WO1997028262A1 - 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

DESCRIPTION

LYST1 AND LYST2 GENE COMPOSITIONS

AND METHODS OF USE

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 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 5P30-AR 41943 from the National Institutes of Health. 1.1 Field of the Invention

The present invention relates generally to the field of molecular biology. More particularly, certain embodiments concern methods and compositions comprising novel DNA segments, and proteins derived from mammalian species. More particularly, the invention provides Lyst1 and Lyst2 gene compositions from murine origins and the homologous LYST1 and LYST2 gene compositions from human origins. Various methods for making and using these LYST/Lyst DNA segments, native peptides and synthetic protein derivatives are disclosed, such as, for example, the use of DNA segments as diagnostic probes and templates for protein production, and the use of LYST1, Lyst1, LYST2, and Lyst2 proteins, fusion protein carriers and Lyst-derived peptides in various pharmacological and immunological applications. 1.2 Description of the Related Art

1.2.1 Chediak-Higashi (CH) Syndrome

Chediak-Higashi syndrome (CHS) is an autosomal recessive, immune deficiency disease that maps on chromosome (Chr) Iq42-q43 (Goodrich and Holcombe, 1995; Barrat et al. 1996; Fukai etai, 1996). Affected individuals have giant, perinuclear lysosomes, defective granulocyte, NK and cytolytic T cell function, and die prematurely of infection or malignancy (Beguez Cesar, 1943, Blume et al., 1968; Wolff et al., 1972; Blume and Wolff, 1972, Root et al., 1972; Roder et al., 1982; Baetz et al., 1995). CHS patients also exhibit partial oculocutaneous albinism, platelet storage pool deficiency and neurologic defects such as peripheral neuropathy and ataxia (Windhorst et al., 1968; Meyers et al., 1974; Maeda et al., 1989; Pettit and Berdal, 1984; Misra et al., 1991). Recently it was demonstrated that intracellular protein transport to and from the lysosome is disordered in CHS (Baetz et al., 1995 ; Brandt et al., 1975; Burkhardt et al., 1993; Zhao et al., 1994). Such functional defects in secretory lysosomes of granular cells (leukocytes, melanocytes,

megakaryocytes and cerebellar Purkinje cells) provide a unifying hypothesis that can explain the diverse clinical features of CHS (Griffiths, 1996).

As an antecedent to identification of the human CHS gene, the inventors undertook positional cloning of the mouse mutation beige (bg), which had long been considered homologous 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 1q42-q43 (the position of the CHS locus) (Jenkins et al., 1991). Additional evidence that human CHS and bg mice were homologous disorders came from interspecific genetic complementation studies, which demonstrated that fusion of bg mouse and human CHS fibroblasts failed to reverse lysosomal 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 et al., 1996a). However, the reported bg candidate cDNA sequences (Lyst and BG) were different. Both sequences were isolated from the same yeast artificial chromosome (YAC) clone. This YAC had been authenticated by mapping within the bg critical region and by restoration of normal lysosomal morphology to bg fibroblasts upon transfection

(Perou et al., 1996a; Perou et al., 1996b). Furthermore, both of the candidate gene sequences contained mutations in different bg alleles.

1.3 Deficiencies in the Prior Art

Methods for the treatment and diagnosis of Chediak-Higashi Syndrome have not been developed because the sequence of the CH gene has not been identified in mice or humans.

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, et al., 1986) . There is some evidence that indicate that the CH mutation is located in the same gene in mouse, mink and human (Perou and Kaplan, 1993); however, except for the beige mouse, the locus of the mutation has not been identified. CHS patients have been reported to suffer from several serious medical conditions, including impaired natural killer cell activity (Haliotis et al., 1980) and defective lymphocyte- mediated antibody dependent cell mediated leukocyte mediated ADCC against tumor cell targets (Klein, et al., 1980). Despite the recognition of these deficiencies, little progress in treatment has been achieved, mainly because the gene harboring the mutation leading to these impairments has not yet been identified.

Chediak-Higashi Syndrome occurs only in a small minority of the population. However, there is a growing realization of the potential role of the CH gene product (LYST1) in developing treatments for conditions such as systemic autoimmune disease and possibly certain types of malignancy related to the regulation of protein trafficking within cells by the CH gene (LYST1). Therefore, what is lacking in the prior art is the isolation and characterization of the CH gene from mice and humans, useful in the development of treatments and assays for autoimmune diseases such as CHS and certain forms of cancer.

2. Summary of the Invention

Positional cloning of the mouse CHS homologous is facilitated 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 induced by radiation. The present invention addresses one or more of the foregoing or other problems associated with the detection of Chediak-Higashi Syndrome in humans. Both the mouse gene and the homologous human have been cloned and sequenced. The isolation and sequencing of the Chediak-Higashi gene (LYST1) 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 segments and/or the encoded proteins or polypeptides. In addition to the practical value, the gene provides a tool for understanding and controlling mechanisms of regulation of protein trafficking to lysosomes, and particularly to the contribution of vesicular sorting to diverse cellular functions. An immediate result of the identification of the LYST1 gene is the ability to perform linkage analysis and to identify individuals at risk to have progeny carrying the mutated gene. The inventors have shown that the murine gene, Lyst1, and BG sequences are derived from a single gene with alternatively spliced mRNAs. In an important embodiment, the inventors have also identified the human homolog of the bg gene (Lyst1), LYST1. LYST1 maps within the CHS critical region and is mutated in several CHS patients. 2.1 LYST and Lyst Gene Compositions

As used herein, the term "DNA segment" 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 segment 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 segment is obtained. Included within the term "DNA segment", are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like. Preferred LYST genes are the LYST1 and LYST2 genes from human origin, while preferred Lyst genes are the Lyst1 and Lyst2 genes from murine origin.

Similarly, a DNA segment comprising an isolated or purified LYST/Lyst gene refers to a DNA segment including 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 term "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 includes both genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides. Such segments 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 LYST1 and LYST2 genes being particularly preferred, or one or more Lyst genes, with murine Lyst1 and Lyst2 genes being particularly preferred.

"Isolated substantially 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 segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.

In particular embodiments, the invention concerns isolated DNA segments and

recombinant vectors incorporating DNA sequences that encode a LYST/Lyst species that includes within its amino acid sequence an amino acid 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 ID NO: 14. In other particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that include within their sequence a nucleotide sequence essentially 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: 1 1, 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 ID 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 ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids 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. The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein (for example, see Illustrative Embodiments). Accordingly, sequences 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 identical 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 "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 ID NO: 14".

In certain other embodiments, the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially 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 "essentially 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: 1 1, or SEQ ID NO: 13" is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion 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 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: 1 1, or SEQ ID NO: 13. Again, DNA segments that encode proteins exhibiting LYST, Lyst, LYST-like, or Lyst-like activity will be most preferred. 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 still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance 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 flanking either of the 5' or 3' portions of the coding region or may include various upstream or downstream regulatory or structural genes.

Naturally, the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence 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: 1 1, or SEQ ID NO: 13. Nucleic acid sequences 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 sequences" means nucleic acid sequences that are substantially complementary, as may be assessed 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: 1 1, or SEQ ID NO: 13 under relatively stringent conditions such as those described herein.

The nucleic acid segments 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 segments, and the 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 preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared that include a short contiguous stretch identical to or complementary 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, such as about 14 nucleotides, and that are up to about 10,000 or about 5,000 base pairs in length, with segments of about 3,000 being preferred in certain cases DNA segments with total lengths of about 2,000, about 1,000, about 500, about 200, about 100 and about 50 base pairs in length (including all intermediate lengths) are also contemplated to be useful. It will be readily understood that "intermediate lengths", in these contexts, means any length between the quoted ranges, such as 14, 15, 16, 17, 18, 19, 20, etc., 21, 22, 23, etc., 30, 31, 32, etc., 50, 51, 52, 53, etc., 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc., including 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: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO;1 1, 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 T0, SEQ ID NO: 12, or SEQ ID NO: 14. Recombinant vectors and isolated DNA segments may therefore 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 immunodetection 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, 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 LYST1, Lyst1, LYST2, or Lyst2 gene, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment, for example, using recombinant cloning and/or PCR™ technology, in connection with the compositions disclosed herein. In other embodiments, 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 intended to refer to a promoter that is not normally 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 promoters isolated from any bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will be important to employ a promoter that effectively directs the expression 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., 1989. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, 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 expression vectors and promotor sequences such as those obtained from tac, trp, lac, lacUV5 or T7. When expression of the recombinant LYST1 LYST2, Lyst1 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 expression vector system (Pharmacia LKB Biotechnology). In connection with expression embodiments to prepare recombinant recombinant LYST1

LYST2, Lyst1 or Lyst2 proteins and peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire LYST1 LYST2, Lyst1 or Lyst2 or functional domains, epitopes, ligand binding domains, subunits, etc. being most preferred.

However, it will be appreciated that the use of shorter DNA segments to direct the expression of LYST1 LYST2, Lyst1 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 segments that encode peptide antigens from about 15 to about 100 amino acids in length, or more preferably, from about 15 to about 50 amino acids in length are contemplated to be particularly useful. The LYST or Lyst genes and DNA segments may also be used in connection with somatic expression in an animal or in the creation of a transgenic animal. Again, in such embodiments, the use of a recombinant vector that directs the expression of the full length or active LYST/Lyst protein is particularly contemplated. Expression of a LYST/Lyst transgene in animals is particularly contemplated to be useful in the production of anti-LYST/Lyst antibodies for use in passive immunization methods, the detection of LYST/Lyst proteins, and the purification of

LYST/Lyst protein in large quantity. In addition to their use in directing the expression of LYST/Lyst, the nucleic acid sequences disclosed herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments 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 SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1, or SEQ ID NO: 13 will find particular utility. Longer contiguous identical or complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) and even up to full length sequences will also be of use in certain embodiments.

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 complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or so, identical or

complementary to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1, or SEQ ID NO: 13 are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. This would allow LYST/Lyst structural or regulatory genes to be analyzed, both in diverse cell types and also in various bacterial cells. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous

complementary region may be varied, such as between about 14 and about 100 nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences 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

complementary sequences over stretches greater than 14 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality 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 herein. All that is required is to review the sequence 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: 1 1, or SEQ ID NO: 13 and to select any continuous portion of the sequence, from about 14-25 nucleotides in length up to and including 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 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 includes a contiguous sequence from within SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1, or SEQ ID NO: 13, may alternatively be described as preparing a nucleic acid fragment. Of course, fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCR™ technology of U. S. Patent 4,683,202 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant 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 complementary stretches of the entire LYST/Lyst gene or gene fragments. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring 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 NaCl at temperatures of 50°C to 70°C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating LYST or Lyst genes.

Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to 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

hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, 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 embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental 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 complementary nucleic acid-containing samples. 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 embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected 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 on the particular circumstances 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 probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantitated, by means of the label.

2.2 Recombinant Host Cells and Vectors

Particular aspects of the invention concern the use of plasmid vectors for the cloning and expression of recombinant peptides, and particular peptide epitopes comprising either native, or site-specifically mutated LYST or Lyst proteins, peptides, or epitopes. The generation of recombinant vectors, transformation of host cells, and expression of recombinant proteins is wellknown to those of skill in the art. Prokaryotic hosts are preferred for expression of the peptide compositions of the present invention. An example of a preferred prokaryotic host is E. coli, and in particular, E. coli strains JM101, XL1-Blue™, RR1, LE392, B, X1776 (ATCC31537), and W3110 (F-, λ-, prototrophic, ATCC273325). Alternatively, other Enterobacteriaceae species such as Salmonella typhimurium and Serratia marcescens, or even other Gram-negative hosts including various Pseudomonas species may be used in the recombinant expression of the genetic constructs disclosed herein. In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli may be typically transformed using vectors such as pBR322, or any of its derivatives (Bolivar et al., 1977). pBR322 contains 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 expression of endogenous proteins.

In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts.

