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WO1999058669A1 - Genes rhoh et leurs utilisations - Google Patents

Genes rhoh et leurs utilisations Download PDF

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Publication number
WO1999058669A1
WO1999058669A1 PCT/US1999/010061 US9910061W WO9958669A1 WO 1999058669 A1 WO1999058669 A1 WO 1999058669A1 US 9910061 W US9910061 W US 9910061W WO 9958669 A1 WO9958669 A1 WO 9958669A1
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Prior art keywords
rhoh
polypeptide
seq
sequence
gene
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PCT/US1999/010061
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English (en)
Inventor
Maxine J. Allen
Raquel Vega
Marc Rutter
Ken Abel
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Axys Pharmaceuticals, Inc.
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Priority to AU39757/99A priority Critical patent/AU3975799A/en
Publication of WO1999058669A1 publication Critical patent/WO1999058669A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4722—G-proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out

Definitions

  • GTP-binding proteins represent a class of small, approximately 20 to 30 kDa, monomeric, receptor-coupled GTPases, which mediate signal transduction in eukaryotic cells. GTP-binding proteins act as molecular switches, alternating between an active GTP-bound state and an inactive GDP-bound state. Subfamily members of the GTP-binding proteins include ras proteins, associated with the regulation of cell proliferation and differentiation, rho and rac proteins, associated with the regulation of cytoskeletal assembly, and rab, arf, sar and ran proteins, associated with the regulation of vesicular transport (Bourne et al. (1991 ), Nature. 349: 17-127; Hall and Zerial (1995) General introduction.
  • the rho sub-family of small ras-related GTPase proteins has been shown to regulate a wide range of cellular processes, ranging from cytoskeletal assembly in response to external stimuli, through membrane trafficking, cell growth control and development (reviewed in Aelst et al.(1997) Genes Dev. 11:2295-2322).
  • the mammalian rho subfamily comprises of at least ten proteins: Rho A, B, C, D, E and G, Rad and Rac2, CDC42Hs and TC10. These ten proteins share approximately 30% identity with p21H-ras and over 50% identity with one another.
  • Rho proteins function as molecular switches cycling between an active GTP-bound form and an inactive GDP-bound form (Bourne et al., 1991 , supra). This cycling is catalyzed by at least three classes of proteins: guanine nucleotide exchange factors (GEFs), which promote exchange between bound GDP and cytoplasmic GTP; GTPase-activating proteins (GAPs), which stimulate the low intrinsic GTPase activity of small GTPases resulting in the inactive GDP bound state; and guanine nucleotide dissociation inhibitors (GDIs), which can inhibit both the exchange of GTP and the hydrolysis of bound GTP (Boguski and McCormick (1993) Nature 366:643-654; Aelst et al. (1997), supra).
  • GEFs guanine nucleotide exchange factors
  • GAPs GTPase-activating proteins
  • GDIs guanine nucleotide dissociation inhibitors
  • Rho proteins Close homology of the Rho proteins is confined to four domains known to be involved in GTP binding and hydrolysis in ras and conserved in all small GTP-binding proteins. All Rho proteins contain two prolines (in positions 71 and 75 in RhoA) within the ⁇ 2helix of p21Hras, resulting in structural differences between Rho and Ras protein subfamilies. Like ras, rho family members also include a C-terminal CAAX motif, used as a signal for posttranslational modification.
  • ras-subfamily proto-oncogenes such as K-ras and TC21 have long been associated with cellular transformation resulting in tumor formation. More recently in vitro studies have shown that overexpression of the rho-subfamily members rhoA, rhoB, and rad can enhance the process of ras-mediated cellular transformation, and that dominant negative mutants of rhoA (Asn19) inhibit transformation by oncogenic ras (Qui et al.(1995) Nature 374:457 9; Khosravi-Far et al. (1995) Mol Cell Biol 15(11):6443-6453).
  • Rho GTPases and the enzymes modifying their activities are known to have central roles in pathways underlying several human diseases.
  • DFNA1 non-syndromic familial deafness
  • the defect appears to be in the regulation of actin polymerization in inner ear hair cells.
  • the gene product encoded by DFNA1 was identified as an effector of Rho, indicating the critical importance of Rho in normal cytoskeletal assembly (Lynch et al. (1997) Science 278:1315- 1318).
  • Rho/Rac guanine nucleotide exchange factor GEF
  • Rho GEF's have been identified as oncogenes in transfection assays; however one of these, VAV, is similar to FGD1 in that it was also shown to be essential for embryonic development in mice.
  • VAV the normal cycling between GDP- and GTP-bound states for at least some Rho family members appears essential for both cellular growth control and for normal development.
  • Rho gene products potentially either represent direct targets themselves, or point to their modifying enzymes or effectors as targets for therapeutic intervention.
  • novel rho proteins which increases the number of known members of this small sub-family of vitally important signaling molecules, is of crucial importance in elucidating the role these proteins may play in human disease.
  • EST sequences present within the RHOH polynucleotide sequence of the invention are summarized in Table 1 below.
  • the nucleotide residues of the RHOH sequence to which there is greater than 90% identity to a provided EST is indicated in the last column.
  • Rho proteins The function of Rho proteins has been the subject of several recent reviews.
  • Rho GTPases The role of Rho GTPases in regulation of the actin cytoskeleton is reviewed in Hall (1998) Science 279:509-14; Dharmawardhane et al. (1997) Curr. Opin. Hematol. 4:12-18; and Hochtin et al. (1996) Cancer Surv. 27:311-22 (also discussing a role of Rho in tumor formation).
  • Rho in regulation of proliferation and apoptosis is reviewed in Lacal (1997) FEBS Lett 410:73-7.
  • Rho The role of Rho in cytoskeletal regulation, as well as its possible role in metastasis and invasiveness of carcinoma cells, is discussed in Takai et al. (1994) P ⁇ ncess Takamatsu Symp. 24:338-50; and reviewed in Aelst et al.(1997) Genes Dev. 11:2295-2322.
