scholarly journals Temperature-Sensitive Chinese Hamster Fibroblast Mutant with a Defect in RNA Metabolism

1982 ◽  
Vol 2 (12) ◽  
pp. 1558-1573 ◽  
Author(s):  
Eric A. Wong ◽  
Immo E. Scheffler
Keyword(s):  
Protein Synthesis ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Temperature Sensitive ◽  
Chinese Hamster Fibroblast ◽  
Rna And Protein Synthesis ◽  
Shock Proteins ◽  
Short Time ◽  
Biosynthetic Machinery ◽  
Temperature Sensitive Phenotype

We describe a new temperature-sensitive mutant of Chinese hamster cell fibroblasts. After a shift to the nonpermissive temperature of 40.5°C, the rates of DNA, RNA, and protein synthesis declined rapidly (to ≤50% within 12 h) and the progression of unsynchronized cells through the cell cycle was affected. We believe that DNA synthesis came to a halt after a short time, because cells no longer entered the S phase. The decrease in protein synthesis at 40.5°C was shown to be a consequence of a decrease in the number of polysomes, whereas free 80S ribosomes accumulated. We concluded that the components of the protein biosynthetic machinery were intact (ribosomes and soluble factors), but synthesis was limited by a shortage of mRNA. The decline in mRNA production had a significant effect on the synthesis of proteins (e.g., heat shock proteins) translated from short-lived messages. We observed that both polyadenylated and nonpolyadenylated RNA syntheses declined at 40.5°C, whereas the synthesis of small RNAs (4 to 5S) was less reduced. The argument is made that the temperature-sensitive phenotype is the result of a defect affecting mRNA synthesis.

Temperature-Sensitive Chinese Hamster Fibroblast Mutant with a Defect in RNA Metabolism

1982 ◽  
Vol 2 (12) ◽  
pp. 1558-1573
Author(s):  
Eric A. Wong ◽  
Immo E. Scheffler
Keyword(s):  
Protein Synthesis ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Temperature Sensitive ◽  
Chinese Hamster Fibroblast ◽  
Rna And Protein Synthesis ◽  
Shock Proteins ◽  
Short Time ◽  
Biosynthetic Machinery ◽  
Temperature Sensitive Phenotype

We describe a new temperature-sensitive mutant of Chinese hamster cell fibroblasts. After a shift to the nonpermissive temperature of 40.5°C, the rates of DNA, RNA, and protein synthesis declined rapidly (to ≤50% within 12 h) and the progression of unsynchronized cells through the cell cycle was affected. We believe that DNA synthesis came to a halt after a short time, because cells no longer entered the S phase. The decrease in protein synthesis at 40.5°C was shown to be a consequence of a decrease in the number of polysomes, whereas free 80S ribosomes accumulated. We concluded that the components of the protein biosynthetic machinery were intact (ribosomes and soluble factors), but synthesis was limited by a shortage of mRNA. The decline in mRNA production had a significant effect on the synthesis of proteins (e.g., heat shock proteins) translated from short-lived messages. We observed that both polyadenylated and nonpolyadenylated RNA syntheses declined at 40.5°C, whereas the synthesis of small RNAs (4 to 5S) was less reduced. The argument is made that the temperature-sensitive phenotype is the result of a defect affecting mRNA synthesis.

Cloning of a human S-phase cell cycle gene: use of transient expression for screening

1987 ◽  
Vol 7 (2) ◽  
pp. 775-779
Author(s):  
A Fainsod ◽  
G Diamond ◽  
M Marcus ◽  
F H Ruddle
Keyword(s):  
Cell Cycle ◽  
Genomic Library ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
S Phase ◽  
Phase Cell ◽  
Cell Cycle Gene ◽  
Temperature Sensitive ◽  
Specific Cell ◽  
Restrictive Temperature

We report here the cloning of a human cell cycle gene capable of complementing a temperature-sensitive (ts) S-phase cell cycle mutation in a Chinese hamster cell line. Cloning was performed as follows. A human genomic library in phage lambda containing 600,000 phages was screened with labeled cDNA synthesized from an mRNA fraction enriched for the specific cell cycle gene message. Plaques containing DNA inserts which hybridized to the cDNA were picked, and their DNAs were assayed for transient complementation in DNA transformation experiments. The transient complementation assay we developed is suitable for most cell cycle genes and indeed for many genes whose products are required for cell proliferation. Of 845 phages screened, 1 contained an insert active in transient complementation of the ts cell cycle mutation. Introduction of this phage into the ts cell cycle mutant also gave rise to stable transformants which grew normally at the restrictive temperature for the ts mutant cells.

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.

scholarly journals Identification and characterization of a third complementation group of emetine-resistant Chinese hamster cell mutants.

1981 ◽  
Vol 1 (1) ◽  
pp. 58-65 ◽  
Author(s):  
J J Wasmuth ◽  
J M Hill ◽  
L S Vock
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Resistant Cell ◽  
Complementation Group ◽  
Ovary Cells ◽  
Complementation Groups ◽  
Resistant Mutants ◽  
Biosynthetic Machinery

We have isolated emetine-resistant cell lines from Chinese hamster peritoneal fibroblasts and have shown that they represent a third distinct class or complementation group of emetine-resistant mutants, as determined by three different criteria. These mutants, like those belonging to the two other complementation groups we have previously defined, which were isolated from Chinese hamster lung and Chinese hamster ovary cells, have alterations that directly affect the protein biosynthetic machinery. So far, there is absolute cell line specificity with respect to the three complementation groups, in that all the emetine-resistant mutants we have isolated from Chinese hamster lung cells belong to one complementation group, all those we have isolated from Chinese hamster ovary cells belong to a second complementation group, and all those isolated from Chinese hamster peritoneal cells belong to a third complementation group. Thus, in cultured Chinese hamster cells, mutations in at least three different loci, designated emtA, emtB, and emtC, encoding for different components of the protein biosynthetic machinery, can give rise to the emetine-resistant phenotype.

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