For example, bacteriophage such as λGEM™-1 1 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 commonly used in recombinant DNA construction include the β-lactamase (penicillinase) and lactose promoter systems (Chang et al., 1978, Itakura et al., 1977; Goeddel et al., 1979) or the tryptophan (trp) promoter system (Goeddel et al., 1980). The use of recombinant 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 preferred embodiment expression 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 microorganisms, although a number of other species may also be employed for such eukaryotic expression systems. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al., 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 detecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman 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-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3' of the 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 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 containing a yeast-compatible promoter, an origin of replication, and termination sequences is suitable. In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts in the routine practice of the disclosed methods. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture . However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has 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 W138, BHK, 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, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expression vectors are often 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., 1978). Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the HindIII site toward the BglI site located in the viral origin of replication

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 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 mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

It will be further understood that certain of the polypeptides may be present in quantities below the detection limits of the Coomassie brilliant blue staining procedure usually employed in the analysis of SDS/PAGE gels, or that their presence may be masked by an inactive polypeptide of similar M_r. Although not necessary to the routine practice of the present invention, it is contemplated that other detection techniques may be employed advantageously in the

visualization of particular polypeptides of interest. Immunologically-based techniques such as Western blotting using enzymatically-, radiolabel-, or fluorescently-tagged antibodies described herein are considered to be of particular use in this regard . Alternatively, the peptides of the present invention may be detected by using antibodies of the present invention in combination with secondary antibodies having affinity for such primary antibodies. This secondary antibody may be enzymatically- or radiolabeled, or alternatively, fluorescently-, or colloidal gold-tagged. Means for the labeling and detection of such two-step secondary antibody techniques are well- known to those of skill in the art. 2.3 Recombinant Expression of one or more LYST Gene Products

As used throughout, a "LYST/Lyst" gene is intended to mean a LYST or Lyst gene from a mammalian source, with human LYST and murine Lyst genes being most preferred. In keeping with the genetic nomenclature schemes known to those of skill in the art, "LYST" genes are those genes derived from human sources while "Lyst" genes are those genes derived from murine sources. Thus, LYST1 and LYST2 genes are two genes of the "LYST/Lyst" family which are isolated from humans, while Lyst1 and Lyst2 represent two genes of the "LYST/Lyst" family which are their murine homologs, respectively.

In analogous fashion, a "LYST/Lyst" protein is intended to mean a LYST or Lyst protein isolated from a mammalian source, with human and murine peptides being most preferred In keeping with the genetic nomenclature schemes known to those of skill in the art, "LYST" proteins are those proteins encoded by LYST genes 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 designations of two proteins of the "LYST/Lyst" protein family which are isolated from humans, while Lyst1 and Lyst2 represent the two homologous proteins of the 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 "Lyst1 isoform I," "Lyst1 isoform II," and so forth to distinguish between the two isoforms. Such isoform designations may also be abbreviated as "Lyst1 -I" or "Lyst1-II," and so forth. Human protein isoforms may be referred to in corresponding manner "LYST1-I" and "LYST1-isoform I" describe the long isoform of the human protein, while

"LYST1-II" and "LYST1-isoform II" are terms used to described the short isoform of the human proteins. Therefore, Lyst1-I and Lyst1-II are terms used to represent two isoforms of the murine isoforms of Lyst1 , and LYST1 -I and LYST1-II are terms used to represent two isoforms of the human LYST1. Similarly, Lyst2-I and Lyst2-II would represent two isoforms of the murine Lyst2 protein, while LYST2-I and LYST2-II would represent two isoforms of the human LYST2 protein. The present invention also concerns recombinant host cells for expression of an isolated LYST1, Lyst1, LYST2, or Lyst2 gene. It is contemplated that virtually any host cell may be employed for this purpose, but certain advantages may be found in using a bacterial host cell such as E. coli, S. typhimurium, B. subtilis, or others. Expression in eukaryotic cells is also contemplated such as those derived from yeast, insect, or mammalian cell lines. These recombinant host cells may be employed in connection with "overexpressing" the LYST1, Lyst1, LYST2, or Lyst2 protein, that is, increasing the level of expression over that found naturally in mammalian cells. As is well known to those of skill in the art, many such vectors and host cells are readily available for the recombinant expression of proteins, one particular detailed example of a suitable vector for expression in mammalian 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, Lyst1, LYST2, or Lyst2 protein) and does not include any coding or regulatory sequences that would have an adverse effect on cells. Therefore, it will also be understood that useful nucleic acid sequences may include additional residues, such as additional non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various regulatory sequences.

After identifying an appropriate 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 and production of the protein or peptide epitope of interest (e.g., the LYST1, Lyst1, 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 of the promoter which is naturally associated with a LYST1-, Lyst1-, LYST2-, or Lyst2-encoding nucleic acid segment, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment, for example, using recombinant cloning and/or PCR™ technology, in connection with the compositions disclosed herein. Direct amplification of nucleic acids using the PCR™ 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. In certain embodiments, it is contemplated that particular advantages will be gained by positioning the LYST1-, Lyst1-, LYST2-, or Lyst2-encoding DNA segment under the control of a recombinant, 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 LYST1, Lyst1, LYST2, or Lyst2-encoding DNA segment in its natural environment. Such promoters may include those normally associated with other genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the particular cell containing the vector comprising the LYST1-, Lyst1-, LYST2-, or Lyst2-encoding nucleic acid segment.

The use of recombinant 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 appropriate conditions to direct high level or regulated expression of the introduced DNA segment. For eukaryotic expression, the currently preferred promoters are those such as CMV, RS V LTR, the SV40 promoter alone, and the SV40 promoter in combination with the SV40 enhancer. In certain embodiments, the expression of recombinant LYST1, Lyst1, LYST2, or Lyst2 protein is carried out using prokaryotic expression systems, and in particular bacterial systems such as E. coli. Such 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 expression vectors and promotor sequences such as those obtained from tac, trp, lac, lacUV5 or T7 promotors.

For the expression of the LYST1, Lyst1, LYST2, or Lyst2 protein and LYST1-, Lyst1-, 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 preparation of the LYST1, Lyst1, LYST2, or Lyst2 protein or peptides derived from one or more of the LYST1, Lyst1, LYST2, or Lyst2 proteins. The engineering of DNA segment(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 expression of LYST1, Lyst1, LYST2, or Lyst2 proteins or epitopes derived from such proteins. 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 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 DNA segments encoding such epitopes will provide a convenient means for obtaining the protein or peptide of interest.

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 expression of the proteins of the present invention, or of peptides or epitopes derived from such proteins, e.g., baculovirus-based, glutamine synthase-based or dihydrofolate reductase-based systems may be employed. In preferred embodiments it is contemplated that plasmid vectors incorporating an origin of replication and an efficient 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 adjacent to and under the control of the promoter. It is understood in the art that to bring a coding sequence under the control of such a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame of the protein between about 1 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 LYST/Lsyt gene product or LYST/Lyst-derived peptides, an appropriate polyadenylation site (e.g.,

5'-AATAAA-3') if one was not contained within the original cloned segment. Typically, the poly- A addition site is placed about 30 to 2000 nucleotides "downstream" of the termination site of the protein at a position prior to transcription termination.

It is contemplated that virtually any of the commonly employed host cells can be used in connection with the expression of the LYST1, Lyst1, LYST2, or Lyst2 proteins and epitopes derived therefrom 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. It is further contemplated that the proteins, peptides, or epitopic peptides derived from native or recombinant LYST1, Lyst1, LYST2, or Lyst2 proteins may be "overexpressed", i.e., expressed in increased levels relative to its natural expression in human cells, or even relative to the expression of other proteins in a recombinant host cell containing LYST1 -, Lyst1-, LYST2-, or Lyst2-encoding DNA segments. Such overexpression may be assessed by a variety of methods, lincluding radiolabeling and/or protein purification. However, facile and direct methods are preferred, for example, those involving SDS/PAGE and protein staining or Western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot. A specific increase in the level of the recombinant protein or peptide in comparison to the level in natural LYST1-, Lyst1-, LYST2-, or Lyst2-producing cells is indicative of overexpression, as is a relative abundance of the 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 intended to refer to a cell into which a recombinant gene, such as a gene encoding LYST1, Lyst1, LYST2, or Lyst2 has been introduced. Therefore, engineered cells are distinguishable 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 flanking DNA, or an operon or other functional nucleic acid segment which may also include genes positioned either upstream and/or downstream 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 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 a high, constant level of expression of the introduced genes. The inventors have noticed that the level of expression from the introduced genes of interest can vary in different clones, or genes isolated from different strains or bacteria. Thus, the level of expression of a particular recombinant gene can be chosen by evaluating different clones derived from each transfection study, once that line is chosen, the constitutive promoter ensures that the desired level of expression is permanently maintained. It may also be possible to use promoters that are specific for cell type used for engineering, such as the insulin promoter in insulinoma cell lines, or the 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 immunological 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 -encoding nucleic acid segments in clinical samples using nucleic acid compositions are also obtained 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/Lyst nucleic acid segments within a clinical sample from a patient suspected of having such a condition. In a preferred embodiment, nucleic acid sequences as disclosed in SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO.5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 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 immune response in an animal. The method generally involves administering to an animal a pharmaceutical composition comprising an immunologically 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, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.

The invention also encompasses LYST/Lyst and LYST/Lyst -derived peptide antigen 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 including those raised in heterologous donor animals or human volunteers immunized with the LYST/Lyst gene product, monoclonal antibodies (mAbs) resulting from hybridomas derived from fusions of B cells from immunized animals or humans with compatible myeloma cell lines, so-called "humanized" mAbs resulting 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-containing 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 vaccination 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 immune response, which methods generally comprise administering to an animal, a pharmaceutically-acceptable composition comprising an immunologically effective amount of a LYST/Lyst peptide composition. Preferred animals include mammals, and particularly humans Other preferred animals include murines, bovines, equines, porcines, canines, and felines. The composition may include partially or significantly purified LYST/Lyst peptide epitopes, obtained from natural or recombinant sources, which proteins or peptides may be obtainable naturally or either chemically synthesized, or alternatively produced in vitro from recombinant host cells expressing DNA segments encoding such epitopes. Smaller peptides that include reactive epitopes, such as those between about 10 and about 50, or even between about 50 and about 100 amino acids in length will often be preferred. The antigenic proteins or peptides may also be combined with other agents, such as other LYST/Lyst -related peptides or nucleic acid

compositions, if desired.

By "immunologically effective amount" is meant an amount of a peptide composition that is capable of generating an immune response in the recipient animal. This includes both the generation of an antibody response (B cell response), and/or the stimulation of a cytotoxic immune response (T cell response). The generation of such an immune response will have utility in both the production of useful bioreagents, e.g., CTLs and, more particularly, reactive antibodies, for use in diagnostic embodiments, and will also have utility in various prophylactic or therapeutic embodiments. Therefore, although these methods for the stimulation of an immune response include vaccination regimens and treatment regimens, it will be understood that achieving either of these end results is not necessary for practicing these aspects of the invention Further means contemplated by the inventors for generating an immune response in an animal includes administering to the animal, or human subject, a pharmaceutically-acceptable composition comprising an immunologically effective amount of a nucleic acid composition encoding a LYST/Lyst epitope, or an immunologically effective amount of an attenuated live organism that includes and expresses such a nucleic acid composition. The "immunologically effective amounts" are those amounts capable of stimulating a B cell and/or T cell response.

Immunoformulations of this invention, whether intended for vaccination, treatment, or for the generation of antibodies useful in the detection of CHS, may comprise native, or synthetically-derived antigenic peptide fragments from these proteins. As such, antigenic functional equivalents 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 immunologically cross-reactive with one or more epitopes derived from any of the particular proteins disclosed. Antigenically functional equivalents, or epitopic sequences, may be first designed or predicted and then tested, or may simply be directly tested for cross-reactivity. The identification 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 detection 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 identification and preparation of epitopes from amino acid sequences 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 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 of these "epitopic core sequences" may then be readily incorporated into peptides, either through the application of peptide synthesis or recombinant 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 circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution. In still further embodiments, the present invention concerns immunodetection 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 peptides. Either type of kit may be used in the immunodetection of compounds, present within clinical samples, that are indicative of CHS The kits may also be used in antigen or antibody purification, as appropriate.

In general, the preferred immunodetection methods will include first obtaining a sample suspected of containing a LYST/Lyst -reactive antibody, such as a biological sample from a patient, and contacting the sample with a first LYST/Lyst protein or peptide under conditions effective to allow the formation of an immunocomplex (primary immune complex). One then detects the presence of any primary immunocomplexes that are formed. Preferable LYST/LYST proteins include LYST1 and LYST2 from human origins, and Lyst1 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) immune 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 time sufficient to allow the added antigens to form immune complexes with, i.e., to bind to, any antibodies present within the sample. After this time, the sample composition, such as a tissue section, ELISA 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 immune complexes to be detected.

