Showing papers by "David Beach published in 1988"

Four mating-type genes control sexual differentiation in the fission yeast.

Abstract: The mating-type region of fission yeast consists of three components, mat1, mat2-P and mat3-M, each separated by 15 kb. Cell-type is determined by the alternate allele present at mat1, either P in an h+ or M in an h- cell. mat2-P and mat3-M serve as donors of information that is transposed to mat1 during a switch of mating type. We have determined the nucleotide sequence of each component of mat. The P and M specific regions are 1104 and 1128 bp, respectively, and bounded by sequences common to each mating-type cassette (H1; 59 bp and H2; 135 bp). A third sequence is present at mat2-P and mat3-M but absent at mat1 (H3; 57 bp), and may be involved in transcriptional repression of these cassettes. mat1-P and mat1-M each encode two genes (Pc; 118 amino acids, Pi; 159 amino acids, Mc; 181 amino acids and Mi; 42 amino acids). Introduction of opal or frame-shift mutations into the open-reading-frame of each gene revealed that Pc and Mc are necessary and sufficient for mating and confer an h+ or h- mating type respectively. All four genes are required for meiotic competence in an h+/h- diploid. The transcription of each mat gene is strongly influenced by nutritional conditions and full induction was observed only in nitrogen-free medium. The predicted product of the Pi gene contains a region of homology with the homeobox sequence, suggesting that this gene encodes a DNA binding protein that directly regulates the expression of other genes.

Human cdc2 protein kinase is a major cell-cycle regulated tyrosine kinase substrate.

Abstract: cdc2 is a catalytic subunit of a protein kinase complex, called the M-phase promoting factor, that induces entry into mitosis and is universal among eukaryotes. In HeLa cells, cdc2 is shown to be the most abundant phosphotyrosine-containing protein and its phosphotyrosine content is subject to cell-cycle regulation. One site of cdc2 tyrosine phosphorylation in vivo is selectively phosphorylated by pp60c-src in vitro.

Involvement of cdc13+ in mitotic control in Schizosaccharomyces pombe: possible interaction of the gene product with microtubules.

Abstract: Previous genetic studies have shown that the fission yeast cdc13+ gene product interacts closely with the cdc2+ protein kinase during mitosis. Here, we have cloned the cdc13+ gene from a S. pombe gene bank by complementation of the temperature-sensitive defect of a cdc13-117 mutant strain. The complementing activity was localized to a 1.9-kb XbaI-NsiI DNA fragment, and nucleotide sequencing revealed a 1446-bp open reading frame. The predicted amino acid sequence contained 482 residues and was not homologous to any protein in a protein database. The cdc13+ gene function was confirmed to be essential for cell division since cells carrying a cdc13 null allele arrested with a cdc phenotype. However, unlike any existing temperature-sensitive cdc13 mutants, cdc13 null mutants arrested in G2 without septa or condensed chromosomes indicating that cdc13+ gene function is required at or prior to the initiation of mitotis. cdc13-117 mutant strains were found to be hypersensitive to the tubulin inhibitor thiabendazole. This observation suggests that the cdc13+ gene product, which is required for mitotic initiation, may interact with microtubules.

A specific inhibitor of the ran1+ protein kinase regulates entry into meiosis in Schizosaccharomyces pombe.

Abstract: In fission yeast, meiosis is initiated by transcriptional activation of the mei3+ gene, under the combined influence of the four mating-type genes. The product of the mei3+ gene acts as a critical meiotic inducer by binding non-covalently to a newly identified protein kinase encoded by the ran1+ gene and inhibiting its enzymatic activity. Inactivation of the ran1+ protein kinase is both necessary and sufficient to divert a vegetative cell from mitotic division to meiotic differentiation.

Synthesis of p34, the mammalian homolog of the yeast cdc2+/CDC28 protein kinase, is stimulated during adenovirus-induced proliferation of primary baby rat kidney cells

Abstract: The homolog of the cdc2+/CDC28+ encoded protein kinases has previously been identified in HeLa cells by immunological methods. Here we have studied the distribution and synthesis of this 34 kd protein in rat tissues and cell lines. p34 was found in a variety of organs, including some such as brain that are not highly active in cell division. Kidney has very low levels of p34. However, proliferative activation of baby rat kidney cells with adenovirus caused rapid induction of p34 synthesis. Induction was dependent on the E1A gene of the virus but not E1B, and was not prevented by inhibition of cellular DNA synthesis with hydroxyurea. Increased synthesis of p34 is due, at least in part, to an increase in abundance of translatable p34 mRNA. These data are consistent with the possibility that p34 plays a role in cell division in higher vertebrates.

Multiple phosphorylated forms of the product of the fission yeast cell division cycle gene cdc2

Abstract: The 34 kilodalton protein product (p34) of the cdc2+ cell cycle control gene of Schizosaccharomyces pombe was expressed in bacteria. Monoclonal antibodies raised against this protein are capable of immunoprecipitating p34cdc2 from yeast lysates. Immunoprecipitates of [35S]methionine- and [32P]orthophosphate-labeled p34cdc2 were analyzed by two-dimensional gel electrophoresis. The cdc2+ gene product is homogeneous in size but resolves into seven species of differing charge. At least four of these species are phosphorylated. Phosphoamino acid analysis reveals that phosphorylation occurs mainly on threonine residues. The pattern of p34 phosphorylation is unaltered at the nonpermissive temperature in strains carrying temperature sensitive alleles of weel-50 and ran1-114 or in a strain overproducing the ran1+ gene product.

Cell Cycle Control in Eukaryotes

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Regulation of the vertebrate cell cycle by the cdc2 protein kinase.

Abstract: A homolog of the cdc2/CDC28 protein kinase of yeast is found in all vertebrate species that have been investigated. Human cdc2 exists as a complex with a 13-kD protein that is homologous to the suc1 gene product of fission yeast. In both human and fission yeast cells, the protein kinase also exists in a complex with a 62-kD polypeptide that has not been identified genetically but acts as a substrate in vitro. We have studied the properties of the protein kinase in rat and human cells, as well as in Xenopus eggs. We find that in baby rat kidney (BRK) cells, which are quiescent in cell culture, the cdc2 protein is not synthesized. However, synthesis is rapidly induced in response to proliferative activation by infection with adenovirus. In human HeLa cells, the protein kinase is present continuously. It behaves as a cell-cycle oscillator that is inactive in G1 but displays

p34, a protein kinase involved in cell cycle regulation in eukaryotic cells.

Abstract: Investigation of the biochemical basis of cellular transformation is a major area of current research directed towards understanding the problem of tumorigenesis and human cancer. Much progress has been made in identifying primary oncogenic agents, both chemical and viral, in addition to the discovery of cellular oncogenes (Bishop, 1985). Biochemical activities have been associated with the products of some viral and cellular oncogenes, most notably those which belong to the protein kinase family (Erikson and Erikson, 1980).