Glu Asn Ile Phe Phe Pro Ser Lys Trp Gln His Leu Val Leu Thr Tyr Ile Gln His Pro Gln Gly Lys ~ys Asn Val His Gly Glu Ile Ser Ile Trp Val Ser Gly Gln Arg Lys ~hr Asp Val Ile Leu Asp Phe Val Leu Pro Arg Lys Thr Ser Leu Ser Ser Asp Ser Asn Lys Thr Phe Cys Met Ile Gly His Cys Leu Thr Ser Gln Glu Glu Ser Leu Gln Leu Ala Gly Lys Trp Asp Leu Gly Asn Leu Leu Leu Phe Asn Gly Ala Lys Ile Gly Ser Gln Glu Ala Phe Phe Leu ~yr Ala Cys Gly Pro Asn Tyr Thr Ser Ile Met Pro Cys Lys Tyr Gly ~ln Pro Val Ile Asp Tyr Ser Lys Tyr Ile Asn Lys Asp Ile Leu Arg Cys Asp Glu Ile Arg Asp Leu Phe Met Thr Lys Lys Glu Val Asp Val Gly Leu Leu Ile Glu Ser Leu Ser Val Val Tyr Thr Thr Cys Cys Pro Ala Gln Tyr Thr Ile Tyr Glu Pro Val Ile Arg Leu Lys Gly Gln Val ~ys Thr Gln Pro Ser Gln Arg Pro Phe Ser Ser Lys Glu Ala Gln Ser ~le Leu Leu Glu Pro Ser Gln Leu Lys Gly Leu Gln Pro Thr Glu Cys Lys Ala Ile Gln Gly Ile Leu His Glu Ile Gly Gly Ala Gly Thr Phe Val Phe Leu Phe Ala Arg Val Val Glu Leu Ser Ser Cys Glu Glu Thr Gln Ala Leu Ala Leu Arg Val Ile Leu Ser Leu Ile Lys Tyr Ser Gln ~ln Arg Thr Gln Glu Leu Glu Asn Cys Asn Gly Leu Ser Met Ile His ~ln Val Leu Val Lys Gln Lys Cys Ile Val Gly Phe His Ile Leu Lys ~hr Leu Leu Glu Gly Cys Cys Gly Glu Glu Val Ile His Val Ser Glu W O 97/28262 PCT~US97/01748 5~5 ~ 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 Ile Arg Val Glu His His Gln Gln Gln Phe Asn Ile Lys Gln Leu Leu Asn ALa His Val Val His His Phe Leu Leu Thr Cys Gln Val Leu Gln Glu His Arg Glu Gly Gln Leu Thr Ser Met Pro Arg Glu Val Cys Arg Ser Phe Val Lys Ile Ile ALa Glu Val Leu Gly Ser Pro Pro Asp Leu Glu Leu Leu Thr Val Ile Phe Asn Phe Leu Leu Ala Val His Pro Pro Thr Asn Thr Tyr Val Cys His Asn Pro Thr Asn Phe Tyr Phe Ser Leu His ILe Asp Gly Lys Ile Phe Gln Glu Lys VaL Gln Ser Leu Ala Tyr Leu Arg His Ser Ser Ser Gly Gly Gln Ala Phe Pro Ser Pro Gly Phe Leu Val Ile Ser Pro Ser Ala Phe Thr Ala Ala Pro Pro Glu Gly Thr Ser Ser Ser Asn Ile Val Pro Gln Arg Met Ala ALa GLn Met Val Arg Ser Arg Ser Leu Pro Ala Phe Pro Thr Tyr Leu Pro Leu Ile Arg ALa Gln Lys Leu Ala ALa Ser Leu Gly Phe Ser Val Asp Lys Leu Gln Asn Ile ALa Asp Ala Asn Pro Glu Lys Gln Asn Leu Leu Gly Arg Pro Tyr ALa Leu Lys Thr Ser Lys Glu Glu Ala Phe Ile Ser Ser Cys Glu Ser Ala Lys Thr Val Cys Glu Met Glu Ala Leu Leu Gly Ala His Ala Ser Ala Asn Gly Val Ser Arg Gly Ser Pro Arg Phe Pro Arg ALa Arg Val ~ Asp His Lys Asp Val Gly Thr Glu Pro Arg Ser Asp Asp Asp Ser Pro _ 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 W097/28262 PCT~US97/01748 l~lo 222~ 2230 2235 2240 Pro Ser Gln Asn Gly Ser Ala Val Ala Ser Arg Trp Pro Ser Leu Val 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 Cys Leu Ile Pro Ile Cys Cys Gly Leu Tyr Glu Leu Leu Ser Gly Val Leu Leu Val Leu Pro Asp Ala Met Leu Glu Asp Val Met Asp Arg Ile Ile Gln Ala Asp Ile Leu Leu Val Leu Val Asn His Pro Ser Pro Ala Ile Gln Gln Gly Val Ile Lys Leu Leu His Ala Tyr Ile Asn Arg Ala Ser Lys Glu Gln Lys Asp Lys Phe Leu Lys Asn Arg Gly Phe Ser Leu Leu Ala Asn Gln Leu Tyr Leu His Arg Gly Thr Gln Glu Leu Leu Glu 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 Ser Lys Val Ala Asp Met Leu Leu Asp Asn Gly Leu Leu Tyr Val Leu Cys Asn Thr Val Ala Ala Leu Asn Gly Leu Glu Lys Asn Ile Pro Val Asn Glu Tyr Lys Leu Leu Ala Cys Asp Ile Gln Gln Leu Phe Ile Ala Val Thr Ile His Ala Cys Ser Ser Ser Gly Thr Gln Tyr Phe Arg Val Ile Glu Asp Leu Ile Val Leu Leu Gly Tyr Leu ~is Asn Ser Lys Asn Lys Arg Thr Gln Asn Met Ala Leu Ala Leu Gln Leu Arg Val Leu Gin Ala Ala Leu Glu Phe Ile Arg Ser Thr Ala Asn His Asp Ser Glu Ser Pro Val His Ser Pro Ser Ala His Arg His Ser Val Pro Pro Lys Arg W 097/28262 PCT~US97101748 1~
Arg Ser Ile Ala Gly Ser Arg Lys Phe Pro Leu Ala Gln Thr Glu Ser Leu Leu Met Lys Met Arg Ser Val Ala Ser Asp Glu Leu His Ser Met Met Gln Arg Arg Met Ser Gln Glu His Pro Ser Gln Ala Ser Glu Ala Glu Leu Ala Gln Arg Leu Gln Arg Leu Thr Ile Leu Ala Val Asn Arg Ile Ile Tyr Gln Glu Leu Asn Ser Asp Ile Ile Asp Ile Leu Arg Thr Pro Glu Asn Thr Ser Gln Ser Lys Thr Ser Val Ser Gln Thr Glu Ile Ser Glu Glu Asp Met His His Glu Gln Pro Ser Val Tyr Asn Pro Phe Gln Lys Glu Met Leu Thr Tyr Leu Leu Asp Gly Phe Lys Val Cys Ile Gly Ser Ser Lys Thr Ser Val Ser Lys Gln Gln Trp Thr Lys Ile Leu Gly Ser Cys Lys Glu Thr Leu Arg Asp Gln Leu Gly Arg Leu Leu Ala His Ile Leu Ser Pro Thr His Thr Val Gln Glu Arg Lys Gln Ile Leu Glu