scholarly journals Enrichment and screening of heat-sensitive mutants of Physarum polycephalum

1978 ◽  
Vol 31 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Peter Sudbery ◽  
Kari Haugli ◽  
Finn Haugli
Keyword(s):  
Protein Synthesis ◽  
Physarum Polycephalum ◽  
New Method ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Temperature Sensitive Mutants ◽  
Nuclear Cycle ◽  
Dna And Protein Synthesis

SUMMARYA new method for the isolation of temperature-sensitive mutants of Physarum polycephalum is described. It involves enrichment and prescreening of mutagenized amoebae followed by screening at both the plasmodial and amoebal stage. A total of 74 temperature-sensitive strains were recovered of which 26 were temperature-sensitive only as plasmodia, 35 only as amoebae and 13 in both stages. After a shift to the nonpermissive temperature, DNA and protein synthesis were followed in temperature-sensitive plasmodia to discover if the lesion affected functions of the nuclear cycle.

scholarly journals A mutation in the tRNA nucleotidyltransferase gene promotes stabilization of mRNAs in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (12) ◽  
pp. 5778-5784
Author(s):  
S W Peltz ◽  
J L Donahue ◽  
A Jacobson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Steady State ◽  
Relative Number ◽  
Mrna Turnover ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
Temperature Sensitive Mutants ◽  
Steady State Levels

To identify trans-acting factors involved in mRNA decay in the yeast Saccharomyces cerevisiae, we have begun to characterize conditional lethal mutants that affect mRNA steady-state levels. A screen of a collection of temperature-sensitive mutants identified ts352, a mutant that accumulated moderately stable and unstable mRNAs after a shift from 23 to 37 degrees C (M. Aebi, G. Kirchner, J.-Y. Chen, U. Vijayraghavan, A. Jacobson, N.C. Martin, and J. Abelson, J. Biol. Chem. 265:16216-16220, 1990). ts352 has a defect in the CCA1 gene, which codes for tRNA nucleotidyltransferase, the enzyme that adds 3' CCA termini to tRNAs (Aebi et al., J. Biol. Chem., 1990). In a shift to the nonpermissive temperature, ts352 (cca1-1) cells rapidly cease protein synthesis, reduce the rates of degradation of the CDC4, TCM1, and PAB1 mRNAs three- to fivefold, and increase the relative number of ribosomes associated with mRNAs and the overall size of polysomes. These results were analogous to those observed for cycloheximide-treated cells and are generally consistent with models that invoke a role for translational elongation in the process of mRNA turnover.

scholarly journals A mutation in the tRNA nucleotidyltransferase gene promotes stabilization of mRNAs in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (12) ◽  
pp. 5778-5784 ◽  
Author(s):  
S W Peltz ◽  
J L Donahue ◽  
A Jacobson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Steady State ◽  
Relative Number ◽  
Mrna Turnover ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
Temperature Sensitive Mutants ◽  
Steady State Levels

To identify trans-acting factors involved in mRNA decay in the yeast Saccharomyces cerevisiae, we have begun to characterize conditional lethal mutants that affect mRNA steady-state levels. A screen of a collection of temperature-sensitive mutants identified ts352, a mutant that accumulated moderately stable and unstable mRNAs after a shift from 23 to 37 degrees C (M. Aebi, G. Kirchner, J.-Y. Chen, U. Vijayraghavan, A. Jacobson, N.C. Martin, and J. Abelson, J. Biol. Chem. 265:16216-16220, 1990). ts352 has a defect in the CCA1 gene, which codes for tRNA nucleotidyltransferase, the enzyme that adds 3' CCA termini to tRNAs (Aebi et al., J. Biol. Chem., 1990). In a shift to the nonpermissive temperature, ts352 (cca1-1) cells rapidly cease protein synthesis, reduce the rates of degradation of the CDC4, TCM1, and PAB1 mRNAs three- to fivefold, and increase the relative number of ribosomes associated with mRNAs and the overall size of polysomes. These results were analogous to those observed for cycloheximide-treated cells and are generally consistent with models that invoke a role for translational elongation in the process of mRNA turnover.

scholarly journals LOCALIZED MUTAGENESIS FOR THE ISOLATION OF TEMPERATURE-SENSITIVE MUTANTS OF ESCHERICHIA COLI AFFECTED IN PROTEIN SYNTHESIS

Genetics ◽  
1979 ◽  
Vol 91 (2) ◽  
pp. 215-227
Author(s):  
W Scott Champney
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Temperature Sensitive ◽  
Chromosomal Locus ◽  
Ribosomal Subunits ◽  
Prolonged Incubation ◽  
Temperature Sensitive Mutants ◽  
Inhibition Of Growth ◽  
Localized Mutagenesis

ABSTRACT Two variations of the method of localized mutagenesis were used to introduce mutations into the 72 min region of the Escherichia coli chromosome. Twenty temperature-sensitive mutants, with linkage to markers in this region, have been examined. Each strain showed an inhibition of growth in liquid medium at 44°, and 19 of the mutants lost viability upon prolonged incubation at this temperature. A reduction in the rate of in vivo RNA and protein synthesis was observed for each mutant at 44°, relative to a control strain. Eleven of the mutants were altered in growth sensitivity or resistance to one or more of three ribosomal antibiotics. The incomplete assembly of ribosomal subunits was detected in nine strains grown at 44°. The characteristics of these mutants suggest that many of them are altered in genes for translational or transcriptional components, consistent with the clustering of these genes at this chromosomal locus.

