tsBN75 and tsBN423, temperature-sensitive X-linked mutants of the BHK21 cell line, can be complemented by the ubiquitin-activating enzyme E1 cDNA

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.

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

Cell Transplantation ◽

10.1177/096368979700600306 ◽

1997 ◽

Vol 6 (3) ◽

pp. 231-238 ◽

Cited By ~ 5

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.6.6.2027 ◽

1986 ◽

Vol 6 (6) ◽

pp. 2027-2032 ◽

Cited By ~ 42

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.92.3.629 ◽

1982 ◽

Vol 92 (3) ◽

pp. 629-633 ◽

Cited By ~ 5

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.

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Transfer of a mutant viral thymidine kinase gene results in temperature-sensitive mouse cells

Molecular and Cellular Biology ◽

10.1128/mcb.3.3.490-494.1983 ◽

1983 ◽

Vol 3 (3) ◽

pp. 490-494

Author(s):

M M Manos ◽

S D Munroe ◽

E Przybytkowska ◽

L A McReynolds

Keyword(s):

Cell Line ◽

Thymidine Kinase ◽

Growth Conditions ◽

Selective Medium ◽

Temperature Sensitive ◽

Sensitive Cell ◽

Kinase Gene ◽

Vitro Analysis ◽

Simplex Virus

Temperature-sensitive cell lines were obtained by DNA-mediated transfer of the thymidine kinase (TK) gene from a mutant, ts1117, of herpes simplex virus type 1. The cells died at 39 degrees C in selective medium which contained low levels (1 microgram/ml) of thymidine. In this lethal condition, no revertants were detected among 10(8) cells. It was shown by in vitro analysis of the TK activity that the temperature-sensitive cell line contains an enzyme whose activity is temperature sensitive and relatively unaffected by dTTP. The viral enzyme has these properties. The effect of the lethal growth conditions in the cell line was characterized by cell cycle analysis and rescue experiments which involved a shift to the permissive conditions. The successful transfer of the mutant viral TK activity to cells provides an additional selective marker for gene transfer.

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Cyclic AMP regulates PDGF-stimulated signal transduction and differentiation of an immortalized optic-nerve-derived cell line

Journal of Experimental Biology ◽

10.1242/jeb.202.4.461 ◽

1999 ◽

Vol 202 (4) ◽

pp. 461-473

Author(s):

R.I. Cohen ◽

R. Mckay ◽

G. Almazan

Keyword(s):

Signal Transduction ◽

Optic Nerve ◽

Growth Factor ◽

Cyclic Amp ◽

Cell Line ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Temperature Sensitive ◽

Permissive Temperature

To facilitate the study of the molecular events underlying the development of optic-nerve-derived oligodendrocytes and their growth-factor-related signal transduction events, we immortalized perinatal rat optic nerve cells with a temperature-sensitive simian virus 40 large T-antigen, carrying the tsA58 and U19 mutations, via a retrovirus vector. The line, tsU19-9, was selected on the basis of the expression of the neural precursor marker nestin. At the permissive temperature, 33 degreesC, tsU19-9 cells had a flat epithelial morphology. In contrast, following exposure to platelet-derived growth factor (PDGF), a factor important in the lineage progression of oligodendrocytes, or in the presence of dibutyryl cyclic AMP at 39 degreesC (the non-permissive temperature), the cells underwent morphological and antigenic differentiation to cells characteristic of the oligodendrocyte lineage. We used this cell line to investigate the binding characteristics of PDGF and related signalling cascades. Competition binding, phosphoinositide hydrolysis and intracellular Ca2+ mobilization assays all demonstrated that the three different isoforms of PDGF (AA, AB and BB) bound to and acted on the cell line. Overnight exposure to forskolin, a treatment that initiated morphological and phenotypic progression into an oligodendrocyte lineage, decreased PDGF-BB-induced intracellular Ca2+ mobilization and inhibited basal and PDGF-stimulated [3H]thymidine incorporation. Our results demonstrate that tsU19-9 may serve as a resource to study early optic-nerve oligodendrocyte development.

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Isolation and characterization of a variant myoblast cell line that is temperature sensitive for differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2335-2341.1988 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2335-2341

Author(s):

R J Akhurst ◽

N B Flavin ◽

J Worden

Keyword(s):

Cell Line ◽

Progenitor Cell ◽

Temperature Shift ◽

Temperature Sensitive ◽

Isolation And Characterization ◽

New Variant ◽

Myoblast Cell Line ◽

Myoblast Cell ◽

Progenitor Cell Line

A new variant rat myogenic cell line, ts485, was isolated by subcloning the cell line ts3b2 (H. T. Nguyen, R. M. Medford, and B. Nadal-Ginard, Cell 34:281-293, 1983). Unlike the progenitor cell line, ts485 was thermosensitive for differentiation. Experiments with conditioned medium suggested that diffusible extracellular factors were not involved in dictating the differential phenotypes of ts485 cells cultured at the permissive and nonpermissive temperatures. Temperature shift experiments performed on cultures of ts485 cells indicated that the temperature-sensitive lesion was in a factor active during the growth phase and required to trigger a cascade of events leading to terminal differentiation.

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