Ile Val His Glu Pro Ala His Glrl Asp Ile Leu Arg Asp Cys Leu Ser Pro Ser Pro Gln His Gly Ala Lys Leu Val Leu Tyr Leu Ser Glu Leu Ile His Asn His Gln Asp Glu Leu Ser Glu Glu Glu Met Asp Thr Ala Glu Leu Leu Met Asn Ala Leu Lys Leu Cys Gly His Lys Cys Ile Pro Pro Ser Ala Pro Ser Lys Pro Glu Leu Ile Lys Ile Ile Arg Glu Glu Gln Lys Lys Tyr Glu Ser Glu Glu Ser Val Ser Lys Gly Ser Trp Gln Lys Thr Val Asn Asn Asn Gln Gln Ser Leu Phe Gln Arg Leu Asp Phe Lys Ser Lys Asp Ile Ser Lys Ile Ala Ala Asp Ile Thr Gln Ala Val Ser Leu Ser Gln Gly Ile Glu Arg Lys Lys Val Ile Gln His Ile Arg Gly Met Tyr Lys Val Asp Leu Ser Ala Ser Arg His Trp Gln Glu Cys Ile Gln Gln Leu Thr His Asp Arg Ala Val Trp Tyr Asp Pro Ile WO 97/28262 PCT~US97/01748 1~
Tyr Tyr Pro Thr Ser Trp Gln Leu Asp Pro Thr Glu Gly Pro Asn Arg Glu Arg Arg Arg Leu Gln Arg Cys Tyr Leu Thr Ile Pro Asn Lys Tyr Leu Leu Arg Asp Arg Gln Lys Ser Glu Gly Val Leu Arg Pro Pro Leu Ser Tyr Leu Phe Glu Asp Lys Thr His Ser Ser Phe Ser Ser Thr Val Lys Asp Lys Ala Ala Ser Glu Ser Ile Arg Val Asn Arg Arg Cys Ile Ser Val Ala Pro Ser Arg Glu Thr Ala Gly Glu Leu Leu Leu Gly Lys Cys Gly Met Tyr Phe Val Glu Asp Asn Ala Ser Asp Ala Val Glu Ser Ser Ser Leu Gln Gly Glu Leu Glu Pro Ala Ser Phe Ser Trp Thr Tyr Glu Glu Ile Lys Glu Val His Arg Arg Trp Trp Gln Leu Arg Asp Asn Ala Val Glu Ile Phe Leu Thr Asn Gly Arg Thr Leu Leu Leu Ala Phe Asp Asn Asn Lys Val Arg Asp Asp Val Tyr Gln Ser Ile Leu Thr Asn Asn Leu Pro Asn Leu Leu Glu Tyr Gly Asn Ile Thr Ala Leu Thr Asn Leu Trp Tyr Ser Gly Gln Ile Thr Asn Phe Glu Tyr Leu Thr His Leu Asn Lys His Ala Gly Arg Ser Phe Asn Asp Leu Met Gln Tyr Pro Val Phe Pro Phe Ile Leu Ser Asp Tyr Val Ser Glu Thr Leu Asp Leu Asn Asp Pro Ser Ile Tyr Arg Asn Leu Ser Lys Pro Ile Ala Val Gln Tyr Lys Glu Lys Glu Asp Arg Tyr Val Asp Thr Tyr Lys Tyr Leu Glu Glu Glu Tyr Arg Lys Gly Ala Arg Glu Asp Asp Pro Met Pro Pro Val Gln Pro Tyr His Tyr Gly Ser His Tyr Ser Asn Ser Gly Thr Val Leu His Phe Leu Val Arg Met Pro Pro Phe Thr Lys Met Phe Leu Ala Tyr Gln Asp Gln Ser Phe Asp Ile Pro Asp Arg Thr Phe His Ser Thr Asn Thr Thr Trp Arg Leu Ser Ser Phe Glu Ser Met Thr Asp Val Lys Glu Leu CA 02244744 l998-07-29 W O 97/28262 PCT~US97/01748 - l~q Ile Pro Glu Phe Phe Tyr Leu Pro Glu Phe Leu Val Asn Arg Glu Gly Phe Asp Phe Gly Val Arg Gln Asn Gly Glu Arg Val Asn His Val Asn Leu Pro Pro Trp Ala Arg Asn Asp Pro Arg Leu Phe Ile Leu Ile His Arg Gln Ala Leu Glu Ser Asp His Val Ser Gln Asn Ile Cys His Trp Ile Asp Leu Val Phe Gly Tyr Lys Gln Lys Gly Lys Ala Ser Val Gln Ala Ile Asn Val Phe His Pro Ala Thr Tyr Phe Gly Met Asp Val Ser Ala Val Glu Asp Pro Val Gln Arg Arg Ala Leu Glu Thr Met Ile Lys Thr Tyr Gly Gln Thr Pro Arg Gln Leu Phe His Thr Ala His Ala Ser Arg Pro Gly Ala Lys Leu Asn Ile Glu Gly Glu Leu Pro Ala Ala Val Gly Leu Leu Val Gln Phe Ala Phe Arg Glu Thr Arg Glu Pro Val Lys Glu Val Thr His Pro Ser Pro Leu Ser Trp Ile Lys Gly Leu Lys Trp Gly Glu Tyr Val Gly Ser Pro Ser Ala Pro Val Pro Val Val Cys Phe Ser Gln Pro His Gly Glu Arg Phe Gly Ser Leu Gln Ala Leu Pro Thr Arg Ala Ile Cys Gly Leu Ser Arg Asn Phe Cys Leu Leu Met Thr Tyr Asn Lys Glu Gln Gly Val Arg Ser Met Asn Asn Thr Asn Ile Gln Trp Ser Ala Ile Leu Ser Trp Gly Tyr Ala Asp Asn Ile Leu Arg Leu Lys Ser Lys Gln Ser Glu Pro Pro Ile Asn Phe Ile Gln Ser Ser Gln Gln His Gln Val Thr Ser Cys Ala Trp Val Pro Asp Ser Cys Gln Leu Phe Thr Gly Ser Lys Cys Gly Val Ile Thr Ala Tyr Thr Asn Arg Leu Thr Ser Ser Thr Pro Ser Glu Ile Glu Met Glu Ser Gln Met His Leu Tyr Gly His Thr Glu Glu Ile Thr Gly Leu Cys Val Cys Lys Pro Tyr Ser CA 02244744 l998-07-29 W 097/28262 PCTnUS97/0l748 1~
Val Met Ile Ser Val Ser Arg Asp Gly Thr Cys Ile Val Trp Asp Leu Asn Arg Leu Cys Tyr Val Gln Ser Leu Ala Gly His Lys Ser Pro Val Thr Ala Val Ser Ala Ser Glu Thr Ser Gly Asp Ile Ala Thr Val Cys Asp Ser Ala Gly Gly Gly Ser Asp Leu Arg Leu Trp Thr Val Asn Gly Asp Leu Val Gly His Val His Cys Arg Glu Ile Ile Cys Ser Val Ala Phe Ser Asn Gln Pro Glu Gly Val Ser Ile Asn Val Ile Ala Gly Gly Leu Glu Asn Gly Ile Val Arg Leu Trp Ser Thr Trp Asp Leu Lys Pro Val Arg Glu Ile Thr Phe Pro Lys Ser Asn Lys Pro Ile Ile Ser Leu Thr Phe Ser Cys Asp Gly His His Leu Tyr Thr Ala Asn Ser Glu Gly Thr Val Ile Ala Trp Cys Arg Lys Asp Gln Gln Arg Val Lys Leu Pro Met Phe Tyr Ser Phe Leu Ser Ser Tyr Ala Ala Gly (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:

