WO2000058464A2 - Rab genes and their uses - Google Patents

Abstract

Isolated nucleotide compositions and sequences are provided for RAB genes. The RAB nucleic acid compositions find use in identifying homologous or related genes; in producing compositions that modulate the expression or function of its encoded protein, RAB; for gene therapy; mapping functional regions of the protein; and in studying associated physiological pathways. In addition, modulation of the gene activity in vivo is used for prophylactic and therapeutic purposes, such as treatment of cancer, identification of cell type based on expression, and the like.

Description

RAB GENES AND THEIR USES

INTRODUCTION Background Eukaryotic cells contain a number of small GTPases, which act as intermediates in signal transduction, and which comprise a large superfamily of small, approximately 20 to 30 kDa, monomeric GTP-binding proteins. This large superfamily of GTP-binding proteins is divided into a number of subfamilies. Members of the Ras subfamily function to regulate cellular proliferation and differentiation. Members of the Rho and Rac subfamily of proteins serve to regulate cytoskeletal assembly, while members of the Rab, Arf, Sar and Ran subfamilies regulate vesicular transport (Bourne et al. (1991), Nature 349:117-127; Hall and Zerial (1995) General introduction. In: "Guidebook to the Small GTPases" (M. Zerial and LA Huber Eds.), pg. 3-11 , Sambrook and Tooze, Oxford University Press).

Small GTPases have been shown to function as molecular switches within signaling pathways, cyclically regulating signal transmission to a downstream effector by alternating between an active GTP bound form and an inactive GDP bound form. This cycling is catalyzed by at least two classes of proteins; guanine nucleotide exchange factors (GEFs) which promote exchange between bound GDP and cytoplasmic GTP and by GTPase activating proteins (GAPs) which stimulate the low intrinsic GTPase activity of small GTPases resulting in the inactive GDP bound state (Boguski and McCormick (1993) Nature 366; 643- 654; Feig (1993) Science 260; 767-768).

In eukaryotic cells, protein transport is thought to occur by the budding and fusion of transport vesicles. Because mutations in rab genes have been shown to have severe consequences in blocking protein transport along the exocytic or endocytic pathway and in altering cell morphology (Simons and Zerial (1993) Neuron 11 :789-799; Pfeffer (1994) Curr. Opin. Cell Biol. 6; 522-526; Novick and Zerial (1997) Curr. Opin. Cell Biol. 9;496-504), these proteins appear to play a key role in membrane trafficking. Rab proteins constitute the largest family of small GTPases, with over 40 (including isoforms) identified to date.

The complexity of the mammalian rab protein family reflects the diversity of vesicle transport routes and distinct intracellular membrane sorting processes, displayed in different mammalian cell types. Rab GTPases are found in two pools, one cytosolic and the other membrane bound. They are associated with the cytoplasmic surface of distinct exocytic and endocytic organelles, and with transport vesicles connecting these compartments. The conformational changes brought about by GTP binding and hydrolysis are thought to regulate interactions with other components of the transport machinery.

Most rab genes are ubiquitously expressed, but a few exhibit tissue or cell-type specificity. For example, rab17 is expressed primarily in epithelial cells (Lutcke et al. 11993) J. Cell Biol. 121 ; 553-564), the human rab 'S10' or rab33, is expressed exclusively in lymphoid cell lines (Koda and Kakinuma (1993) FEBS lett. 328; 21-24), rab27b is mainly expressed in testis (Chen et al. (1996) Biochemi. and Mol. Med. 60; 27-37) and rab15 is highly brain specific (Elferlink et al. (1992) J.Biol. Chem. 267;1-8). These restricted expression profiles may reflect specific involvement with transport processes or downstream effectors present in the expressing tissues.

Rab proteins contain four highly conserved peptide sequences designated I to IV involved in GTP binding and hydrolysis. They also contain a consensus carboxy-terminal isoprenylation motif encoding two cysteine residues. Rab geranylgeranyltransferase (GGTase), a heterodimer of α and β subunits, transfers a geranylgeranyl group to (usually) two cysteines at the carboxy-terminus of the protein, enabling rab proteins to anchor to the plasma membrane or to intracellular vesicular membranes (Casey and Seabra (1996) J. Biol. Chem. 271; 3692-3698). The process of isoprenylation may represent another opportunity for regulating rab function.

The number of human genes encoding Rab-like proteins is currently unknown. It is possible that yet-undiscovered Rab genes play important roles in the regulation of transport processes in normal cells, or may be implicated in human disease. Towards this aim the discovery of novel Rab-related proteins and characterization of their specific roles in cellular processes, especially in tumor development, is of particular interest.

SUMMARY OF THE INVENTION

Isolated nucleotide compositions and sequences are provided for RAB genes. The

RAB nucleic acid compositions find use in identifying homologous or related genes; in producing compositions that modulate the expression or function of its encoded protein, RAB; for gene therapy; mapping functional regions of the protein; and in studying associated physiological pathways. In addition, modulation of the gene activity in vivo is used for prophylactic and therapeutic purposes, such as treatment of cancer, identification of cell type based on expression, and the like. DESCRIPTION OF THE SPECIFIC EMBODIMENTS Nucleic acid compositions encoding RAB related genes are provided. They are used in identifying homologous or related genes; in producing compositions that modulate the expression or function of its encoded protein; for gene therapy; mapping functional regions of the protein; and in studying associated physiological pathways. The RAB gene products are members of the Rab subfamily of small GTPases, which have a role in regulation of protein and/or vesicular transport.

Modulation of RAB gene activity in vivo is used for prophylactic and therapeutic purposes, investigation of Rab signaling pathway function, identification of cell type based on expression, and the like. The protein is useful as an immunogen for producing specific antibodies, in screening for biologically active agents that act in the Rab signaling pathway and for therapeutic and prophylactic purposes.

CHARACTERIZATION OF RAB Many components of the Rab signaling pathway have been identified and characterized, including other GTPases, Raf kinases, p190 binding protein, etc. The availability of isolated genes and gene products in this pathway allows the in vitro reconstruction of the pathway and its regulation, using native or genetically altered molecules, or a combination thereof.

