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.

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 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 the human gene, CCG2, that complements the BHK-derived temperature-sensitive cell cycle mutant tsBN63: identity of CCG2 with the human X chromosomal SCAR/RPS4X gene

1991 ◽  
Vol 100 (1) ◽  
pp. 35-43
Author(s):  
M. Watanabe ◽  
N. Furuno ◽  
M. Goebl ◽  
M. Go ◽  
K. Miyauchi ◽  
...  
Keyword(s):  
Cell Line ◽  
Genomic Dna ◽  
Human Gene ◽  
G1 Phase ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
G2 Phase ◽  
Synchronized Cultures

A temperature-sensitive mutant tsBN63 cell line was isolated by the fluorodeoxyuridine method from the BHK21/13 cell line after mutagenesis with nitrosoguanidine. When cultures of tsBN63 cells growing asynchronously at 33.5 degrees C were shifted to 39.5 degrees C, a nonpermissive temperature, the ability for protein synthesis was rapidly reduced and cell proliferation stopped mainly at G1 phase, and partly at G2 phase. Synchronized cultures of tsBN63 cells did not commence DNA synthesis when shifted up in G1 phase. The human gene complementing the tsBN63 mutation was cloned by DNA-mediated gene transfer and its cDNA of 1.1 kb conferring ts+ phenotype on tsBN63 cells was isolated from the cDNA library of Raj (mer+) cells with a frequency of 10(−3). On the basis of the determined nucleotide sequence, the isolated human gene turned out to be the X chromosomal RPS4X encoding the ribosomal protein S4. The size of the CCG2 gene was estimated to be about 12 kb by complementation analysis of the tsBN63 mutation with cloned genomic DNA.

scholarly journals Molecular cloning of a human cDNA encoding a novel protein, DAD1, whose defect causes apoptotic cell death in hamster BHK21 cells

1993 ◽  
Vol 13 (10) ◽  
pp. 6367-6374
Author(s):  
T Nakashima ◽  
T Sekiguchi ◽  
A Kuraoka ◽  
K Fukushima ◽  
Y Shibata ◽  
...  
Keyword(s):  
Cell Line ◽  
Apoptotic Cell ◽  
Protein Synthesis Inhibitor ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Synthesis Inhibitor ◽  
Base Sequences ◽  
Encoding Gene ◽  
Induced Apoptosis ◽  
Mutant Lines

The tsBN7 cell line, one of the mutant lines temperature sensitive for growth which have been isolated from the BHK21 cell line, was found to die by apoptosis following a shift to the nonpermissive temperature. The induced apoptosis was inhibited by a protein synthesis inhibitor, cycloheximide, but not by the bcl-2-encoded protein. By DNA-mediated gene transfer, we cloned a cDNA that complements the tsBN7 mutation. It encodes a novel hydrophobic protein, designated DAD1, which is well conserved (100% identical amino acids between humans and hamsters). By comparing the base sequences of the parental BHK21 and tsBN7 DAD1 cDNAs, we found that the DAD1-encoding gene is mutated in tsBN7 cells. The DAD1 protein disappeared in tsBN7 cells following a shift to the nonpermissive temperature, suggesting that loss of the DAD1 protein triggers apoptosis.

scholarly journals Molecular cloning of a human cDNA encoding a novel protein, DAD1, whose defect causes apoptotic cell death in hamster BHK21 cells.

1993 ◽  
Vol 13 (10) ◽  
pp. 6367-6374 ◽  
Author(s):  
T Nakashima ◽  
T Sekiguchi ◽  
A Kuraoka ◽  
K Fukushima ◽  
Y Shibata ◽  
...  
Keyword(s):  
Cell Line ◽  
Apoptotic Cell ◽  
Protein Synthesis Inhibitor ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Synthesis Inhibitor ◽  
Base Sequences ◽  
Encoding Gene ◽  
Induced Apoptosis ◽  
Mutant Lines

The tsBN7 cell line, one of the mutant lines temperature sensitive for growth which have been isolated from the BHK21 cell line, was found to die by apoptosis following a shift to the nonpermissive temperature. The induced apoptosis was inhibited by a protein synthesis inhibitor, cycloheximide, but not by the bcl-2-encoded protein. By DNA-mediated gene transfer, we cloned a cDNA that complements the tsBN7 mutation. It encodes a novel hydrophobic protein, designated DAD1, which is well conserved (100% identical amino acids between humans and hamsters). By comparing the base sequences of the parental BHK21 and tsBN7 DAD1 cDNAs, we found that the DAD1-encoding gene is mutated in tsBN7 cells. The DAD1 protein disappeared in tsBN7 cells following a shift to the nonpermissive temperature, suggesting that loss of the DAD1 protein triggers apoptosis.

scholarly journals Temperature-sensitive adult liver cell line dependent on glucocorticoid for differentiation.