AACTCTTGGA CAGTACCTTG TCCATGGACG AGGATTTCTG TTACTTACCA AACTA~ATTC 360 TCGAAAAATC AGGCGACAGC GTAAAAGTAC CCATCGTTAT TcTGTAAGAG ATGCAAGAAA 660 W 097/28262 PCTrUS97/01748 1~

GGTTTCAGCT TTTAAAAATC AG~lll~lAA AAGCCCCTTT GAAGAGACCG CAGAGGGAGA 1800 TCTACCTGTC CTACTTAAAT CCAGGGTA~T AAGAGATTTG TTTTTAAGTT GTAATGGAGT 2580 W 097/28262 PCTrUS97/01748 TGAAACTCTG ATTGTCAGCC TAGGGGAACA ACAGAAAGAT GCTGCAGTTC TAGACGTCGA 2700 r CA 02244744 l998-07-29 WO 97/28262 PCTrUS97/01748 1~3 ACACCTGTCA GAAGGATTTA GTGTTTCTCT ~GTGGGTTTA ATGTGGAATA CATCCAATGA 4620 CACCAATGGC CCCTTCTAGT TACTGTCCCT GATCATTTAT ATGTA~CAGT CCAAAGTTAG 5100 TTTGTTCTCT T~ll~ AA GCAATCAACA ~l"l"l~l"l~AAG TCATATAGCA GCTAGAGGAA 5220 ATGGCATGTG CTCCCTGGGC CATGGTCAGT TGTGTTAGAG TGACCCAATC CAACA~AAGC 5760 (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 _ 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 W O 97/28262 PCT~US97/01748 - 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 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 Arg Gln Arg Lys Ser Thr His Arg Tyr Ser Val Arg Asp Ala Arg Lys Thr Gln Leu Ser Thr Ser Asp Ser Glu Gly Asn Ser Asp Glu Lys Ser Thr Val Val Ser Lys His Arg Arg Leu His Ala Leu Pro Arg Phe Leu Thr Gln Ser Pro Lys Glu Gly His Leu Val Ala Lys Pro Asp Pro Ser Ala Thr Lys Glu Gln Val Leu Ser Asp Thr Met Ser Val Glu Asn Ser 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 Leu Cys His Val Leu Leu Ser Leu Leu Glu Lys Val Cys Lys Phe Asp Ile Ala Leu Asn His Asn Ser Ser Leu Ala Leu Ser Val Val Pro Thr Leu Thr Glu Phe Leu Ala Gly Phe Gly Asp Cys Cys Asn Gln Ser Asp Thr Leu Glu Gly Gln Leu Val Ser Ala Gly Trp Thr Glu Glu Pro Val Ala Leu Val Gln Arg Met Leu Phe Arg Thr Val Leu His Leu Met Ser Val Asp Val Ser Thr Ala Glu Ala Met Pro Glu Ser Leu Arg Lys Asn Leu Thr Glu Leu Leu Arg Ala Ala Leu Lys Ile Arg~la Cys Leu Glu Lys Gln Pro Glu Pro Phe Ser Pro Arg Gln Lys Lys Thr Leu Gln Glu W097/28262 PCT~US97/01748 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 ~' 405 410 415 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 Ile Asn Ser Val Ile Lys Ile Met Ser Thr Val Lys Lys Val Lys Ser Glu Gln Leu His His Ser Met Cys Thr Arg Lys Arg His Arg Arg Cys Glu Tyr Ser His Phe Met Gln His His Arg Asp Leu Ser Gly Leu Leu Val Ser Ala Phe Lys Asn Gln Leu Ser Lys Ser Pro Phe Glu Glu Thr Ala Glu Gly Asp Val Gln Tyr Pro Glu Arg Cys Cys Cys Ile Ala Val Cys Ala His Gln Cys Leu Arg Leu Leu Gln Gln Val Ser Leu Ser Thr Thr Cys Val Gln Ile Leu Ser Gly Val His Ser Val Gly Ile Cys Cys Cys Met Asp Pro Lys Ser Val Ile Ala Pro Leu Leu His Ala Phe Lys Leu Pro Ala Leu Lys Ala Phe Gln Gln His Ile Leu Asn Val Leu Ser Lys Leu Leu Val Asp Gln Leu Gly Gly Ala Glu Leu Ser Pro Arg Ile Lys Lys Ala Ala Cys Asn Ile Cys Thr Val Asp Ser Asp Gln Leu Ala Lys Leu Gly Glu Thr Leu Gln Gly Thr Leu Cys Gly Ala Gly Pro Thr Ser Gly Leu Pro Ser Pro Ser Tyr Arg Phe Gln Gly Ile Leu Pro Ser Ser Gly Ser Glu Asp Leu Leu Trp Lys Trp Asp Ala Leu Glu Ala Tyr Gln Ser Phe Val Phe Gln Glu Asp Arg Leu His Asn Ile Gln Ile Ala Asn His Ile Cys Asn Leu Leu Gln Lys Gly Asn Val Val Val Gln Trp Lys Leu Tyr Asn Tyr Ile Phe Asn CA 02244744 l998-07-29 W O 97/28262 PCT~US97/01748 f~
Pro Val Leu Gln Arg Gly VaL Glu Leu Val His His Cys Gln Gln Leu Ser Ile Pro Ser Ala Gln Thr His Met Cys Ser Gln Leu Lys Gln Tyr Leu Pro Gln Glu Val Leu Gln Ile Tyr Leu Lys Thr Leu Pro Val Leu Leu Lys Ser Arg Val Ile Arg Asp Leu Phe Leu Ser Cys Asn Gly Val ~sn His Ile Ile Glu Leu Asn Tyr Leu Asp Gly Ile Arg Ser His Ser ~eu Lys Ala Phe Glu Thr Leu Ile Val Ser Leu Gly Glu Gln Gln Lys Asp Ala Ala Val Leu Asp Val Asp Gly Leu Asp Ile Gln Gln Glu Leu Pro Ser Leu Ser Val Gly Pro Ser Leu His Lys Gln Gln Ala Ser Ser Asp Ser Pro Cys Ser Leu Arg Lys Phe Tyr Ala Ser Leu Arg Glu Pro ~sp Pro Lys Lys Arg Lys Thr Ile His Gln Asp Val His Ile Asn Thr ~le Asn Leu Phe Leu Cys Val Ala Phe Leu Cys Val Ser Lys Glu Ala Asp Ser Asp Arg Glu Ser Ala Asn Glu Ser Glu Asp Thr Ser Gly Tyr Asp Ser Pro Pro Ser Glu Pro Leu Ser His Met Leu Pro Cys Leu Ser Leu Glu Asp Val Val Leu Pro Ser Pro Glu Cys Leu His His Ala Ala ~sp Ile Trp Ser Met Cys Arg Trp Ile Tyr Met Leu Asn Ser Val Phe ~ln Lys Gln Phe His Arg Leu Gly Gly Phe Gln Val Cys His Glu Leu Ile Phe Met Ile Ile Gln Lys Leu Phe Arg Ser His Thr Glu Asp Gln Gly Arg Arg Gln Gly Glu Met Ser Arg Asn Glu Asn Gln Glu Leu Ile Arg Ile Ser Tyr Pro Glu Leu Thr Leu Lys Gly Asp Val Ser Ser Ala ~hr Ala Pro Asp Leu Gly Phe Leu Arg Lys Ser Ala Asp Ser Val Arg ~ly Phe Gln Ser Gln Pro Val Leu Pro Thr Ser Ala Glu Gln Ile Val ~la Thr Glu Ser Val Pro Gly Glu Arg Lys Ala Phe Met Ser Gln Gln W O 97/28262 PCT~US97/01748 15~

Ser Glu Thr Ser Leu Gln Ser Ile Arg Leu Leu Glu Ser Leu Leu Asp Ile Cys Leu His Ser Ala Arg Ala Cys Gln Gln Lys Met Glu Leu Glu Leu Pro Ser Gln Gly Leu Ser Val Glu Asn Ile Leu Cys Glu Leu Arg Glu His Leu Ser Gln Ser Lys Val Ala Glu Thr Glu Leu Ala Lys Pro Leu Phe Asp Ala Leu Leu Arg Val Ala Leu Gly Asn His Ser Ala Asp Leu Gly Pro Gly Asp Ala Val Thr Glu Lys Ser His Pro Ser Glu Glu Glu Leu Leu Ser Gln Pro Gly Asp Phe Ser Glu Glu Ala Glu Asp Ser Gln Cys Cys Ser Leu Lys Leu Leu Gly Glu Glu Glu Gly Tyr Glu Ala Asp Ser Glu Ser Asn Pro Glu Asp Val Asp Thr Gln Asp Asp Gly Val Glu Leu Asn Pro Glu Ala Glu Gly Phe Ser Gly Ser Ile Val Ser Asn Asn Leu Leu Glu Asn Leu Thr His Gly Glu Ile Ile Tyr Pro Glu Ile 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 Glu Lys Asn Ile Ser Leu Leu Ile Gln Gln Gly Thr Val Lys Ile Leu Leu Gly Gly Phe Leu Asn Ile Leu Thr Gln Thr Asn Ser Asp Phe Gln Ala Cys Gln Arg Val Leu Val Asp Leu Leu Val Ser Leu Met Ser Ser Arg Thr Cys Ser &lu Asp Leu Thr Leu Leu Trp Arg Ile Phe Leu Glu Lys Ser Pro Cys Thr Glu Ile Leu Leu Leu Gly Ile His Lys Ile Val Glu Ser Asp Phe Thr Met Ser Pro Ser Gln Cys Leu Thr Phe Pro Phe Leu His Thr Pro Ser Leu Ser Asn Gly Val Leu Ser Gln Lys Pro Pro Gly Ile Leu Asn Ser Lys Ala Leu Gly Leu Leu Arg Arg Ala Arg Ile W 0 97/28262 PCT~US97/01748 - 1~
~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 Asn Cys Trp Pro His Leu Ser Glu Gly Phe Ser Val Ser Leu Val Gly Leu Met Trp Asn Thr Ser Asn Glu Ser Glu Ser Ala Ala Glu Arg Gly Lys Arg Val Lys Lys Arg Asn Lys Pro Ser Val Leu Glu Asp Ser Ser Phe Glu Gly Ala Gly Met Met Ala Gly Ser Asp Leu Tyr Thr Lys Ile Leu Gln Ile Ala Ala Cys Leu Ser Phe Lys His Ile Trp Gln Tyr Phe Asn Val Phe Phe Lys Cys Tyr Ser Pro (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:

AAGCTA~ATT CTATAATTGA TCAGGCATTG ACATGTAGAG AAGAACTCCT GACTCTTCTT 780 CTGTCTCTCC TTCCACTGGT ATGGA~GATA CCTGTCCAAG AAGAAAAGGC AACAGATTTT 840 CA 02244744 l998-07-29 WO 97/28262 PCTrUS97/01748 i '~q AATTTAATCC AGA~AGGCAA TATAGTTGTT CAGTGGAAAT TATATAATTA CATATTTAAT 2760 W O97/2B262 ~ PCT~US97/01748 1~

T~l~lll~ATC ATCAGCAAGC TTATTCAGAT TCTCCTCAGA GTCTCAGCAA ATTTTATGCT 3180 ACAATAAACC TATTCCTCTG TGTGGCTTTT TTATGCGTAA GTA~AGAAGC AGAGTCTGAC 3300 CA 02244744 l998-07-29 WO 97/28262 PCTrUS97/~l748 )lcl ATTTTAAACA GTAAGGCCAT GGGTTTATTG AGAAGAGCAC GAGTTTCACG GAGCAAGA~A 4860 ~ ATAAAGAAAA GAAACAAATC ATTAATTTTA CQGATAGCA GTTTTGATGG TACAGAGAGC S100 TTGCGATGTG AACAAATCAG AGAA'l~ ATGACCAAGA AAGATGTGGA TATTGGTCTC 5760 GGACTTTCTA TGATTCATCA GGTGTTGATC AAACAAA~AT GCATTGTTGG GTTTTACATT 6180 CTACTTCTTC ACTACTAACG CAATCACAAA AACTGACTGG AAGTTTGGGT TGTAGTATcG 6780 WO 97/28262 PCT~US97/01748 li9~

(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 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 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 Arg Gln Arg Lys Ile Thr His Arg Tyr Ser Val Arg Asp Ala Arg Lys Thr Gln Leu Ser Thr Ser Asp Ser Glu Ala Asn Ser Asp Glu Lys Gly 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 A~a Thr Lys Glu Gln Thr Pro Pro Asp Ala Met Ala Leu Glu Asn Ser Arg Glu Ile Ile P~o Arg Gln Gly Ser Asn Thr Asp Ile Leu Ser Glu CA 02244744 l998-07-29 W O 97/28262 PCTrUS97/01748 i~3 - Pro Ala Ala Leu Ser Val Ile Ser Asn Met Asn Asn Ser Pro Phe Asp Leu Cys His Val Leu Leu Ser Leu Leu Glu Lys Val Cys Lys Phe Asp Val Thr Leu Asn His Asn Ser Pro Leu Ala Ala Ser Val Val Pro Thr Leu Thr Glu Phe Leu Ala Gly Phe Gly Asp Cys Cys Ser Leu Ser Asp Asn Leu Glu Ser Arg Val Val Ser Ala Gly Trp Thr Glu Glu Pro Val Ala Leu Ile Gln Arg Met Leu Phe Arg Thr Val Leu His Leu Leu Ser Val Asp Val Ser Thr Ala Glu Met Met Pro Glu Asn Leu Arg Lys Asn Leu Thr Glu Leu Leu Arg Ala Ala Leu Lys Ile Arg Ile Cys Leu Glu Lys Gln Pro Asp Pro Phe Ala Pro Arg Gln Lys Lys Thr Leu Gln Glu Val Gln Glu Asp Phe Val Phe Ser Lys Tyr Arg His Arg Ala Leu Leu Leu Pro Glu Leu Leu Glu Gly Val Leu Gln Ile Leu Ile Cys 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 His Gly Phe Asn Leu Phe Glu Thr Ala Val Leu Gln Met Glu Trp Leu Val Leu Arg Asp Gly Val Pro Pro Glu Ala Ser Glu His Leu Lys Ala Leu Ile Asn Ser Val Met Lys Ile Met Ser Thr Val Lys Lys Val Lys Ser Glu Gln Leu His His Ser Met Cys Thr Arg Lys Arg His Arg Arg Cys Glu Tyr Ser His Phe Met His His His Arg Asp Leu Ser Gly Leu Leu Val Ser Ala Phe Lys Asn Gln Val .. Ser Lys Asn Pro Phe Glu Glu Thr Ala Asp Gly Asp Val Tyr Tyr Pro Glu Arg Cys Cys Cys Ile Ala Val Cys Ala His Gln Cys Leu Arg Leu Leu Gln Gln Ala Ser Leu Ser Ser Thr Cys Val Gln Ile Leu Ser Gly CA 02244744 l998-07-29 W 097/28262 PCT~US97/01748 1~
Val His Asn Ile Gly Ile Cys Cys Cys Met Asp Pro Lys Ser Val Ile .
Ile Pro Leu Leu His Ala Phe Lys Leu Pro Ala Leu Lys Asn Phe Gln Gln His Ile Leu Asn Ile Leu Asn Lys Leu Ile Leu Asp Gln Leu Gly Gly Ala Glu Ile Ser Pro Lys Ile Lys Lys Ala Ala Cys Asn Ile Cys ~hr Val Asp Ser Asp Gln Leu Ala Gln Leu Glu Glu Thr Leu Gln Gly ~sn Leu Cys Asp Ala Glu Leu Ser Ser Ser Leu Ser Ser Pro Ser Tyr Arg Phe Gln Gly Ile Leu Pro Ser Ser Gly Ser Glu Asp Leu Leu Trp Lys Trp Asp Ala Leu Lys Ala Tyr Gln Asn Phe Val Phe Gly Glu Asp Arg Leu His Ser Ile Gln Ile Ala Asn His Ile Cys Asn Leu Ile Gln ~ys Gly Asn Ile Val Val Gln Trp Lys Leu Tyr Asn Tyr Ile Phe Asn ~ro Val Leu Gln Arg Gly Val Glu Leu Ala His His Cys Gln His Leu Ser Val Thr Ser Ala Gln Ser His Val Cys Ser His His Asn Gln Cys Leu Pro Gln Asp Val Leu Gln Ile Tyr Val Lys Thr Leu Pro Ile Leu Leu Lys Ser Arg Val Ile Arg Asp Leu Phe Leu Ser Cys Asn Gly Val ~er Gln Ile Ile Glu Leu Asn Cys Leu Asn Gly Ile Arg Ser His Ser ~eu Lys Ala Phe Glu Thr Leu Ile Ile Ser Leu Gly Glu Gln Gln Lys Asp Ala Ser Val Pro Asp Ile Asp Gly Ile Asp Ile Glu Gln Lys Glu Leu Ser Ser Val His Val Gly Thr Ser Phe His His Gln Gln Ala Tyr Ser Asp Ser Pro Gln Ser Leu Ser Lys Phe Tyr Ala Gly Leu Lys Glu ~la Tyr Pro Lys Arg Arg Lys Thr Val Asn Gln Asp Val His Ile Asn ~hr Ile Asn Leu Phe Leu Cys Val Ala Phe Leu Cys Val Ser Lys Glu ~la Glu Ser Asp Arg Glu Ser Ala Asn Asp Ser Glu Asp Thr Ser Gly W O 97/28262 PCT~US97/01748 ~,, Tyr Asp Ser Thr Ala Ser Glu Pro Leu Ser His Met Leu Pro Cys Ile Ser Leu Glu Ser Leu Val Leu Pro Ser Pro Glu His Met His Gln Ala Ala Asp Ile Trp Ser Met Cys Arg Trp Ile Tyr Met Leu Ser Ser Val Phe Gln Lys Gln Phe Tyr Arg Leu Gly Gly Phe Arg Val Cys His Lys Leu Ile Phe Met Ile Ile Gln Lys Leu Phe Arg Ser His Lys Glu Glu Gln Gly Lys Lys Glu Gly Asp Thr Ser Val Asn Glu Asn Gln Asp Leu Asn Arg Ile Ser Gln Pro Lys Arg Thr Met Lys Glu Asp Leu Leu Ser Leu Ala Ile Lys Ser Asp Pro Ile Pro Ser Glu Leu Gly Ser Leu Lys Lys Ser Ala Asp Ser Leu Gly Lys Leu Glu Leu Gln His Ile Ser Ser Ile Asn Val Glu Glu Val Ser Ala Thr Glu Ala Ala Pro Glu Glu Ala Lys Leu Phe Thr Ser Gln Glu Ser Glu Thr Ser Leu Gln Ser Ile Arg Leu Leu Glu Ala Leu Leu Ala Ile Cys Leu His Gly Ala Arg Thr Ser Gln Gln Lys Met Glu Leu Glu Leu Pro Asn Gln Asn Leu Ser Val Glu Ser Ile Leu Phe Glu Met Arg Asp His Leu Ser Gln Ser Lys Val Ile Glu Thr Gln Leu Ala Lys Pro Leu Phe Asp Ala Leu Leu Arg Val Ala Leu Gly Asn Tyr Ser Ala Asp Phe Glu His Asn Asp Ala Met Thr Glu Lys Ser Hls Gln Ser Ala Glu Glu Leu Ser Ser Gln Pro Gly Asp Phe Ser Glu Glu Ala Glu Asp Ser Gln Cys Cys Ser Phe Lys Leu Leu Val 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 Glu Ile Ile Tyr Pro Glu Ile Cys Met Leu Glu Leu Asn Leu Leu Ser WO 97/28262 PC~US97/01748 1~

