Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-12-03
2002-05-07
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C530S350000
Reexamination Certificate
active
06383792
ABSTRACT:
BACKGROUND OF THE INVENTION
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.
349:117-127; Hall and Zerial (1995) General introduction. In: “
Guidebook to the Small GTPases
” (M. Zerial and L. A. Huber Eds.), pp. 3-11, Sambrook and Tooze, Oxford University Press).
The Ras subfamily members share four conserved domains which, as demonstrated through both mutagenic studies and X-ray crystallography, are involved in the binding and hydrolysis of guanine nucleotides. Many Ras proteins also share a fifth conserved carboxy-terminal domain required for posttranslational modification of the Ras proteins prior to membrane localization (Boguski and McCormick (1993)
Nature
366:643-654; Hall and Zerial (1995), supra).
At least nine of the sixty or so members of the Ras-related have greater than about 50% amino acid identity to H-, K-, and N-ras oncogenes (Hall and Zerial (1995), supra). These include R-ras1 and its related proteins R-ras-2(TC21) and the recently identified R-ras3 (Hall and Zerial (1995), supra; Kimmelmann et al. (1997),
Oncogene
15(22):2675-2685) which share about 55% amino acid identity with H-ras, including an identical effector domain, and are about 70% identical to one another. The Rap proteins (Rap1a and b, Rap2a and b) also share a conserved effector domain and about 50% protein identity with H-ras. The RaIA and RaIB proteins also belong to the ras subfamily, with about 50% peptide identity to H-ras, although there is a one residue difference in their effector domain (Hall and Zerial (1995).
Recently two small GTPases, termed Rin and Rit, were identified. Rin and Rit share about 50% identity and four conserved GTP-binding motifs of Ras proteins,(Lee et al. (1996)
J. Neurosci.
16(21):6784-6794). However, these two proteins were unusual in that they lacked the known CAAX recognition signal for C-terminal lipidation found in each of the other Ras subfamily members.
The Ras GTP-binding protein is coupled to a tyrosine kinase receptor. The formation of an agonist-receptor complex facilitates GTP binding to the Ras protein, whereupon the protein bound GTP is hydrolyzed to GDP via the intrinsic GTPase activity of the Ras protein. The GDP dissociates from the Ras protein and it reverts to its inactive form. This cycling between inactive and active states initiates a mitogen-activated kinase cascade which leads to the phosphorylation of a number of transcription factors in the nucleus which culminates in cell proliferation and differentiation.
GTP binding and its hydrolysis to GDP are catalyzed by at least two classes of proteins: guanine nucleotide exchange factors, which promote exchange between bound GDP and cytoplasmic GTP, and GTPase activating proteins, which stimulate the low intrinsic GTP hydrolysis by the GTPases (Boguski and McCormick (1993),
Nature.
366, pp 643-654; Feig (1993),
Science.
260, pp 767-768). Mutations in the GTP-binding proteins affecting the nucleotide exchange or GTP hydrolysis can stabilize the GTP- or GDP-bound conformation and thereby cause the GTP-binding proteins to be constitutively active or inactive. For example, Ras genes with point mutations are known, wherein the intrinsic GTPase activity of Ras GTP-binding protein is insensitive to GTPase activating proteins.
The link between mutations in Ras genes and cancer is well established. For example, there is a strong association between abnormal signal transduction involving activated Ras genes and the development of a variety of tumors. In fact, mutations in Ras genes are found in 30% of all human tumors and in some the mutation frequency approaches 100%, e.g., pancreatic adenocarcinoma. The number of genes encoding Ras or Ras-related proteins is unknown, but it is clear that their role in cellular processes is of crucial importance. Accordingly, the discovery of novel Ras-related genes will advance the understanding and treatment of human disease.
Relevant Literature
EST sequences present within the RAQ 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 are indicated in the last column.
TABLE 1
Relevant EST sequences.