IDENTIFICATION OF RAB SEQUENCES Homologs of RAB are identified by any of a number of methods. A fragment of the provided cDNA may be used as a hybridization probe against a cDNA library from the target organism of interest, where low stringency conditions are used. The probe may be a large fragment, or one or more short degenerate primers.

Novel nucleic acid compositions of the invention of particular interest comprise a sequence set forth in any one of SEQ ID NOS: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23 or 25 or an identifying sequence thereof. An "identifying sequence" is a contiguous sequence of residues at least about 10 nt to about 20 nt in length, usually at least about 50 nt to about 100 nt in length, that uniquely identifies the provided sequence.

The nucleic acids of the invention also include nucleic acids having sequence similarity or sequence identity. Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50°C and 10XSSC (0.9 M NaCI/0.09 M sodium citrate) and remain bound when subjected to washing at 55°C in 1XSSC. Sequence identity can be determined by hybridization under stringent conditions, for example, at 50°C or higher and OJXSSC (9 mM NaCI/0.9 mM sodium citrate). Hybridization methods and conditions are well known in the art, see, e.g., U.S. Patent No. 5,707,829. Nucleic acids that are substantially identical to the provided nucleic acid sequences, e.g. allelic variants, genetically altered versions of the gene, etc., bind to the provided nucleic acid sequences (SEQ ID NOS:1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23 or 25) under stringent hybridization conditions. By using probes, particularly labeled probes of DNA sequences, one can isolate homologous or related genes. The source of homologous genes can be any species, e.g. primate species, particularly human; rodents, such as rats and mice, canines, felines, bovines, ovines, equines, yeast, nematodes, etc.

Preferably, hybridization is performed using at least 15 contiguous nucleotides of at least one of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 or 25. That is, when at least 15 contiguous nucleotides of one of the disclosed SEQ ID NOs. is used as a probe, the probe will preferentially hybridize with a nucleic acid or mRNA comprising the complementary sequence, allowing the identification and retrieval of the nucleic acids of the biological material that uniquely hybridize to the selected probe. Probes of more than 15 nucleotides can be used, e.g. probes of from about 25 nucleotides to not more than about 100 nucleotides, but 15 nucleotides generally represents sufficient sequence for unique identification.

The nucleic acids of the invention also include naturally occurring variants of the nucleotide sequences, e.g. degenerate variants, allelic variants, etc. Variants of the nucleic acids of the invention are identified by hybridization of putative variants with nucleotide sequences disclosed herein, preferably by hybridization under stringent conditions. For example, by using appropriate wash conditions, variants of the nucleic acids of the invention can be identified where the allelic variant exhibits at most about 25-30% base pair mismatches relative to the selected nucleic acid probe. In general, allelic variants contain 15-25% base pair mismatches, and can contain as little as even 5-15%, or 2-5%, or 1-2% base pair mismatches, as well as a single base-pair mismatch.

The invention also encompasses homologs corresponding to the nucleic acids of SEQ ID NOS: 1, 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23 or 25, where the source of homologous genes can be any related species within the same genus or group. Within a group, homologs have substantial sequence similarity, e.g. at least 75% sequence identity, usually at least 90%, more usually at least 95% between nucleotide sequences. Sequence similarity is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. A reference sequence will usually be at least about 18 contiguous nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al., J. Mol. Biol. (1990) 215:403-10.

In general, variants of the invention have a sequence identity greater than at least about 65%, preferably at least about 75%, more preferably at least about 85%, and can be greater than at least about 90%, 95% or more, as determined by the Smith-Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular). For the purposes of this invention, a preferred method of calculating percent identity is the Smith- Waterman algorithm, using the following. Global DNA sequence identity must be greater than 65% as determined by the Smith- Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular) using an affine gap search with the following search parameters: gap open penalty, 12; and gap extension penalty, 1.

RAB NUCLEIC ACID COMPOSITIONS Nucleic acids encoding RAB may be cDNA or genomic DNA or a fragment thereof.

The term "RAB gene" shall be intended to mean the open reading frame encoding specific RAB polypeptides, and RAB introns, as well as adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression, up to about 20 kb beyond the coding region, but possibly further in either direction. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into a host genome.

The term "cDNA" as used herein is intended to include all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3' and 5' non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns, when present, removed by nuclear RNA splicing, to create a continuous open reading frame encoding a RAB protein.

A genomic sequence of interest comprises the nucleic acid present between the initiation codon and the stop codon, as defined in the listed sequences, including all of the introns that are normally present in a native chromosome. It may further include the 3' and 5' untranslated regions found in the mature mRNA. It may further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 1 kb, but possibly more, of flanking genomic DNA at either the 5' or 3' end of the transcribed region. The genomic DNA may be isolated as a fragment of 100 kbp or smaller; and substantially free of flanking chromosomal sequence. The genomic DNA flanking the coding region, either 3' or 5', or internal regulatory sequences as sometimes found in introns, contains sequences required for proper tissue and stage specific expression.

The sequence of the 5' flanking region may be utilized for promoter elements, including enhancer binding sites, that provide for developmental regulation in tissues where RAB is expressed. The tissue specific expression is useful for determining the pattern of expression, and for providing promoters that mimic the native pattern of expression. Naturally occurring polymorphisms in the promoter region are useful for determining natural variations in expression, particularly those that may be associated with disease.

Alternatively, mutations may be introduced into the promoter region to determine the effect of altering expression in experimentally defined systems. Methods for the identification of specific DNA motifs involved in the binding of transcriptional factors are known in the art, e.g. sequence similarity to known binding motifs, gel retardation studies, etc. For examples, see Blackwell et al. (1995), Mol. Med. 1:194-205; Mortlock et al. (1996), Genome Res. 6:327-33; and Joulin and Richard-Foy (1995), Eur. J. Biochem. 232:620-626. The regulatory sequences may be used to identify cis acting sequences required for transcriptional or translational regulation of RAB expression, especially in different tissues or stages of development, and to identify cis acting sequences and trans-acting factors that regulate or mediate RAB expression. Such transcription or translational control regions may be operably linked to a RAB gene in order to promote expression of wild type or altered RAB or other proteins of interest in cultured cells, or in embryonic, fetal or adult tissues, and for gene therapy.