Ala Ser Lys Ala Lys Leu Asp Val Leu Ala His Val Phe Glu Ser Phe "

Leu Lys Ile Ile Arg Gln Lys Glu Lys Asn Val Phe Leu Leu Met Gln Gln Gly Thr Val Lys Asn Leu Leu Gly Gly Phe Leu Ser Ile Leu Thr Gln Asp Asp Ser Asp Phe Gln Ala Cys Gln Arg Val Leu Val Asp Leu Leu Val Ser Leu Met Ser Ser Arg Thr Cys Ser Glu Glu Leu Thr Leu Leu Leu Arg Ile Phe Leu Glu Lys Ser Pro Cys Thr Lys Ile Leu Leu Leu Gly Ile Leu Lys Ile Ile Glu Ser Asp Thr Thr Met Ser Pro Ser G- n Tyr Leu Thr Phe Pro Leu Leu His Ala Pro Asn Leu Ser Asn Gly Val Ser Ser Gln Lys Tyr Pro Gly Ile Leu Asn Ser Lys Ala Met Gly Leu Leu Arg Arg Ala Arg Val Ser Arg Ser Lys Lys Glu Ala Asp Arg Glu Ser Phe Pro His Arg Leu Leu Ser Ser Trp His Ile Ala Pro Val His Leu Pro Leu Leu Gly Gln Asn Cys Trp Pro His Leu Ser Glu Gly Phe Ser Val Ser Leu Trp Phe Asn Val Glu Cys Ile His Glu Ala Glu Ser Thr Thr Glu Lys Gly Lys Lys Ile Lys Lys Arg Asn Lys Ser Leu Ile Leu Pro Asp Ser Ser Phe Asp Gly Thr Glu Ser Asp Arg Pro Glu Gly Ala Glu Tyr Ile Asn Pro Gly Glu Arg Leu Ile Glu Glu Gly Cys Ile His Ile Ile Ser Leu Gly Ser Lys Ala Leu Met Ile Gln Val Trp Ala Asp Pro His Asn Ala Thr Leu Ile Phe Arg Val Cys Met Asp Ser Asn Asp Asp Met Lys Ala Val Leu Leu Ala Gln Val Glu Ser Gln Glu Asn Ile Phe Leu Pro Ser Lys Trp Gln His Leu Val Leu Thr Tyr Leu ~.

Gln Gln Pro Gln Gly Lys Arg Arg Ile His Gly Lys Ile Ser Ile Trp W O 97/28262 PCTrUS97/01748 f~
Val Ser Gly Gln Arg Lys Pro Asp Val Thr Leu Asp Phe Met Leu Pro Arg Lys Thr Ser Leu Ser Ser Asp Ser Asn Lys Thr Phe Cys Met Ile Gly His Cys Leu Ser Ser Gln Glu Glu Phe Leu Gln Leu Ala Gly Lys Trp Asp Leu Gly Asn Leu Leu Leu Phe Asn Gly Ala Lys Val Gly Ser Gln Glu Ala Phe Tyr Leu Tyr Ala Cys Gly Pro Asn His Thr Ser Val Met Pro Cys Lys Tyr Gly Lys Pro Val Asn Asp Tyr Ser Lys Tyr Ile Asn Lys Glu Ile Leu Arg Cys Glu Gln Ile Arg Glu Phe Phe Met Thr Lys Lys Asp Val Asp Ile Gly Leu Leu Ile Gly Val Phe Gln Leu Phe Ile Gln Leu Thr Val Leu Leu Gln Tyr Thr Ile Tyr Glu Pro Val Ile Arg Leu Lys Gly Gln Met Lys Thr Gln Leu Ser Gln Arg Pro Phe Ser Ser Lys Glu Val Gln Ser Ile Leu Leu Glu Pro His His Leu Lys Asn Leu Gln Pro Thr Glu Tyr Lys Thr Ile Gln Gly Ile Leu His Glu Ile Gly Gly Thr Gly Ile Phe Val Phe Leu Phe Ala Arg Val Val Glu Leu Ser Ser Cys Glu Glu Thr Gln Ala Leu Ala Leu Arg Val Ile Leu Ser Leu Ile Lys Tyr Asn Gln Gln Arg Val His Glu Leu Glu Asn Cys Asn Gly Leu Ser Met Ile His Gln Val Leu Ile Lys Gln Lys Cys Ile Val Gly Phe Tyr Ile Leu Lys Thr Leu Leu Glu Gly Cys Cys Gly Glu Asp 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 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 Thr Cys Gln Val Leu Gln Glu Tyr Lys Glu Gly Gln Leu Thr Pro Met Pro Arg Glu Met Ala Arg Ser Phe Arg Arg Lys Cys Gly Gln Ser Cys 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:

GTTTCATCTA GTTTATGAGT CCAAATGATA TAGACTGTAA ATGTCACAGC AGTGGTGA~A 540 TCCACTCAGG A~AAATTACA TTTAGAAGGA AGTGCCCTGT CTAGTCAGGT TTCTGCAAAA 960 GTAAATGTTT TTCGAAAAAG CAGACGACAG CGTA~AATTA CCCATCGCTA TTCTGTAAGA 1020 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 CA 02244744 l998-07-29 W 097/28262 PCT~US97/01748 GTAGATGTTA GTACTGCAGA GATGATGCCA GAAAATCTTA GGA~AAATTT AACTGAATTG 1620 TGCTTGCGCT TACTGCAGCA GGCTTCCTTG AGCAGCACTT GTGTCCAGAT CCTATCGGGT ~280 CA 02244744 l998-07-29 W097/28262 PCTrUS97/01748 1~

GAAGTTTCAG CTACTGAAGC'CGCTCCCGAG GAAGCAAAGC TATTTACAAG TCAAGAAAGT 3840 GAGCTAACCC 'l"l'~ GAG AATATTTCTG GAGAAATCTC CTTGTACAAA AATTCTTCTT 4680 W O 97/28262 PCTrUS97/01748 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 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 Ser Ser Gln Arg Ser Thr Gln Glu Lys Leu His Leu Glu Gly Ser Ala Leu Ser Ser Gln Val Ser Ala Lys Val Asn Val Phe Arg Lys Ser Arg Arg Gln Arg Lys Ile Thr His Arg Tyr Ser Val Arg Asp Ala Arg Lys Thr Gln Leu Ser Thr Ser Asp Ser Glu Ala Asn Ser Asp Glu Lys Gly Ile Ala Met Asn Lys His Arg Arg Pro His Leu Leu His His Phe Leu Thr Ser Phe Pro Lys Gln Asp His Pro Lys Ala Lys Leu Asp Arg Leu Ala Thr Lys Glu Gln Thr Pro Pro Asp Ala Met Ala Leu Glu Asn Ser 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 -~ Leu Cys His Val Leu Leu Ser Leu Leu Glu Lys Val Cys Lys Phe Asp Val Thr Leu Asn His Asn Ser Pro Leu Ala Ala Ser Val Val Pro Thr W O 97/28262 PCT~US97/01748 ~'1~