  • the invention features polynucleotides encoding novel GTP-binding polypeptides, hereinafter referred to as RHOH polypeptides; expression vectors comprising a RHOH polynucleotide of the invention; isolated cells comprising a RHOH-encoding vector; a transgenic non-human animal comprising an alteration in a RHOH gene; and the use of RHOH polynucleotides in detecting in an individual the presence of a genetic polymorphism of a RHOH gene.
  • the invention further features novel RHOH polypeptides; monoclonal antibodies specific for RHOH polypeptides; and a method for making RHOH polypeptides.
  • FIG. 1A shows the nucleotide sequence of RHOH (SEQ ID NO:3).
  • the coding region (SEQ ID NO:1) is enclosed; the translational start and stop sites are underlined.
  • the 5' UTR is 327 nt, the 3' UTR is 420 nt.
  • Fig. 1B shows the predicted polypeptide sequence of RHOH (SEQ ID NO:2).
  • Fig. 2 is a sequence alignment of the RHOH protein with related Rho-GTPases TC10 (human), CDC42 (canine), RhoA (human), RhoB (human), RhoC (human), RhoD (mouse), RhoE (human), Rac1 (human), Rac2 (human), and RhoG (human). Alignments were performed using the PILEUP program (Wisconsin Package, version 9.1 , Genetics Computer Group, Madison, WI)Dots represent spaces to optimize alignment. Residues occurring in 9 or more of these proteins are capitalized and comprise the consensus sequence.
  • Fig. 3 is a dendrogram showing the phylogenetic relationship between RHOH and other small GTPase proteins selected from the Rho-subfamily.
  • RHOH gene is intended to generically refer to both the wild-type and allelic forms of the sequence, unless specifically denoted otherwise.
  • gene is intended to refer to the genomic region encompassing 5' UTR, exons, introns, and 3' UTR. Of the 5' UTR and 3'UTR, those sequences involved in the regulation of expression are of particular interest; such sequences are positioned generally up to about 20 kb beyond the coding region, but possible further in either the 5' or 3' direction. Individual segments may be specifically referred to, e.g.
  • RHOH polynucleotide is meant to include any polynucleotide having substantial identity to a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment, variant, or analog thereof, as well as any polynucleotide encoding a polypeptide having substantial identity to the amino acid sequence of SEQ ID NO:2 NO:3 or a fragment or variant thereof.
  • RHOH polynucleotide is meant to refer to RHOH RNA, cDNA, RHOH genomic DNA, and fragments, variants, and analogs thereof, as well as polynucleotides encoding RHOH polypeptide fragments or variants, unless specifically indicated otherwise.
  • RHOH polypeptide is meant to include any polypeptide having substantial identity to the amino acid sequence of SEQ ID NO:2 or a fragment, variant, or analog thereof, or a polypeptide encoded by a polynucleotide having substantial identity to the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment, variant, or analog thereof.
  • RHOH polypeptide is meant to refer to the full-length polypeptide, as well as fragments, variants, and analogs thereof, particularly those that fragments, variants, and analogs, especially those that retain biological activity, (e.g., biologically active fragments (e.g., fragments corresponding to functional domains such as GTP-binding domains), fusion proteins comprising all or a portion of a RHOH polypeptide, and the like) unless specifically indicated otherwise.
  • biologically active fragments e.g., fragments corresponding to functional domains such as GTP-binding domains
  • fusion proteins comprising all or a portion of a RHOH polypeptide, and the like
  • “Substantial identity” when referring to the RHOH polynucleotides of this Invention, means polynucleotides having at least about 80%, typically at least about 90% and preferably at least about 95% sequence identity to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:3. Sequence identity 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.
  • -5- reference sequence will usually be at least about 18 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. (1990) J Mol Biol 215:403-10.
  • the DUST filter is described at http://www.ncbi.nlm.nih.gov/ BLAST/filtered.html.
  • Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50°C and 6XSSC (0.9 M saline/0.09 M sodium citrate) and remain bound when subjected to washing at 55°C in 1XSSC (0.15 M sodium chloride/0.015 M sodium citrate).
  • Sequence identity may be determined by hybridization under stringent conditions, for example, at 50°C or higher and 0.1XSSC (15 mM sodium chloride/0.15 mM sodium citrate).
  • probes particularly labeled probes of DNA sequences
  • the source of homologous genes may be any species, e.g. primate species, particularly human; rodents, such as rats and mice, canines, felines, bovines, ovines, equines, yeast, Drosophila, Caenhorabditis, etc.
  • the DUST filter is described at http://www.ncbi.nlm.nih.gov/BLAST/filtered.html. For a discussion of the sequence identity of Rho subfamily members, see Kahn et al. (1992) FASEB J. 6:2512-3.
  • RHOH polynucleotides and polypeptides of this invention include, without limitation, RhoH polypeptides and polynucleotides found in primates, rodents, canines, felines, equines, nematodes, yeast and the like, and the natural and non-natural variants thereof.
  • Bio activities of a RHOH polypeptide include, but are not necessarily limited to, for example, regulatory or biochemical functions, (e.g., GTPase activity, GTP-binding, interaction with a polypeptide as in a signaling pathway, and the like), antigenic activity, to be
  • RHOH polypeptide -6- bound by an immunoglobulin or T cell antigen receptor specific for a RHOH polypeptide, and the like), structural motifs, and other activities associated with naturally occurring RHOH polypeptide.
  • cDNA means a polynucleotide having a nucleotide sequence corresponding to the wild-type mRNA, i.e., the same arrangement of exons and 3' and 5' non-coding regions.