The detection of immunocomplex 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., (1987), 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 alkaline phosphatase, urease, horseradish peroxidase and glucose oxidase being suitable. The particular antigen employed may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of bound antigen present in the composition to be determined. Alternatively, the primary immune complexes may be detected by means of a second binding ligand that is linked to a detectable 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 immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies and the remaining bound label is then detected.

For diagnostic purposes, it is proposed that virtually any sample suspected of containing 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 contemplated that such embodiments may have application to non-clinical samples, such as in the titering of antibody samples, in the selection of 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 embodiments, 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 speaking, kits in accordance with the present invention will include a suitable protein or peptide together with an immunodetection reagent, and a means for containing the protein or peptide and reagent.

The immunodetection reagent will typically comprise a label associated with a LYST/Lyst protein or peptide, or associated with a secondary binding ligand. Exemplary ligands might 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 known in the art and all such labels may be employed in connection with the present invention. 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 conjugated 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. Where a second binding ligand is provided, the kit 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 containing the vials in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained. 2.6 Formulation as Vaccines

It is expected that to achieve an "immunologically effective formulation" it may be desirable to administer LYST- or Lyst-encoding proteins to the human or animal subject in a pharmaceutically acceptable composition comprising an immunologically effective amount of LYST or Lyst proteins or peptides mixed with other excipients, carriers, or diluents which may 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. Immunostimulatory excipients, often referred to as adjuvants, may include salts of aluminum (often referred to as Alums), simple or complex fatty acids and sterol compounds, physiologically acceptable oils, polymeric carbohydrates, chemically or genetically modified 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 0.001 to 0.1 milligrams, or even more preferably less than 0.1 milligrams per dose.

It is also contemplated that attenuated organisms may be engineered to express

recombinant LYST or Lyst proteins or peptides, and the organisms themselves be delivery vehicles for the invention. Pox-, polio-, adeno-, or other viruses, and bacteria such as Salmonella, 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 immunization, has been shown to be suitable for protection against infectious organisms. Such DNA segments could be used in a variety of forms including naked DNA and plasmid DNA, and may administered to the subject in a variety of ways including 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 immunization techniques is thus proposed to be useful as a vaccination strategy against Lyme disease.

It is recognized by those skilled in the art that an optimal dosing schedule of a vaccination regimen may include as many as five to six, but preferably three to five, or even more preferably one to three administrations of the immunizing 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 LYSTI PEPTIDES/APTAMERS As PHARMACEUTICALS THAT BLOCK OR MIMIC LYSTI FUNCTION

Lyst regulates degranulation of lysosomes, late endosomes and acidic secretory granules primarily in leukocytes. Blockade of such degranulation using dominant-negatively acting truncated Lyst peptides may reasonably be expected to be efficacious in inflammatory and autoimmune diseases such as asthma, urticaria, inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, systemic vasculitis, glomerulonephritis, multiple sclerosis, post-angioplasty restenosis. Proof of this principal is documented in Clark et al., 1982, who demonstrated that bg mice are protected from lupus nephritis.

2.8 USE OF PHARMACEUTICAL COMPOUNDS THAT BLOCK OR MIMIC LYSTI FUNCTION Lyst regulates degranulation of lysosomes, late endosomes and acidic secretory granules primarily in leukocytes. Blockade of such degranulation using dominant-negatively acting truncated Lyst peptides may reasonably be expected to be efficacious in inflammatory and autoimmune diseases such as asthma, urticaria, inflammatory bowel disease systemic lupus erythematosus, rheumatoid arthritis, psoriasis, systemic vasculitis, glomerulonephritis, multiple sclerosis, post-angioplasty restenosis. Proof of this principal is documented in Clark et al., (1982) who demonstrated that bg mice are protected from lupus nephritis.

Lyst peptides that mimic or augment Lyst function may reasonably be expected to be efficacious in the treatment of neoplasia. Proof of this principle is documented in Aboud et al. (1993) and Hayakawa et al. (1986), who demonstrate that bg mice and CHS patients are susceptible to development off neoplasia, and have more aggressive neoplasms with accelerated metastases.

2.9 USE OF LYST2 PEPTIDES/APTAMERS AS PHARMACEUTICAL AGENTS THAT BLOCK LYST2 FUNCTION OR REPRODUCE LYST2 FUNCTIONS

Lyst2 is thought to act to regulate degranulation of vesicles within cells in the brain and kidney. Bblockade of such degranulation using dominant-negatively acting truncated Lyst2 peptides may reasonably be expected to be efficacious for the treatment of neurologic and renal degenerative diseases such as Alzheimer's disease, motor neuron disease, Parkinson's disease, acute tubular necrosis, glomerulonephritis and glomerulosclerosis. 2.10 USE OF PHARMACEUTICAL COMPOUNDS THAT BLOCK OR MIMIC LYST2 FUNCTIONS

Drugs that mimic the action of dominant-negatively acting truncated 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 truncated Lyst2 peptides may reasonably be expected to be efficacious for the treatment of neurologic and renal degenerative diseases such as Alzheimer's disease, motor neuron disease, Parkinson's disease, acute tubular necrosis, glomerulonephritis and glomerulosclerosis.

3. BRIEF DESCRIPTION OF THE DRAWINGS

The drawings form part of the present specification and are included to further 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.

4. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

4.1 LYST-ENCODING NUCLEIC ACID SEGMENTS

As used herein, the term "LYST1 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 "LYST1 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 entail the administration of one, two, three, or more, genes or gene segments. The maximum number of genes that may be used is limited only by practical considerations, such as the effort involved in simultaneously preparing a large number of gene constructs or even the possibility of eliciting 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 LYST2 proteins or peptides may be used in the methods and compositions of the invention. The nucleic acid compositions and methods disclosed herein may entail the administration of one, two, three, or more, genes or gene segments. The maximum number of genes that may be used is limited only by practical considerations, such as the effort involved in simultaneously preparing 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 of the same of different types.

Thus, an almost endless combination of different genes and genetic constructs may be employed.

Certain gene combinations may be designed to, or their use may otherwise result in, achieving synergistic effects on formation of an immune response, or the development of antibodies to gene products encoded by such nucleic acid segments, or in the production of diagnostic and treatment protocols for, among other things, Chediak-Higashi Syndrome. Any and all such combinations are intended to fall within the scope of the present invention. Indeed, many synergistic effects have been described in the scientific literature, so that one of ordinary skill in the art would readily be able to identify likely synergistic gene combinations, or even gene-protein combinations.

It will also be understood that, if desired, the nucleic segment or gene could be administered in combination with further agents, such as, e.g., proteins or polypeptides or various pharmaceutically 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 agents do not cause a significant adverse effect upon contact with the target cells or tissues.

4.2 THERAPEUTIC AND DIAGNOSTIC KITS

Therapeutic kits comprising, in suitable container means, a LYST or Lyst composition of the present invention in a pharmaceutically acceptable formulation represent another aspect of the invention. The LYST or Lyst composition may be native LYST or Lyst protein, truncated LYST or Lyst protein, site-specifically mutated LYST or Lyst-encoding DNAs, or LYST- or Lyst-derived peptide epitopes, or alternatively antibodies which bind the native LYST or Lyst gene product, truncated LYST or Lyst protein, site-specifically mutated LYST or Lyst protein, or LYST- or Lyst-encoded peptide epitopes. In other embodiments, the LYST or Lyst composition may be nucleic acid segments encoding one or more native LYST or Lyst proteins, truncated LYST or Lyst proteins, site-specifically mutated LYST or Lyst proteins, or peptide epitope derivatives of LYST or Lyst. Such nucleic acid segments may be DNA or RNA, and may be either native, recombinant, 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 pharmaceutically acceptable sterile excipient, having associated with it, the LYST or Lyst composition and, optionally, a detectable label or imaging agent. The formulation may be in the form of a gelatinous 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 administered as needed. However, the single container means may contain a dry, or lyophilized, mixture 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 a sterile DNA solution or in a lyophilized form, and the other container would include the matrix, which may or may not itself be pre-wetted with a sterile solution, or be in a gelatinous, liquid or other syringeable form.

The kits may also comprise a second or third container means for containing a sterile, pharmaceutically acceptable buffer, diluent or solvent. Such a solution may be required to formulate the LYST or Lyst component into a more suitable form 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 (lyophilized), 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. The kits may also comprise a second or third container means for containing a pharmaceutically acceptable detectable imaging agent or composition.

The container means will generally be a container such as a vial, test tube, flask, bottle, syringe or other container 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 kits of the present invention may also include a means for containing the individual containers in close confinement for commercial 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 assisting with the placement of the ultimate LYST or Lyst 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 ACID DELIVERY AND DNA TRANSFECTION

In certain embodiments, it is contemplated that the nucleic acid segments disclosed herein will be used to transfect appropriate host cells. Technology for introduction of DNA into cells is well-known to those of skill in the art Four general methods for delivering a nucleic segment into cells have been described:

(1) chemical methods (Graham and VanDerEb, 1973);

(2) physical methods such as microinjection (Capecchi, 1980), electroporation (Wong and Neumann, 1982, Fromm et al., 1985) and the gene gun (Yang et al., 1990);

(3) viral vectors (Clapp, 1993; Eglitis and Anderson, 1988); and

(4) receptor-mediated mechanisms (Curiel et al., 1991 ; Wagner et al., 1992).

4.4 LIPOSOMES AND NANOCAPSULES

In certain embodiments, the inventors contemplate the use of liposomes and/or nanocapsules for the introduction of particular peptides or nucleic acid segments into host cells. Such formulations may be preferred for the introduction of pharmaceutically-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 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-Michelland et al., 1987). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) should be designed 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 al., 1977, 1988).

Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles

(MLVs). MLVs generally have diameters of from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 C, containing an aqueous solution in the core. In addition to the teachings of Couvreur et 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 preferred structure. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low 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 increase in permeability to ions, sugars and drugs.

Liposomes interact with cells via four different mechanisms: Endocytosis 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 simultaneous 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 mechanism is operative and more than one may operate at the same time.

4.5 METHODS FOR PREPARING ANTIBODY COMPOSITIONS

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. Reference to antibodies throughout the specification includes whole polyclonal and monoclonal antibodies (mAbs), and parts thereof, either alone or conjugated with other moieties. Antibody parts include Fab and F(ab)₂ fragments and single chain antibodies. The antibodies may be made in vivo in suitable laboratory animals or in vitro using recombinant DNA techniques. 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 immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized 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 conventional immunization techniques, as will be generally known to those of skill in the art. A composition containing antigenic epitopes of particular proteins can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against LYST- or Lyst-derived peptides. Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.

The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen, as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, 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 immunized 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 different lineage. mAbs, by contrast, are defined as coming from antibody-producing cells with a common B-cell ancestor, hence their "mono" clonality.

When peptides are used as antigens 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) conformations. 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, more stable in an immunologic 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 80%. Even higher clonality - 90%, 95% or greater - is contemplated .

To obtain mAbs, one would also initially immunize an experimental animal, often preferably a mouse, with a LYST- or Lyst-containing composition. One would then, after a period of time sufficient to allow antibody generation, obtain a population of spleen or lymph cells from the animal. The spleen or lymph cells can then be fused with cell lines, such as human or 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 immunization, spleen cells are removed and fused, using a standard fusion protocol with plasmacytoma cells to produce hybridomas secreting mAbs against the LYST or 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 immunochemical 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-LYST/Lyst 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. The antibodies disclosed herein may be employed in antibody cloning protocols to obtain cDNAs or genes encoding LYST/Lyst proteins from other species or organisms, or to identify proteins having significant 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 Anti- LYST/Lyst antibodies will also be useful in immunolocalization studies to analyze the distribution of cells expressing LYST/Lyst protein during particular cellular activities, or for example, to determine the cellular or tissue-specific distribution of LYST/Lyst under different physiological conditions. A particularly useful application of such antibodies is in purifying native or recombinant LYST/Lyst proteins, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.

