scholarly journals Characterization of a cell cycle mutant derived from hamster fibroblast: reversion analysis.

1982 ◽  
Vol 92 (3) ◽  
pp. 629-633 ◽  
Author(s):  
D J Scharff ◽  
A M Delegeane ◽  
A S Lee
Keyword(s):  
Cell Line ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Molecular Weights ◽  
A Cell ◽  
Ts Mutant ◽  
Spontaneous Revertant

K12 is a temperature-sensitive (ts) mutant cell line derived from Chinese hamster fibroblasts. When incubated at the nonpermissive temperature, K12 cells exhibit the following properties: (a) the cells cannot initiate DNA synthesis;o (b) the synthesis of cytosol thymidine kinase is suppressed; and (c) the synthesis of three cellular proteins of molecular weights 94, 78, and 58 kdaltons is greatly enhanced. Here we characterize a spontaneous revertant clone, R12, derived from the K12 cells. We selected the revertant clone for its ability to grow at the nonpermissive temperature. Our results indicate that all the traits which constitute the K12 mutant phenotype are simultaneously reverted to the wild type in the revertant cell line, suggesting that the ts mutation of the K12 cells is of regulatory nature and exerts multiple effects on the expressed phenotypes.

Molecular cloning of a human gene that regulates chromosome condensation and is essential for cell proliferation

1986 ◽  
Vol 6 (6) ◽  
pp. 2027-2032
Author(s):  
R Kai ◽  
M Ohtsubo ◽  
M Sekiguchi ◽  
T Nishimoto
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Human Gene ◽  
Chromosome Condensation ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Hindiii Fragment ◽  
Human Dna ◽  
Ts Mutant ◽  
Tsbn2 Cells

The tsBN2 cell line, a temperature-sensitive (ts) mutant of baby hamster kidney cell line BHK21/13, seems to possess a mutation in the gene that controls initiation of chromosome condensation. At the nonpermissive temperature (39.5 degrees C), the chromatin of tsBN2 cells is prematurely condensed, and the cells die. Using tsBN2 cells as a recipient of DNA-mediated gene transfer, we investigated a human gene that is responsible for regulation of chromosome condensation and cell proliferation. We found that the human gene complementing the tsBN2 mutation resides in the area of the 40- to 50-kilobase HindIII fragment, derived from HeLa cells. Based on this finding, we initiated cloning of a human gene complementing the tsBN2 mutation. From lambda and cosmid libraries carrying partial digests of DNA from the secondary transformants, the 41.8-kilobase HindIII fragment containing the human DNA was isolated. The cloned human DNA was conserved in ts+ transformants through primary and secondary transfections. Two cosmid clones convert the ts- phenotype of tsBN2 cells to ts+ with more than 100 times a higher efficiency, compared with cases of transfection with total human DNA. Thus, the cloned DNA fragments contain an active human gene that complements the tsBN2 mutation.

scholarly journals Molecular cloning of a human gene that regulates chromosome condensation and is essential for cell proliferation.

1986 ◽  
Vol 6 (6) ◽  
pp. 2027-2032 ◽  
Author(s):  
R Kai ◽  
M Ohtsubo ◽  
M Sekiguchi ◽  
T Nishimoto
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Human Gene ◽  
Chromosome Condensation ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Hindiii Fragment ◽  
Human Dna ◽  
Ts Mutant ◽  
Tsbn2 Cells

The tsBN2 cell line, a temperature-sensitive (ts) mutant of baby hamster kidney cell line BHK21/13, seems to possess a mutation in the gene that controls initiation of chromosome condensation. At the nonpermissive temperature (39.5 degrees C), the chromatin of tsBN2 cells is prematurely condensed, and the cells die. Using tsBN2 cells as a recipient of DNA-mediated gene transfer, we investigated a human gene that is responsible for regulation of chromosome condensation and cell proliferation. We found that the human gene complementing the tsBN2 mutation resides in the area of the 40- to 50-kilobase HindIII fragment, derived from HeLa cells. Based on this finding, we initiated cloning of a human gene complementing the tsBN2 mutation. From lambda and cosmid libraries carrying partial digests of DNA from the secondary transformants, the 41.8-kilobase HindIII fragment containing the human DNA was isolated. The cloned human DNA was conserved in ts+ transformants through primary and secondary transfections. Two cosmid clones convert the ts- phenotype of tsBN2 cells to ts+ with more than 100 times a higher efficiency, compared with cases of transfection with total human DNA. Thus, the cloned DNA fragments contain an active human gene that complements the tsBN2 mutation.

Revertants of a Chinese Hamster Ovary Cell Mutant with an Altered β-Tubulin: Evidence that the Altered Tubulin Confers Both Colcemid Resistance and Temperature Sensitivity on the Cell

1982 ◽  
Vol 2 (6) ◽  
pp. 720-729
Author(s):  
Fernando Cabral ◽  
Irene Abraham ◽  
Michael M. Gottesman
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Chinese Hamster Ovary Cell ◽  
Temperature Sensitivity ◽  
Chinese Hamster Ovary ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Temperature Sensitive ◽  
Β Tubulin