  • RNA splicing 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
  • Polypeptide fragment when referring to RHOH polypeptides of the invention, means any portion of a RHOH polypeptide, preferably one that retains substantially the same biological activity as that of the polypeptide having the amino acid sequence of SEQ ID NO:2.
  • Exemplary RHOH polypeptide fragments are RHOH polypeptide fragments corresponding to functional domains, e.g., GTP-binding domains, GTPase catalytic domains and the like, of a RHOH polypeptide (e.g., SEQ ID NO:2).
  • Polypeptide fragments of the invention are typically greater than 8 amino acids, usually about 12 to 20 amino acids, more usually about 50 to 100 amino acids, generally 150 amino acids or more, up to at least about 90-95% of the full-length polypeptide.
  • polypeptide fragments that are greater than about 150 to 160 amino acids (e.g., at least about 151 amino acids), usually greater than about 160 to 180 amino acids, normally about 200 amino acids, in length.
  • Polynucleotide fragment when referring to RHOH polynucleotides of the invention, means polynucleotide fragments that are useful as probes, primers, antisense sequences for inhibition of transcription and/or translation, as well as coding sequences for the production of RHOH polypeptides and fragments thereof, and the like. Of particular interest are polynucleotide fragments of the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.
  • Polynucleotide fragments comprising the RHOH polynucleotides of the invention typically have greater than 95% identity to a RHOH polynucleotide of the invention and are greater than about 420 to 450 nucleotides (e.g., at least about 421 nucleotides), usually 450 to 480 nucleotides or 500 to 640 nucleotides, in size.
  • Polynucleotides fragments of interest also include fragments of the RHOH coding sequence of greater than about 420 nucleotides (e.g., at least about 421 nucleotides), usually greater than bout 450 to about 500 nucleotides, up to the entire RHOH coding sequence.
  • Genomic DNA means a polynucleotide having substantially the same nucleotide sequence, i.e., the same initiation codon, the stop codon and all intervening exons and introns, present in a corresponding wild type chromosome. Of particular interest is a genomic sequence comprising the nucleic acid present between the initiation codon and the stop codon,
  • Genomic DNA may include 3' and 5' untranslated regions found in mature mRNA. Genomic DNA may include transcriptional and translational regulatory sequences, such as promoters, enhancers, and the like, and may include approximately 1 kb, or more, of flanking genomic DNA at the 5' or 3' end of the transcribed region, i.e., sequences required for proper tissue and stage specific expression. Alternatively, the genomic DNA may comprise a fragment of 100 kbp, or less, and substantially free of flanking genomic DNA. The genomic DNA flanking the coding region, either 3' or 5', or internal regulatory sequences as sometimes found in introns, may contain sequences required for proper tissue and stage specific expression.
  • isolated means separated from some or all other material present in the natural environment of the RHOH polynucleotide or RHOH polypeptide.
  • RHOH polynucleotides of the invention are generally isolated as other than an intact chromosome. Molecules so isolated can be introduced into vectors, host cells or whole organisms or occur in composition with other ingredients and still be considered isolated, as such term is used herein.
  • RHOH polynucleotides and polypeptides of the Invention are isolated in substantial purity. Substantial purity, when referring to polynucleotides, means generally at least about 50%, typically at least about 90%, pure.
  • RHOH polynucleotides of the invention include "recombinant" polynucleotides, i.e. a RHOH sequence flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome.
  • Antigenic activity means the capacity to elicit an immune response (e.g., B cell- or T cell-mediated response) and/or be bound by polypeptide-specific antibodies, (e.g., RHOH polypeptide-specific antibodies).
  • Substantially the same biological activity means that activity which would be considered meaningful, qualitatively and/or quantitatively, by one of ordinary skill in the relevant art.
  • Variant means a polynucleotide or polypeptide which differs from a provided polynucleotide or polypeptide by one or more residues (e.g., nucleotide residue or amino acid residue), and include polynucleotides or polypeptides having residue substitutions, deletions and/or insertions relative to a reference polynucleotide or polypeptide sequence.
  • residues e.g., nucleotide residue or amino acid residue
  • “Naturally occurring variants” means polynucleotides and polypeptides derived from natural sources and "non-natural variants" means variants that have been artificially produced.
  • non-natural variants may differ from the polynucleotide or polypeptide referred to by at least two, but not more than about ten, nucleotides or amino acids.
  • variants refers to polynucleotides or polypeptides that are changed in their sequence relative to a reference sequence, but that have substantially no change in the chemical composition of the individual residues or the backbone of the molecule.
  • Analog means a polynucleotide or polypeptide that differs from a provided polynucleotide or polypeptide due to the presence of modifications in the chemical composition of one or more residues (e.g., an amino acid residue or a nucleotide residue) and/or modifications to the chemistry of the backbone of the molecule. Analogs of amino acids and polynucleotides, and methods of production of same, are well known in the art. Chemical “derivatives” (e.g., as “derivative” is used in the chemical arts) are meant to be encompassed by the use of the term"analog" in accordance with the invention.
  • polynucleotides of the invention can be modified so as to contain synthetic nucleotide analogs. Such analogs may be preferred for use as probes because of superior stability under assay conditions.
  • Modifications in the native structure including alterations in the backbone, sugars or heterocyclic bases, have been shown to increase intracellular stability and binding affinity.
  • 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 analogs include 3'-0'-5'-S-phosphorothioate, 3'-S-5'-0- phosphorothioate, 3'-CH 2 -5'-0-phosphonate and 3'-NH-5'-0-phosphoroamidate.
  • Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage.
  • Sugar modifications are also used to enhance stability and affinity.
  • the a-anomer of deoxyribose may be used, where the base is inverted with respect to the natural b-anomer.
  • the 2'-OH of the ribose sugar may be altered to form 2'-0- methyl or 2'-0-allyl sugars, which provides resistance to degradation without comprising affinity.
  • 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.
  • wild-type may be used to refer to the most common allele in a population.
