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Biomedical subjects

M Wigler

Publications and source records attributed to M Wigler.

At least 73 records · Page 4Linked to original sources
PubMed1987

Expression in Escherichia coli of BCY1, the regulatory subunit of cyclic AMP-dependent protein kinase from Saccharomyces cerevisiae. Purification and characterization.

The regulatory (R) subunit of cAMP-dependent protein kinase from the yeast Saccharomyces cerevisiae was expressed in Escherichia coli by engineering the gene for yeast R, BCY1, into an E. coli expression vector that contained a promoter from phage T7. Oligonucleotide-directed mutagenesis was used to create an NdeI restriction site at the natural ATG of the yeast R. This facilitated construction of the T7 expression vector so that the sequence of the protein produced was identical to the natural R subunit. Yeast R was highly expressed in a soluble form. 20 mg of purified yeast R was obtained from 4 liters of E. coli. N-terminal amino acid sequencing revealed that the expressed protein began with the natural sequence. 60% of the molecules contained an N-terminal methionine, and 40% initiated with valine, the second amino acid of yeast R. The protein produced in E. coli migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000. The yeast R bound 2 mol of cAMP/mol of R monomer with a Kd of 76 nM. The protein was treated with urea to remove bound cAMP. Sedimentation values before and after the urea treatment were identical (s20,w = 5.1). Addition of purified R subunit to a preparation of yeast C subunit (TPK1) rendered catalytic activity cAMP-dependent with an activity ratio of 4.6. The yeast R was autophosphorylated by yeast C to a level of 0.8 mol of phosphate/mol of R monomer. By these criteria, the R subunit produced in E. coli was structurally and functionally identical to the natural yeast R subunit and similar to mammalian type II R subunits.

Base Sequence↗
PubMed1987

The S. cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway.

The gene corresponding to the S. cerevisiae cell division cycle mutant cdc25 has been cloned and sequenced, revealing an open reading frame encoding a protein of 1589 amino acids that contains no significant homologies with other known proteins. Cells lacking CDC25 have low levels of cyclic AMP and decreased levels of Mg2+-dependent adenylate cyclase activity. The lethality resulting from disruption of the CDC25 gene can be suppressed by the presence of the activated RAS2val19 gene, but not by high copy plasmids expressing a normal RAS2 or RAS1 gene. These results suggest that normal RAS is dependent on CDC25 function. Furthermore, mutationally activated alleles of CDC25 are capable of inducing a set of phenotypes similar to those observed in strains containing a genetically activated RAS/adenylate cyclase pathway, suggesting that CDC25 encodes a regulatory protein. We propose that CDC25 regulates adenylate cyclase by regulating the guanine nucleotide bound to RAS proteins.

Adenylyl Cyclases↗
PubMed1987

Expression and rearrangement of the ROS1 gene in human glioblastoma cells.

The human ROS1 gene, which possibly encodes a growth factor receptor, was found to be expressed in human tumor cell lines. In a survey of 45 different human cell lines, we found ROS1 to be expressed in glioblastoma-derived cell lines at high levels and not to be expressed at all, or expressed at very low levels, in the remaining cell lines. The ROS1 gene was present in normal copy numbers in all cell lines that expressed the gene. However, in one particular glioblastoma line, we detected a potentially activating mutation at the ROS1 locus.

DNA, Neoplasm↗
PubMed1987

Rigorous feedback control of cAMP levels in Saccharomyces cerevisiae.

We have isolated and characterized normal and mutant alleles of many of the genes of the RAS/adenylyl cyclase pathway of the yeast Saccharomyces cerevisiae. Manipulation of those genes has revealed a system for feedback control that can modulate cAMP levels over at least a 10,000-fold range. The feedback control depends upon the activity of the cAMP-dependent protein kinases and requires the presence of the CDC25 and RAS proteins. The capacity for such dramatic control of cAMP levels raises fundamental questions about the normal mechanism of action of the cAMP signaling system in yeast.

Adenylyl Cyclases↗
PubMed1987

Cloning and characterization of the low-affinity cyclic AMP phosphodiesterase gene of Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains two genes which encode cyclic AMP (cAMP) phosphodiesterase. We previously isolated and characterized PDE2, which encodes a high-affinity cAMP phosphodiesterase. We have now isolated the PDE1 gene of S. cerevisiae, which encodes a low-affinity cAMP phosphodiesterase. These two genes represent highly divergent branches in the evolution of phosphodiesterases. High-copy-number plasmids containing either PDE1 or PDE2 can reverse the growth arrest defects of yeast cells carrying the RAS2(Val-19) mutation. PDE1 and PDE2 appear to account for the aggregate cAMP phosphodiesterase activity of S. cerevisiae. Disruption of both PDE genes results in a phenotype which resembles that induced by the RAS2(Val-19) mutation. pde1- pde2- ras1- ras2- cells are viable.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae.

