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M Wigler

Publications and source records attributed to M Wigler.

At least 91 records · Page 5Linked to original sources
PubMed1985

Differential activation of yeast adenylate cyclase by wild-type and mutant RAS proteins.

In these experiments we demonstrate that purified RAS proteins, whether derived from the yeast RAS1 or RAS2 or the human H-ras genes, activate yeast adenylate cyclase in the presence of guanine nucleotides. These results confirm the prediction of earlier genetic and biochemical data and for the first time provide a complete biochemical assay for RAS protein function. Furthermore, we observe a biochemical difference between the RAS2 and RAS2val19 proteins in their ability to activate adenylate cyclase after preincubation with GTP.

Adenylyl Cyclases↗
PubMed1985

Purification and characterization of human H-ras proteins expressed in Escherichia coli.

The full-length normal and T24 mutant human H-ras proteins and two truncated derivatives of the T24 mutant were expressed efficiently in Escherichia coli. The proteins accumulated to 1 to 5% of total cellular protein, and each was specifically recognized by anti-ras monoclonal antibodies. The two full-length proteins as well as a carboxyl-terminal truncated derivative (deleted for 23 amino acid residues) were soluble upon cell lysis and were purified to 90% homogeneity without the use of denaturants. In contrast, an amino-terminal truncated ras derivative (deleted for 22 amino acid residues) required treatment with urea for its solubilization. The guanine nucleotide binding activity of these four proteins was assessed by a combination of ligand binding on proteins blots, immunoprecipitation, and standard filter binding procedures. The full-length proteins showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The carboxyl-terminal truncated protein also bound GTP, but to a reduced extent, whereas the amino-terminal truncated protein did not have binding activity. Apparently, the carboxyl-terminal domain of ras, although important for transforming function, does not play a critical role in GTP binding.

DNA, Recombinant↗
PubMed1984

Genes in S. cerevisiae encoding proteins with domains homologous to the mammalian ras proteins.

The ras genes, which were first identified by their presence in RNA tumor viruses and which belong to a highly conserved gene family in vertebrates, have two close homologs in yeast, detectable by Southern blotting. We have cloned both genes (RAS1 and RAS2) from plasmid libraries and determined the complete nucleotide sequence of their coding regions. They encode proteins with nearly 90% homology to the first 80 positions of the mammalian ras proteins, and nearly 50% homology to the next 80 amino acids. Yeast RAS1 and RAS2 proteins are more homologous to each other, with about 90% homology for the first 180 positions. After this, at nearly the same position that the mammalian ras proteins begin to diverge from each other, the two yeast ras proteins diverge radically. The yeast ras proteins, like the proteins encoded by the mammalian genes, terminate with the sequence cysAAX, where A is an aliphatic amino acid. Thus the yeast ras proteins have the same overall structure and interrelationship as the family of mammalian ras proteins. The domains of divergence may correspond to functional domains of the ras proteins. Monoclonal antibody directed against mammalian ras proteins immunoprecipitates protein in yeast cells containing high copy numbers of the yeast RAS2 gene.

Amino Acid Sequence↗
PubMed1984

Genetic analysis of yeast RAS1 and RAS2 genes.

We present a genetic analysis of RAS1 and RAS2 of S. cerevisiae, two genes that are highly homologous to mammalian ras genes. By constructing in vitro ras genes disrupted by selectable genes and introducing these by gene replacement into the respective ras loci, we have determined that neither RAS1 nor RAS2 are by themselves essential genes. However, ras1 - ras2 - spores of doubly heterozygous diploids are incapable of resuming vegetative growth. We have determined that RAS1 is located on chromosome XV, 7 cM from ade2 and 63 cM from his3; and RAS2 is located on chromosome XIV, 2 cM from met4 . We have also constructed by site-directed mutagenesis a missense mutant, RAS2val19 , which encodes valine in place of glycine at the nineteenth amino acid position, the same sort of missense mutation that is found in some transforming alleles of mammalian ras genes. Diploid yeast cells that contain this mutation are incapable of sporulating efficiently, even when they contain wild-type alleles.

Animals↗
PubMed1984

Analysis of the transforming potential of the human H-ras gene by random mutagenesis.

Some tumor cells contain mutant ras genes that are capable of transforming NIH 3T3 cells. Those genes that have been analyzed arise from the wild-type, non-transforming ras genes by mutations producing single amino acid substitutions at position 12 or 61 of the encoded protein. We have performed random bisulfite-induced mutagenesis on the cloned wild-type human H-ras gene to find if mutations at other positions can activate the transforming potential of that gene. Most mutations are not activating, but mutations that specify single amino acid substitutions at position 12, 13, 59, or 63 of the encoded protein do activate the transforming potential of the H-ras gene. Some, but not all, mutant ras proteins show an altered electrophoretic mobility in NaDodSO4/polyacrylamide gels.