The nucleic acid compositions of the subject invention may encode all or a part of the subject polypeptides. Double or single stranded fragments may be obtained of the DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, etc. For the most part, DNA fragments will be of at least 15 nt, usually at least 18 nt or 25 nt, and may be at least about 50 nt. Such small DNA fragments are useful as primers for PCR, hybridization screening probes, etc. Larger DNA fragments, i.e. greater than 100 nt are useful for production of the encoded polypeptide. For use in amplification reactions, such as PCR, a pair of primers will be used. The exact composition of the primer sequences is not critical to the invention, but for most applications the primers will hybridize to the subject sequence under stringent conditions, as known in the art. It is preferable to choose a pair of primers that will generate an amplification product of at least about 50 nt, preferably at least about 100 nt. Algorithms for the selection of primer sequences are generally known, and are available in commercial software packages. Amplification primers hybridize to complementary strands of DNA, and will prime towards each other.

The RAB genes are isolated and obtained in substantial purity, generally as other than an intact chromosome. Usually, the DNA will be obtained substantially free of other nucleic acid sequences that do not include a RAB sequence or fragment thereof, generally being at least about 50%, usually at least about 90% pure and are typically "recombinant", i.e. flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.

The DNA may also be used to identify expression of the gene in a biological specimen. The manner in which one probes cells for the presence of particular nucleotide sequences, as genomic DNA or RNA, is well established in the literature and does not require elaboration here. DNA or mRNA is isolated from a cell sample. The mRNA may be amplified by RT-PCR, using reverse transcriptase to form a complementary DNA strand, followed by polymerase chain reaction amplification using primers specific for the subject DNA sequences. Alternatively, the mRNA sample is separated by gel electrophoresis, transferred to a suitable support, e.g. nitrocellulose, nylon, etc., and then probed with a fragment of the subject DNA as a probe. Other techniques, such as oligonucleotide ligation assays, in situ hybridizations, and hybridization to DNA probes arrayed on a solid chip may also find use. Detection of mRNA hybridizing to the subject sequence is indicative of RAB gene expression in the sample.

The sequence of a RAB gene, including flanking promoter regions and coding regions, may be mutated in various ways known in the art to generate targeted changes in promoter strength, sequence of the encoded protein, etc. The DNA sequence or protein product of such a mutation will usually be substantially similar to the sequences provided herein, i.e. will differ by at least one nucleotide or amino acid, respectively, and may differ by at least two but not more than about ten nucleotides or amino acids. The sequence changes may be substitutions, insertions, deletions, or a combination thereof. Deletions may further include larger changes, such as deletions of a domain or exon. Other modifications of interest include epitope tagging, e.g. with the FLAG system, HA, etc. For studies of subcellular localization, fusion proteins with green fluorescent proteins (GFP) may be used.

Techniques for in vitro mutagenesis of cloned genes are known. Examples of protocols for site specific mutagenesis may be found in Gustin et al. (1993), Biotechniques 14:22; Barany (1985), Gene 37:111-23; Colicβlli et al. (1985), Mol. Gen. Genet. 199:537-9; and Prentki et al. (1984), Gene 29:303-13. Methods for site specific mutagenesis can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp. 15.3- 15.108; Werner et al. (1993), Gene 126:35-41; Sayers et al. (1992), Biotechniques 13:592-6; Jones and Winistorfer (1992), Biotechniques 12:528-30; Barton et al. (1990), Nucleic Acids Res 18:7349-55; Marotti and Tomich (1989), Gene Anal. Tech. 6:67-70; and Zhu (1989), Anal Biochem 177:120-4. Such mutated genes may be used to study structure-function relationships of RAB, or to alter properties of the protein that affect its function or regulation.

RAB POLYPEPTIDES The subject gene may be employed for producing all or portions of RAB polypeptides. By "RAB polypeptide" or "RAB protein" is meant an amino acid sequence encoded by an open reading frame (ORF) of the RAB gene, including the full-length native RAB polypeptide and fragments thereof, particularly biologically active fragments and/or fragments corresponding to functional domains, e.g. GTP-binding domains, etc; and including fusions of the subject polypeptides to other proteins or parts thereof. For expression, an expression cassette may be employed. The expression vector will provide a transcriptional and translational initiation region, which may be inducible or constitutive, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region. These control regions may be native to a RAB gene, or may be derived from exogenous sources. Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present.

Expression vectors may be used for the production of fusion proteins, where the exogenous fusion peptide provides additional functionality, i.e. increased protein synthesis, stability, reactivity with defined antisera, an enzyme marker, e.g. -galactosidase, etc.

Expression cassettes may be prepared comprising a transcription initiation region, the gene or fragment thereof, and a transcriptional termination region. Of particular interest is the use of sequences that allow for the expression of functional epitopes or domains, usually at least about 8 amino acids in length, more usually at least about 15 amino acids in length, to about 25 amino acids, and up to the complete open reading frame of the gene. After introduction of the DNA, the cells containing the construct may be selected by means of a selectable marker, the cells expanded and then used for expression.

RAB polypeptides may be expressed in prokaryotes or eukaryotes in accordance with conventional ways, depending upon the purpose for expression. For large scale production of the protein, a unicellular organism, such as E. coli, B. subtilis, S. cerevisiae, insect cells in combination with baculovirus vectors, or cells of a higher organism such as vertebrates, particularly mammals, e.g. COS 7 cells, may be used as the expression host cells. In some situations, it is desirable to express the RAB gene in eukaryotic cells, where the RAB protein will benefit from native folding and post-translational modifications. Small peptides can also be synthesized in the laboratory. Polypeptides that are subsets of the complete RAB sequence may be used to identify and investigate parts of the protein important for function, such as the GTP binding domain(s), or to raise antibodies directed against these regions. With the availability of the protein or fragments thereof in large amounts, by employing an expression host, the protein may be isolated and purified in accordance with conventional ways. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. The purified protein will generally be at least about 80% pure, preferably at least about 90% pure, and may be up to and including 100% pure. Pure is intended to mean free of other proteins, as well as cellular debris.

