Transformation of temperature-sensitive growth mutant of BHK21 cell line to wild-type phenotype with hamster and mouse DNA

1983 ◽  
Vol 9 (6) ◽  
pp. 673-680 ◽  
Author(s):  
Ryosuke Kai ◽  
Takeshi Sekiguchi ◽  
Katsumi Yamashita ◽  
Mutsuo Sekiguchi ◽  
Takeharu Nishimoto
Keyword(s):  
Cell Line ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Bhk21 Cell ◽  
Wild Type Phenotype

Growth Properties of Neural Cell Lines Immortalized with the Tsa58 Allele of Sv40 Large T Antigen

1997 ◽  
Vol 6 (3) ◽  
pp. 231-238 ◽  
Author(s):  
M.E. Truckenmiller ◽  
Ora Dillon-Carter ◽  
Carlo Tornatore ◽  
Henrietta Kulaga ◽  
Hidetoshi Takashima ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Cell Lines ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Large T Antigen ◽  
Wild Type ◽  
Sv40 Large T Antigen ◽  
Growth Properties

In vitro growth properties of three CNS-derived cell lines were compared under a variety of culture conditions. The M213-20 and J30a cell lines were each derived from embryonic CNS culture with the temperature-sensitive (ts) allele of SV40 large T antigen, tsA58, while the A7 cell line was immortalized using wild-type SV40 large T antigen. Cells immortalized with tsA58 SV40 large T proliferate at the permissive temperature, 33° C, while growth is expected to be suppressed at the nonpermissive temperature, 39.5°C. Both the M213-20 and J30a cell lines were capable of proliferating at 39.5°C continuously for up to 6 mo. All three cell lines showed no appreciable differences in growth rates related to temperature over a 7-day period in either serum-containing or defined serum-free media. The percentage of cells in S-phase of the cell cycle did not decrease or was elevated at 39.5°C for all three cell lines. After 3 wk at 39.5°C, the three cell lines also showed positive immunostaining using two monoclonal antibodies reacting with different epitopes of SV40 large T antigen. Double strand DNA sequence analyses of a 300 base pair (bp) fragment of the large T gene from each cell line, which included the ts locus, revealed mutations in both the J30a and M213-20 cell lines. The J30a cell line ts mutation had reverted to wild type, and two additional loci with bp substitutions with predicted amino acid changes were also found. While the ts mutation of the M213-20 cells was retained, an additional bp substitution with a predicted amino acid change was found. The A7 cell line sequence was identical to the reference wild-type sequence. These findings suggest that (a) nucleic acid sequences in the temperature-sensitive region of the tsA58 allele of SV40 large T are not necessarily stable, and (b) temperature sensitivity of cell lines immortalized with tsA58 is not necessarily retained.

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells

1985 ◽  
Vol 5 (4) ◽  
pp. 902-905
Author(s):  
M Narkhammar ◽  
R Hand
Keyword(s):  
Cell Line ◽  
Nuclear Extract ◽  
Cell Extract ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Whole Cell ◽  
Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

scholarly journals Restoration of endogenous wild-type p53 activity in a glioblastoma cell line with intrinsic temperature-sensitive p53 induces growth arrest but not apoptosis

2001 ◽  
Vol 94 (1) ◽  
pp. 35-43 ◽  
Author(s):  
Jun Ikeda ◽  
Mitsuhiro Tada ◽  
Nobuaki Ishii ◽  
Hideyuki Saya ◽  
Kazuhiko Tsuchiya ◽  
...  
Keyword(s):  
Cell Line ◽  
Growth Arrest ◽  
Temperature Sensitive ◽  
Glioblastoma Cell Line ◽  
Glioblastoma Cell ◽  
Wild Type ◽  
Wild Type P53

scholarly journals Cdc42 Is Required for Proper Growth and Development in the Fungal Pathogen Colletotrichum trifolii

2006 ◽  
Vol 5 (1) ◽  
pp. 155-166 ◽  
Author(s):  
Changbin Chen ◽  
Young-sil Ha ◽  
Ji-young Min ◽  
Stephen D. Memmott ◽  
Martin B. Dickman
Keyword(s):  
Spore Germination ◽  
Fungal Pathogen ◽  
Hyphal Growth ◽  
Dominant Negative ◽  
Temperature Sensitive ◽  
Ras Signaling ◽  
Wild Type ◽  
Colletotrichum Trifolii ◽  
Downstream Effector ◽  
Wild Type Phenotype

ABSTRACT Cdc42 is a highly conserved small GTP-binding protein that is involved in regulating morphogenesis in eukaryotes. In this study, we isolated and characterized a highly conserved Cdc42 gene from Colletotrichum trifolii (CtCdc42), a fungal pathogen of alfalfa. CtCdc42 is, at least in part, functionally equivalent to Saccharomyces cerevisiae Cdc42p, since it restores the temperature-sensitive phenotype of a yeast Cdc42p mutant. Inhibition of CtCdc42 by expression of an antisense CtCdc42 or a dominant negative form of CtCdc42 (DN Cdc42) resulted in appressorium differentiation under noninductive conditions, suggesting that CtCdc42 negatively regulates pathogenic development in this fungus. We also examined the possible linkage between CtCdc42 and Ras signaling. Expression of a dominant active Cdc42 (DA Cdc42) in C. trifolii leads to aberrant hyphal growth under nutrient-limiting conditions. This phenotype was similar to that of our previously reported dominant active Ras (DA Ras) mutant. Also consistent with our observations of the DA Ras mutant, high levels of reactive oxygen species (ROS) were observed in the DA Cdc42 mutant, and proline restored the wild-type phenotype. Moreover, overexpression of DN Cdc42 resulted in a significant decrease in spore germination, virtually no hyphal branching, and earlier sporulation, again similar to what we observed in a dominant negative Ras (DN Ras) mutant strain. Interestingly, coexpression of DA Cdc42 with DN Ras resulted in germination rates close to wild-type levels, while coexpression of DN Cdc42 with the DA Ras mutant restored the wild-type phenotype. These data suggest that CtCdc42 is positioned as a downstream effector of CtRas to regulate spore germination and pathogenic development.

