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Isolation of temperature-sensitive mutants for mRNA capping enzyme in Saccharomyces cerevisiae
Authors:Masahiro Yamagishi   Kiyohisa Mizumoto  Akira Ishihama
Affiliation:(1) Department of Molecular Genetics, National Institute of Genetics, 411 Mishima, Shizuoka, Japan;(2) School of Pharmaceutical Sciences, Kitasato University, 9-1 Shirokane 5 chome, 108 Minato-ku, Tokyo, Japan;(3) Department of Cell Biology, Aichi Cancer Center Research Institute, Chikusa-ku, 464 Nagoya, Aichi, Japan
Abstract:The guanylyltransferase activity of mRNA capping enzyme catalyzes the transfer of GMP from GTP to the 5prime terminus of mRNA. In Saccharomyces cerevisiae, the activity is carried on the agr subunit of capping enzyme, the product of the CEG1 gene. We have isolated 10 recessive, temperature-sensitive mutations of CEG1; nine (cegl-1 to cegl-9) were isolated on a single-copy plasmid and the remaining one (cegl-10) on a multicopy plasmid. The presence of cegl-10 in multiple copies is essential for the viability of cells carrying the mutation, and a shift to the restrictive temperature resulted in rapid growth arrest of cegl-10 cells, while growth rates of other mutants decreased gradually upon temperature upshift. Intragenic complementation was not observed for pairwise combinations of the mutations. Although the majority of the mutations occurred at the amino acid residues conserved between Cegl and the Schizosaccharomyces pombe homologue, none were located in the regions that are also conserved among viral capping enzymes and polynucleotide ligases. Guanylyltransferase activity of the mutant proteins as measured by covalent Ceg1-GMP complex formation was heat-labile. The availability of these mutants should facilitate studies of the structure-function relationships of capping enzyme, as well as the roles and regulation of mRNA capping.
Keywords:Saccharomyces cerevisiae  CEG1  Temperature-sensitive mutants  mRNA capping enzyme  Guanylyltransferase
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