Genetic and biochemical studies of asparagine-linked oligosaccharide assembly

1982 ◽  
Vol 300 (1099) ◽  
pp. 207-223 ◽  
Keyword(s):  
Protein Synthesis ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutants ◽  
Isolation And Characterization ◽  
Oligosaccharide Processing ◽  
Biochemical Studies ◽  
The Common ◽  
Specific Lipid ◽  
Temperature Sensitive Phenotype

The formation of N -glycosidic linkages of eukaryotic glycoproteins involves the assembly of a specific lipid-linked precursor oligosaccharide in the endoplasmic reticulum. This oligosaccharide is transferred from the lipid carrier to appropriate asparagine residues during protein synthesis. The protein-linked oligosaccharide then undergoes processing reactions that include both removal and addition of carbohydrate residues. In this paper we report recent studies from our laboratory on the synthesis of asparagine-linked oligosaccharides. In the first part we describe the isolation and characterization of temperature-sensitive mutants of yeast blocked at specific stages in the assembly of the lipid-linked oligosaccharide. In addition, we are using these mutants to clone the genes for the enzymes in this pathway by complementation of the temperature-sensitive phenotype. The second part deals with the topography of asparagine-linked oligosaccharide assembly. Our studies on the transmembrane movement of sugar residues during the assembly of secreted glycoproteins from cytoplasmic precursors are presented. Finally, experiments on the control of protein-linked oligosaccharide processing are described. Recent data are presented on the problem of how specific oligosaccharides are assembled from the common precursors at individual sites on glycoproteins.

Apoptosis Was Promoted at a Nonpermissive Temperature in DNA Replication-Defective Temperature-Sensitive Mutants of Mouse FM3A Cells

1998 ◽  
Vol 238 (2) ◽  
pp. 317-323 ◽  
Author(s):  
Yukika Yamauchi ◽  
Akiko Tanaka ◽  
Kazunori Yamaguchi ◽  
Masayuki Kobayashi ◽  
Seiichi Shimamura ◽  
...  
Keyword(s):  
Dna Replication ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Temperature Sensitive Mutants ◽  
Fm3a Cells

scholarly journals Temperature-sensitive mutants of Physarum polycephalum: viability, growth, and nuclear replication.

1979 ◽  
Vol 138 (2) ◽  
pp. 499-504 ◽  
Author(s):  
T G Laffler ◽  
A Wilkins ◽  
S Selvig ◽  
N Warren ◽  
A Kleinschmidt ◽  
...  
Keyword(s):  
Physarum Polycephalum ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutants

scholarly journals Nuclear transport defects and nuclear envelope alterations are associated with mutation of the Saccharomyces cerevisiae NPL4 gene.

1996 ◽  
Vol 7 (11) ◽  
pp. 1835-1855 ◽  
Author(s):  
C DeHoratius ◽  
P A Silver
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Protein Import ◽  
Nuclear Protein ◽  
Nuclear Transport ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Temperature Sensitive Mutants ◽  
Nuclear Protein Import

To identify components involved in nuclear protein import, we used a genetic selection to isolate mutants that mislocalized a nuclear-targeted protein. We identified temperature-sensitive mutants that accumulated several different nuclear proteins in the cytoplasm when shifted to the semipermissive temperature of 30 degrees C; these were termed npl (nuclear protein localization) mutants. We now present the properties of yeast strains bearing mutations in the NPL4 gene and report the cloning of the NPL4 gene and the characterization of the Np14 protein. The npl4-1 mutant was isolated by the previously described selection scheme. The second allele, npl4-2, was identified from an independently derived collection of temperature-sensitive mutants. The npl4-1 and npl4-2 strains accumulate nuclear-targeted proteins in the cytoplasm at the nonpermissive temperature consistent with a defect in nuclear protein import. Using an in vitro nuclear import assay, we show that nuclei prepared from temperature-shifted npl4 mutant cells are unable to import nuclear-targeted proteins, even in the presence of cytosol prepared from wild-type cells. In addition, npl4-2 cells accumulate poly(A)+ RNA in the nucleus at the nonpermissive temperature, consistent with a failure to export mRNA from the nucleus. The npl4-1 and npl4-2 cells also exhibit distinct, temperature-sensitive structural defects: npl4-1 cells project extra nuclear envelope into the cytoplasm, whereas npl4-2 cells from nuclear envelope herniations that appear to be filled with poly(A)+ RNA. The NPL4 gene encodes an essential M(r) 64,000 protein that is located at the nuclear periphery and localizes in a pattern similar to nuclear pore complex proteins. Taken together, these results indicate that this gene encodes a novel nuclear pore complex or nuclear pore complex-associated component required for nuclear membrane integrity and nuclear transport.

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