The expressed RAB polypeptides are useful for the production of antibodies, where short fragments provide for antibodies specific for the particular polypeptide, and larger fragments or the entire protein allow for the production of antibodies over the surface of the polypeptide. Antibodies may be raised to the wild-type or variant forms of RAB. Antibodies may be raised to isolated peptides corresponding to these domains, or to the native protein.

Antibodies are prepared in accordance with conventional ways, where the expressed polypeptide or protein is used as an immunogen, by itself or conjugated to known immunogenic carriers, e.g. KLH, pre-S HBsAg, other viral or eukaryotic proteins, or the like.

Various adjuvants may be employed, with a series of injections, as appropriate. For monoclonal antibodies, after one or more booster injections, the spleen is isolated, the lymphocytes immortalized by cell fusion, and then screened for high affinity antibody binding. The immortalized cells, i.e. hybridomas, producing the desired antibodies may then be expanded. For further description, see Monoclonal Antibodies: A Laboratory Manual, Harlow and Lane eds., Cold Spring Harbor Laboratories, Cold Spring Harbor, New York, 1988. If desired, the mRNA encoding the heavy and light chains may be isolated and mutagenized by cloning in E. coli, and the heavy and light chains mixed to further enhance the affinity of the antibody. Alternatives to in vivo immunization as a method of raising antibodies include binding to phage "display" libraries, usually in conjunction with in vitro affinity maturation. DIAGNOSTIC USES The subject nucleic acid and/or polypeptide compositions may be used to analyze a patient sample for the presence of polymorphisms associated with a disease state or genetic predisposition to a disease state. Biochemical studies may be performed to determine whether a sequence polymorphism in a RAB coding region or control regions is associated with disease, particularly diseases associated with defects in protein transport, etc. Disease associated polymorphisms may include deletion or truncation of the gene, mutations that alter expression level, that affect the activity of the protein in binding to GTP, GTPase activity, etc.

Changes in the promoter or enhancer sequence that may affect expression levels of RAB can be compared to expression levels of the normal allele by various methods known in the art. Methods for determining promoter or enhancer strength include quantitation of the expressed natural protein; insertion of the variant control element into a vector with a reporter gene such as β-galactosidase, luciferase, chloramphenicol acetyltransferase, etc. that provides for convenient quantitation; and the like. A number of methods are available for analyzing nucleic acids for the presence of a specific sequence, e.g. a disease associated polymorphism. Where large amounts of DNA are available, genomic DNA is used directly. Alternatively, the region of interest is cloned into a suitable vector and grown in sufficient quantity for analysis. Cells that express RAB may be used as a source of mRNA, which may be assayed directly or reverse transcribed into cDNA for analysis. The nucleic acid may be amplified by conventional techniques, such as the polymerase chain reaction (PCR), to provide sufficient amounts for analysis. The use of the polymerase chain reaction is described in Saiki, et al. (1985), Science 239:487, and a review of techniques may be found in Sambrook, ef al. Molecular Cloning: A Laboratory Manual, CSH Press 1989, ppJ 4.2-14.33. Alternatively, various methods are known in the art that utilize oligonucleotide ligation as a means of detecting polymorphisms, for examples see Riley et al. (1990), Nucl. Acids Res. 18:2887-2890; and Delahunty et al. (1996), Am. J. Hum. Genet. 58:1239-1246.

A detectable label may be included in an amplification reaction. Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, aliophycocyanin, 6-carboxyfluorescein (6-FAM), 2',7'-dimethoxy-4^,,5'-dichloro-6- carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4',7',4,7- hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA), radioactive labels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system, where the amplified DNA is conjugated to biotin, haptens, etc. having a high affinity binding partner, e.g. avidin, specific antibodies, etc., where the binding partner is conjugated to a detectable label. The label may be conjugated to one or both of the primers. Alternatively, the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product. The sample nucleic acid, e.g. amplified or cloned fragment, is analyzed by one of a number of methods known in the art. The nucleic acid may be sequenced by dideoxy or other methods, and the sequence of bases compared to a wild-type RAB sequence. Hybridization with the variant sequence may also be used to determine its presence, by Southern blots, dot blots, etc. The hybridization pattern of a control and variant sequence to an array of oligonucleotide probes immobilized on a solid support, as described in US 5,445,934, or in

WO 95/35505, may also be used as a means of detecting the presence of variant sequences.

Single strand conformational polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), and heteroduplex analysis in gel matrices are used to detect conformational changes created by DNA sequence variation as alterations in electrophoretic mobility. Alternatively, where a polymorphism creates or destroys a recognition site for a restriction endonuclease, the sample is digested with that endonuclease, and the products size fractionated to determine whether the fragment was digested. Fractionation is performed by gel or capillary electrophoresis, particularly acrylamide or agarose gels.

Screening for mutations in RAB may be based on the functional or antigenic characteristics of the protein. Protein truncation assays are useful in detecting deletions that may affect the biological activity of the protein. Various immunoassays designed to detect polymorphisms in RAB proteins may be used in screening. Where many diverse genetic mutations lead to a particular disease phenotype, functional protein assays have proven to be effective screening tools. The activity of the encoded RAB protein in GTP binding, GTPase activity, etc., may be determined by comparison with the wild-type protein.

Antibodies specific for a RAB may be used in staining or in immunoassays. Samples, as used herein, include biological fluids such as semen, blood, cerebrospinal fluid, tears, saliva, lymph, dialysis fluid and the like; organ or tissue culture derived fluids; and fluids extracted from physiological tissues. Also included in the term are derivatives and fractions of such fluids. The cells may be dissociated, in the case of solid tissues, or tissue sections may be analyzed. Alternatively a lysate of the cells may be prepared.