Read
Extent RAQ sequence
Sequence
orien-
(SEQ ID NO:3) having >90%
accession no.
tation
Clone ID
identity (RAQ nt residue numbers)
AA031734
5′
IC470515
1793-2277
AA131239
3′
IC 503581
1776-2308
AA437054
5′
IC 758247
1364-1811
AA663946
3′
IC 855719
1887-2292
AA769749
3′
IC 1324233
1877-2294
AA782027
3′
IC 1376649
1918-2292
N98847
5′
IC 278823
1189-1548
D62865
5′
GEN-333F03
1891-2207
AA742672
3′
IC 1257275
1980-2292
HO1201
3′
IC 150011
1964-2284
R40247
3′
IC 28777
2019-2299
D62925
5′
GEN-340E03
1764-2069
HO1299
5′
IC 150011
1364-1616
D79833
5′
GEN-334H02
1764-2064
Z19345
5′
17C02
1021-1315
AA037415
3′
IC484600
2022-2292
D79915
5′
GEN-354F08
1764-2027
AA663274
3′
IC 853301
2007-2255
F00427
3′
17C02
2043-2273
AA564698
3′
IC993282
2158-2291
R 14297
5′
IC 28777
267-512
AA214398
5′
IC649419
407-620
R55855
5′
IC 40732
651-1020
AA131358
5′
IC 503581
886-1451
IC = IMAGE clone
The sequence of human genes related to RAQ may be accessed at Genbank at the indicated accession numbers: R-ras2 (TC21), Genbank:M31468 Genbank:Y07565; Rin (human, Genbank accession #=Y07565); Rit, Genbank:U71203; RaIA, Genbank:X15014; and Rap1b, Genbank:X08004. The sequence of
Dictostylium discoideum
genes sharing homology with human RAQ may be accessed at Genbank at the indicated accession numbers: RasS, Genbank:Z14134; and RasD, Genbank:J04160.
The role of ras oncogenes in human cancer is reviewed in Zachos et al. (1997)
Crit Rev Oncol Hematol
26(2):65-75 and in Bos (1989)
Cancer Res.
49:4682-4689. The targeting of small GTPases for cancer therapies is discussed in Symons (1995)
Curr. Opin. Biotechnol.
6:668-74.
The association of the 12p12-13 cytoband region with cancer is described in several references. The possibility of a human pulmonary adenoma susceptibility 1 (Pas1) locus homolog at 12p12-13 is discussed in Manenti et al. (1997)
Carcinogenesis
18(10):1917-1920. Pas1 is a major locus affecting inherited predisposition to lung cancer in mice within the syntenic chromosome region. Johansson et al. ((1993)
Genes, Chromosomes, Cancer.
8:205-218) and Hatta et al. ((1997)
Br J Cancer.
75(9):1256-1262, suggested that a new tumor suppressor gene may lie within the 12p12-13 region.
SUMMARY OF THE INVENTION
The invention features polynucleotides encoding novel GTP-binding polypeptides, hereinafter referred to as RAQ polypeptides; expression vectors comprising a RAQ polynucleotide of the invention; isolated cells comprising a RAQ-encoding vector; a transgenic non-human animal comprising an alteration in a RAQ gene; and the use of RAQ polynucleotides in detecting in an individual the presence of a genetic polymorphism of a RAQ gene.
The invention further features novel RAQ polypeptides; monoclonal antibodies specific for RAQ polypeptides; and a method for making RAQ polypeptides.
REFERENCES:
patent: 5973130 (1999-10-01), Hillman et al.
Bos, J.L., “ras Oncogenes in Human Cancer: A Review,”Cancer Res. 49:4682-4689 (Sep. 1, 1989).
Hatta, et al., “Ovarian Cancer Has Frequent Loss of Heterozygosity at Chromosome 12p12.3-13.1 (Region of TEL and Kip 1 loci) and Chromosome 12q23-ter: Evidence for two New Tumour Suppres
Allen Maxine J.
Buckler Alan J.
Rutter Marc
AxyS Pharmaceuticals, Inc.
Borden Paula A.
Monshipouri M.
Pamela J. Sherwood Bozicevic, Field & Francis, LLP
Prouty Rebecca E.
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