scholarly journals Analysis of Constructed E Gene Mutants of Mouse Hepatitis Virus Confirms a Pivotal Role for E Protein in Coronavirus Assembly

1998 ◽  
Vol 72 (10) ◽  
pp. 7885-7894 ◽  
Author(s):  
Françoise Fischer ◽  
Carola F. Stegen ◽  
Paul S. Masters ◽  
William A. Samsonoff
Keyword(s):  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Wild Type ◽  
E Gene ◽  
E Protein ◽  
Wild Type Phenotype ◽  
Necessary And Sufficient ◽  
Targeted Recombination

ABSTRACT Expression studies have shown that the coronavirus small envelope protein E and the much more abundant membrane glycoprotein M are both necessary and sufficient for the assembly of virus-like particles in cells. As a step toward understanding the function of the mouse hepatitis virus (MHV) E protein, we carried out clustered charged-to-alanine mutagenesis on the E gene and incorporated the resulting mutations into the MHV genome by targeted recombination. Of the four possible clustered charged-to-alanine E gene mutants, one was apparently lethal and one had a wild-type phenotype. The two other mutants were partially temperature sensitive, forming small plaques at the nonpermissive temperature. Revertant analyses of these two mutants demonstrated that the created mutations were responsible for the temperature-sensitive phenotype of each and provided support for possible interactions among E protein monomers. Both temperature-sensitive mutants were also found to be markedly thermolabile when grown at the permissive temperature, suggesting that there was a flaw in their assembly. Most significantly, when virions of one of the mutants were examined by electron microscopy, they were found to have strikingly aberrant morphology in comparison to the wild type: most mutant virions had pinched and elongated shapes that were rarely seen among wild-type virions. These results demonstrate an important, probably essential, role for the E protein in coronavirus morphogenesis.

scholarly journals Accumulation of p53 in a mutant cell line defective in the ubiquitin pathway.

1994 ◽  
Vol 14 (3) ◽  
pp. 1997-2003 ◽  
Author(s):  
D R Chowdary ◽  
J J Dermody ◽  
K K Jha ◽  
H L Ozer
Keyword(s):  
Cell Line ◽  
Mutant Cell ◽  
P53 Gene ◽  
Mouse Cell ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Proteolytic Pathway ◽  
Ubiquitination Pathway ◽  
Wild Type P53

The wild-type p53 gene product plays an important role in the control of cell proliferation, differentiation, and survival. Altered function is frequently associated with changes in p53 stability. We have studied the role of the ubiquitination pathway in the degradation of p53, utilizing a temperature-sensitive mutant, ts20, derived from the mouse cell line BALB/c 3T3. We found that wild-type p53 accumulates markedly because of decreased breakdown when cells are shifted to the restrictive temperature. Introduction of sequences encoding the human ubiquitin-activating enzyme E1 corrects the temperature sensitivity defect in ts20 and prevents accumulation of p53. The data therefore strongly indicate that wild-type p53 is degraded intracellularly by the ubiquitin-mediated proteolytic pathway.

scholarly journals Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells.

1985 ◽  
Vol 5 (4) ◽  
pp. 902-905 ◽  
Author(s):  
M Narkhammar ◽  
R Hand
Keyword(s):  
Cell Line ◽  
Nuclear Extract ◽  
Cell Extract ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Whole Cell ◽  
Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

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 Temperature-sensitive transport of glycoproteins to the surface of a variant mouse lymphoma cell line.

1988 ◽  
Vol 8 (2) ◽  
pp. 833-842 ◽  
Author(s):  
M Nori ◽  
M R Stallcup
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Lymphoma Cell ◽  
Response To Treatment ◽  
Temperature Sensitive ◽  
Lymphoma Cell Line ◽  
Wild Type ◽  
Variant Cell ◽  
Variant Cell Line ◽  
Mouse Lymphoma

The expression of mouse mammary tumor virus (MMTV) glycoproteins on the surface of stably infected mouse lymphoma cell line W7MG1 is dramatically increased by glucocorticoid hormones. A variant cell line, W7M.TS1, was selected from W7MG1 for its lack of expression of MMTV glycoproteins on the cell surface in response to treatment with glucocorticoid. Hormonal stimulation of MMTV RNA levels and hormone-induced cytolysis occurred normally in the variant cells. Furthermore, the rates of production of the precursor and mature forms of MMTV glycoproteins in the presence of glucocorticoid were similar in variant and wild-type cells. However, the accumulation of MMTV glycoproteins on the cell surface after hormone treatment was delayed by about 8 h in the variant relative to wild-type cells. The steady-state level of a constitutively expressed cellular protein, T200, on the variant cell surface was comparable to that on wild-type cells. However, in pulse-chase experiments, the appearance of newly synthesized T200 on the cell surface was delayed in the variant compared with wild-type cells. Another glucocorticoid hormone response, removal of H-2 class I antigens from the cell surface, was also delayed in the variant relative to wild-type cells, suggesting that turnover or internalization of cell surface glycoproteins may also be affected in the variant. The defects in the variant cell line were observed at 37 degrees C, but not at 31 degrees C; the variant cells grew normally at both temperatures. This variant phenotype defines a new genetic entity that is important for transport of glycoproteins between internal microsomal compartments and the cell surface.

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