  • wild-type is merely a convenient label for a common allele, and should not be construed as conferring any particular property on that form of the sequence.
  • a preferred aspect of the invention are isolated RHOH polynucleotides selected from (i) polynucleotides having substantial identity to a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3 and (ii) polynucleotides encoding polypeptides having substantial identity to an amino acid sequence of SEQ ID NO:2. Particularly preferred are those RHOH polynucleotides having the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3 and those RHOH polynucleotides encoding a polypeptide having the amino acid sequence of SEQ ID NO:2.
  • a preferred aspect of the invention are isolated RHOH polypeptides selected from
  • Particularly preferred are those RHOH polypeptides having the nucleotide sequence of SEQ ID NO:2 and those RHOH polypeptides encoded by polynucleotides having the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3; and fragments, variants, and analogs thereof.
  • Human RHOH polynucleotides of the invention comprise the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, and fragments, analogs, and variants thereof.
  • the human gene sequence is provided as SEQ ID NO:3, and the encoded polypeptide product as SEQ ID NO:2.
  • the longest open reading frame (ORF), encoded by SEQ ID NO:1 of the human gene encodes a 214 amino acid polypeptide.
  • the chromosomal location of the human gene has been localized to 14q22-23.
  • the amino acid sequence of SEQ ID NO:2 is consistent with the primary structure of a GTP-binding protein, such as those within the rho GTPase subfamily.
  • RHOH conserved GTP-binding domains characteristic of GTP-binding proteins are found in the full- length RHOH polypeptide (Fig. 2, domains I, III, IV, and V).
  • RHOH also contains the conserved effector domain (Fig. 2, II), suggesting that RHOH may interact with some of the same downstream effector molecules as other Rho subfamily members.
  • RHOH shares the highest level of amino acid sequence identity (approximately 80% identity) with the human Rho protein TC10.
  • RHOH contains the sequence the CAAX motif, CSII (SEQ ID NO:17).
  • Rho proteins except TC10, RhoD, and RhoE contain a CAAX motif ending with a leucine residue, indicating that the protein is a substrate for modification by geranylgeranylation rather than famesylation.
  • the RHOH sequence encodes a CAAX motif but, in contrast to the majority of
  • RHOH CAAX box terminates with an isoieucine rather than a leucine.
  • RHOH expression was detected in most tissues tested by RT-PCR including, adipose, adrenal gland, bladder, brain, cerebellum, cervix, colon, esophagus, fetal brain, fetal liver, heart, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, rectum, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus, trachea, and uterus.
  • RhoH expression was not detectable by RT-PCR using Hela cell RNA. This pattern of expression may reflect the widespread involvement of the RHOH protein in regulating the cellular activities associated with the Rho protein subfamily, and may further indicate that mutations in RHOH may be associated with aberrant pathologies in a variety of tissues.
  • polynucleotides and polypeptides of the invention are useful for detecting in an individual the presence of a genetic polymorphism of a RHOH gene associated with a disease state or genetic predisposition to a disease state.
  • Polymorphisms associated with disease states or genetic predispositions to a disease state include, without limitation, deletion or truncation of the gene and mutations that affect gene expression, the affinity of the RHOH polypeptide for GTP and/or the GTPase activity of the RHOH polypeptide.
  • Fragments comprising the RHOH polynucleotides of the invention are useful as primers for PCR, probes in hybridization screening, and the like.
  • the polynucleotides of the invention also can be used as probes in identifying the level of expression of the RHOH gene in biological specimens. Such methods are well known in the art. In short, DNA or mRNA is isolated from a cell sample, mRNA is amplified by RT-PCR or separated by gel electrophoresis, and probed with a fragment of the RHOH polynucleotide. Oligonucleotide ligation, in situ hybridization and solid chip DNA array techniques also may be used.
  • Polynucleotides comprising the 5'-flanking region of the RHOH gene are useful for their promoter elements, including enhancer binding sites.
  • 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 RHOH is expressed. The tissue specific
  • -11- expression is useful for determining the pattern of expression, and for providing promoters that mimic the native pattern of expression.
  • Naturally occurring variants of polynucleotides containing the promoter region can be used to determine polymorphisms in RHOH gene expression that are associated with a particular disease.
  • 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 e a/. (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 of RHOH may be used to identify cis acting sequences required for transcriptional or translational regulation of RHOH expression, especially in different tissues or stages of development, and to identify cis acting sequences and transacting factors that regulate or mediate RHOH expression.
  • Such transcription or translational control regions may be operably linked to a RHOH gene in order to promote expression of wild type or altered RHOH or other proteins of interest in cultured cells, or in embryonic, fetal or adult tissues, and for gene therapy.
  • variants of RHOH polynucleotides can be used to, for example, detect alteration of expression in experimentally defined systems, and other uses that will be readily apparent to one of ordinary skill in the art.
  • variants include polynucleotide sequences having mutations introduced into a 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 er a/. (1996) Genome Res.
  • fusion proteins comprising FLAGTM epitope tags or green fluorescent protein sequences.
  • Polynucleotides encoding RHOH may be cDNA, genomic DNA, or a fragment, variant, or analog thereof, and can be prepared by methods known to those of ordinary skill in the art provided with the information of the present application.
  • cDNA libraries can be screened with hybridization probes designed to complement a portion of the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.
  • Suitable probes will be of approximately 18 nucleotides to the full length of the gene, more typically approximately 30 nucleotides in length.
  • -12- Low stringency conditions e.g., 50° C and 6XSSC (0.9 M saiine/0.09 M sodium citrate) are used to identify and isolate RHOH polynucleotides having nucleotide sequences similar to that of the probe.
  • High stringency conditions e.g., 50° C or higher and 0.1XSSC (15 mM saline/1.5 mM sodium citrate) are used to identify and isolated RHOH polynucleotides having nucleotides essentially identical to that of the probe.