Leu Thr Glu Phe Leu Ala Gly Phe Gly Asp Cys Cys Ser Leu Ser Asp Asn Leu Glu Ser Arg Val Val Ser Ala Gly Trp Thr Glu Glu Pro Val Ala Leu Ile Gln Arg Met Leu Phe Arg Thr Val Leu His Leu Leu Ser Val Asp Val Ser Thr Ala Glu Met Met Pro Glu Asn Leu Arg Lys Asn Leu Thr Glu Leu Leu Arg Ala Ala Leu Lys Ile Arg Ile Cys Leu Glu Lys Gln Pro Asp Pro Phe Ala Pro Arg Gln Lys Lys Thr Leu Gln Glu Val Gln Glu Asp Phe Val Phe Ser Lys Tyr Arg His Arg Ala Leu Leu Leu Pro Glu Leu Leu Glu Gly Val Leu Gln Ile Leu Ile Cys 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 His Gly Phe Asn Leu Phe Glu Thr Ala Val Leu Gln Met Glu Trp Leu Val Leu Arg Asp Gly Val Pro Pro Glu Ala Ser Glu His Leu Lys Ala Leu Ile Asn Ser Val Met Lys Ile Met Ser Thr Val Lys Lys Val Lys Ser Glu Gln Leu His His Ser Met Cys Thr Arg Lys Arg His Arg Arg Cys Glu Tyr Ser His Phe Met His His His Arg Asp Leu Ser Gly Leu Leu Val Ser Ala Phe Lys Asn Gln Val Ser Lys Asn Pro Phe Glu Glu Thr Ala Asp Gly Asp Val Tyr Tyr Pro Glu Arg Cys Cys Cys Ile Ala Val Cys Ala His Gln Cys Leu Arg Leu Leu Gln Gln Ala Ser Leu Ser Ser Thr Cys Val Gln Ile Leu Ser Gly Val His Asn Ile Gly Ile Cys Cys Cys Met Asp Pro Lys Ser Val Ile Ile Pro Leu Leu His Ala Phe Lys Leu Pro Ala Leu Lys Asn Phe Gln Gln His Ile Leu Asn Ile Leu Asn Lys Leu Ile Leu Asp Gln Leu Gly WO 97/28262 PCTrUS97/01748 ll73 Gly Ala Glu Ile Ser Pro Lys Ile Lys Lys Ala Ala Cys Asn Ile Cys ~hr Val Asp Ser Asp Gln Leu Ala Gln Leu Glu Glu Thr Leu Gln Gly Asn Leu Cys Asp Ala Glu Leu Ser Ser Ser Leu Ser Ser Pro Ser Tyr Arg Phe Gln Gly Ile Leu Pro Ser Ser Gly Ser Glu Asp Leu Leu Trp Lys Trp Asp Ala Leu Lys Ala Tyr Gln Asn Phe Val Phe Gly Glu Asp Arg Leu His Ser Ile Gln Ile Ala Asn His Ile Cys Asn Leu Ile Gln Lys Gly Asn Ile Val Val Gln Trp Lys Leu Tyr Asn Tyr Ile Phe Asn Pro Val Leu Gln Arg Gly Val Glu Leu Ala His His Cys Gln His Leu Ser Val Thr Ser Ala Gln Ser His Val Cys Ser His His Asn Gln Cys Leu Pro Gln Asp Val Leu Gln Ile Tyr Val Lys Thr Leu Pro Ile Leu 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 Leu Lys Ala Phe Glu Thr Leu Ile Ile Ser Leu Gly Glu Gln Gln Lys Asp Ala Ser Val Pro Asp Ile Asp Gly Ile Asp Ile Glu Gln Lys Glu Leu Ser Ser Val His Val Gly Thr Ser Phe His His Gln Gln Ala Tyr Ser Asp Ser Pro Gln Ser Leu Ser Lys Phe Tyr Ala Gly Leu Lys Glu Ala Tyr Pro Lys Arg Arg Lys Thr Val Asn Gln Asp Val His Ile Asn Thr Ile Asn Leu Phe Leu Cys Val Ala Phe Leu Cys Val Ser Lys Glu Ala Glu Ser Asp Arg Glu Ser Ala Asn Asp Ser Glu Asp Thr Ser Gly 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 W O 97/28262 PCTnUS97/01748 Phe Gln Lys Gln Phe Tyr Arg Leu Gly Gly Phe Arg Val Cys His Lys Leu Ile Phe Met Ile Ile Gln Lys Leu Phe Arg Ser His Lys Glu Glu Gln Gly Lys Lys Glu Gly Asp Thr Ser Val Asn Glu Asn Gln Asp Leu Asn Arg Ile Ser Gln Pro Lys Arg Thr Met Lys Glu Asp Leu Leu Ser Leu Ala Ile Lys Ser Asp Pro Ile Pro Ser Glu Leu Gly Ser Leu Lys Lys Ser Ala Asp Ser Leu Gly Lys Leu Glu Leu Gln His Ile Ser Ser Ile Asn Val Glu Glu Val Ser Ala Thr Glu Ala Ala Pro Glu Glu Ala Lys Leu Phe Thr Ser Gln Glu Ser Glu Thr Ser Leu Gln Ser Ile Arg Leu Leu Glu Ala Leu Leu Ala Ile Cys Leu His Gly Ala Arg Thr Ser Gln Gln Lys Met Glu Leu Glu Leu Pro Asn Gln Asn Leu .Ser Val Glu Ser Ile Leu Phe Glu Met Arg Asp His Leu Ser Gln Ser Lys Val Ile Glu Thr Gln Leu Ala Lys Pro Leu Phe Asp Ala Leu Leu Arg Val Ala Leu Gly Asn Tyr Ser Ala Asp Phe Glu His Asn Asp Ala Met Thr Glu 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 Glu Thr Gln Asp Asp Gly Val Asp Leu Lys Ser Glu Thr Glu Gly Phe Ser Ala Ser Ser Ser Pro Asn Asp Leu Leu Glu Asn Leu Thr Gln Gly Glu Ile Ile Tyr Pro Glu Ile Cys Met Leu Glu Leu Asn Leu Leu Ser Ala Ser Lys Ala Lys Leu Asp Val Leu Ala His Val Phe Glu Ser Phe Leu Lys Ile Ile Arg Gln Lys Glu Lys Asn Val Phe Leu Leu Met Gln Gln Gly Thr Val Lys Asn Leu Leu Gly Gly Phe Leu Ser Ile Leu Thr W 097l28262 PCTAUS97/01748 1'7~

- Gln Asp Asp Ser Asp Phe Gln Ala Cys Gln Arg Val Leu Val Asp Leu Leu Val Ser Leu Met Ser Ser Arg Thr Cys Ser Glu Glu Leu Thr Leu Leu Leu Arg Ile Phe Leu Glu Lys Ser Pro Cys Thr Lys Ile Leu Leu Leu Gly Ile Leu Lys Ile Ile Glu Ser Asp Thr Thr Met Ser Pro Ser Gln Tyr Leu Thr Phe Pro Leu Leu His Ala Pro Asn Leu Ser Asn Gly Val Ser Ser Gln Lys Tyr Pro Gly Ile Leu Asn Ser Lys Ala Met Gly Leu Leu Arg Arg Ala Arg Val Ser Arg Ser Lys Lys Glu Ala Asp Arg Glu Ser Phe Pro His Arg Leu Leu Ser Ser Trp His Ile Ala Pro Val His Leu Pro Leu Leu Gly Gln Asn Cys Trp Pro His Leu Ser Glu Gly Phe Ser Val Ser Leu Trp Phe Asn Val Glu Cys Ile His Glu Ala Glu Ser Thr Thr Glu Lys Gly Lys Lys Ile Lys Lys Arg Asn Lys Ser Leu Ile Leu Pro Asp Ser Ser Phe Asp Gly Thr Gly Met Met Thr Gly Leu Ser Asp Leu Tyr Thr Lys Ile Val Phe Arg Leu (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 TTCCCCCTTG GGC~AAAA CCTGAAGACT TTGTGCGGAT CAACAGGATG GCCCTAGAAA 180 CA 02244744 l998-07-29 W097/28262 PCT~US97/~1748 1~

