Saccharomyces cerevisiae synthesizes proteins related to the p21 gene product of ras genes found in mammals.

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The cellular homologs of the Harvey and Kirsten murine sarcoma virus oncogenes comprise a multigene family, ras, that displays striking evolutionary conservation. We recently reported [DeFeo-Jones et al., Nature (London) 306:707-709, 1983] the cloning of two ras homologs from the yeast Saccharomyces cerevisiae. The nucleotide sequences of these genes predict polypeptides that show remarkable homology to p21, the mammalian ras gene product. We have also round proteins in yeast lysates with serological cross-reactivity to p21 (Papageorge et al., Mol. Cell. Biol. 4:23-29, 1984). In this work, we explored the relationship between the immunoprecipitated proteins and the yeast ras genes. We show that both ras genes are expressed in the wildtype cell. Furthermore, we demonstrate by in vitro translation of hybrid-selected RAS"l mRNA and immunoprecipitation of the translation products that the cloned RAS"' gene encodes the proteins immunoprecipitated from yeast lysates by anti-p21 monoclonal antibody. Finally, we used anti-p21 monoclonal antibodies to detect a guanine nucleotide binding activity in yeast lysates. The structural and biochemical homologies between ras gene products of S. cerevisiae and mammalian cells suggest that information obtained by genetic analysis of ras function in a lower eucaryote should be applicable to higher organisms as well.The ras oncogenes are a multigene family (11) present in a variety of vertebrate and invertebrate species (10,14,21). Alteration in the form or expression of the mammalian ras gene product, p21, has been implicated in transformation of cells in culture (3) and in some forms of human cancer (2,5,18,23,24,26).The role of the ras genes in normal cells is unknown. In an effort to understand the function of ras genes, we sought to study them in a simple organism easily amenable to genetic manipulation. We have recently reported the cloning of two ras homologs (4) and the identification of ras-related protein products (17) Strain SR140-6A, designated "parent," was a gift from S. Roeder; a derived strain, SR140-6A-486-5, containing multiple copies of the RASsCl gene, is designated "overproducer" and will be described in detail in a subsequent publication.Growth of yeasts. Yeast cells were grown in synthetic minimal medium as previously described (17).Preparation of RNA. Total yeast RNA was prepared from 1-liter cultures that had been grown to late-log phase (absorbance at 600 nm = 1.0 optical density unit). The RNA extraction protocol was a modification of a procedure in EIder et al. (7). Cells were harvested by centrifugation, washed once in sterile distilled water, and resuspended in RNA extraction buffer (0.5 M NaCl, 0.2 M Tris-hydrochloride [pH 7.5], 0.1 M EDTA, 1% sodium dodecyl sulfate [SDS]). One volume of phenol-chloroform-isoamyl alcoholwater (25:24:1:50, vol/vol) and 0.5 volume of 0.4-mm-diameter, acid-washed, glass beads were added, and the mixture was vortexed at top speed for 2 min. One additional volume each of RNA extraction buffer and phenol-chloroform-isoamyl ...

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confidence: 68%

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confidence: 99%

Expression and characterization of ras mRNAs from Saccharomyces cerevisiae.

Temeles¹,

Defeo-Jones²,

Tatchell³

et al. 1984

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“…Monoclonal antibody (Y13-259) prepared against Harvey murine sarcoma virus ras proteins is known to react with both cellular and viral (Harvey and Kirsten) ras gene products as well as with yeast ras proteins (15,29,32,47). Because of the observed conservation of ras genes in eucaryotes, it is possible to remove endogenous ras proteins from Xenopus oocytes via microinjection of Y13-259 antibody (19,36).…”

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confidence: 99%

Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 ras proteins.

Deshpande¹,

Kung²

1987

174

Microinjection of transforming p21 ras protein induces maturation of Xenopus laevis oocytes, and the induction is blocked by coinjection of monoclonal antibody (Y13-259) against p21 ras proteins. Similar to other inducing agents, the effect of p21 ras protein is mediated via the appearance of maturation or meiosispromoting factor activity. In addition, the neutralizing antibody markedly reduces oocyte maturation after insulin induction, whereas it fails to inhibit progesterone induction. Our results suggest that insulin induces maturation of oocytes via a different pathway than that of steroidal agents. The induction by insulin is ras dependent, and the action of ras may be directed at the steps before meiosis-promoting factor autocatalytic activation. These results suggest a role of p21 ras protein in the events associated with amphibian oocyte maturation.The ras family forms a group of closely related transforming genes that are highly conserved in eucaryotes (5,11,18,28,34,48). Normal (cellular) ras genes acquire transforming properties by single point mutations within their coding sequences (4,32,33,41,(45)(46)(47)54), and these mutated ras genes have been detected in a significant fraction of human cancers as well as in experimentally induced animal tumors (45,48,54). It has been speculated that p21 ras proteins are essential components of normal cells that are involved in cell division and differentiation (30,31). Yet, little is known regarding the mechanisms by which p21 proteins induce malignant transformation, and even less is known regarding their role in normal cells.It has been demonstrated that p21 proteins can induce transformation of NIH 3T3 cells when introduced by microinjection (13,44) and that the transforming activity of the ras proteins can be blocked by coinjection with monoclonal antibody Y13-259 (19). Recently, Birchmeier et al. (3) have shown that ras proteins can induce meiosis in Xenopus laevis oocytes. Fully grown oocytes can be easily injected and analyzed biochemically. Oocytes surgically removed from adult Xenopus ovaries are arrested in the prophase of meiosis and can be triggered to undergo meiosis by treatment with progesterone, insulin, and a variety of other agents. In the present study, we determined the effect of injected neutralizing antibody (Y13-259) on the maturation of oocytes.Gravid X. laevis females were obtained from Nasco Co. (Fort Atkinson, Wis.) and maintained under laboratory conditions (9). Ovarian fragments were surgically removed from frogs that were anesthetized by hypothermia. Fully grown stage VI oocytes were manually dissected, and follicle cells were removed after collagenase treatment of ovarian fragments at 21°C following the procedure of Eppig and Dumont (12) in modified Barth medium (MBSH) buffered with 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.6 (22). The selected stage VI oocytes were thoroughly washed in MBSH and equilibrated for at least 2 h before use. Oocytes were cultured in 30-mm Falcon * Corresponding author. ster...

“…Previous studies have indicated that Y13-259 recognizes a site distal to position 33 in the 189-amino acid chain (20). To precisely localize the epitope, we utilized a strategy based on the generation of a series of p21 deletion mutants expressed in bacteria.…”

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confidence: 99%

Monoclonal antibody Y13-259 recognizes an epitope of the p21 ras molecule not directly involved in the GTP-binding activity of the protein.

Lacal¹,

Aaronson²

1986

The p21 products of ras proto-oncogenes are GTP-binding proteins with associated GTPase activity. Recent studies have indicated that ras p21 may be required for the initiation of normal cell DNA synthesis, since microinjection of a monoclonal antibody, Y13-259, blocks serum stimulation of DNA synthesis in quiescent cell cultures (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We localized the structural domain within the p21 molecule recognized by the Y13-259 monoclonal antibody. By analysis of a series of bacterially expressed p21 deletion mutants, the monoclonal antibody was found to interact with a region between positions 70 and 89 in the p21 amino acid sequence. By comparison of the coding sequences of different p21 proteins recognized by this monoclonal antibody, a highly conserved amino acid region between positions 70 and 81 was found to be the most likely site for the epitope detected by the Y13-259 antibody. This monoclonal antibody was further shown not to interfere directly with in vitro biochemical functions of the molecule, including GTP binding, GTPase, and autokinase activities.