  • the RHOH polynucleotides may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into a host genome.
  • the RHOH polynucleotides of the invention may be obtained as double or single stranded fragments by conventional means, i.e., chemical synthesis, restriction enzyme digestion, PCR amplification, and the like.
  • small DNA fragments such as those useful as primers for PCR, hybridization screening probes, etc., will be of at least 15 nt, usually at least 18 nt or 25 nt, and may be at least about 50 nt.
  • PCR amplification requires a pair of primers, typically a pair which will generate an amplification product of at least 50 nucleotides, preferably at least 100 nucleotides in length. Suitable primers hybridize to the target polynucleotide under stringent conditions.
  • primer sequences are 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. Software designed for selecting suitable sequences for primers are commercially available. Larger DNA fragments, i.e. greater than 100 nt are useful for production of the encoded polypeptide.
  • Variants of RHOH polynucleotides of the invention can be prepared by methods known in the art. For example, techniques for site specific in vitro mutagenesis are found in Gustin er al. (1993), Biotechniques 14:22; Barany (1985), Gene 37:111-23; Colicelli er al. (1985), Mol. Gen. Genet. 199:537-9; and Prentki et al., (1984), Gene 29:303-13. Molecular Clonin ⁇ : A Laboratory Manual. CSH Press 1989, pp 15.3-108; Weiner et al. (1993), Gene 126:35-41 ; Sayers et al.
  • the resulting variants may, for example, contain mutations in the RHOH gene sequence, which may include flanking promoter regions and coding regions.
  • variants of the invention may include variants having 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
  • 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., as well as production of fusion proteins, e.g., using green fluorescent proteins (GFP).
  • GFP green fluorescent proteins
  • a GST (Glutathione S-Transferase) fusion protein of RHOH can be prepared using the pGEX vector (Pharmacia) encoding the full length 199 residue RHOH protein.
  • the resulting fusion protein can be purified using the GST-moiety, and the purified protein used in assays to test the intrinsic GTPase activity of RHOH using radiolabeled P32- ⁇ - GTP retention assays as described (Lee et al. (1996) J. Neurosci. 16(21):6784-94).
  • the RHOH polypeptides of this invention can be prepared by methods known to those of ordinary skill in the art once provided the information in the present application.
  • the polynucleotides of the Invention can be used to construct expression vectors, and can be used to produce all or a portion of RHOH polypeptides.
  • 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 RHOH 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.
  • the cells containing the construct may be selected by means of a selectable marker, the cells expanded and then used for expression.
  • RHOH polypeptides may be expressed in prokaryotes or eukaryotes in accordance with conventional ways, depending upon the purpose for expression. For large scale production
  • 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.
  • a higher organism such as vertebrates, particularly mammals, e.g. COS 7 cells
  • Small peptides can also be synthesized in the laboratory. Polypeptides that are subsets of the complete RHOH 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.
  • 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.
  • RHOH 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 RHOH.
  • 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.
  • 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.
  • 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.
  • Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50°C and 6XSSC (0.9 M saline/0.09 M sodium citrate) and remain bound when subjected to washing at 55°C in 1XSSC (0.15 M sodium chloride/0.015 M sodium citrate). Sequence identity may be determined by hybridization under stringent conditions, for example, at50°C or higher and 0.1XSSC (15 mM sodium chloride/01.5 mM sodium citrate). Nucleic acids having a region of substantial identity to the provided RHOH sequences, e.g. allelic variants, genetically altered versions of the gene, etc., bind to the provided RHOH sequences under stringent hybridization conditions.
  • homologous or related genes By using probes, particularly labeled probes of DNA sequences, one can isolate homologous or related genes.
  • the source of homologous genes may be any species, e.g., primate species, particularly human; rodents, such as rats and mice, canines, felines, bovines, ovines, equines, yeast, nematodes, etc.
  • homologs have substantial sequence similarity, e.g. at least 75% to 80% sequence identity, usually at least 90%, more usually at least 95% identity between nucleotide sequences.
  • Sequence identity is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc.
  • a reference sequence will usually be at least about 18 nt long, more usually at least about 30 nt long, and may extend to the complete sequence that is being compared.
  • the sequences provided herein are essential for recognizing RHOH-related and homologous proteins in database searches.
  • the DNA of the invention may be used to identify expression of the RHOH gene in a biological specimen.
  • 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.
  • 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
  • -16- 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 RHOH gene expression in the sample.
  • 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 RHOH coding region or control regions is associated with disease. 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 RHOH 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 ⁇ -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.
  • the region of interest is cloned into a suitable vector and grown in sufficient quantity for analysis.
  • Cells that express RHOH 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.
  • PCR polymerase chain reaction
  • 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, et al. Molecular Clonin ⁇ : A Laboratory Manual. CSH Press 1989, pp.14.2-14.33.
  • a detectable label may be included in an amplification reaction.
  • Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin,
  • 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. 32 P, ⁇ S, 3 H; 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.
  • 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 RHOH 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.
  • 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 acryiamide or agarose gels.
  • Screening for mutations in RHOH 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 RHOH 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 RHOH protein in GTP binding, GTPase activity, etc., may be determined by comparison with the wild-type protein.
  • Antibodies specific for a RHOH may be used in staining or in immunoassays.
  • Samples 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
  • 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 RHOH 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.
  • the primary antibody may be conjugated to biotin, with horseradish peroxidase-conjugated avidin added as a second stage reagent.
  • 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 a/., supra.
  • RHOH genes, gene fragments, or the encoded RHOH protein or protein fragments are useful in gene therapy to treat disorders associated with RHOH defects.
  • Expression vectors may be used to introduce the RHOH 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,
  • lentivirus adenovirus
  • 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 RHOH gene or RHOH 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 RHOH DNA, then bombarded into skin cells.
  • Antisense molecules can be used to down-regulate expression of RHOH 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.