AGAT Q CAGA CCTCGTTGAC CAGAGTATAC A~ATCAATGC ACATTGTTTT GTGGTAACAG 780 TGAAGATAAA GGAAGAAC Q AAAGCCAAGT TA~AGCTGAG GGCACAAGTG CTGCATGGAA 1620 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 W 097/28262 PCTrUS97/01748 - 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 Ala Met Glu Ala Gln Ile Gln Asn Phe Gly Gln Thr Pro Ser Gln Leu Leu Ile Glu Pro His Pro Pro Arg Asn Ser Ala Met His Leu Cys Phe 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 Ala Asn Thr Leu Pro His Leu Thr Ile Pro Ala Val Val Thr Val Thr Cys Ser Arg Leu Phe Ala Val Asn Arg Trp His Asn Thr Val Gly Leu Arg Gly Ala Pro Gly Tyr Ser Leu Asp Gln Ala His His Leu Pro Ile Glu Met Asp Pro Leu Ile Ala Asn Asn Ser Gly Val Asn Lys Arg Gln Ile Thr Asp Leu Val Asp Gln Ser Ile Gln Ile Asn Ala His Cys Phe Val Val Thr Ala Asp Asn Arg Tyr Ile Leu Ile Cys Gly Phe Trp Asp Lys Ser Phe Arg Val Tyr Thr Thr Glu Thr Gly Lys Leu Thr Gln Ile Val Phe Gly His Trp Asp Val Val Thr Cys Leu Ala Arg Ser Glu Ser Tyr Ile Gly Gly Asp Cys Tyr Ile Val Ser Gly Ser Arg Asp Ala Thr Leu Leu Leu Trp Tyr Trp Ser Gly Arg His His Ile Ile Gly Asp Asn Pro Asn Ser Ser Asp Tyr Pro Ala Pro Arg Ala Val Leu Thr Gly His CA 02244744 l998-07-29 WO 97t28262 PCT/US97/01748 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 Asp Leu Leu Arg Ala Leu Glu Gly Pro Glu Asn Cys Leu Phe Pro Arg Leu Ile Ser Val Ser Ser Glu Gly His Cys Ile Ile Tyr Tyr Glu Arg Gly Arg Phe Ser Asn Phe Ser Ile Asn Gly Lys Leu Leu Ala Gln Met Glu Ile Asn Asp Ser Thr Arg Ala Ile Leu Leu Ser Ser Asp Gly Gln Asn Leu Val Thr Gly Gly Asp Asn Gly Val Val Glu Val Trp Gln Ala Cys Asp Phe Lys Gln Leu Tyr Ile (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:

WO 97/28262 PCT~US97/01748 Iflq TGTGTTCCCT TCCACAGAGC CCACTCATGT TCA~AGATCA GATGCAGCAG GATGTGATCA 1080 CGGAGTCCTA CATTGGTGGA GACTGCTACA TAGTGTCTGG ATCTCGGGAC GCCACCTTGC lS60 CGATGGATTT ATCCCATGAC CA~AGGACTC TGATCACTGG CATGGCTTCC GGCAGCATTG 2100 GCAGCAGAAG.CCACATTCAA GTGAGAGCAC AAGTGCTTCT GTGGAAAGGC AGTATCTCTG 2220 -(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 703 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear CA 02244744 l998-07-29 W097/28262 PCT~US97/01748 1~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
- Ser Arg Ala Asn Arg Thr Ser Val Met Phe Asn Phe Pro Asp Gln Ala 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 His Leu Ser Lys Pro Lys Gly Ala Leu Asn Pro Lys Arg Ala Val Phe Tyr Ala Glu Arg Tyr Glu Thr Trp Glu Glu Asp Gln Ser Pro Pro Phe His Tyr Asn Thr His Tyr Ser Thr Ala Thr Ser Pro Leu Ser Trp Leu Val Arg Ile Glu Pro Phe Thr Thr Phe Phe Leu Asn Ala Asn Asp Gly Lys Phe Asp His Pro Asp Arg Thr Phe Ser Ser Ile Ala Arg Ser Trp Arg Thr Ser Gln Arg Asp Thr Ser Asp Val Lys Glu Leu Ile Pro Glu Phe Tyr Tyr Val Pro Glu Met Phe Val Asn Ser Asn Gly Tyr His 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 Ala Met Val Ala Gln Ile Gln Asn Phe Ala Gln Thr Pro Ser Gln Leu Leu Ile Glu Pro His Pro Pro Arg Thr Ser W O 97/28262 PCT~US97/~1748 1~\
Ala Met His Leu Cys Ser Leu Pro Gln Ser Pro Leu Met Phe Lys Asp Gln Met Gln Gln Asp Val Ile Met Val Leu Lys Phe Pro Ser Asn Ser Pro Val Thr His Val Ala Ala Asn Thr Leu Pro His Leu Thr Ile Pro Ala Val Val Thr Val Thr Cys Ser Arg Leu Phe Ala Val Asn Arg Trp His Asn Thr Val Gly Leu Arg Gly Ala Pro Gly Tyr Ser Leu Asp Gln Ala His His Leu Pro Ile Glu Met Asp Pro Leu Ile Ala Asn Asn Ser Gly Val Asn Lys Arg Gln Ile Thr Asp Leu Val Asp Gln Ser Ile Gln Ile Asn Ala His Cys Phe Val Val Thr Ala Asp Asn Arg Tyr Ile Leu Ile Cys Gly Phe Trp Asp Lys Ser Phe Arg Val Tyr Ser Thr Glu Thr Gly Lys Leu Thr Gln Ile Val Phe Gly His Trp Asp Val Val Thr Cys Leu Ala Arg Ser Glu Ser Tyr Ile Gly Gly Asp Cys Tyr Ile Val Ser Gly Ser Arg Asp Ala Thr Leu Leu Leu Trp Tyr Trp Ser Gly Arg His His Ile Ile Gly Asp Asn Pro Asn Ser Ser Asp Tyr Pro Ala Pro Arg Ala Val Leu Thr Gly His Asp His Glu Val Val Cys Val Ser Val Cys Ala Glu Leu Gly Leu Val Ile Ser Gly Ala Lys Glu Gly Pro Cys Leu Val His Thr Ile Thr Gly Asn Leu Leu Lys Ala Leu Glu Gly Pro Glu 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 Lys Leu Leu Ala Gln Met Glu Ile Asn Asp Ser Thr Arg Ala Ile Leu Leu Ser Ser Asp Gly Gln Asn Leu Val Thr Gly Gly Asp Asn Gly Val Val Glu Val Trp Gln Ala Cys Asp Phe Lys Gln Leu Tyr Ile Tyr Pro Gly Cys Asp Ala Gly Ile Arg Ala Met Asp Leu Ser His Asp Gln Arg W O 97/28262 PCTrUS97/01748 Thr Leu Ile Thr Gly Met Ala Ser Gly Ser Ile Val Leu Leu Ile (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:

(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:

(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:

(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
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(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 WO 97/28262 PCT~US97/01748 ~3 (xi) SEQUENCE DESC~IPTION: SEQ ID NO: 19:

(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
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(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
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(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:

(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:

~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 W097l28262 PCT~US97/01748 (xi) SEQUENCE DESCRIPTICN: SEQ ID NO: 24:

(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:

(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:

(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:

(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:

(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 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:

(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:

(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:

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 W 097/28262 PCT~US97/01748 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

(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:

(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:

(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:

(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:

(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 CA 02244744 l998-07-29 W O 97/28262 PCTrUS97/01748 (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:

(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:

(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:

(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:

-(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 CA 02244744 l998-07-29 W O g7/28262 PCTrUS97/01748 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:

(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:

(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:

(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:

(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 CA 02244744 l998-07-29 W O 97/28262 PCT~US97/01748 1~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:

(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:

(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:

(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:

(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:

(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 CA 02244744 l998-07-29 WO 97/28Z62 PCTrUS97/01748 ~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 CA 02244744 l998-07-29 (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:

(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:

(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:

(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:

(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 W 0 97/28262 ~CT~US97/01748 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:

(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:

(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:

(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:

(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 W097/28262 PCTAUS97/0~748 Iq'~
(~i) SEQUENCE DESCRIPTION: SEQ ID NO: 69:

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:

(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:

(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:

(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:

(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 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:

(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:

(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:

(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:

(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:

CA 02244744 l998-07-29 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.

Claims (65)

CLAIMS:

1. A purified mammalian LYST1, Lyst1, LYST2, or Lyst2 protein.

2. The protein according to claim 1, wherein said protein is isolated from a mouse or human.

3. The protein according to claim 1, comprising the amino acid sequence of 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.

4. A purified nucleic acid segment encoding a LYST1, Lyst1, LYST2, or Lyst2 protein.

5. The nucleic acid segment of claim 4, wherein said segment encodes a human LYST1 or LYST2 protein, or a murine Lyst1 or Lyst2 protein.

6. The nucleic acid segment of claim 4, further defined as encoding a protein comprising the amino acid sequence of 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.

7. The nucleic acid segment of claim 4, further defined as comprising the nucleic acid sequence 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, or the complements thereof, or a sequence which hybridizes to the sequence 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.