4.6 RECOMBINANT EXPRESSION OF "LYST FAMILY" PEPTIDES

Recombinant clones expressing the "LYST family" nucleic acid segments may be used to prepare purified recombinant LYST protein (rLYST), purified rLYST-derived peptide antigens as well as mutant or variant recombinant protein species in significant quantities. 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, and/or antibodies against such rLYSTs may also be used in immunoassays to detect the presence of LYST or as vaccines or immunotherapeutics to treat CHS and related disorders. Additionally, by application of techniques such as DNA mutagenesis, the present invention allows the ready preparation of socalled "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 antigen, such as a particular antigenic/immunogenic epitopic core sequence. Epitopic sequences can be obtained from relatively short molecules prepared from knowledge of the peptide, or encoding DNA sequence information. Such variant molecules may not only be derived from selected immunogenic/ 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. 4.7 ANTIBODY COMPOSITIONS AND FORMULATIONS THEREOF

Means for preparing 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 immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized 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 necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred 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-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine. mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U. S. Patent 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified protein, polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized 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 immunized mouse contains approximately about 5 × 10⁷ to about 2 × 10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render 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 immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-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-HMy2 and UC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.

Methods for generating hybrids of antibody-producing spleen or lymph 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 (chemical or electrical) that promote the fusion 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 Gefter et al. (1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies, about 1 × 10^-6 to about 1 × 10^-8. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), 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 supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding 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 indefinitely 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 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 concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

4.8 IMMUNOASSAYS

As noted, it is proposed that native and synthetically-derived peptides and peptide epitopes of the invention will find utility as immunogens, e.g., in connection with vaccine development, or as antigens in immunoassays for the detection of reactive antibodies. Turning first to immunoassays, in their most simple and direct sense, preferred immunoassays of the invention include the various types of enzyme linked immunosorbent assays (ELISAs), as are known to those of skill in the art. However, it will be readily appreciated that the utility of LYST-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 preferred ELISA assays, proteins or peptides incorporating LYST, rLYST, or LYST-derived protein antigen sequences are immobilized 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, one would then generally desire to bind or coat a nonspecific protein that is known to be antigenically 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. After 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 contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation. Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween™. 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 4 hours, at temperatures preferably on the order of about 25° to about 27°. Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween™, or borate buffer. Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and the amount of immunocomplex 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 preferably be an antibody having specificity for human antibodies. To provide a detecting means, the second antibody will preferably have an associated detectable label, such as an enzyme label, that will generate a signal, such as color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the antisera-bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions that favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween™).

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-benzthiazoline)-6-sulfonic acid (ABTS) and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation 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 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 immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

4.9 IMMUNOPRECIPITATION

The anti-LYST protein antibodies of the present invention are particularly useful for the isolation of LYST protein antigens by immunoprecipitation. Immunoprecipitation involves the separation of the target antigen component -from a complex mixture, and is used to discriminate or isolate minute 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 WESTERN BLOTS

The compositions of the present invention will find great use in immunoblot 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 combinations thereof. In conjunction with immunoprecipitation, followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background. This is especially useful when the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the detecting agent, or they migrate at the same relative molecular weight as a cross-reacting signal. Immunologically-based detection methods in conjunction with Western blotting (including enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety) are considered to be of particular use in this regard.

4.11 PHARMACEUTICAL COMPOSITIONS

The pharmaceutical compositions disclosed herein may be orally administered, for example, with an inert diluent or with an assimilable 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 administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal 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. The tablets, troches, pills, capsules. and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be 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. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent 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 form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

The active compounds may also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably 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 microorganisms. 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 manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, 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 fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal 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 injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum 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 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 medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary 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 appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate The active ingredient may also be dispersed in dentifrices, including 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 humectants.

The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered 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 solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.

The composition can be formulated in a neutral or salt form. Pharmaceutically-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, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.

For parenteral administration 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, intramuscular, subcutaneous and intraperitoneal administration 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 NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical 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 administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

4.12. EPITOPIC CORE SEQUENCES

The present invention is also directed to protein or peptide compositions, free from total cells and other peptides, which comprise a purified protein or peptide which incorporates an epitope that is immunologically cross-reactive with one or more of the antibodies of the present invention. As used herein, the term "incorporating an epitope(s) that is immunologically cross-reactive with one or more anti-LYST protein antibodies" is intended to refer to a peptide or protein antigen which includes 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 monoclonal or polyclonal antibodies directed against the LYST polypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen. Various immunoassay 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. 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 straightforward matter. For example, one may employ the methods of Hopp, as taught in U.S. Patent 4,554,101, incorporated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences 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 sequences (see, for example, 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. 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 amino acids in length. It is proposed that shorter antigenic peptide sequences will provide advantages in certain circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and 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 preparation of synthetic peptides which include modified and/or extended epitopic/immunogenic core sequences which result in a "universal" epitopic peptide directed to the LYST gene product or LYST-related sequences It is proposed that these regions represent those which are most likely to promote T-cell or B-cell stimulation 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 preferred. 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 identification 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 identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. Moreover, numerous computer programs are available for use in predicting antigenic portions of proteins (see e.g., Jameson and Wolf, 1988, Wolf et al., 1988). Computerized peptide sequence analysis programs (e.g., DNAStar® software, DNAStar, Inc., 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 immunologically 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 sequence, or using more general screens, e.g., of a pool of randomly generated synthetic peptides or protein fragments. The screening assays, may 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 immunohistochemical assays, ELISAs, RIAs, 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 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 detectable label, such as, e.g., biotin or an enzymatic, radioactive or fluorogenic label, to enable subsequent identification. One would then incubate the labeled antigen with the other, test, antigen to be examined at various ratios (e.g., 1 : 1, 1:10 and 1:100) 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 attaching 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 included in the incubation. The assay may be any one of a range of immunological 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 detecting 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 significantly reduce LYST binding, as evidenced by a reduction in the amount of label detected.

The reactivity of the labeled antigen, 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 significant reduction in labeled antigen reactivity in the presence of a test 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., consistently observed) reduction in binding.

In addition to the peptidyl compounds described herein, the inventors also contemplate that other sterically similar compounds may be formulated 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 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 synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptide antigens synthesized in this 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 aqueous solution without appreciable degradation or loss of antigenic activity. However, where extended aqueous storage is contemplated it will generally be desirable to include agents including buffers such as Tris or phosphate buffers to maintain 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 extended storage in an aqueous state it will be desirable to store the solutions at 4°C, or more preferably, frozen. Of course, where the peptides are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g., in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled) or buffer prior to use.

4.13 SITE-SPECIFIC MUTAGENESIS

Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the 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 mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent 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 10 residues on both sides of the junction of the sequence being altered. 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 M13 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 eliminates the step of transferring 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 includes within its sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli 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-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encoding DNA segments 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 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 sequence variants Specific details regarding these methods and protocols are found in the teachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each incorporated herein by reference, for that purpose.

4.14 BIOLOGICAL FUNCTIONAL EQUIVALENTS

Modification and changes may be made in the structure of the peptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. The amino acid changes may be achieved by changing the codons of the DNA sequence, according to the codon chart listed in TABLE 1.

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 example, 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 sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned 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); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-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 similar biological 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 preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. 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 the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

As detailed in U. S. Patent 4,554, 101, the following hydrophilicity values have been assigned to amino acid residues arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); 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 immunologically equivalent protein In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, 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 lysine, glutamate and aspartate, serine and threonine, glutamine and asparagine, and valine, leucine and isoleucine.

* * * * * * * * * *

5. EXAMPLES

The following examples are included to demonstrate preferred 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 preferred 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.

5.1 EXAMPLE 1 - MAPPING OF THE BG CRITICAL REGION ON MOUSE CHR 13

Three mouse mutations whose molecular basis is unknown, beige (bg), crinkled (cr), and progressive motor neuronophathy (pmn), 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 of a 0.24 cM interval of mouse Chr 13 which corresponds to the bg critical region. 1 1 Yeast-artificial chromosomes (YACs) and 2 P1 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 PCR™ and direct selection 2400-kb of the bg-critical region was isolated in YAC and P1 clones. Expressed sequence tags were identified from a bg-critical region YAC clone by direct selection, and represent potential candidates for bg and cr.

Positional cloning represents an approach to disease gene identification based solely upon chromosomal location. In the 10 years since its inception, positional cloning has become established as a general, relatively efficient mode of identification of genes causing mammalian Mendelian disorders (Collins, 1995). Recently developed techniques and resources have both disencumbered 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 (STS), precise genetic maps, and establishment 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 adjacent to the extra-toes (Xt) locus is rich in mutant phenotypes, and represents an interval where a regional approach to disease gene identification may be synergistic. Xt is homologous to the human disorder Greig cephalopolysyndactyly; using a positional candidate approach, mutations in a zinc-finger gene (Gli3) were shown to underlie Xt (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 muscular 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 et al., 1967). Finally, beige (bg), the homolog of human 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 existence of numerous bg alleles facilitates candidate gene mutation analysis;

(2.) bg is associated with a characteristic cellular phenotype (giant, perinuclear, dysfunctional lysosomes) offering the possibility of screening candidate genes by genetic complementation; and

(3.) direct selection can be utilized to identify transcribed sequences which are candidates for bg from YAC clones since all cell types are affected in bg homozygotes.

Positional cloning of bg has been performed as an antecedent to identification of the homologous human gene, which is probably defective in human Chediak-Higashi syndrome. Using backcross mice, bg was previously located to a 0.24 cM interval on Chr 13. The example illustrates the further characterization of the bg critical region with 20 novel sequence tagged sites (STS), and the isolation of overlapping YAC and P1 clones which encompass most of this region of mouse Chr 13.

5.1.1 MATERIALS AND METHODS 5.1.1.1 YAC MANIPULATION

A mouse genomic DNA library constructed in the vector pYAC4 (Kusumi et al., 1994; Research Genetics Inc.) was screened by PCR™ with primers derived from STS flanking bg. False positive PCR™ products were minimized by raising annealing temperatures, and addition of an enhancer of polymerase specificity as necessary (Perfect Match, Stratagene, La Jolla, CA). Veracity of PCR™ 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 containing YACs of interest were isolated on plates and frozen in 50% glycerol to prevent occurrence of microdeletions. YAC clones were grown in liquid YPD medium, converted to spheroplasts at exponential growth using Zymolase (ICN Pharmaceuticals, Costa Mesa, CA), and chromosomal DNA purified in agarose. YAC DNA was separated from host yeast chromosomes using preparative pulsed field electrophoresis (PFGE) with low melting point agarose (SeaPlaque™ GTG, FMC Bioproducts, Rockland, ME), and excised with a sterile blade.

5.1.1.2 Pi CLONES

A mouse genomic DNA library constructed in the vector P1 (Pierce et al., 1992; Genome Systems Inc., St. Louis, MO) was screened by PCR™ with primers derived from STS flanking 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 FIELD ELECTROPHORESIS

Preparation of high molecular weight DNA in agarose blocks, restriction enzyme digestion, PFGE, and Southern transfer were performed as previously described (Kingsmore et al., 1989). In brief, mouse splenocytes, lymph node cells, or yeast spheroplasts, were suspended in 0.5% low-melting point agarose (InCert®, FMC BioProducts) at 1-2 × 10⁷ cells per ml (mammalian cells) or 1-2 × 10¹⁰ cells per ml (yeast). DNA was prepared by incubation of agarose blocks in 500 mM EDTA (pH 9.0), 1% sodium lauroyl sarcosinate, 2% proteinase K at 50°C twice for 24 h. Blocks were then washed, treated with phenylmethylsulfonylfluoride, washed again, and digested with 2-10 units/μgDNA of restriction endonucleases (Boehringer-Mannheim Biochemicals, Indianapolis, IN), if necessary. PFGE was carried out in 1% agarose gels (Fastlane, FMC BioProducts) at 14°C in 1X 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 ethidium bromide to visualize molecular size standards (oligomers of λ phage, and chromosomes of Saccharomyces cerevisiae [FMC BioProducts]). Southern transfer of DNA onto Zeta-probe™ membranes (Bio-Rad Laboratories), and filter hybridizations were performed as previously described (Kingsmore et al., 1989). Assignment of two probes to a common restriction fragment was based on sequential 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 "-[³²P]dCTP as previously described (Kingsmore et al., 1989). Restriction endonuclease fragments representing ends of YAC clones were identified by Southern blot hybridization with pBR322 (which hybridizes efficiently to pYAC4); YAC clone internal restriction endonuclease fragments were identified by hybridization with a mouse B1 repetitive element probe.