  • ODN antisense oligonucleotides
  • 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.
  • 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.
  • 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.
  • phosphorothioates 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'-0'-5'-S-phosphorothioate, 3'-S-5'-0-phosphorothioate, 3'-CH2-5'-0- phosphonate and 3'-NH-5'-0-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 ⁇ -anomer of deoxyribose may be used, where the base is inverted with respect to the natural ⁇ -anomer.
  • the 2'-OH of the ribose sugar may be altered to form 2'-0-methyl or 2'-0-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.
  • 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).
  • 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.
  • 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 RHOH locus is altered.
  • a nucleic acid may 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 RHOH locus is altered.
  • a nucleic acids may 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 RHOH locus is altered.
  • a nucleic acid may be used to generate transgenic, non-human animals or site specific gene modifications in cell lines.
  • 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 RHOH function and regulation. For example, a series of small deletions and/or substitutions may be made in the host's native RHOH gene to determine the role of different exons in oncogenesis, signal transduction, etc.
  • RHOH a series of small deletions and/or substitutions may be made in the host's native RHOH gene to determine the role of different exons in oncogenesis, signal transduction, etc.
  • RHOH to construct transgenic animal models for cancer, where expression of RHOH is specifically reduced or absent.
  • Specific constructs of interest include anti-sense RHOH, which will block RHOH expression, expression of dominant negative RHOH mutations, and over-expression of RHOH genes.
  • the introduced sequence may be either a complete or partial sequence of a RHOH gene native to the host, or may be a complete or partial RHOH sequence that is exogenous to the host animal, e.g., a human RHOH sequence.
  • a detectable marker such as lac Z may be introduced into the RHOH locus, where upregulation of RHOH expression will result in an easily detected change in phenotype.
  • One may also provide for expression of the RHOH 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 RHOH protein in cells in which it is not normally produced, one can induce changes in cell behavior, e.g.
  • DNA constructs for homologous recombination will comprise at least a portion of the human RHOH gene or of a RHOH 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.
  • 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).
  • LIF leukemia inhibiting factor
  • ES or embryonic cells 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
  • 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.
  • 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.
  • the availability of a number of components in the Ras superfamily signaling pathway 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 RHOH protein.
  • One can identify ligands or substrates that bind to, modulate or mimic the action of RHOH.
  • Crosstalk between the ras and rho signaling pathways indicates that agents that modulate RHOH signaling may be useful in modulating ras-mediated signaling, which in turn can involve regulation of normal and transformed (e.g., cancerous) cell growth and proliferation.
  • Drug screening identifies agents that provide a replacement for RHOH function in abnormal cells that are defective or decreased in RHOH function, or agents that inhibit aberrant RHOH function in abnormal cells in which RHOH is overexpressed or otherwise functioning at super-normal levels.
  • screening assays for agents that have a low toxicity for human cells are of particular interest.
  • assays may be used for this purpose, including labeled in vitro protein-protein binding assays, eiectrophoretic mobility shift assays,
  • 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.
  • agent as used herein describes any molecule, e.g. protein or pharmaceutical, with the capability of altering or mimicking the physiological function of RHOH. 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.
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.
  • 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.
  • 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
  • the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures.
  • 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-microbiai agents, etc. may be used. The mixture of components are added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4 and 40 ° C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0.1 and 1 hours will be sufficient.
  • an expression construct comprising a RHOH gene may be introduced into a cell line under conditions that allow expression.
  • the level of RHOH activity is determined by a functional assay, as previously described.
  • candidate agents are added in combination with GTP, and activity in conversion of GTP to GDP is detected.
  • the ability of candidate agents to inhibit or enhance RHOH function is determined.
  • candidate agents are added to a cell that lacks functional RHOH, and screened for the ability to reproduce RHOH 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 RHOH function in, for example, wound healing, etc.
  • 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.
  • 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.%.
  • 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.
  • nucleic acid compositions encoding RHOH can be used for a variety of purposes, including, but not necessarily limited to, identification of 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 RHOH gene product is encoded by a novel sequence that is most closely related to members of the Rho subfamily of small GTPases, which play a role in the regulation of a variety of cellular processes including cytoskeletal assembly, membrane trafficking, cell motility, and cellular growth control and development.
  • Modulation of RHOH gene activity in vivo is used for prophylactic and therapeutic purposes, such as treatment of disease, investigation of Rho 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 Rho signaling pathway and for therapeutic and prophylactic purposes.
  • association of RHOH with tumorigenesis may provide for use of RHOH as a marker for tumor metastasis in selected tissues. Recently (Sula et al. (1998) Brit. J.
  • GenBank expressed sequence tag (EST) database http://www. ncbi.nlm.nih.gov/BLAST/blast_databases.html
  • EST The GenBank expressed sequence tag (EST) database (http://www. ncbi.nlm.nih.gov/BLAST/blast_databases.html) was searched for ESTs showing similarity to 34 known Rho-related proteins (see Table 2) using the "basic local alignment search tool" program, TBLASTN, with default settings as described at (http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html).
  • Rho-related defined as p ⁇ 0.0001
  • NR GenBank non-redundant
  • Table 2 List of protein probes used in database mining experiments.
  • ESTs that had top human hits with >95% identity over 100 amino acids were discarded. This was based upon the inventors' experience that these sequences were usually identical to the starting probe sequences, with the differences due to sequence error.
  • the remaining BLASTN and BLASTX outputs for each EST were examined manually, i.e., ESTs were removed from the analysis if the inventors determined that the variation from the known Rho- related probe sequence was a result of poor database sequence. Poor database sequence was usually identified as a number of 'n' nucleotides in the database sequence for a BLASTN search and as a base deletion or insertion in the database sequence, resulting in a peptide frameshift, for a BLASTX output. ESTs for which the highest scoring match was to non-Rho-related sequences were also discarded at this stage.
