The analysis of JB7-K, a temperature-sensitive mutant of Chinese hamster ovary cells.
Item
-
Title
-
The analysis of JB7-K, a temperature-sensitive mutant of Chinese hamster ovary cells.
-
Identifier
-
AAI9009729
-
identifier
-
9009729
-
Creator
-
Du, Hong.
-
Contributor
-
Adviser: Harvey L. Ozer
-
Date
-
1989
-
Language
-
English
-
Publisher
-
City University of New York.
-
Subject
-
Biology, Molecular | Biology, Genetics | Chemistry, Biochemistry
-
Abstract
-
Over 200 CHO cell temperature-sensitive mutants have ben isolated in the laboratory and 10% of them show a tsDNA phenotype. Five of them are defective in polyoma viral DNA replication at the nonpermissive temperature (NPT), whereas only one out of the five is also defective in adenovirus DNA replication at the NPT. General growth properties have been studied for those mutants selectively defective for polyoma DNA replication (JB3-O, JB7-K, JB8-D and JB11-J). One of these mutants, JB7-K, has been studied in more detail. DNA synthesis in this mutant decreased 60% after 12 h at NPT, but both RNA and protein syntheses are normal for more than 16 h at the NPT. Cell cycle analysis shows that JB7-K cell arrested predominantly in G1 at the NPT. The rate as well as level of polyoma DNA replication has been found to be reduced at the NPT. Molecular cloning of the wild type human gene that corrects the ts defect of JB7-K has been undertaken. Primary ts{dollar}\sp+{dollar} transfectants (5A1) were isolated following protoplast fusion with a human genomic cosmid library (pCV103Kgpt). A secondary ts{dollar}\sp+{dollar} transfectant (2{dollar}\sp\circ{dollar}5D) was isolated by DNA mediated gene transfer from 5A1. A phage genomic library of 2{dollar}\sp\circ{dollar}5D which contains a single copy of human Alu and gpt sequences was constructed. Unique human gene sequences (2.8 kb and 1.4 kb EcoR I fragments) were isolated by screening of the phage library using a human Alu sequence as a probe and restriction enzyme digestion of the recombinant phage DNA. This 2.8 kb sequence has been mapped to human chromosome 2 and located at 2q11-2p23 by Southern analysis of human-rodent hybrid cells. Transfection of JB7-K by the recombinant phage results in partial correction of the ts phenotype. This 2.8 kb fragment has also been used as a probe to screen a human cDNA library.
-
Type
-
dissertation
-
Source
-
PQT Legacy CUNY.xlsx
-
degree
-
Ph.D.