5.1.1.5 INTERSPERSED REPETITIVE ELEMENT-POLYMERASE CHAIN REACTION

IRE-PCR™ was performed essentially as described using mouse B1 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 B1). Inter-B1 PCR™ was performed with the forward primer alone, the reverse primer alone, or both primers together. PCR™ amplification reactions were performed using 40 ng of YAC DNA, 1 μM of each primer, and 200 μM of each dNTP in a 20 μl 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™ products were isolated either by band excision from low-melting agarose gels, or by TA subcloning (Invitrogen). IRE-PCR™ products were sequenced, screened for the 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 DIRECT SELECTION

Direct selection was performed as previously described (Lovett et al., 1991 ; Lovett, 1994). Briefly, cDNA was generated from mouse spleen by reverse transcription using random-and oligo(dT)-priming, ligated to amplification cassettes, and PCR™ amplified. Preparative PFGE was used to purify YAC 195A8 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 C₀t=20. YAC DNA (with annealed cDNAs) was captured on streptavidin-coated beads, washed at high stringency, and encoded cDNAs eluted. Eluted cDNAs were PCR™-amplified, and subjected to a further round of direct selection. Selected cDNAs were reamplified by PCR™, subcloned into λgt10, and individual clones picked into SM buffer in 96-well plates. Direct selection products were amplified from phage-containing supernatents by PCR™ with the following primers: ; and

Direct selection amplicons were cycle sequenced with standard M13 forward and reverse primers. Oligonucleotides suitable for STS were designed using direct selection product sequences. 5.1.1.7 STS PCR™

PCR™ amplification reactions were performed using 40 ng of template DNA (YAC clone, P1 clone, S. cerevisiae strain 1380, or C57BL/6J genomic DNA), 1 μM of each primer, and 200 μM of each dNTP in a 20 μl 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 20 sec. Amplification products were separated on 3% agarose gels, and visualized by ethidium bromide staining, or by end-labeling one of the primers using [γ-[^3.P]ATP and T4 polynucleotide kinase, and separation of products on 6% denaturing polyacrylamide gels, with autoradiographic visualization. Simple sequence length polymorphism (SSLP) primers were as described (Dietrich et al., 1994; Research Genetics Inc., Hunstsville, AL). Novel STS primer sequences, amplicon sizes, and annealing temperatures are summarized in Table 2.

5.1.2 RESULTS AND DISCUSSION

5.1.2.1 ISOLATION OF YACS AND P1s

1 1 YAC clones and 2 P1 clones were isolated from mouse YAC and P1 libraries by

PCR™ using markers genetically 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 examined for chimerism, microdeletions, and overlaps by STS content mapping. Previously described SSLP were the first source of STS to be utilized. The genomic region encompassing bg is particularly rich in such SSLP (38 have been localized within a 2 cM interval containing bg; Dietrich et al., 1994). Additional proximal chromosome 13 STS were generated using IRE-PCR™ and direct selection.

5.1.2.2 NOVEL CHR 13 STS DERIVED BY IRE-PCR™

IRE-PCR™ represents a rapid and facile method with which to saturate a genomic region with novel STS for initial characterization of YAC clones and contig development (Hunter et al., 1993; Simmler et al., 1991). IRE-PCR™ 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-PCR™ products were subcloned, sequenced, and nonrepetitive regions used to design oligonucleotides suitable for sequence tagged sites. 12 novel STS (D13Sfk1-D13Sfk12) were developed by this method (Table 2), and physically assigned to Chr 13 YAC and P1 clones by PCR™ (FIG. 2).

5.1.2.3 NOVEL CHR 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 chromosomes. 192 candidate cDNA fragments were eluted from YAC195A8 following two rounds of direct selection with mouse splenocyte cDNA. 56 of these direct selection products were sequenced. Comparison with DNA sequence databases revealed 2 (4%) nidogen (Nid), 32 (57%) novel, 12 (21%) repetitive elements (B 1=2, B2=1, LINE1=4, IAP=2, XL30=1, MT=1, (satellite=1), and 9 (16%) contaminants (rRNA=3, actin=1, Nip2=1, plasmid=4). The presence of Nid cDNA fragments 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 PCR™ analysis (D13Sfk13-D13Sfk19; Table 2, FIG. 2). D13Sfk13 and D13Sfk18 also hybridized sufficiently well to Southern blots to permit physical mapping adjacent to Nid on a polymorphic NotI fragment (1 100-kb in DBA/2J DΝA and 1150-kb in SB/LeJ DΝA). D13Sfk13 was also genetically mapped within the bg critical region in 504 backcross mice [C57BL/6J- bg^J X (C57BL/6J-bg^J × CAST/Ei)F₁] using a TaqI polymorphism.

5.1.2.4 ARRANGEMENT OF PROXIMAL CHR 13 YAC AND P1 CLONES IN CONTIGS

YAC and P1 clones were typed for the presence or absence of STS derived from SSLP, IRE-PCR™ amplicons, and direct selection products. STS content mapping enabled examination 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 D13Mit44 is located within the Nid gene, the portion of YAC 64F5 derived from Chr 13 was concluded to be less than 80-kb.

YAC clone (84A8) contained an internal deletion which included D13Sfk6 (FIG. 2). Furthermore, the physical size of 84A8 (370-kb) was considerably smaller than expected: the distance between the other genetic markers it encompassed was approximately 600-kb, confirming a substantial genomic deletion within this YAC. Some YAC clones have been reported to be unstable in culture, and become progressively smaller with time (Nehls et al., 1995). YAC 84 A8 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 P1 clones, extended from D13Sfk19 to D13Sfk2, and was approximately 1150-kb in length. The orientation this contig with respect to centromere was not established. The second contig 2 consisted of 2 YAC clones. It extended from D13Mit207 (proximal) to D13Sfk10 (distal), and was approximately 1000-kb in length. Contig 2 spanned the crossover defining the distal border of the bg critical region (FIG. 2). Despite STS content mapping, 2 additional critical region YAC clones remained unlinked with these contigs (165F7 and 148E11). Isolation of YAC end clones will be necessary to definitively evaluate whether overlaps exist between these YACs and contig 1 or 2.

Efforts to identify YAC clones corresponding to one critical region genetic marker (D13Mit114) and the two STS which define the proximal border of the bg critical region (D13Mit172 and D13Mit239) were unsuccessful, furthermore, these STS were not present in any of the Chr 13 YAC/P1 clones identified. These data suggest that a region of the nonrecombinant interval remains unrepresented in the present YAC and P1 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 estimated to be at least 2400-kb in length. Direct selection products identified from YAC 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- PCR™, 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, retrotranspositions, 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 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, approximately 2400-kb of the bg critical region has been physically mapped and isolated in the form of YAC and P1 clones. These studies represent an necessary intermediate step in positional cloning of bg, and may also be of value in positional cloning of cr and pmn.

5.2 EXAMPLE 2 - MAPPING OF THE BEIGE LOCUS TO MOUSE LYST 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 studies precisely localize bg on Chr 13, and provide a foundation for YAC contig development and efficient screening of candidate genes for bg.

5.2.1 MATERIALS AND METHODS

5.2.1.1 MICE

C57BL/6J-bg^J X (C51BL/6J-bg^J × CAST/EiJ)F₁ backcross mice were bred and maintained as described (Barbosa et al., 1995). (C57BL/6J-bg^J × PWK)F₁ X C57BL/6J-bg^J backcross mice, and (C51BL/6J-bg^J × PAC)Fi X C57BL/6J-bg ^J backcross mice used have been described (Holcombe et al., 1991).

5.2.1.2 SOUTHERN HYBRIDIZATION

DNA was isolated from mouse organs using standard techniques and digested with restriction endonucleases, and 10 μg samples were subjected to electrophoresis on 0.9% agarose gels. DNA was transferred to Zeta-probe membranes (Bio-Rad Laboratories, Hercules, CA), and filter hybridizations were performed as previously described (Barbosa et al., 1995).

5.2.1.3 NORTHERN BLOT ANALYSIS

20 μg of total RNA prepared from liver, spleen and kidney of C57BL/6J-+/+, C57BL/6J-bg^J, SB/LeJ-bg, and C3H/HeJ-bg^2J mice using standard techniques, was separated on formaldehyde agarose gels, transferred to Zeta-probe membranes (Bio-Rad Laboratories), and hybridized as previously described (Kingsmore et al., 1994).

5.2.1.4 RT- PCR™ ASSAYS

Total RNA was prepared from liver of C57BL/6J-+/+, C57BL/6J-bg^J, SB/LeJ-bg and C3H/HeJ-bg^2J mice by extraction with phenol / guanidine isothiocyanate (TRIzol7, Gibco BRL,

Gaithersburg, MD). The template for quantitative RT- PCR™ assays was 1-10 ng of first-strand cDNA, which had been synthesized 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- PCR™ correspond to bp 3805-3822, and bp 3938-3955 of the mouse Nid cDNA (Durkin et al., 1988). The Estm9 primers used were:

These correspond to the 5' and 3' ends, respectively, of an Estm9 cDNA (Bettenhausen and Gossler, 1995). RT-PCR7 products were amplified from bg, bg^J, bg^2J, and +/+ RNA with Nid primers or Estm9 primers F1-R1 or F2-R2. Quantitative RT-PCR7 of aldolase A, which is constitutively expressed, was also performed, to ensure that equal amounts of bg, bg^J, bg^2J, and +/+ template were used

PCR™ reactions were performed in a 50 μl volume containing 1-20 ng of cDNA, 1 μM of each primer, 200 μM each dNTP, 10 mM Tris-HCl, pH 8.8, 50 mM KCl, 1.5 mM MgCl₂, 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™ products were separated by electrophoresis on agarose gels, and quantified by intensity of ethidium bromide staining.

5.2.1.5 SSLP PCR™

PCR™ amplification reactions were performed using 40 ng of genomic DNA, 1 μM of each primer (Dietrich et al., 1994; Research Genetics, Inc., Huntsville, AL), and 200 μM of each dNTP in a 20 μl 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 possible, amplification products (20 μl) were separated on 3% agarose gels, and visualized by ethidium bromide staining. SSLP with allele sizes differing among strains by less than 8 bp were typed by end-labeling one of the primers using [γ³²P]ATP and T4 polynucleotide kinase, separation of amplification products (4 μl) on 6% denaturing polyacrylamide gels, and visualization by autoradiography. SSLP allele sizes are summarized 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 electrophoresis (PFGE), and Southern transfer were performed as previously described (Kingsmore 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 digested with 2-10 units/μg DNA of restriction endonucleases (Boehringer Mannheim Biochemicals). PFGE was carried out in 1% agarose gels (Fastlane, FMC BioProducts) at 14EC in 1X 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 ethidium bromide to visualize molecular size standards (oligomers of λ phage, and chromosomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe [FMC BioProducts]). Southern transfer of DNA onto Zeta-probe^® membranes (Bio-Rad Laboratories), and filter hybridizations were performed as previously described (Kingsmore et al., 1989). Assignment of two probes to a common restriction fragment was based on sequential hybridization of a filter and exhibition of identity by double- or partial-digests.

5.2.1.7 MOLECULAR PROBES

All probes were labeled by the hexanucleotide technique with "-[³²P]dCTP as previously described (Kingsmore et al., 1989). The nidogen (Nid) probe used was pN-5 (Jenkins et al., 1991). The glioblastoma oncogene homolog-3 (Gli3) probe was derived from pGli3a (Hui and Joyner, 1993). The probes used for the T cell receptor ( chain (Tcrg), and the mid-gestation embryo cDNA ESTM9, have been described previously (Holcombe et al., 1991). Informative

CAST/EiJ RFLV sizes are summarized in FIG. 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/HeJ × C57BL/6J-bg^J)F₁ X C51BL/6J-bg ^J ], and [(C57BL/6J - W^sh-bg^J × Mus domesticus PAC)F₁ X C57BL/6J-bg^J ], and an intersubspecific backcross [(C57BL/6J-W^sh-bg^J x Mus musculus PWK)Ε₁ X C57BL/6J-bg^J]}, have shown bg to lie proximal to Tcrg on mouse Chr 13 (Holcombe et al., 1987, 1991). In order to assess candidate genes for linkage to bg and as a precedent to positional cloning, the inventors have now generated a high-resolution linkage map of proximal mouse Chr 13 using the latter 2 backcrosses and a third, novel backcross.