8. The nucleic acid segment of claim 4, further defined as an RNA segment.

9. A DNA segment comprising an isolated LYST1, Lyst1, LYST2, or Lyst2 gene.

10. The DNA segment of claim 9, comprising an isolated LYST1, Lyst1, LYST2, or Lyst2 gene.

11. The DNA segment of claim 10, comprising an isolated human LYST1 or LYST2 gene or an isolated murine Lyst1 or Lyst2 gene.

12. The DNA segment of claim 11, comprising an isolated human LYST1 or LYST2 gene, or murine Lyst1 or Lyst2 gene that encodes a protein or peptide that includes a contiguous amino acid sequence from 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.

13. The DNA segment of claim 9, comprising an isolated human LYST1 or LYST2 gene, or murine Lyst1 or Lyst2 gene that includes a contiguous nucleic acid sequence 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.

14. The DNA segment of claim 9, comprising an isolated human LYST1 or LYST2 gene, or murine Lyst1 or Lyst2 gene that encodes a protein of from about 15 to about 50 amino acids in length.

15. The DNA segment of claim 9, comprising an isolated human LYST1 or LYST2 gene, or murine Lyst1 or Lyst2 gene that encodes a protein of from about 50 to about 150 amino acids in length.

16. The DNA segment of claim 9, comprising an isolated human LYST1 or LYST2 gene, or murine Lyst1 or Lyst2 gene that encodes a protein of about 1185 amino acids in length.

17. The DNA segment of claim 9, defined further as a recombinant vector.

18. The DNA segment of claim 17, defined further as recombinant vector pCH.

19. The DNA segment of claim 9, wherein said DNA is operatively linked to a promotor, said promoter expressing the DNA segment.

20. A recombinant host cell comprising the DNA segment of claim 9.

21. The recombinant host cell of claim 20, defined further as being a prokaryotic cell.

22. The recombinant host cell of claim 21, further defined as a bacterial cell.

23. The recombinant host cell of claim 20, defined further as being a eukaryotic cell.

24. The recombinant host cell of claim 23, further defined as a yeast cell or an animal cell.

25. The recombinant host cell of claim 24, wherein said cell is a mammalian cell.

26. The recombinant host cell of claim 25, wherein said cell is a human cell.

27. The recombinant host cell of claim 20, wherein said DNA segment is introduced into the cell by means of a recombinant vector.

28. The recombinant host cell of claim 20, wherein said host cell expresses the DNA segment to produce a LYST1, Lyst1, LYST2, or Lyst2 protein or peptide.

29. The recombinant host cell of claim 28, wherein said LYST1, Lyst1, LYST2, or Lyst2 protein or peptide comprises a contiguous amino acid sequence from 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.

30. A method of using a DNA segment that encodes an isolated human LYST1 or LYST2 protein or murine Lyst1 or Lyst2 protein, comprising the steps of:

(a) preparing a recombinant vector in which a LYST1-, Lyst1-, LYST2-, or Lyst2- encoding DNA segment is positioned under the control of a promoter;

(b) introducing said recombinant vector into a host cell;

(c) culturing said host cell under conditions effective to allow expression of the encoded protein or peptide; and (d) collecting said expressed protein or peptide.

31. An isolated nucleic acid segment characterized as:

(a) a nucleic acid segment comprising a sequence region that consists of at least 14 contiguous nucleotides that have the same sequence as, or are complementary to, 14 contiguous nucleotides of 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, or (b) a nucleic acid segment of from 14 to about 10,000 nucleotides in length thathybridizes to the nucleic acid segment of 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; or the complements thereof, under standard hybridization conditions.

32. The nucleic acid segment of claim 31, further defined as comprising a sequence region that consists of at least 14 contiguous nucleotides that have the same sequence as, or are complementary to, 14 contiguous nucleotides 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.

33. The nucleic acid segment of claim 31, further defined as comprising a nucleic acid segment of from 14 to about 10,000 nucleotides in length that hybridizes to the nucleic acid segment of 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, or the complements thereof, under standard hybridization conditions.

34. The nucleic acid segment of claim 33, wherein the segment comprises a sequence region of at least about 20 nucleotides; or wherein the segment is about 20 nucleotides in length.

35. The nucleic acid segment of claim 34, wherein the segment comprises a sequence region of at least about 30 nucleotides; or wherein the segment is about 30 nucleotides in length.

36. The nucleic acid segment of claim 35, wherein the segment comprises a sequence region of at least about 50 nucleotides; or wherein the segment is about 50 nucleotides in length.

37. The nucleic acid segment of claim 36, wherein the segment comprises a sequence region of at least about 100 nucleotides; or wherein the segment is about 100 nucleotides in length.

38. The nucleic acid segment of claim 37, wherein the segment comprises a sequence region of at least about 200 nucleotides; or wherein the segment is about 200 nucleotides in length.

39. The nucleic acid segment of claim 38, wherein the segment comprises a sequence region of at least about 500 nucleotides; or wherein the segment is about 500 nucleotides in length.

40. The nucleic acid segment of claim 39, wherein the segment comprises a sequence region of at least about 1000 nucleotides; or wherein the segment is about 1000 nucleotides in length.

41. The nucleic acid segment of claim 40, wherein the segment comprises a sequence region of 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.

42. The nucleic acid segment of claim 31, wherein the segment is up to 10,000 basepairs in length.

43. The nucleic acid segment of claim 42, wherein the segment is up to 5,000 basepairs in length.

44. The nucleic acid segment of claim 43, wherein the segment is up to 4,000 basepairs in length.

45. The nucleic acid segment of claim 44, wherein the segment is up to 3,000 basepairs in length.

46. The nucleic acid segment of claim 45, wherein the segment is about 3514 basepairs in length.

47. A method for detecting a nucleic acid sequence encoding a LYST1, Lyst1, LYST2. or Lyst2 protein, comprising the steps of:

(a) obtaining sample nucleic acids suspected of encoding a LYST1, Lyst1, LYST2, or Lyst2 protein;

(b) contacting said sample nucleic acids with an isolated nucleic acid segment encoding said protein under conditions effective to allow hybridization of substantially complementary nucleic acids; and (c) detecting the hybridized complementary nucleic acids thus formed.

48. The method of claim 47, wherein the sample nucleic acids contacted are located within a cell.

49. The method of claim 47, wherein the sample nucleic acids are separated from a cell prior to contact.

50. The method of claim 47, wherein the isolated protein-encoding nucleic acid segment comprises a detectable label and the hybridized complementary nucleic acids are detected by detecting said label.

51. A nucleic acid detection kit comprising, in suitable container means, an isolated LYST1, Lyst1, LYST2 or Lyst2 nucleic acid segment and a detection reagent.

52. The nucleic acid detection kit of claim 51, wherein the detection reagent is a detectable label that is linked to said nucleic acid segment.

53. The nucleic acid detection kit of claim 51, further comprising a restriction enzyme.

54. A peptide composition, free from total cells, comprising a LYST1, Lyst1, LYST2, or Lyst2 protein that includes a contiguous amino acid sequence of 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.

55. The composition of claim 54, comprising a peptide that includes an about 15 to about 50 amino acid long sequence from of 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.

56. The composition of claim 54, comprising a peptide that includes an about 50 to about 150 amino acid long sequence from of 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.

57. The composition of claim 54, comprising a peptide that includes an about 150 to about 300 amino acid long sequence from of 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.

58. The composition of claim 54, wherein the protein or peptide is a recombinant protein or peptide.

59. A purified antibody that binds to a LYST1, Lyst1, LYST2, or Lyst2 protein or peptide.

60. The antibody of claim 59, wherein the antibody is linked to a detectable label.

61. The antibody of claim 60, wherein the antibody is linked to a radioactive label, a fluorogenic label, a nuclear magnetic spin resonance label, biotin or an enzyme that generates a colored product upon contact with a chromogenic substrate.

62. The antibody of claim 61, wherein the antibody is linked to an alkaline phosphatase, hydrogen peroxidase or glucose oxidase enzyme.

63. The antibody of claim 59, wherein said antibody is a monoclonal antibody.

64. A method for diagnosing Chediak-Higashi Syndrome, comprising identifying a Lyst1 or LYST1 nucleic acid segment or a Lyst1 or LYST1 protein or peptide present within a clinical sample from a patient suspected of having such a syndrome.

65. A transgenic animal having incorporated into its genome a transgene that encodes a LYST1, Lyst1, LYST2, or Lyst2 protein or peptide.