Diagnosis may be performed by a number of methods to determine the absence or presence or altered amounts of normal or abnormal RAB in patient cells. For example, detection may utilize staining of cells or histological sections, performed in accordance with conventional methods. Cells are permeabilized to stain cytoplasmic molecules. The antibodies of interest are added to the cell sample, and incubated for a period of time sufficient to allow binding to the epitope, usually at least about 10 minutes. The antibody may be labeled with radioisotopes, enzymes, fluorescers, chemiluminescers, or other labels for direct detection. Alternatively, a second stage antibody or reagent is used to amplify the signal. Such reagents are well known in the art. For example, the primary antibody may be conjugated to biotin, with horseradish peroxidase-conjugated avidin added as a second stage reagent. Alternatively, the secondary antibody conjugated to a flourescent compound, e.g. fluorescein, rhodamine, Texas red, etc. Final detection uses a substrate that undergoes a color change in the presence of the peroxidase. The absence or presence of antibody binding may be determined by various methods, including flow cytometry of dissociated cells, microscopy, radiography, scintillation counting, etc.

Diagnostic screening may also be performed for polymorphisms that are genetically linked to a disease predisposition, particularly through the use of microsatellite markers or single nucleotide polymorphisms. Frequently the microsatellite polymorphism itself is not phenotypically expressed, but is linked to sequences that result in a disease predisposition.

However, in some cases the microsatellite sequence itself may affect gene expression.

Microsatellite linkage analysis may be performed alone, or in combination with direct detection of polymorphisms, as described above. The use of microsatellite markers for genotyping is well documented. For examples, see Mansfield et al. (1994), Genomics 24:225-233; Ziegle et al. (1992), Genomics 14:1026-1031 ; Dib et al., supra.

MODULATION OF RAB GENE EXPRESSION The RAB genes, gene fragments, or the encoded RAB protein or protein fragments are useful in gene therapy to treat disorders associated with RAB defects. Expression vectors may be used to introduce the RAB gene into a cell. Such vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences. Transcription cassettes may be prepared comprising a transcription initiation region, the target gene or fragment thereof, and a transcriptional termination region. The transcription cassettes may be introduced into a variety of vectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like, where the vectors are able to transiently or stably be maintained in the cells, usually for a period of at least about one day, more usually for a period of at least about several days to several weeks. The gene or RAB protein may be introduced into tissues or host cells by any number of routes, including viral infection, microinjection, or fusion of vesicles. Jet injection may also be used for intramuscular administration, as described by Furth et al. (1992), Anal Biochem 205:365-368. The DNA may be coated onto gold microparticles, and delivered intradermally by a particle bombardment device, or "gene gun" as described in the literature (see, for example, Tang et al. (1992), Nature 356:152-154), where gold microprojectiles are coated with the RAB DNA, then bombarded into skin cells.

Antisense molecules can be used to down-regulate expression of RAB in cells. The anti-sense reagent may be antisense oligonucleotides (ODN), particularly synthetic ODN having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA. The antisense sequence is complementary to the mRNA of the targeted gene, and inhibits expression of the targeted gene products. Antisense molecules inhibit gene expression through various mechanisms, e.g. by reducing the amount of mRNA available for translation, through activation of RNAse H, or steric hindrance. One or a combination of antisense molecules may be administered, where a combination may comprise multiple different sequences.

Antisense molecules may be produced by expression of all or a part of the target gene sequence in an appropriate vector, where the transcriptional initiation is oriented such that an antisense strand is produced as an RNA molecule. Alternatively, the antisense molecule is a synthetic oligonucleotide. Antisense oligonucleotides will generally be at least about 7, usually at least about 12, more usually at least about 20 nucleotides in length, and not more than about 500, usually not more than about 50, more usually not more than about 35 nucleotides in length, where the length is governed by efficiency of inhibition, specificity, including absence of cross-reactivity, and the like. It has been found that short oligonucleotides, of from 7 to 8 bases in length, can be strong and selective inhibitors of gene expression (see Wagner et al. (1996), Nature Biotechnol. 14:840-844).

A specific region or regions of the endogenous sense strand mRNA sequence is chosen to be complemented by the antisense sequence. Selection of a specific sequence for the oligonucleotide may use an empirical method, where several candidate sequences are assayed for inhibition of expression of the target gene in an in vitro or animal model. A combination of sequences may also be used, where several regions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methods known in the art (see Wagner et al. (1993), supra, and Milligan et al., supra.) Preferred oligonucleotides are chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases. Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3'-O'-5'-S-phosphorothioate, 3'-S-5'-O-phosphorothioate, 3'-CH2-5'-O- phosphonate and 3'-NH-5'-O-phosphoroamidate. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage. Sugar modifications are also used to enhance stability and affinity. The α-anomer of deoxyribose may be used, where the base is inverted with respect to the natural β-anomer. The 2'-OH of the ribose sugar may be altered to form 2'-O-methyl or 2'-O-allyl sugars, which provides resistance to degradation without comprising affinity. Modification of the heterocyclic bases must maintain proper base pairing. Some useful substitutions include deoxyuridine for deoxythymidine; 5-methyl-2'- deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine. 5- propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and deoxycytidine, respectively.

As an alternative to anti-sense inhibitors, catalytic nucleic acid compounds, e.g. ribozymes, anti-sense conjugates, etc. may be used to inhibit gene expression. Ribozymes may be synthesized in vitro and administered to the patient, or may be encoded on an expression vector, from which the ribozyme is synthesized in the targeted cell (for example, see International patent application WO 9523225, and Beigelman et al. (1995), Nucl. Acids Res. 23:4434-42). Examples of oligonucleotides with catalytic activity are described in WO 9506764. Conjugates of anti-sense ODN with a metal complex, e.g. terpyridylCu(ll), capable of mediating mRNA hydrolysis are described in Bashkin et al. (1995), Appl. Biochem. Biotechnol. 54:43-56.