5.2.2.1 PHENOTYPIC ANALYSIS OF BG BACKCROSS MICE

Three backcrosses segregating for bg were utilized, Phenotypic analysis of 109 (C57BL/6J -W^sh-bg^J × Mus domesticus PAC)F₁ X C57BL/6J-bg^J backcross mice, and 11 1 (C57BL/6J-W^sh-bg^J × Mus musculus PWK)F₁ X C57BL/6J-bg ^J backcross mice has been reported previously (Holcombe et al., 1991). The third backcross was established between C57BL/6J-bg^J mice and Mus castaneus (CAST/EiJ), and 504 [C57BL/6J-bg^J X (C57BL/6J-bg^J × CAST/EiJ)F₁ ] progeny were generated. Mus castaneus was chosen as the second parent in the latter intrasubspecific backcross due to the increased likelihood of detection 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

Informative RFLV were ascertained by hybridizing gene probes to Southern blots containing genomic DNA from C57BL/6J-bg^J 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. PWK and PAC RFLV for Tcrg have been described previously (Holcombe et al., 1991); CAST/EiJ RFLV for Estm9 have been described previously. Informative SSLP were ascertained by PCR™ of genomic DNA from C57BL/6J-bg^J and CAST/EiJ, PAC, and PWK parental mice. Approximate sizes of SSLP- PCR™ products are listed in Table 3

5.2.2.3 PRECISE GENETIC MAPPING OF BG ON PROXIMAL MOUSE CHR 13

111 (C57BL/6J -W^sh-bg^J × Mus domesticus PAC)F₁ X C57BL/6J-bg¹ backcross mice, 111 (C57BL/6J-W^sh-bg^J × Mus musculus PWK)F₁ X C57BL/6J-bg ^J backcross mice, and 504 [C57BL/6J-bg^J X (C57BL/6J-bg^J × CAST/EiJ)F₁ ] backcross mice were genotyped for a total of 23 SSLPs and 3 RFLVs known to map to proximal mouse Chr 13. At each locus, backcross DNA displayed either the homozygous or heterozygous F₁ pattern. Linkage relationships were determined using segregation analysis (Green, 1981), and the best gene order decided by minimization of crossover events and elimination 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 detected. 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 excluded from subsequent analysis. Upon exclusion of these animals, no significant differences in gene order or recombination frequencies were found among the three crosses.

The best gene order and recombination frequency (± standard deviation) for the [C57BL/6J-bg^J X (C57BL/6J-bg^J × CAST/EiJ)F₁ ] backcross was: centromere - D13Mit158, D13Mit172, D13Mit205, D13Mit206, D13Mit239 - 0.20 ± 0.20 cM - bg^J, Nid, Estm9, D13Mit44, D13Mit114, D13Mit134, D13Mit207 - 0.20 ± 0.20 cM - Gli3, D13Mit56, D13Mit162, D13Mit174, D13Mit237, D13Mit240, D13Mit305 - 0.20 ± 0.20 cM - D13Mit218, D13Mit219, D13Mit271 - 0.40 ± 0.28 cM - D13Mit3, D13Mit133 - telomere.

The best gene order and recombination frequency (± standard deviation) for the [(C57BL/6J -W^sh-bg^J × Mus domesticus PAC)F₁ X C57BL/6J-bg ^J] backcross was centromere - D13Mit79 - 5.4 ± 2.1 cM - D13Mit1 - 0.9 ± 0.9 cM - bg^J, D13Mit44, D13Mit134, D13Mit174,

D13Mit205 - 0.9 ± 0.9 cM - Tcrg, D13Mit218, D13Mit219 - 3.6 ± 1.8 cM - D13Mit3 - telomere.

The best gene order and recombination frequency (± standard deviation) for the [(C57BL/6J-W^sh-bg^J × Mus musculus PWK)F₁ X C57BL/6J-bg^J ] backcross was: centromere - D13Mit79 - 5.4 ± 2.1 cM - D13Mit1 - 0.9 ± 0.9 cM - bg^J, D13Mit44, D13Mit134, D13Mit205, D13Mit237 - 0.9 ± 0.9 cM - D13Mit174 - 0.9 ± 0.9 cM - Tcrg, 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 containing bg to a 0.24 ± 0.17 interval on Chr 13, flanked proximally by the genetic markers D13Mit172 and D13Mit239, and distally by Gli3, D13Mit56, D13Mit162, D13Mit237, D13Mit240, and D13Mit305. bg cosegregated with 6 genetic markers (Nid, Estm9, D13Mit44, D13Mit114, D13Mit134 and D13Mit207). Backcross mice with recombination events which define the bg nonrecombinant interval were derived from the [C57BL/6J-6g^J X (C57BL/6J- bg^J × CAST/EiJ)F₁ ] backcross.

5.2.2.4 EVALUATION OF THE CANDIDACY OF NID AND ESTM9 FOR CAUSALITY IN BG

Given the availability of numerous bg alleles, it was reasoned that northern, Southern, and RT- PCR™ analyses would be effective modalities for initial evaluation of the candidacy of Nid and Estm9 for causality in bg. Southern blots were generated with DNA from 6 bg alleles: SB/LeJ-bg, C57BL/6J-bg ^J,

C3H/HeJ-bg ^ZJ, DBA/2J-bg ^8J, C57BL/6J-bg ^10J, C57BL/6J-bg ^{11 J}, and from appropriate +/+ coisogenic controls using 5 restriction endonucleases (EcoRI, HindIII, BamHI, MspI, and TaqI)No restriction fragment length differences were observed between bg alleles and coisogenic controls upon hybridization with Nid or Estm9, excluding a deletion or insertion in these genes from causality in these bg alleles.

Expression of Nid and Estm9 in bg mice was examined by northern blot analysis and quantitative RT-PCR7. Hybridization of northern blots of liver and kidney RNA from +/+, bg, bg^J , and bg ^2J with probes for Nid and Estm9, yielded signals of similar size and intensity in bg and +/+ RNA. Furthermore, no difference in amplicon size or amount was observed upon quantitative RT-PCR7 using liver or kidney RNA from +/+, bg, bg^J , and bg ^2J mice and oligonucleotides for Nid or Estm9, indicating expression of Nid and Estm9 to be grossly intact in bg. 5.2.2.5 PHYSICAL MAPPING OF PROXIMAL MOUSE CHR 13 IN THE VICINITY OF BG

Cytogenetic and physical mapping studies have demonstrated mouse mutations induced by gonadal x-irradiation to be frequently associated with genomic rearrangements (typically deletions or translocations). The SB/LeJ-bg allele was discovered among the offspring of a male which had received such treatment In order to examine 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 infrequently. 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 Glι3). 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 DNA 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 endonucleases (NotI, MluI, NruI, and SrfI complete digests, NaeI partial digest, and NotI/MluI double digest), differences were observed between DBA/2 and the other DΝAs (C57BL/6J-bg^J, CAST/EiJ and SB/LeJ-bg). In each case, the DBA/2 fragment was 25-50kb smaller than the band identified in C57BL/6J- bg^J, SB/LeJ-bg, or CAST/EiJ DΝA (FIG 3A, FIG 3B, FIG 3C, and FIG 3D, Table 4). No differences in Nid band sizes were evident among other mouse strains examined (C57BL/6J-bg^J, SB/LeJ-bg, and CAST/EiJ). Other restriction endonucleases, which identify smaller fragments when probed with Nid (BssHII, ClaI, NaeI, SmaI, XhoI) were identical in all strains tested (FIG. 3 A, FIG.3B, FIG. 3C, FIG.3D, Table 4). Nid fragment size differences were observed using both methylation-sensitive and -insensitive restriction endonucleases.

5.2.3 DISCUSSION

Previous studies have localized bg to proximal Chr 13. Lyon et al.,(1969) demonstrated bg to be 0.5 cM proximal to the mutation Xt, which corresponds to the Gli3 gene. Several groups have demonstrated tight linkage between bg and Tcrg (Holcombe et al., 1987, 1991 ; Justice et al., 1990). Jenkins et al., (1991) found bg to cosegregate with Nid in 123 meiotic events. Precise genetic mapping of bg has been undertaken with respect to these genes and recently identified SSLP markers (Dietrich et al., 1994) as an antecedent to generation of a YAC contig of the genomic region encompassing bg. These results are in agreement with previous 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 distances between markers were observed among the present 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 1q42.1-1q43, which represent the approximate limits of this conserved linkage group (Jenkins et al., 1991; Mattei et 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 assumed 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 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 lysosomal trafficking, nidogen is a component of basement membranes, a specialized extracellular matrix structure limited to certain tissues (Durkin et al., 1988). The candidacy 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 (Bettenhausen and Gossler, 1995). Comparison of partial Estm9 cDNA sequences with DNA and peptide databases demonstrate significant sequence similarity only with uncharacterized human ESTs. While the function of Estm9 is unknown, expression analysis reveals it to be constitutively expressed, temporally and spatially, in the mouse (Bettenhausen and Gossler, 1995).

Initial genetic evaluation of the candidacy of Nid and Estm9 for bg by northern and Southern blot hybridization or quantitative RT- PCR™, revealed no differences between several bg alleles and coisogenic controls. These studies do not definitively exclude Nid or Estm9 from candidacy for bg. A more robust method of evaluation for bg candidate genes would be genetic 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 to examine 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-induced SB-bg allele for the presence of a gross genomic rearrangement. SB-bg -specific restriction fragment length differences were not observed with Nid, Estm9, or Gli3 gene probes. Furthermore, all critical region SSLP amplicons (D13Mit44, D13Mit114, D13Mit134 and D13Mit207) were present in SB-bg DNA.. 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, 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-Southern blots with Gli3 or Estm9. Since fragment size differences were observed with many rare-cutting restriction endonucleases, including several which are methylation-insensitive, it is unlikely that they are merely interstrain differences in DNA methylation or point mutations. Instead, it is suggested that a genomic rearrangement has occurred in the DBA/2 mouse at a distance of less than 900 kb from Nid (FIG. 3D). The rearrangement may represent a small (25-50 kb) genomic deletion in the DBA/2 mouse. The functional significance of such a putative rearrangement is uncertain Interestingly, 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 SalI, nidogen identified polymorphic band sizes in Caucasian populations. In 2 CHS patients that have been examined to date, homozygosity for one NID allele was observed, suggesting the possibility of linkage of human CHS and NID (Goodrich and Holcombe, 1995). Definitive mapping of human CHS, however, must await identification of the mouse bg gene. On a practical note, the interstrain differences in pulsed field restriction fragment length provide a physical landmark within the bg nonrecombinant interval. Thus bg candidate genes can be easily screened for physical linkage of with Nid as a means of determining whether or not they lie within the bg nonrecombinant interval.

In summary, the bg locus has been localized, which is the mouse homolog of human CHS, to a genomic 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.

5.3 EXAMPLE 3 - IDENTIFICATION OF THE HOMOLOGOUS BEIGE AND CHS 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. Candidate cDNAs for bg were isolated from YAC 195A8, which contains 650 kb of the bg non-recombinant interval using direct cDNA selection with mouse spleen cDNA (FIG. 11). Of 56 candidate cDNA clones analyzed from a direct-selection study, evidence for causality in bg was found in one (see below), and this gene was designated Lyst (lysosomal trafficking regulator). As this clone was 132 nucleotides long, additional Lyst sequences were sought by screening three mouse cDNA libraries and performing polymerase chain reaction (PCR™) amplification of cDNA ends (Kingsmore et al., 1994). Ten overlapping Lyst clones were identified, representing ~7 kb (Genbank accession number, L77889). These were physically assigned 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 number, MGD-PMEX-14). The Lyst probes identified the same polymorphic PFGE restriction fragments as nidogen (Nid), indicating that Lyst and Nid are clustered within 650 kb. Lyst was also mapped genetically in 504[C57BL/6-bg^J × (C57BL/6J-bg^J × CAST/ELJ)F₁] backcross mice by means of three TaqI restriction fragment length polymorphisms (RFLPs). The Lyst RFLPs cosegregated with bg (and Nid), confirming their colocalization on proximal mouse chromosome 13 (MGD accession number, MGD-CREX-615).

Evidence for Lyst mutations was found in two bg alleles A 5-kb genomic deletion that contained the 3' end of Lyst exon β, and exons γ and δ, was identified in bg^11J DNA (FIG. 12). The bg^11J deletion corresponds to the loss of -400 internal amino acids of the predicted Lyst peptide. Furthermore, whereas the 5' end of the bg^11J deletion occurs within Lyst exon β, the 3' end is intronic. Therefore the truncated Lyst mRNA in bg¹¹ J mice is also anticipated to splice incorrectly, terminate prematurely, and lack polyadenylation.