  • This IMAGE clone was shown to be 80% identical to the human Rho- related protein TC10 by BLASTX analysis.
  • the IMAGE clone 341923 was sequenced using standard ABI dye-primer and dye- terminator chemistry on a 377 automated DNA sequencer. Sequence was analyzed using the SEQUENCHER program (GENE CODES Corp.). The 3' end of the 341923 clone terminated in a premature poly-A motif due to probable mispriming of the poly-dT oligo in an A-rich nucleotide stretch. For this reason a second, overlapping IMAGE clone 795958 was identified by BLASTN screening of the EST database with the 3' terminal 50 nt. of the first IMAGE clone.
  • the 795958 IMAGE clone overlapped with the C-terminal 388 nt of the 341923 IMAGE clone. Sequencing of the 795958 clone extended the sequence by 466 nt, through the A-rich nucleotide stretch (nt 918-932), and contained the final 15 coding residues and the full length 3'UTR to the true poly-A tail.
  • the gene was designated RHOH.
  • the full length mRNA as deduced from the two overlapping IMAGE clone sequences is 1392 nt, with a 326 nt 5' UTR and a 421 nt 3'UTR (Fig. 1A; SEQ ID NO:3).
  • the RHOH open reading frame (SEQ ID NO:1) encodes a predicted amino acid sequence (SEQ ID NO:2) of 214 residues.
  • An in-frame stop codon is present in the sequence 36 nt upstream of the predicted start methionine (Fig. 1B).
  • RT-PCR was performed on a panel of 31 RNAs from different tissues; adipose, adrenal gland, bladder, brain, cerebellum, cervix, colon, esophagus, fetal brain, fetal liver, heart, Hela cell, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, rectum, salivary gland, skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus, trachea, and uterus.
  • the primers used for amplification were 341923.RHR and 341923.RHF (above). Cycling conditions were 95°C for 1 cycle of 12 minutes, 94°C for 30s, 58°C for 30s, 72°C for 30s for 30 cycles, 72 °C for 5 minutes. This analysis showed that RHOH
  • the RHOH primers resulted in production of RT-PCR products from each of the tissues tested. These data show that, like other Rho subfamily members, expression of the RHOH gene is ubiquitous.
  • the alignment of RHOH with its most closely related proteins is shown in Fig. 2.
  • the alignment was performed using the PILEUP program. Dots represent spaces to optimize alignment; residues occurring in 9 or more of the aligned proteins are capitalized and comprise the consensus sequence.
  • the five conserved domains (I, III, IV, and V) characteristic of small GTP-binding proteins are underlined; the effector loop is underlined and indicated as "II.”
  • RhoH shares the highest level of protein identity (80%) with TC10.
  • the Rho versions of the four conserved GTP-binding domains found in all small GTPase proteins can be identified within the RhoH sequence (residues 38-44, 75-80, 131-138, 184-187 of the sequence shown in Fig. 2), implying that RHOH has activity in cycling GDP/GTP.
  • a conserved Rho effector domain [Y-X-PTVF-XX-Y (SEQ ID NO:6), at residues 50-58 of the sequence of Fig. 2] is also present in RHOH, indicating that it may interact with many of the same downstream effector molecules as other Rho subfamily members.
  • the RHOH protein also contains a CAAX motif, CSII.
  • the CAAX motif of the majority of Rho proteins ends with a leucine residue, indicating that it is a substrate for modification by geranylgeranylation rather than famesylation.
  • the CAAX box of rhoH does not end with a leucine residue, but with an isoieucine residue, an arrangement which is not observed in any of the other rho proteins.. By analogy to ras it is likely that additional sequences at the carboxy-terminal end of the protein are also important for rhoH
  • Rab3c rat, from the Ran-subfamily
  • RhoH is a member of the Rho-subfamily of small GTPases rather than the ras, ran or rab subfamilies.
  • the RhoH protein is most closely related phylogenetically to the rho proteins TC10, rhoD and rhoE.
  • a bacterial expression construct encoding the full length RHOH protein was made using the pGEX system (Pharmacia). First, the RHOH coding sequence from the ATG start site to the TAG stop codon was amplified using PCR primers. The primers included BamHI "tails," so that the resulting PCR product could be subsequently cloned. Sequencing of the construct confirmed that no errors in the sequence were introduced during PCR.
  • the rhoH protein is expressed in E. coli as a fusion protein with the Schistosoma japonicum glutathione-S-transferase (GST) gene.
  • the fusion protein is purified by affinity chromatography using glutathione agarose or sepharose beads.
  • GTP hydrolysis studies to ascertain the intrinsic GTPase properties of the rhoH protein are performed using GTPyS labeled RHOH protein as described by Brauers et al. (1996) Eur J Biochem. 237; 833-840.
  • the bacterially expressed RHOH is used in GTPase activating (GAP) assays to ascertain if the rhoGAPs, P190, Bcr and rho-GAP can stimulate GTP hydrolysis of GTP-bound GTPase activating (GAP) assays to ascertain if the rhoGAPs, P190, Bcr and rho-GAP can stimulate GTP hydrolysis of GTP-bound
  • MYC-tagged mammalian expression construct expressing full length RHOH under the control of the CMV promoter was also constructed.
  • the PCR product comprising the RHOH coding sequence and the BamHI "tails" as described above was inserted into the BamHI sites of a CMV-driven expression vector.
  • the sequence encoding the MYC ( EQKLISEEDL ) Tag was incorporated upstream of the RHOH protein-encoding insert. Sequencing of the construct confirmed that no errors in the sequence were introduced during PCR.
  • pRK5-MYC The resulting construct, termed pRK5-MYC is transfected into mammalian cells (Cos, Swiss 3T3, BHK21 or NIH 3T3). Immunohistochemistry to localize the overexpressed rhoH protein using anti-MYC antibody is performed as described by Takaishi K. et al., (1995) Oncogene 11; 39-48 and Daniels et al., (1998) EMBO J. 17; 754-764.