We have cloned a gene (BCY1) from the yeast Saccharomyces cerevisiae that encodes a regulatory subunit of the cyclic AMP-dependent protein kinase. The encoded protein has a structural organization similar to that of the RI and RII regulatory subunits of the mammalian cyclic AMP-dependent protein kinase. Strains of S. cerevisiae with disrupted BCY1 genes do not display a cyclic AMP-dependent protein kinase in vitro, fail to grow on many carbon sources, and are exquisitely sensitive to heat shock and starvation.

Amino Acid Sequence↗
PubMed1987

Guanine nucleotide activation of, and competition between, RAS proteins from Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, yeast RAS proteins are potent activators of adenylate cyclase. In the present work we measured the activity of adenylate cyclase in membranes from Saccharomyces cerevisiae which overexpress this enzyme. The response of the enzyme to added RAS2 proteins bound with various guanine nucleotides and their analogs suggests that RAS2 proteins are active in their GTP-bound form and are virtually inactive in their GDP-bound form. Also, active RAS2 protein is not inhibited by inactive RAS2, suggesting that the inactive form does not compete with the active form in binding to its effector.

Adenylyl Cyclases↗
PubMed1987

Human ros1 and mas1 oncogenes located in regions of chromosome 6 associated with tumor-specific rearrangements.

Oncogenes have been implicated in tumorigenesis based on their localization to chromosomal sites associated with tumor-specific structural rearrangements. We have mapped the human ros1 (formerly mcf3) and mas1 oncogenes to the distal half of chromosome 6q, within a region frequently rearranged in malignant cells. Chromosomal mapping of these two new human transforming genes may help elucidate the involvement of the long arm of chromosome 6 in diverse tumor types.

Cell Line↗
PubMed1986

RAM, a gene of yeast required for a functional modification of RAS proteins and for production of mating pheromone a-factor.

We have identified a gene (SUPH) of S. cerevisiae that is required for both RAS function and mating by cells of a mating type. supH is allelic to ste16, a gene required for the production of the mating pheromone a-factor. Both RAS and a-factor coding sequences terminate with the potential acyltransferase recognition sequence Cys-A-A-X, where A is an aliphatic amino acid. Mutations in SUPH-STE16 prevent the membrane localization and maturation of RAS protein, as well as the fatty acid acylation of it and other membrane proteins. We propose the designation RAM (RAS protein and a-factor maturation function) for SUPH and STE16. RAM may encode an enzyme responsible for the modification and membrane localization of proteins with this C-terminal sequence.

Acylation↗
PubMed1986

Isolation and characterization of a new cellular oncogene encoding a protein with multiple potential transmembrane domains.

We have cloned and sequenced a new human oncogene and have named it mas. This oncogene was detected by its tumorigenicity in nude mice using the cotransfection and tumorigenicity assay previously described. The mas oncogene has a weak focus-inducing activity in transfected NIH 3T3 cells. A DNA rearrangement in the 5' noncoding sequence, which occurred during transfection, is probably responsible for activation of the mas gene. The cDNA sequence of the mas oncogene reveals a long open reading frame that codes for a 325 amino acid protein. This protein is very hydrophobic and has seven potential transmembrane domains. In this respect, the structure of the mas protein is novel among cellular oncogene products and may reflect a new functional class of oncogenes.

Animals↗
PubMed1986

Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae.

A gene, PDE2, has been cloned from the yeast Saccharomyces cerevisiae that, when present in high copy, reverses the phenotypic effects of RAS2Val19, a mutant form of the RAS2 gene that renders yeast cells sensitive to heat shock and starvation. It has previously been shown that the RAS proteins are potent activators of yeast adenylate cyclase. We report here that PDE2 encodes a high-affinity cAMP phosphodiesterase that shares sequence homology with animal cell phosphodiesterases. These results therefore imply that the effects of RAS2Val19 are mediated through its changes in cAMP concentration.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Characterization of an activated human ros gene.