Quantitative reverse transcription (RT)-PCR™ demonstrated a moderate decrease in Lyst mRNA in bg and bg¹ liver, and a gross reduction in bg^2J (Lyst ΔOD after normalization for β-actin mRNA; +/+, 1.00, bg²/bg^2J, 0.19; bg/bg, 0.28; bg^J/bg^J, 0.40). A commensurate reduction in bg^2J transcript abundance was noted by using several primer pairs derived from different regions of the Lyst cDNA. Aberrant Lyst RT-PCR™ products were not observed. The particularly striking (more than fivefold) reduction in Lyst expression evident in bg^2J homozygotes suggested the existence of a mutation in bg^2J Lyst that results in decreased transcription or mRNA instability. The molecular basis of the decrease in Lyst mRNA in bg^2J is not yet known, but it is reminiscent of the leaky ablation of mature message associated with an intronic retrotransposition event (Kingsmore et al., 1994).

The predicted open reading frame (ORF) of Lyst was 4,635 nucleotides, encoding a protein of 1,545 amino acids and relative molecular mass 172,500 (M_r 172.5K) (FIG. 13α).

Nucleotides 51-74 are rich in CG nucleotides, a common feature of the 5' region of housekeeping 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' untranslated region (UTR), 91% in the ORF, and amin-acid identity was 97%, 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 identified restriction fragments that were indistinguishable from mouse Lyst1; physical mapping of the human clones to the same region of the mouse genome as Lyst indicates that they are indeed homologous to Lyst. It has been suggested that CHS and bg represent homologous disorders, as their clinical features(Blume and Wolff, 1972) and defects in lysosomal transport (Burkhardt et al., 1993) are identical. Homology of bg and CHS is supported by genetic complementation studies; fusion of fibroblasts from bg mice and CHS patients failed to reverse lysosomal 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 1q43 and the bg region on mouse Chromosome 13. Therefore LYST mutations in CHS patients were sought by sequencing LYST lymphoblast and fibroblast cDNAs corresponding to these ESTs from 10 CHS patients. In one patient, a single-base insertional mutation was found at nucleotides 117-118 of the LYST coding domain, resulting in a frame shift and termination 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 stathmin (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 × 10^-6). The domain is stathmin that matches Lyst is helical and has heptad repeats that participate in coiled-coil interactions with other proteins (Sobel, 1991; Maucuer et al., 1995). The stathmin-like 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 stathmin, suggesting 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-mediated lysosome transport. Although Lyst is no predicted to have transmembrane helices, the C-terminal tetrapeptide (CYSP; amino acids 1,542-1,545) is strikingly similar to known prenylation sites, which could provide attachment to lysosomal/late endosomal membranes through thioester linkage with the cysteine.

Previous studies of bg leukocytes have shown correction of microtubule function (as assessed by Concavalin A capping) and natural killer activity when treated with inhibitors of protein kinase C (PKC) breakdown (Sato et al., 1990; Ito et al., 1989), suggesting that bg might be regulated 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 protein kinase, and one by tyrosine kinase (FIG. 136). Almost half of the predicted helices outside the stathmin-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 stathmin (Sobel, 1991; Maucuer et al., 1995). Furthermore, phosphorylation of these positions could provide a control mechanism by causing a conformational shift in the bundles, thereby affecting interactions with other molecules.

Northern analysis and RT-PCR™ indicated that Lyst is ubiquitously transcribed, both temporally and spatially, in mouse and 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 leukaemia HL-60, and several leukaemia lines, the 12-14 kb isoform was either undetectable or barely detectable, but smaller Lyst transcripts were abundant (FIG. 14). Given the significance for bg mice and CHS patients of defects in the lysosomal and late-endosomal compartments of granulocytes, NK cells and cytolytic T lymphocytes (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 functional significance. Probes derived from the 5' or 3' ends of the Lyst

5.4 EXAMPLE 4 - - MUTATION ANALYSIS AND PHYSICAL AND GENETIC MAPPING

ESTABLISH HUMAN LYST AS THE CHS GENE

5.4.1 MATERIALS AND METHODS

5.4.1.1 CLONING OF THE HUMAN LYST GENE

Segments of the human LYST sequence were obtained by an anchored, nested PCR™

(5' RACE-PCR™) using liver cDNA as a template (Clontech Laboratories, Palo Alto, CA), by

RT-PCR™ using total RNA and by sequencing of human ESTs similar in sequence to mouse Lyst.

For the 5' RACE-PCR™ two nested primers were used that were derived from a human EST

(GenBank accession number W26957) and had the following nucleotide sequence: and

.

For RT-PCR™ experiments, total RNA was prepared from the promyelocytic HL-60 cell line. Reverse transcription was performed with Expand (Boehringer Mannheim, Meylan France) with the following primer pairs:

The primers used to amplify the cDNA between bp 1891 and 3050 were derived from the mouse Lyst sequence. Human primers were designed from the sequence of the PCR™ product (1159 bp) and used to amplify the flanking sequences.

5.4.1.2 DNA SEQUENCING AND SEQUENCE ANALYSIS

PCR™ products were cloned using a TA cloning kit (Invitrogen 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 Information database were performed using the BLAST network server (Altschul et al., 1990) (National Library of Medicine, via INTERNET) and the Whitehead Institute Sequence Analysis Programs (MIT, Cambridge, Massachusetts).

5.4.1.3 SOUTHERN AND NORTHERN BLOT ANALYSIS

Preparation of mouse, human and yeast DNA samples, digestion with restriction endonucleases, agarose gel electrophoresis and Southern transfers were performed using standard techniques (Maniatis et al., 1984). The EcoRI monochromosomal somatic cell hybrid blot was obtained from BIOS Laboratories (New Haven, Connecticut). Isolation of poly(A)⁺RNA from fibroblast and EBV-transformed B lymphoblast cell lines, formaldehyde agarose gel electrophoresis and Northern blotting were performed according to standard procedures (Maniatis et al., 1984). Membranes were hybridization with various LYST or actin probes labeled with a³²P-dCTP. Mouse genetic mapping analyses were performed as described (Barbosa et al., 1995). 5.4.1.4 SSCP ANALYSIS

Detection of nucleotide changes by SSCP was performed as described by Orita et al.

(1989). Briefly, each PCR™ product was mixed with an equal volume of denaturing buffer and heated to 95°C for 3 min., after which the samples were loaded onto 0.8 mm thick, 10% native polyacrylamide gels. Gels were run at ambient temperature at 9 W for 6-10 hours, depending on the size of the PCR™ product. Bands were visualized by silver- staining (Beidler et al., 1982).

5.4.1.5 ALLELE-SPECIFIC OLIGONUCLEOTIDE ANALYSIS

PCR™ products spanning the mutation site in patient 371 were transferred to nylon membranes using a slot blot apparatus. Approximately 5 ng of each PCR™ product was treated with a denaturing solution (0.5 M NaOH, 1 .5 M NaCl), split in half and loaded in duplicate. Two 17 mer oligonucleotides were synthesized that span the region containing the mutation. One contained the sequence of the normal allele (5'-CGCACATGGCAACCCTT-3')(SEQ 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 γ³²P-dATP using T4 polynucleotide kinase and hybridized to the membranes at 50°C. Hybridization and wash buffers were as described (Church 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.

5.4.2 RESULTS

5.4.2.1 A QUESTION OF TWO BG GENES

In order to resolve the dilemma created by the existence of two different bg candidate genes (Lyst and BG), the inventors isolated and sequenced additional mouse cDNA and genomic clones corresponding to the 3' end of Lyst. An anchored, nested PCR™ (3'RACE-PCR™) from this region yielded two fragments (1.25 kb and 2 kb). The 1.25 kb clone contained the previously published 3' end of Lyst, while the 2 kb clone contained sequences derived from Lyst (at the 5' end) and from BG (at the 3' end). Reverse transcription and PCR™ (RT-PCR™) confirmed 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 1-4706 of Lyst, the 2 kb 3 'RACE-PCR™ clone and 6824 nucleotides of BG cDNA. This 11,817 bp cDNA sequence (Lyst-I, Genbank accession number U70015) corresponds to the largest mRNA observed in Northern blots (~12 kb) (Goodrich and Holcombe, 1995).

Analysis of a P1 genomic clone (number 8592) containing Lyst and BG revealed that the 11,817bp Lyst-I cDNA results from splicing of Lyst exon σ (containing nucleotide 4706) to downstream exon τ (FIG. 15b). Incomplete splicing and reading through the intron σ' interposed between exons σ and τ yields the 5893 bp cDNA described by Barbosa et al. (1996) (Lyst-II, FIG. 15b, Genbank accession number L77884). Intron σ' encodes 37 in-frame amino acids followed by a stop codon and a polyadenylation signal. Lyst-II corresponds to a smaller (~4kb) mRNA 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 (M_r 425K) while that of Lyst-II is predicted to be of M_r 172.5K.

5.4.2.2 SEQUENCE OF HUMAN LYST1 AND LYST2 cDNAs

cDNAs corresponding to LYST1, the human homolog of Lyst1-isoform 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 Lyst1 by database searches (Genbank accession numbers L77889, W26957 and H51623). Intervening cDNA sequences were isolated using RT-PCR™ with primers derived from mouse Lyst1 sequence and adjacent ESTs. The partial LYST1 cDNA sequence (Genbank Accession number U70064; 7.1 kb) was assembled by alignment of these clones with mouse Lyst1 cDNA. Human LYST1 has 82% predicted amino acid identity with mouse Lyst1 over 1,990 amino acids. The predicted human LYST1 amino acid sequence contains a 6 amino acid insertion relative to mouse Lyst1 at residue 1,039. Recently, another group has published the sequence of the human LYST1 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 (LYST1 -isoform I) observed on northern blots of human tissues (caption in FIG. 2). These northern blots also demonstrated the existence of a smaller LYST isoform (~4.5 kb, designated

LYST-isoform II) that was similar in size to the smaller mouse Lyst1 mRNA, and that appeared to differ in distribution of expression in human tissues from LYST1 -isoform I. Assuming that the genomic derivation of human LYST1 -isoform II was the same as mouse Lyst1-isoform II, the sequence of the 3' end of the human LYST1 -II isoform was sought by cloning human LYST1 intron F' using PCR™ of human genomic DNA with primers derived from LYST1 exon F and mouse intron F' (caption in FIG. 2). The sequence of the 5' end of human LYST1 intron F' contained 17 codons in frame with LYST1 exon F, followed by a stop codon. By amplification of a LYST1-isoform II cDNA from human peripheral blood RNA by RT-PCR™ with primers from a 5' LYST1 exon and LYST1 intron F', it was demonstrated that this intron was indeed retained in human LYST1 -isoform II mRNA. Nucleotides 1-5905 of human LYST1 -isoform II cDNA are identical to LYST1-isoform I, and are followed by intron F' sequence (Genbank accession number U84744)(FIG. 2). The predicted intron-encoded amino termini of the mouse Lyst1 -isoform II and human LYST1- isoform II peptides shared 65% identity.

The only significant sequence similarity of LYST1- isoform II to known proteins was with the stathmin family. Identity with mouse Lyst1 -isoform II in this region (amino acids 376-540) was 92% (and similarity was 99%)(FIG. 5).

5.4.2.3 GENETIC AND PHYSICAL MAPPING OF LYST

A 2 kb human LYST probe was assigned 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 containing human chromosome 1 DNA. In order to precisely map LYST on human chromosome 1, LYST probes were hybridized to

YAC clones encompassing the CHS critical region (FIG. 16b and FIG. 16c) (Barrat et al. 1996). Three probes, derived from different segments of the LYST cDNA each hybridized to five CHS critical region YACs (FIG. 16d), confirming localization to the correct interval.

Genetic mapping in 504 [C57BL/6J-bg^J × (C57BL/6J-bg^J × CAST/EiJ)F₁] backcross mice 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 cosegregate with bg and Lyst on mouse Chromosome 13. 5.4.2.4 MUTATION ANALYSIS

As an initial screen for LYST mutations in CHS patients, we analyzed northern blots of poly(A)⁺ RNA from CHS patients. The largest LYST mRNA species (LYST-I, approximately 12 kb) was greatly reduced in abundance or absent in lymphoblastoid mRNA of patients P1 and P3, respectively (FIG.4a), while the smaller LYST transcript (LYST-lI, approximately 4 4 kb) was both present and undiminished in abundance. Rehybridization of this 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 LYST-II mRNA was essentially unaltered in abundance (103-147% of control).