  • Constitutively activated mutant forms of RHOH are created using site directed mutagenesis to incorporate the DNA changes into the mammalian RHOH vector constructs. These constructs are overexpressed in a fibroblast cell line (Swiss 3T3 cells, NIH3T3 cells or BHK21 cells). After fixing, the cells are permeabilized and stained (green fluorescence) for expression of the myc-tagged proteins with an anti-myc monoclonal (9E10) followed by FITC-conjugated goat anti-mouse IgG and TRITC phalloidin (to stain actin fibres red) as described by Daniels et al., (1998) EMBO J. 17; 754-764.
  • a fibroblast cell line Swiss 3T3 cells, NIH3T3 cells or BHK21 cells.
  • the cells are permeabilized and stained (green fluorescence) for expression of the myc-tagged proteins with an anti-myc monoclonal (9E10) followed by FITC-conju
  • This assay allows detection of morphological changes in the fibroblast cells. These constructs are also transfected into cell lines expressing activated ras to determine if RHOH has a synergistic effect on ras-mediated transformation as described (Khosravi-Far et al., (1996) Mol Cell Biol 15; 6443-6453)
  • RhoH is a novel member of the Rho-subfamily of small GTPases. Rho proteins play crucial roles in regulating a wide variety of crucial cellular processes. The identification of RHOH is an important step in the elucidation of the signaling pathways which this protein may regulate as a small GTPase and its characterization as a potential disease gene or possible proto-oncogene.

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Abstract

L'invention concerne des polynucléotides codant de nouveaux polypeptides de liaison à la GTP (ci-après dénommés polypeptides RHOH), des vecteurs d'expression comprenant un polynucléotide RHOH de l'invention, des cellules isolées comprenant un vecteur codant RHOH, un animal non humain transgénique présentant une altération dans un gène RHOH, et l'utilisation de polynucléotides RHOH pour détecter chez un individu la présence d'un polymorphisme génétique d'un gène RHOH. L'invention concerne en outre de nouveaux polypeptides RHOH, des anticorps monoclonaux spécifiques pour les polypeptides RHOH, et des procédés de production de polypeptides RHOH.
PCT/US1999/010061 1998-05-11 1999-05-06 Genes rhoh et leurs utilisations WO1999058669A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001055420A1 (fr) * 2000-01-26 2001-08-02 Biodoor Gene Technology Ltd. Shanghai Nouveau polypeptide, site 27 actif de la famille rho des enzymes gtp, et polynucleotide codant pour ce polypeptide
WO2006067506A2 (fr) 2004-12-24 2006-06-29 Immunoclin Limited Genes de resistance
US10746739B2 (en) 2015-09-14 2020-08-18 Leukemia Therapeutics, LLC Identification of novel diagnostics and therapeutics by modulating RhoH

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998011212A1 (fr) * 1996-09-13 1998-03-19 Winfried Siffert Proteines g sensibles a l'anatoxine coquelucheuse, leur preparation et leur utilisation
WO1998018942A2 (fr) * 1996-10-29 1998-05-07 Incyte Pharmaceuticals, Inc. Nouvelles proteines humaines rab
WO1999015554A2 (fr) * 1997-09-22 1999-04-01 Incyte Pharmaceuticals, Inc. Proteine de type ras

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998011212A1 (fr) * 1996-09-13 1998-03-19 Winfried Siffert Proteines g sensibles a l'anatoxine coquelucheuse, leur preparation et leur utilisation
WO1998018942A2 (fr) * 1996-10-29 1998-05-07 Incyte Pharmaceuticals, Inc. Nouvelles proteines humaines rab
WO1999015554A2 (fr) * 1997-09-22 1999-04-01 Incyte Pharmaceuticals, Inc. Proteine de type ras

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DALLERY-PRUDHOMME E ET AL: "Genomic structure and assignment of the RhoH/TTF small GTPase gene (ARHH) to 4p13 by in situ hybridization.", GENOMICS, vol. 43, no. 1, 1 July 1997 (1997-07-01), pages 89 - 94, XP002115827 *
DRIVAS ET AL., MOL CELL BIOL, vol. 10, 1990, pages 1793 - 1798 *
DRIVAS ET AL.: "GTP-binding protein TC10", EMBL SEQUENCE DATABASE, 1 August 1990 (1990-08-01), HEIDELBERG DE, XP002114862 *
HILLIER ET AL.: "WashUMerck EST Project 1997", EMBL SEQUENCE DATABASE, 13 June 1997 (1997-06-13), HEIDELBERG DE, XP002114860 *
HILLIER ET AL.: "WashU-NCI human EST Project", EMBL SEQUENCE DATABASE, 1 June 1997 (1997-06-01), HEIDELBERG DE, XP002114861 *
ROUMIER ET AL.: "Non-random chromosomal rearrangement, at 4p13, of the RhoH/TTF gene, encoding a small GTPase restrictively expressed in hematopoietic tissues, in non-Hodgkin's lymphoma and multiple myeloma.", BLOOD, vol. 90, no. 10 Suppl. 1 part 1, 15 November 1997 (1997-11-15), pages 557a, XP002115826 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001055420A1 (fr) * 2000-01-26 2001-08-02 Biodoor Gene Technology Ltd. Shanghai Nouveau polypeptide, site 27 actif de la famille rho des enzymes gtp, et polynucleotide codant pour ce polypeptide
WO2006067506A2 (fr) 2004-12-24 2006-06-29 Immunoclin Limited Genes de resistance
WO2006067506A3 (fr) * 2004-12-24 2007-02-08 Immunoclin Ltd Genes de resistance
US10746739B2 (en) 2015-09-14 2020-08-18 Leukemia Therapeutics, LLC Identification of novel diagnostics and therapeutics by modulating RhoH

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