GENETICALLY ALTERED CELL OR ANIMAL MODELS FOR RAB FUNCTION

The subject nucleic acids can be used to generate transgenic, non-human animals or site specific gene modifications in cell lines. Transgenic animals may be made through homologous recombination, where the normal RAB locus is altered. Alternatively, a nucleic acid construct is randomly integrated into the genome. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like. The modified cells or animals are useful in the study of RAB function and regulation. For example, a series of small deletions and/or substitutions may be made in the host's native RAB gene to determine the role of different exons in protein transport, signal transduction, etc. Specific constructs of interest include anti-sense RAB, which will block RAB expression, expression of dominant negative RAB mutations, and over-expression of RAB genes. Where a RAB sequence is introduced, the introduced sequence may be either a complete or partial sequence of a RAB gene native to the host, or may be a complete or partial RAB sequence that is exogenous to the host animal, e.g., a human RAB sequence. A detectable marker, such as lac Z may be introduced into the RAB locus, where upregulation of RAB expression will result in an easily detected change in phenotype.

One may also provide for expression of the RAB gene or variants thereof in cells or tissues where it is not normally expressed, at levels not normally present in such cells or tissues, or at abnormal times of development. By providing expression of RAB protein in cells in which it is not normally produced, one can induce changes in cell behavior, e.g. through RAB-mediated intracellular signaling.

DNA constructs for homologous recombination will comprise at least a portion of the human RAB gene or of a RAB gene native to the species of the host animal, wherein the gene has the desired genetic modification(s), and includes regions of homology to the target locus.

DNA constructs for random integration need not include regions of homology to mediate recombination. Conveniently, markers for positive and negative selection are included. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For various techniques for transfecting mammalian cells, see Keown et al. (1990), Meth. Enzymol. 185:527-537.

For embryonic stem (ES) cells, an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, e.g. mouse, rat, guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder layer or grown in the presence of leukemia inhibiting factor (LIF). When ES or embryonic cells have been transformed, they may be used to produce transgenic animals. After transformation, the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be detected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct. Those colonies that are positive may then be used for embryo manipulation and blastocyst injection. Blastocysts are obtained from 4 to 6 week old superovulated females. The ES cells are trypsinized, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting offspring screened for the construct. By providing for a different phenotype of the blastocyst and the genetically modified cells, chimeric progeny can be readily detected. The chimeric animals are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allogeneic or congenic grafts or transplants, or in in vitro culture. The transgenic animals may be any non-human mammal, such as laboratory animals, domestic animals, etc. The transgenic animals may be used in functional studies, drug screening, etc., e.g. to determine the effect of a candidate drug on Ras or related gene activation, oncogenesis, etc.

IN VITRO MODELS FOR RAB FUNCTION The availability of a number of components in the Rab signaling pathway, as previously described, allows in vitro reconstruction of the pathway. Two or more of the components may be combined in vitro, and the behavior assessed in terms of activation of transcription of specific target sequences; modification of protein components, e.g. proteolytic processing, phosphorylation, methylation, etc.; ability of different protein components to bind to each other; utilization of GTP, etc. The components may be modified by sequence deletion, substitution, etc. to determine the functional role of specific domains.

Drug screening may be performed using an in vitro model, a genetically altered cell or animal, or purified RAB protein. One can identify ligands or substrates that bind to, modulate or mimic the action of RAB. Drug screening identifies agents that provide a replacement for RAB function in abnormal cells. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, and the like. The purified protein may also be used for determination of three-dimensional crystal structure, which can be used for modeling intermolecular interactions, such as GTP binding, etc. The term "agent" as used herein describes any molecule, e.g. protein or pharmaceutical, with the capability of altering or mimicking the physiological function of RAB.

Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.

Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

Where the screening assay is a binding assay, one or more of the molecules may be joined to a label, where the label can directly or indirectly provide a detectable signal. Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e.g. magnetic particles, and the like. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc. For the specific binding members, the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures. A variety of other reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used. The mixture of components are added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4 and 40^°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0J and 1 hours will be sufficient. Other assays of interest detect agents that mimic RAB function, such as GTP binding properties, GTPase activity, etc. For example, an expression construct comprising a RAB gene may be introduced into a cell line under conditions that allow expression. The level of RAB activity is determined by a functional assay, as previously described. In one screening assay, candidate agents are added in combination with GTP, and activity in conversion of GTP to GDP is detected. In another assay, the ability of candidate agents to inhibit or enhance RAB function is determined. Alternatively, candidate agents are added to a cell that lacks functional RAB, and screened for the ability to reproduce RAB in a functional assay.

The compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host for treatment of cancer, etc. The compounds may also be used to enhance RAB function. The inhibitory agents may be administered in a variety of ways, orally, topically, parenterally e.g. subcutaneously, intraperitoneally, by viral infection, intravascularly, etc. Topical treatments are of particular interest. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt.%.

The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds. Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.

EXPERIMENTAL

All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing, for example, the compounds and methodologies that are described in the publications which might be used in connection with the presently described invention. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.

Methods and Materials;

GENBANK Database searching;

Peptide sequences representative of 74 known Rab proteins from a variety of plant and animal species (Table 1) were used in TBLASTN searches to screen the Expressed Sequence TAG (EST) database (dbEST). EST homologies which had a p-score >0.0001 or were non-human, were discarded. The remaining ESTs were used in a BLASTN screen against the NR database. ESTs which had top human hits with >95% identity over 100 nucleotides were discarded. This was based upon our experience which has shown that these sequences were usually identical to starting probe sequences, with the differences due to sequence error. The remaining ESTs were BLASTX screened against the NR database. These outputs, in concert with the corresponding BLASTN outputs, were manually assessed to identify ESTs representing potential novel human rab-related genes. These ESTs were used to screen the UNIGENE database. All ESTs from matching contigs were recovered and aligned using the SEQUENCHER program (GENE CODES Corp.). Consensus sequences from SEQUENCHER contigs were submitted for BLASTN and BLASTX analyses. Representative IMAGE clones (Research Genetics) from each rab-related contig were chosen for sequence analyses (Table 2). Table 1 List of the protein probes used in experiments to identify human Rab sequences

Table 2 Summary of representative IMAGE and BAC clones, pius mapping data for human Rab sequences.