Amino Acids↗
PubMed1984

A product of yeast RAS2 gene is a guanine nucleotide binding protein.

Yeast Saccharomyces cerevisiae contains two genes, RAS1 and RAS2, which show remarkable homology to mammalian ras genes. To characterize these gene products, we have expressed the RAS2 gene in yeast using an inducible GAL10 promoter. After labeling with [35S]methionine and immunoprecipitating with a monoclonal antibody Y13-259, which reacts with p21 encoded by mammalian ras genes, a major band having an apparent molecular weight of 41,000 is detected. This band has also been identified in cell-free translation products of polyadenylated RNA extracted from yeast cells grown in the presence of galactose. Crude extracts of cells expressing the RAS2 gene exhibit guanine nucleotide binding activity. This is detected by incubation with [3H]GDP followed by immunoprecipitation with the antibody Y13-259. The binding of labeled GDP is inhibited by a 20-fold excess of GDP, GTP, and, to a lesser extent, by UTP, a characteristic similar to that possessed by the mammalian ras proteins. However, the activity of the yeast protein differs from that of the mammalian proteins in its strong dependence on temperature. The guanine nucleotide binding activity provides an assay to purify the yeast protein.

Cloning, Molecular↗
PubMed1984

New human transforming genes detected by a tumorigenicity assay.

We have developed a sensitive bioassay for transforming genes based on the tumorigenicity of cotransfected NIH3T3 cells in nude mice. The assay differs substantially from the NIH3T3 focus assay. Using it, we have detected the transfer of three transforming genes from the DNA of MCF-7, a human mammary carcinoma cell line. One of these is N-ras, which is amplified in MCF-7 DNA. The other two, which we have called mcf2 and mcf3, do not appear to be related to known oncogenes. We cannot detect their transfer by using the NIH3T3 focus assay. We do not yet know whether either mcf2 or mcf3 is associated with genetic abnormalities in MCF-7 cells.

Animals↗
PubMed1983

Structure and activation of the human N-ras gene.

The normal human N-ras gene has been cloned. In structure and sequence it closely resembles the human H-ras and K-ras genes. The three genes share regions of nucleotide homology and nucleotide divergence within coding sequences and have a common intron/exon structure, indicating that they have evolved from a similarly spliced ancestral gene. The N-ras gene of SK-N-SH neuroblastoma cells has transforming activity, while the normal N-ras gene does not, the result of a single nucleotide change substituting lysine for glutamine in position 61 of the N-ras gene product. From previous studies we conclude that amino acid substitutions in two distinct regions can activate the transforming potential of ras gene products.

Amino Acid Sequence↗
PubMed1983

Chromosomal assignment of a family of human oncogenes.

A family of human transforming genes, previously shown to share homology with the ras family of viral oncogenes, maps to three different human chromosomes. A well-characterized mouse-human hybrid cell panel, combined with Southern blotting, was used in this study. The transforming gene of the T24 bladder carcinoma cell line maps to human chromosome 11. An oncogene isolated from the lung carcinoma cell line SK-Calu-1 maps to human chromosome 12. The third ras-related gene, cloned from SK-N-SH, a neuroblastoma cell line, maps to human chromosome 1.

Animals↗
PubMed1983

Isolation and preliminary characterization of the transforming gene of a human neuroblastoma cell line.

DNA from the human neuroblastoma cell line SK-N-SH is capable of inducing foci of transformed NIH 3T3 cells after DNA-mediated gene transfer. Using genetic selection with the Escherichia coli sup F gene, we have isolated human sequences from mouse cells responsible for the oncogenic transformation. These sequences are present in all human DNAs surveyed and no gross rearrangements of these sequences are found in SK-N-SH cells. Although clearly distinct from two other human transforming genes present in bladder, lung, and colon carcinoma cell lines, all three transforming gene sequences may be related members of the ras gene family.

Animals↗
PubMed1983

Sequence and structure of the coding region of the human H-ras-1 gene from T24 bladder carcinoma cells.

We have molecularly cloned and sequenced cDNA to the transcript of H-ras-1, the transforming gene of the T24 human bladder carcinoma cell line. The transcript derives from at least five exons in the H-ras-1 gene, and RNA splicing occurs at sites typical of exon-intron junctions. T24 H-ras-1 RNA has an AUG-initiated open reading frame of 567 nucleotides, which can encode a protein of mass comparable to the apparent molecular weight of the T24 H-ras-1 gene product. The T24 H-ras-1 gene product is nearly identical to v-H-ras p21, the transforming protein encoded by the genome of Harvey sarcoma virus. We discuss the implications of this sequence conservation in the structure-function relationships of ras proteins.