Single-strand conformation polymorphism (SSCP) analysis was undertaken using cDNA samples derived from lymphoblastoid or fibroblast cells lines from CHS patients. Anomalous bands were detected in PCR™ products from the 5' end of the LYST ORF in two unrelated CHS patients different from those with aberrant northern blot patterns (371 and 373, FIG. 4b).

Subsequent sequence analysis identified a C to T transition at nucleotide 148 of the coding domain in patient 373 (FIG.4c). Four of nine cDNA clones derived from patient 373 contained this mutation. Restriction enzyme digestion confirmed this mutation. TaqI digestion of LYST cDNA. (nucleotide 520 to 808) showed loss of this 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 et 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 homozygous or hemizygous for this mutation.

Mutations were identified in three other CHS patients cDNA isolated from

EBV-transformed lymphoblasts 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 1 104 (R1 104X) [Nagle et al., 1996]. Patient 370 contained a homozygous C to T transition at nucleotide 3085 of the coding domain, that created a stop codon at amino acid 1029 (Q1029X). Patient 369 had a heterozygous frame shift mutation. Nucleotides 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 of these patients (369, 370, 371, 372, 373, P1 and P3) contain the giant perinuclear lysosomal 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 cosanguinous. In contrast, the clinical course of patients 372 and 373 was milder: Lymphoblasts were immortalized from patient 372 at 27 years of age. He had oculocutaneous albinism, recurrent skin infections, and peripheral neuropathy. Patient 373 has not had systemic infections and is alive at age 37. Patient 373 does, however, have hypopigmented hair and irides as well as peripheral neuropathy.

5.4.2.5 EXPRESSION OF LYST-I AND LYST-II IN HUMAN TISSUES

Analysis of northern blots of mouse mRNA had suggested that the relative abundance of mouse Lyst-I and Lyst-II transcripts differed from tissue to tissue (Barbosa et al., 1996). The relative abundance of LYST mRNA isoforms in human tissues at different developmental stages was examined by sequential hybridization of a poly(A)⁺ RNA dot blot with several LYST cDNA 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, hypoxanthine guanine phosphoribosil transferase, $-actin, and ubiquitin) to allow estimation of the relative abundance of LYST mRNA isoforms in different tissues.

Using a probe that hybridized only to LYST-I transcripts (the largest LYST isoform) on northern blots (Barbosa et al., 1996), 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 detected in fetal brain. Negligible LYST-I transcription was also apparent in heart, lung, kidney, or liver at any developmental stage.

A somewhat different pattern of expression was evident upon rehybridization of the blot with a probe derived from the 5' end of the coding domain of LYST, a region that hybridized to both LYST-I and LYST-II mRNAs on northern blots (Barbosa et al., 1996). Consonant with the pattern of LYST-I transcription was abundant expression detected with this probe in peripheral blood leukocytes, thymus (adult and fetal), and bone marrow, and negligible expression detected in skeletal muscle. However, several tissues with abundant LYST-I transcripts, exhibited considerably less hybridization signal with the LYST-I + LYST-II probe, including most regions of the adult brain, fetal and adult thymus, and spleen. Furthermore, several tissues with negligible LYST-I transcription exhibited intense hybridization with the LYST-I + LYST-II 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 (Lysosomal trafficking regulator), was identified from a bg critical region YAC and showed that it was mutated in two bg alleles. The 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. Simultaneously, another group published a partial cDNA sequence (BG) that had been isolated from the same YAC (Perou et al., 1996a). This partial cDNA was mutated in two other bg alleles, but was different in sequence from Lyst. The inventors have resolved this bg gene dilemma by demonstrating that Lyst 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 different 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-PCR™ products, the inventors have shown that nucleotides 1-4706 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 σ (containing nucleotide 4706) to exon τ results in an mRNA (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 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 et 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 different. The C-terminus of Lyst-I contains a 'WD'-repeat domain that is similar to the β-subunit of heterotrimeric G proteins and which may assume a propeller-like secondary structure (Lambright et al., 1996). In contrast, Lyst-II has a C-terminal prenylation motif that could provide attachment to the lysosomal 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 transmembrane 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 LYST 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 identified two human ESTs homologous to mouse Lyst 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 LYST isoforms (LYST-I), and identified 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, designated LYST-II, encodes a protein of 1531 amino acids that is homologous to mouse Lyst-II. Like the latter, human LYST-II mRNA arises through incomplete splicing and retention of a transcribed intron that encodes the C-terminus of the predicted LYST-II protein. The mouse and human LYST-II -specific codons share 65 % predicted amino acid identity. The stop codon, however, is not precisely conserved between human and mouse LYST-II. While mouse Lyst-II is predicted to contain a C-terminal prenylation motif (CYSP), translation of human LYST-II 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 stathmin (amino acids 376-540). While mouse and human LYST had an overall amino acid identity of 81%, identity in the stathmin-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 intracellular signal transduction (Sobel 1991; Belmont and Mitchison, 1996). This region of LYST may encode a coiled-coil protein interaction domain and may regulate microtubule-mediated lysosome trafficking.

Intriguingly, a defect in microtubule dynamics has previously been documented in CHS (Oliver et al., 1975) and intact microtubules are required for maintenance of lysosomal morphology and trafficking (Matteoni and Kreis, 1987; Swanson et al., 1987; Swanson et al., 1992, Oka and

Weigel, 1983). Other putative structural features of 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 hypothesized to have a signal transduction function similar to stathmin. The conserved phosphorylation sites have been hypothesized 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 candidacy of LYST for CHS, segments of the LYST sequence were mapped in the human genome. The CHS locus was recently assigned to human chromosome 1q42-43 (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 containing the bg locus and human chromosome 1q42-q43 (Beguez-Cesar, 1943). D1S2680 and D1S163 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 LYST PCR™ products to CHS critical region YACs also precluded the possibility that the LYST sequence 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 LYST-II), however, was present in mRNA from these patients in normal abundance. These results suggest that, at least in some patients, CHS results from loss of the protein encoded by LYST-I rather than LYST-II. This result is surprising since previous Northern blots had suggested that the major LYST mRNA in granular cells was LYST-II, while LYST-I was either undetectable or barely detectable 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 mRNA represented the transcript of primary functional significance. In this context, it is interesting to note that the bg^8J mutation results in the generation of a premature stop codon in Lyst-l 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-II expression alone cannot compensate for loss of LYST-I. Mutations were identified within the coding domain of LYST in five CHS patients, two of which have been reported previously (Barbosa et al., 1996; Nagle et al., 1996). The genetic lesions in three CHS (patients 370, 372 and 373) were C to T transitions that resulted in premature termination (Q1029X, R1 104X and R50X, respectively)[Nagle et al., 1996]. Two other patients had coding domain frame shift mutations that induced premature termination. One of these, patient 371, had a G insertion at nucleotide 1 18 of the coding domain, leading to premature termination at codon 63 (Barbosa et al., 1996). Allele-specific oligonucleotide analysis indicated that this mutation was either homozygous or that mRNA corresponding to this region is not produced from the other allele (hemizygosity). Patient 369 was heterozygous for a dinucleotide deletion that results in premature termination at codon 1030. Interestingly, all bg and CHS mutations identified to date are predicted to result in the production of either truncated 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 truncated LYST proteins (which may or may not be stable) with clinical features or disease severity in CHS 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 different developmental stages revealed an overlapping but distinct pattern of expression. A quantitative estimate of the expression of the smaller LYST mRNA 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 LYST transcripts. LYST-I transcripts predominated in thymus, fetal 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 LYST isoforms were expressed in several tissues, including heart, fetal heart, aorta, thyroid gland, salivary gland, kidney, liver, fetal liver, appendix, lung, fetal lung, and fetal brain. The developmental pattern of LYST mRNA isoform expression in brain was particularly interesting, since only the smaller LYST isoforms were expressed in fetal brain, whereas the largest isoform (LYST-I) predominated in many regions of the adult brain.

In summary, the inventors have shown that the same gene is mutated in human CHS and bg mice. Without bone marrow transplantation, CHS patients typically die in childhood of infection and malignancy. The existence of an animal model of CHS with a similar genetic lesion will assist efforts to develop novel therapies for this disease. 5.5 EXAMPLE 5╌ DNA SEQUENCES OF MOUSE LYST1

5.6 EXAMPLE 6 - - DEDUCED AMINO ACID SEQUENCES OF MOUSE LYST1 PROTEINS

5.7 EXAMPLE 7╌ DNA SEQUENCES OF HUMAN LYST1 GENE

5.8 EXAMPLE 8╌ DEDUCED AMINO ACID SEQUENCES OF HUMAN LYST1 PROTEIN

5.9 EXAMPLE 9 - - IDENTIFICATION OF A DNA SEGMENT ENCODING LYST2

Lyst2 was identified in a search for human genes similar in sequence to Lyst1 (the CH gene) Mouse Lyst1 cDNA sequence was compared with Genbank sequences, and significant similarity (52%) was noted between residues 3275 to 3413 of Lyst1 (Genbank Accession number U70015) 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 library of human infant brain, and is 1979-bp in length. The clone was designated human LYST2.

5.10 EXAMPLE 10 - DNA SEQUENCE OF THE HUMAN LYST2 GENE

The LYST2 clone was sequenced using standard methodologies. The DNA sequence is given below (SEQ ID NO: 11):

This DNA sequence corresponds to the 3' end of the coding domain of human LYST2 and the 3' untranslated region.

5.11 EXAMPLE 11 - - AMINO ACID SEQUENCE OF THE HUMAN LYST2 PROTEIN

Translation of the DNA of SEQ ID NO: 1 1 provided the deduced amino acid sequence of the LYST2 protein (SEQ ID NO: 12) which is shown below:

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 Lyst1. The C-terminal residues of LYST2 are not similar to LYST1, but do have a similar predicted secondary structure: This region of LYST1 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 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 Lyst1 and human LYST2 occur approximately the same distance from the matching region.

5.12 EXAMPLE 12 - - GENETIC MAPPING OF THE LYST2 GENE

By hybridization to Southern blots of human-rodent somatic cell hybrids, LYST2 was shown to map on human Chromosome 13. This is in contrast to LYST1, 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

[C57BL/6J-bg^J X (C57BL/6J-bg^J x CAST/EiJ)F₁] mice revealed Lyst2 to map to mouse Chromosome3 between D3Mit21 and D3Mit22. This contrasts with Lyst, which maps on mouse Chromosome 13. Pulsed field gel electrophoresis blots of mouse DNA hybridized with a Lyst2 probe showed a single band, indicating that Lyst2 is a single genetic locus.

5.13 EXAMPLE 13 - - EXPRESSION ANALYSIS OF THE LYST2 GENE

Hybridization of northern blots of human and mouse tissues with LYST2 revealed the 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. Lyst2 is not expressed in mouse spleen or liver. The largest (and most prominent) band observed on northern blots was 13kb in size (very similar to the largest Lyst mRNA). Additional transcripts on 6kb and 5kb were evident in mouse brain RNA. 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, lymph node, thymus, and appendix. No expression was detected in peripheral blood leucocyte, bone marrow, fetal liver, lung carcinoma, or leukemia cell lines (K562, MOLT4, Raji, HL60).

The major transcript was 13-kb in size in human RNA.

In summary, LYST2 appears to be similar in size to the largest LYST1 mRNA, but has a very different tissue distribution of expression, being abundantly expressed only in brain. LYST2 appears to be a brain-specific homologue of LYST1, and may function to regulate protein trafficking to the lysosome and late endosome within the brain.

The relative abundance of LYST2 mRNA isoforms in human tissues at different 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 normalized to eight housekeeping genes (phospholipase, ribosomal protein S9, tubulin, a highly basic 23-kDa protein, glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine guanine phosphoribosil transferase, β-actin, and ubiquitin) to allow estimation of the relative abundance of LYST2 mRNA isoforms in different tissues.

Abundant LYST2 transcripts were detected in all brain regions and in kidney. LYST2 transcripts were detected in those regions at all developmental stages. 5.14 EXAMPLE 14 - - IDENTIFICATION OF MOUSE LYST2 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. 5.15 EXAMPLE 15 - DNA SEQUENCE OF THE MOUSE LYST2 GENE

Mouse Lyst2 shares 98% amino acid identity with human LYST

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 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 apparent 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 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

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 L YST2 protein, or a murine Lyst 1 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: 1 1, 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 LYSTI, Lystl, LYST2, or Lyst2 gene.

1 1. 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 1 1, 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: 1 1 or SEQ ID NO : 13, or

(b) 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.

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: 1 1, 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: 1 1 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.