Cloning and sequence analysis;

IMAGE clones were sequenced using standard ABI dye-primer and dye-terminator chemistry on a 377 automated DNA sequencer. When necessary the N-terminus of the gene was recovered using the RACE method (Rapid amplification of cDNA ends) to achieve full length coding sequences. A nested primer strategy, using two gene specific oligos, was used on cDNA with attached RACE linkers (Clontech), from a variety of different tissues. RT-PCR data was used to identify a cDNA source for RACE experiments. RACE PCR products were sub-cloned using the TA cloning kit from INVITROGEN. A third nested oligo was used for RACE product confirmation by colony hybridization or PCR. Gene specific RACE oligo sequences are listed in Table 3a.

Chromosomal localization;

PCR primers were designed in the 3' UTR sequences of rab2b, rab3C, rab4B, rab10, rab18, rab20B, rab22, rab24, rab 25 and rab36, which were used to amplify products in either the Stanford G3 radiation hybrid(RH) panel (http://www-shgc.stanford.edu/RH/index.html) or the GENEBRIDGE 4 RH panel (http://-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl). A BAC clone representing rab14 was isolated by PCR from BAC DNA pool plates (Research Genetics). Since PCR assays could not be designed that clearly distinguished between human and hamster rab14 sequences in the RH mapping panels, RH mapping primers were designed using the non-repetitive end sequence of a rab14 specific human BAC clone. This BAC clone was isolated by PCR from BAC DNA pool plates (Research Genetics).

RH panel DNAs were aliquoted into 96-well trays, dried for storage, and resuspended in PCR buffer prior to PCR amplification. A 20 μl PCR reaction (2.5 mM MgCI₂) was performed using Taq Gold polymerase (Perkin Elmer). The assays were done in duplicate. PCR data were submitted to the appropriate URL for automatic 2 point linkage analysis. Mapping data were correlated with cytoband using the Genetic location database (http://cedar.genetics.soton.ac.uk/public_html/). Gene specific RH mapping oligo sequences are listed in Table 3b. Table 3b, Oligos used in RT-PCR and RH-mapping

Rab34 and 35 were mapped by localizing representative BAC clones by hybridization to human metaphase chromosome spreads in FISH experiments, following standard protocols (Lichter et al., 1990, Selleri et al., 1991). BAC probes were direct labeled with Spectrum Red (VYSIS) and chromosomes counterstained with DAPI (100ng/ml). Additional verification was provided by sequences from rab14, rab34, rab35 were PCR amplified from cognate BAC clones and sequenced, and over stretches ranging from 135-205 bp, all PCR products showed 100% identity to the predicted RAB sequences.

Expression analysis;

Total RNA was purchased (Clontech, Invitrogen) and used to synthesize first strand cDNA using M-MLV reverse transcriptase and the supplied buffer (Gibco-BRL). The 20 μl reaction contained 5 μg total RNA, 100 ng of random hexamer primers, 10 mM DTT, 0.5 mM each dNTP, and an RNAse inhibitor (Gibco-BRL). Identical reactions were set up without reverse transcriptase to control for DNA contamination in the RNA samples. The synthesis reaction proceeded for 1 hour at 37°C followed by 10 minutes at 95°C. The cDNAs were tested with primers with defined expression patterns to verify the presence of amplifiable cDNA in each well. The cDNAs were diluted 1 :5 and 2 μl of each sample were arrayed into 96-well trays, dried, and resuspended in PCR buffer prior to PCR amplification. 20 μl PCR reactions with standard conditions, 2.5 mM MgCI2, Taq Gold (Perkin Elmer) were used to detect expression.

Results and discussion;

Database screening and full length cloning;

After extensive analysis of the BLASTN and BLASTX outputs of ESTs identified during the initial screening, 23 ESTs representing a potential novel human sequence related to a rab protein were identified. IMAGE clones were identified for each of these genes and clone end sequencing was performed. Of these 23 potential novel genes the clone end sequencing proved that 13 were distinct new rab proteins. 5' RACE experiments were utilized to amplify the N-terminus of rab4b, rab20B, rab22 and rab24. The nucleotide and predicted peptide coding regions for each novel rab-related gene are provided in the sequence listing.

Sequence analysis

BLASTX analysis indicated that 2 of these genes were completely novel rab-related proteins, unreported in other species. Their closest matches were <80% amino acid identity to reported rab proteins, which would indicate that they are novel members of the rab subfamily of small GTPases, rather than the ras, rho, ran, arf or sar sub-families. In accordance with the nomenclature guidelines established by Kahn et al., (1992), they were given the next available rab names of rab34 and rab35. The most closely related proteins to rab34 were human and canine rab9 proteins (76% amino acid identity) and the human rab7 protein (56% amino acid identity). Rab35 was most closely related (76% amino acid identity), to an unnamed human GTP-binding protein (GENBANK accession number: X99962). Rab35 also showed high similarity to a C.elegans YPT1-like protein (64% amino acid identity) and to D. melanogaster rab2 (53% amino acid identity). Sequence alignment of rab34 and 35 with the other 11 rab proteins cloned in this study indicated that they encoded the four conserved GTP-binding motifs found in all rab proteins and the C-terminal cysteine residues required for post-translational processing. Another 2 of these genes showed a high similarity to reported rab proteins, but with a 10-15% amino acid divergence. Sequences from the IMAGE clone 938157, showed 80-89% identity to mouse rab20, with the greatest divergence from the mouse protein at the C-terminal end of the gene. In comparison with what is generally seen for rab proteins the true human ortholog of mouse rab20 would be expected to share a 95-100% amino acid identity with mouse rab20. Therefore we have designated the human sequence rab20B, and have renamed the mouse protein as rab20A. Sequences from IMAGE clone 30566, showed 83% identity to human and mouse rab2 sequences. Again the majority of sequence divergence was at the C-terminus of the protein. Therefore we designated our new gene sequence as human rab2B.