Base Sequence↗
PubMed1983

A test of the role of two oncogenes in inherited predisposition to colon cancer.

Inheritance of mutationally altered oncogenes could predispose individuals to the development of specific tumors and account for familial tumor phenotypes. Using adjacent DNA sequence polymorphisms as genetic markers, we have examined two oncogenes, the Kirsten ras2, isolated from a human colon cancer cell line, and the Harvey ras1, isolated from a human bladder cancer cell line, for their role in the genetic etiology of inherited colon cancer in Gardner syndrome. Both oncogene loci have been shown to be unlinked to the Gardner syndrome locus and are, therefore, eliminated as candidates for the Gardner syndrome gene.

Base Sequence↗
PubMed1983

Mutational analysis of the cloned chicken thymidine kinase gene.

We have analyzed the transcription and coding unit of the chicken thymidine kinase (tk) gene. We have constructed a library of mutant chicken tk genes by the in vitro linker insertion method of Heffron et al. A total of 125 mutations within a 3.0 kbp HindIII fragment containing the gene were isolated and mapped. The effect of each mutation upon the thymidine kinase gene was determined by measurement of the transfection efficiency in mouse Ltk- cells. The chicken tk mRNA is about 2 kb and polyadenylated. The direction of transcription was also determined. From these results, we propose a structure for the gene in which at least three small introns separate the amino acid coding region into at least four segments.

Animals↗
PubMed1982

Activation of the T24 bladder carcinoma transforming gene is linked to a single amino acid change.

Several different transforming genes have been observed in the DNA of a variety of tumours and tumour cell lines of human and rodent origin by the ability of these genes to induce morphological transformation in NIH 3T3 cells1-5. The transforming gene found in a human bladder carcinoma cell line, T24, is H-ras-1, the human homologue of the Harvey sarcoma virus oncogene (v-H-ras)6-9. In the present study we have compared the H-ras-1 genes cloned from T24 and normal human DNA. The H-ras-1 gene cloned from T24 DNA induces transformation in NIH 3T3 cells, while the same gene cloned from normal cellular DNA does not. The functionally significant difference between the transforming and normal genes appears to be a single base mutation, which produces an amino acid change in the sequence of the proteins that the genes encode.

Amino Acid Sequence↗
PubMed1982

Isolation and preliminary characterization of a human transforming gene from T24 bladder carcinoma cells.

DNA from T24, a cell line derived from a human bladder carcinoma, can induce the morphological transformation of NIH 3T3 cells. Using techniques of gene rescue to clone the gene responsible for this transformation, we have found that it is human in origin, less than 5 kilobase pairs in size and is homologous to a 1,100-base polyadenylated RNA species found in T24 and HeLa cells. Blot analysis indicates extensive restriction endonuclease polymorphism near this gene, in human DNAs.

Animals↗
PubMed1981

Human-tumor-derived cell lines contain common and different transforming genes.

We have screened different cultured cell lines established from human tumors for the ability of their DNAs to induce transformed foci in NIH/3T3 cells. Based on restriction endonuclease digestions and the presence of human sequences in mouse transformants, we conclude that five of these human tumor cell lines contain a gene or genes capable of transforming mouse cells and that at least three different transforming genes are present in these five lines. Three cell lines, two derived from lung carcinomas and one derived from a colon carcinoma, transfer the same or closely related human genes. If these transforming genes are mediating the tumorigenic state of the human cells, then our results indicate that overlapping pathways leading to tumorigenesis may arise independently.

3T3 Cells↗
PubMed1981

The somatic replication of DNA methylation.

We have tested the hypothesis that DNA methylation patterns are replicated in the somatic cells of vertebrates. Using M-Hpa II, the modification enzyme from Haemophilus parainfluenzae which methylates the internal cytosine residues in the sequence 5'CCGG 3' GGCC, we methylated bacteriophage phi X174 RF DNA and the cloned chicken thymidine kinase (tk) gene in vitro and then introduced these DNAs and unmethylated controls into tk- cultured mouse cells by DNA-mediated transformation. Twenty-five cell generations later, the state of methylation of transferred DNA was examined by restriction endonuclease analysis and blot hybridization. We conclude that methylation at Hpa II sites is replicated by these cultured cells but not with 100% fidelity. We have also noted that methylation of the cloned chicken tk gene decreases its apparent transformation efficiency relative to unmethylated molecules.

Animals↗