We recently described the isolation of a mutant Chinese hamster ovary cell (Cmd 4) resistant to the cytotoxic effects of colcemid (Cabral et al., Cell 20 :29-36, 1980). This mutant carries an altered β-tubulin but still grows normally at 37°C. In the present study we found that Cmd 4 is temperature sensitive for growth at 40.3°C. A class of revertants selected for temperature resistance had simultaneously lost colcemid resistance and the altered β-tubulin. In addition, we isolated a temperature-resistant revertant which carries a further alteration in the mutant β-tubulin polypeptide. This second alteration appears to make the mutant β-tubulin incompetent to assemble into microtubules, resulting in a strain which is again colcemid sensitive. These revertant cell lines provide strong evidence that a mutation in β-tubulin can confer both colcemid resistance and temperature sensitivity on a mammalian cell line. Cellular microtubules studied by indirect immunofluorescence in both mutant and revertant cell lines had an apparently normal distribution at permissive and nonpermissive temperatures, yet mitosis appears to be abnormal in the mutant cell line. We conclude from these studies that incorporation of the altered β-tubulin into microtubules does not affect their distribution but may affect their function during mitosis.

scholarly journals Rescue of the complex temperature-sensitive phenotype of Chinese hamster ovary E36ts20 cells by expression of the human ubiquitin-activating enzyme cDNA

1994 ◽  
Vol 304 (3) ◽  
pp. 1015-1020 ◽  
Author(s):  
P M Handley-Gearhart ◽  
J S Trausch-Azar ◽  
A Ciechanover ◽  
A L Schwartz
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Conjugation System ◽  
Primary Defect ◽  
Ubiquitin Conjugation

The ubiquitin conjugation system is a multi-step pathway in which ubiquitin is activated and conjugated to acceptor proteins, one function of which is to target acceptor proteins for rapid degradation within the cell. The conjugation system is involved in many aspects of cellular functions, including the cell cycle. Several cell-cycle arrest mutant cell lines have been characterized and appear to harbour a mutant ubiquitin-activating enzyme, E1, as their primary defect. One such cell line is ts20, which is derived from Chinese hamster ovary E36 cells. This cell line has been used to characterize some of the potential functions of the ubiquitin conjugation system in vivo, such as its involvement in the maturation of autophagic vacuoles. The present study describes the complete rescue of the complex ts20 phenotype following the expression of the cDNA for human E1. Stable transfectants expressing the human E1 cDNA in the CMVneo expression vector were measured for ubiquitin-conjugation activity, protein degradation and growth in culture at the nonpermissive temperature. This rescue confirms that the phenotype observed in the ts20 cells is due to a defect in the E1 enzyme. Thus, the ts20 cell line will serve as a useful tool to delineate the functions of the ubiquitin system in vivo.

Biochemical and genetic approaches to the study of mammalian mitochondrial tRNAs

1978 ◽  
Vol 56 (6) ◽  
pp. 592-597
Author(s):  
Luke Aujame ◽  
Randall W. Yatscoff ◽  
Karl B. Freeman
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Trna Synthetase ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Cho Cell ◽  
Isolated Mitochondria ◽  
Slab Gels

The possible existence of mammalian mitochondrial asparaginyl-tRNA has been examined using a variety of approaches. [3H]Asparagine was incorporated into protein by mitochondria of the Chinese hamster ovary (CHO) cell line Asn-7, which has a temperature-sensitive cytosolic asparaginyl-tRNA synthetase, either in the presence of cycloheximide or at a nonpermissive temperature. Isolated mitochondria of CHO thymidine kinase minus (TK−) cells also incorporated the amino acid into protein. In each case, the number and electrophoretic mobility of the proteins was the same as mitochondrially synthesized proteins of CHO TK− ceils labelled with [35S]methionine. A tRNAAsn could be charged in isolated CHO TK cell mitochondria and the asparaginyl-tRNA was found to elute before its cytosolic counterpart on an RPC-5 column and to have a higher mobility on polyacryiamide slab gels run under denaturing conditions. This is the first demonstration of a unique mitochondrial asparaginyl-tRNA.

scholarly journals Revertants of a Chinese Hamster Ovary Cell Mutant with an Altered β-Tubulin: Evidence that the Altered Tubulin Confers Both Colcemid Resistance and Temperature Sensitivity on the Cell

1982 ◽  
Vol 2 (6) ◽  
pp. 720-729 ◽  
Author(s):  
Fernando Cabral ◽  
Irene Abraham ◽  
Michael M. Gottesman
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Chinese Hamster Ovary Cell ◽  
Temperature Sensitivity ◽  
Chinese Hamster Ovary ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Temperature Sensitive ◽  
Β Tubulin

We recently described the isolation of a mutant Chinese hamster ovary cell (Cmd 4) resistant to the cytotoxic effects of colcemid (Cabral et al., Cell20:29-36, 1980). This mutant carries an altered β-tubulin but still grows normally at 37°C. In the present study we found that Cmd 4 is temperature sensitive for growth at 40.3°C. A class of revertants selected for temperature resistance had simultaneously lost colcemid resistance and the altered β-tubulin. In addition, we isolated a temperature-resistant revertant which carries a further alteration in the mutant β-tubulin polypeptide. This second alteration appears to make the mutant β-tubulin incompetent to assemble into microtubules, resulting in a strain which is again colcemid sensitive. These revertant cell lines provide strong evidence that a mutation in β-tubulin can confer both colcemid resistance and temperature sensitivity on a mammalian cell line. Cellular microtubules studied by indirect immunofluorescence in both mutant and revertant cell lines had an apparently normal distribution at permissive and nonpermissive temperatures, yet mitosis appears to be abnormal in the mutant cell line. We conclude from these studies that incorporation of the altered β-tubulin into microtubules does not affect their distribution but may affect their function during mitosis.

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