BLASTN and BLASTX analysis of the remaining 9 full length clones indicated that these sequences showed very high (>96%) amino acid identity to rab proteins reported in other species. In accordance with the nomenclature guidelines established by Kahn et al., (1992), these genes were designated the human orthologs of their closest reported matching mammalian counterpart. These included the human orthologs of ; rat and bovine rab3C, rat and canine rab4B, rat, canine and murine rab10, rat and drosophila rab14, murine and snail rabl 8, canine rab22, murine rab24 and rabbit rab25. The top hit for the ninth gene sequence was 96% amino acid identity to an un-named rat GTPase binding protein (M94043). This was given the name rab36, which was the next available novel rab name. Expression data;

The RT-PCR data (Table 4) indicates that these genes are typically widely expressed with the notable exception of rab24 which is exclusively expressed in the adult brain. Murine rab24 has been suggested as a candidate for purkinje cell degeneration in the mouse and this pattern of expression is consistent with that type of phenotype. Rab2B is the only gene which shows a ubiquitous expression in the tissues tested. The data for rab25 come from northern blot analysis using MTN blots I, II and III (Clontech) according to the manufacturers instructions. Certain tissues from the RT-PCR panel were not tested by Northern analysis and are left as blank boxes on Table 3. Other additional Northern data, indicates that rab25 is expressed in the thyroid gland, but not in bone marrow, spinal cord, lymph node, ovary or peripheral blood leukocytes.

= this data is from Northern blot analysis, not from PCR.

Chromosomal localization;

The marker to which RH-mapping indicated closest linkage and the corresponding cytogenetic positions are presented in Table 2. The identity of the BAC clones for rab14, 34 and 35 which were utilized in mapping are also presented in this table. Previously, Barbosa et al. (1995) Genomics 30(3); 439-44 and McMurtie et al., (1997) used interspecific backcross mice to localize murine orthologs of rab4B, rab10, rab18, rab22 and rab24 in the mouse. Using predicted linkage conservation patterns between human and mouse, they proposed the putative human localization of these genes. Our mapping data provide direct experimental evidence to confirm and refine these proposed chromosomal localizations. The results of our RH mapping panel clearly indicate that human rab4B maps to human chromosome 19q13 and that rab22 maps to 20q13. The human map position of 2p23-24 previously proposed for human rab10 was amended to 2p21-22, after consideration of the RH-mapping linkage. Using our RH result for rab24 we were able to narrow the proposed localization of rab24 from all of human 5q to 5q35. In addition we were able to clarify which of two previously proposed locations for human rab18, 10p11 or 18p11-12 was correct. The finding that human rab20B maps to a different location, 13q30, rather than the 8p1 1-p23 predicted by synteny with the mouse rab20A (mapping to proximal mouse chromosome 8), supports the idea that they are different isoforms of rab20 and the sequence divergence between mouse rab20A and human rab20B are not due to species differences alone. This cytoband information, and presence of a linked physical marker for each of these genes (except rab34 and rab35 which were FISH mapped, resulting only in cytoband information), allows us to examine reported disease linkages in each chromosome region, to asses the potential candidacy of these rab genes in Mendelian disease. Since rab proteins are implicated in the regulation of membrane traffic, defects in rab genes could potentially affect several aspects of the intracellular protein transport machinery.

Rab4B maps near the spontaneous mouse mutation reduced pigmentation (rp) (Brilliant et al., 1994 Mamm Genome 5; S 104-23) which is characterized by immune deficiency and a diluted coat color pigmentation. In addition rp mice exhibit morphologic abnormalities in the lysosomes and melanosomes associated with defects in the trafficking of renal lysosomal glycosidases (Gibb et al., 1981). Again, no similar human phenotype has yet been linked to human 19q13, although this does not exclude murine rab4B as a candidate for rp.

Claims

WHAT IS CLAIMED IS:

1. An isolated nucleic acid encoding a mammalian RAB protein.

2. An isolated nucleic acid according to Claim 1 , wherein said RAB protein has the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26.

3. An isolated nucleic acid according to Claim 1 , wherein said RAB protein has an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26..

4. An isolated nucleic acid according to Claim 1 wherein the nucleotide sequence of said nucleic acid is SEQ ID NO:1, 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23 or 25.

5. An isolated nucleic acid comprising at least 18 contiguous nucleotides of the sequence of SEQ ID NO:1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23 or 25.

6. An isolated nucleic acid comprising at least 50 contiguous nucleotides of the sequence σf SEQ ID NO.1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23 or 25.

7. An isolated nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23 or 25.

8. An expression cassette comprising a transcriptional initiation region functional in an expression host, a nucleic acid having a sequence of the isolated nucleic acid according to Claim 1 under the transcriptional regulation of said transcriptional initiation region, and a transcriptional termination region functional in said expression host.

9. A cell comprising an expression cassette according to Claim 8 as part of an extrachromosomal element or integrated into the genome of a host cell as a result of introduction of said expression cassette into said host cell, and the cellular progeny of said host cell.

10. A method for producing mammalian RAB protein, said method comprising: growing a cell according to Claim 9, whereby said mammalian RAB protein is expressed; and isolating said RAB protein substantially free of other proteins.

11. A purified polypeptide composition comprising at least 50 weight % of the protein present as a RAB protein or a fragment thereof.

12. A monoclonal antibody binding specifically to a RAB protein.

13. A non-human transgenic animal model for RAB gene function wherein said transgenic animal comprises an introduced alteration in a RAB gene.

14. The animal model of claim 13, wherein said animal is heterozygous for said introduced alteration.

15. The animal model of claim 13, wherein said animal is homozygous for said introduced alteration.

16. The animal model of claim 13, wherein said introduced alteration is a knockout of endogenous RAB gene expression.