A human oncogene, mcf3, previously detected by a combination of DNA-mediated gene transfer and a tumorigenicity assay, derives from a human homology of the avian v-ros oncogene. Both v-ros and mcf3 can encode a protein with homology to tyrosine-specific protein kinases, and both mcf3 and v-ros encode a potential transmembrane domain N terminal to the kinase domain. mcf3 probably arose during gene transfer from a normal human ros gene by the loss of a putative extracellular domain. There do not appear to be any other gross rearrangements in the structure of mcf3.

Amino Acid Sequence↗
PubMed1986

Exploring the function of RAS oncogenes by studying the yeast Saccharomyces cerevisiae.

The RAS oncogenes comprise a family of genes found to be activated in perhaps 10-20% of human cancers and which have been highly conserved in evolution. Homologs of the mammalian RAS exist in the yeast Saccharomyces cerevisiae (RAS1 and RAS2). We have shown that human ras proteins can complement the loss of RAS1 and RAS2 proteins in yeast, and hence are functionally homologous. Both human and yeast RAS proteins can stimulate the magnesium and guanine nucleotide-dependent adenylate cyclase activity present in yeast membranes. However, RAS proteins do not appear to stimulate adenylate cyclase in vertebrate cells. Our studies indicate that although RAS proteins are essential controlling elements of adenylate cyclase in yeast, they have other essential functions in that organisms. RAS proteins are themselves probably controlled by growth regulatory proteins.

Fungal Proteins↗
PubMed1985

DNA sequence and characterization of the S. cerevisiae gene encoding adenylate cyclase.

We have cloned CYR1, the S. cerevisiae gene encoding adenylate cyclase. The DNA sequence of CYR1 can encode a protein of 2026 amino acids. This protein would contain a central region comprised of over twenty copies of a 23 amino acid repeating unit with remarkable homology to a 24 amino acid tandem repeating unit of a trace human serum glycoprotein. Gene disruption and biochemical experiments indicate that the catalytic domain of adenylate cyclase resides in the carboxyl terminal 400 amino acids. Elevated expression of adenylate cyclase suppresses the lethality that otherwise results from loss of RAS gene function in yeast. Yeast adenylate cyclase, made in E. coli, cannot be activated by added RAS protein.

Adenosine Triphosphatases↗
PubMed1985

ras proteins can induce meiosis in Xenopus oocytes.

Injection of human H-ras protein induces maturation of Xenopus oocytes; that is, progression from prophase to metaphase of meiosis. The oncogenic protein encoded by H-rasval12 is nearly a 100-fold more potent than the protein encoded by the wild-type gene. We do not observe any measurable increase or decrease in cyclic AMP concentration in injected oocytes, and the effects of H-ras protein are only partially blocked by cholera toxin. Our results suggest that not all, if any, of the effects of H-rasval12 protein in this system are mediated by adenylate cyclase.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Functional homology of mammalian and yeast RAS genes.

Yeast spores lacking endogenous RAS genes will not germinate. If such spores contain chimeric mammalian/yeast RAS genes or even the mammalian H-ras gene under the control of the galactose-inducible GAL10 promoter, they will germinate in the presence of galactose and produce viable haploid progeny dependent on galactose for continued growth and viability. These results indicate that the biochemical function of RAS proteins is essential for vegetative haploid yeast and that this function has been conserved in evolution since the progenitors of yeast and mammals diverged.

Animals↗
PubMed1985

In yeast, RAS proteins are controlling elements of adenylate cyclase.

S. cerevisiae strains containing RAS2val19, a RAS2 gene with a missense mutation analogous to one that activates the transforming potential of mammalian ras genes, have growth and biochemical properties strikingly similar to yeast strains carrying IAC or bcy1. Yeast strains carrying the IAC mutation have elevated levels of adenylate cyclase activity. bcy1 is a mutation that suppresses the lethality in adenylate cyclase deficient yeast. Yeast strains deficient in RAS function exhibit properties similar to adenylate cyclase deficient yeast. bcy1 suppresses lethality in ras1- ras2- yeast. Compared to wild-type yeast strains, intracellular cyclic AMP levels are significantly elevated in RAS2val19 strains, significantly depressed in ras2- strains, and virtually undetectable in ras1- ras2- bcy1 strains. Membranes from ras1- ras2- bcy1 yeast lack the GTP-stimulated adenylate cyclase activity present in membranes from wild-type cells, and membranes from RAS2val19 yeast strains have elevated levels of an apparently GTP-independent adenylate cyclase activity. Mixing membranes from ras1- ras2- yeast with membranes from adenylate cyclase deficient yeast reconstitutes a GTP-dependent adenylate cyclase.

Adenylyl Cyclases↗