1983 ◽  
Vol 3 (6) ◽  
pp. 1013-1020 ◽  
Author(s):  
J Y Chou
Keyword(s):  
Growth Rate ◽  
Cell Line ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Adult Liver ◽  
Hepatocyte Cell Line ◽  
Low Levels

A clonal rat adult hepatocyte cell line (RALA255-10G) was shown to be temperature sensitive (ts) for growth and differentiation. Glucocorticoid was necessary to maintain the maximal levels of differentiated functions in these cells. The RALA255-10G cell line was established by transforming primary adult hepatocytes with simian virus 40 tsA255 virus that is temperature sensitive for maintenance of transformation. At the permissive temperature (33 degrees C), RALA255-10G cells showed characteristics of malignant transformation, synthesized low levels of albumin and transferrin, and contained low levels of functional receptors for glucagon. At the nonpermissive temperature (40 degrees C), these cells regain the normal differentiated phenotype, and the levels of these three hepatic functions were increased. Induction of albumin and transferrin production by RALA255-10G cells at 40 degrees C was shown to be the result of the increase in the biosynthesis of these proteins. Furthermore, the albumin and transferrin produced by these cells were immunologically and electrophoretically indistinguishable from authentic rat albumin and transferrin. Glucocorticoid, which reduced the growth rate and saturation density of RALA255-10G cells at 33 degrees C, was absolutely required by these cells to synthesize albumin at both temperatures. This hormone also enhanced transferrin production and glucagon response. Our data indicate that glucocorticoid hormone is one of the factors that maintain adult hepatocytes in a differentiated state.

Temperature-sensitive adult liver cell line dependent on glucocorticoid for differentiation

1983 ◽  
Vol 3 (6) ◽  
pp. 1013-1020
Author(s):  
J Y Chou
Keyword(s):  
Growth Rate ◽  
Cell Line ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Adult Liver ◽  
Hepatocyte Cell Line ◽  
Low Levels

A clonal rat adult hepatocyte cell line (RALA255-10G) was shown to be temperature sensitive (ts) for growth and differentiation. Glucocorticoid was necessary to maintain the maximal levels of differentiated functions in these cells. The RALA255-10G cell line was established by transforming primary adult hepatocytes with simian virus 40 tsA255 virus that is temperature sensitive for maintenance of transformation. At the permissive temperature (33 degrees C), RALA255-10G cells showed characteristics of malignant transformation, synthesized low levels of albumin and transferrin, and contained low levels of functional receptors for glucagon. At the nonpermissive temperature (40 degrees C), these cells regain the normal differentiated phenotype, and the levels of these three hepatic functions were increased. Induction of albumin and transferrin production by RALA255-10G cells at 40 degrees C was shown to be the result of the increase in the biosynthesis of these proteins. Furthermore, the albumin and transferrin produced by these cells were immunologically and electrophoretically indistinguishable from authentic rat albumin and transferrin. Glucocorticoid, which reduced the growth rate and saturation density of RALA255-10G cells at 33 degrees C, was absolutely required by these cells to synthesize albumin at both temperatures. This hormone also enhanced transferrin production and glucagon response. Our data indicate that glucocorticoid hormone is one of the factors that maintain adult hepatocytes in a differentiated state.

scholarly journals Analysis of HCF, the Cellular Cofactor of VP16, in Herpes Simplex Virus-Infected Cells

2000 ◽  
Vol 74 (1) ◽  
pp. 99-109 ◽  
Author(s):  
Sylvie LaBoissière ◽  
Peter O'Hare
Keyword(s):  
Protein Synthesis ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Line ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus (HSV) immediate-early (IE) gene expression is initiated via the recruitment of the structural protein VP16 onto specific sites upstream of each IE gene promoter in a multicomponent complex (TRF.C) that also includes the cellular proteins Oct-1 and HCF. In vitro results have shown that HCF binds directly to VP16 and stabilizes TRF.C. Results from transfection assays have also indicated that HCF is involved in the nuclear import of VP16. However, there have been no reports on the role or the fate of HCF during HSV type 1 (HSV-1) infection. Here we show that the intracellular distribution of HCF is dramatically altered during HSV-1 infection and that the protein interacts with and colocalizes with VP16. Moreover, viral protein synthesis and replication were significantly reduced after infection of a BHK-21-derived temperature-sensitive cell line (tsBN67) which contains a mutant HCF unable to associate with VP16 at the nonpermissive temperature. Intracellular distribution of HCF and of newly synthesized VP16 in tsBN67-infected cells was similar to that observed in Vero cells, suggesting that late in infection the trafficking of both proteins was not dependent on their association. We constructed a stable cell line (tsBN67r) in which the temperature-sensitive phenotype was rescued by using an epitope-tagged wild-type HCF. In HSV-1-infected tsBN67r cells at the nonpermissive temperature, direct binding of HCF to VP16 was observed, but virus protein synthesis and replication were not restored to levels observed at the permissive temperature or in wild-type BHK cells. Together these results indicate that the factors involved in compartmentalization of VP16 alter during infection and that late in infection, VP16 and HCF may have additional roles reflected in their colocalization in replication compartments.

scholarly journals Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210

1992 ◽  
Vol 117 (5) ◽  
pp. 1041-1053 ◽  
Author(s):  
JR Hamaguchi ◽  
RA Tobey ◽  
J Pines ◽  
HA Crissman ◽  
T Hunter ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Phosphorylation Site ◽  
Cyclin A ◽  
Cyclin B1 ◽  
Histone H1 ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature

The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases. H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1. The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo. A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells.

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