Characterization ofts2, a Balb/3T3 temperature-sensitive cell cycle mutant defective in DNA synthesis.
Item
-
Title
-
Characterization ofts2, a Balb/3T3 temperature-sensitive cell cycle mutant defective in DNA synthesis.
-
Identifier
-
AAI9417500
-
identifier
-
9417500
-
Creator
-
Orlian, Martin S.
-
Contributor
-
Adviser: David A. Foster
-
Date
-
1994
-
Language
-
English
-
Publisher
-
City University of New York.
-
Subject
-
Biology, Cell | Biology, Genetics | Biology, Molecular
-
Abstract
-
Ts2, a temperature-sensitive mutant cell line derived from Balb/3T3 cells, was isolated on the basis of the cell's inability to undergo DNA synthesis when incubated at 39{dollar}\sp\circ{dollar}C. {dollar}\sp3{dollar}H-TdR labeling shows that ts2 is defective in DNA synthesis following serum induction at 39{dollar}\sp\circ{dollar}C. Flow cytometry shows that when ts2 cells are maintained at 39{dollar}\sp\circ{dollar}C, if they manage to enter S phase, they arrest in G2 + M. Infection with SV40 virions overcomes the block in DNA synthesis. Cells infected at 39{dollar}\sp\circ{dollar}C accumulate in G2 + M and undergo apoptosis. These data suggest that ts2 cells incubated at 39{dollar}\sp\circ{dollar}C can arrest at two points in the cell cycle: the first prevents them from entering S phase and the second prevents them from undergoing normal cell division. SV40 infection bypasses or overrides the temperature-dependent block at G1/S, but not the block at G2 + M.;Thymidine kinase (TK) mRNA levels were determined in ts2 cells following stimulation by serum addition or SV40 infection. Cells incubated at 39{dollar}\sp\circ{dollar}C show significantly reduced TK mRNA expression following serum stimulation when compared to cells maintained at 33{dollar}\sp\circ{dollar}C. SV40-infected cells show temperature-independent induction of the TK message. Several human TK plasmid constructs were stably transfected into ts2. Analysis of the ts2-derived cell lines show that the human TK gene, when linked to the human TK promoter, shows temperature-dependent cell cycle regulation following serum stimulation. These data suggest that the temperature-dependent induction of human TK mRNA levels following serum stimulation is manifested at the level of the promoter.;A complex phenotype emerges when ts2 cells are incubated at 39{dollar}\sp\circ{dollar}C. The transcription of a least one cell cycle-regulated gene whose expression is associated with the onset of DNA synthesis (TK) is defective. The observation that the genetic defect in ts2 blocks cell cycle progression at two places (G1/S and G2/M)--both major cyclin-dependent regulatory positions of the cell cycle--suggests that ts2 is not a DNA synthesis mutant per se but rather a cell cycle mutant.
-
Type
-
dissertation
-
Source
-
PQT Legacy CUNY.xlsx
-
degree
-
Ph.D.
