Isolation and characterization of a thymidylate synthase-deficient human colon tumor cell line.

T) at nucleotide 647 that predicts the replacement of Ser216 by a leucine residue. That the C18 cell line was homozygous for this mutation was indicated by restriction fragment-length polymorphism analysis and by primer extension analysis. To provide additional evidence that substitution of Ser216 by a leucine residue created a defective protein, a TS-deficient bacterial strain was transformed with an expression vector containing the mutated human TS cDNA. The transformed strain exhibited thymidine auxotrophy, indicating that the mutant TS (Leu216) is nonfunctional. -->
Substance Nomenclature: EC 2.1.1.45 (Thymidylate Synthase)
VC2W18DGKR (Thymidine)
Entry Date(s): Date Created: 19991027 Date Completed: 19991104 Latest Revision: 20190623
Update Code: 20231215
DOI: 10.1016/s0006-2952(99)00237-3
PMID: 10535743
Autor: Hoganson DK; Department of Basic Pharmaceutical Sciences, University of South Carolina, Columbia 29208, USA., Williams AW, Berger SH
Jazyk: angličtina
Zdroj: Biochemical pharmacology [Biochem Pharmacol] 1999 Nov 15; Vol. 58 (10), pp. 1529-37.
DOI: 10.1016/s0006-2952(99)00237-3
Abstrakt: Following mutagenesis of the human colorectal tumor cell line HCT C with ethyl methanesulfonate, clonal sublines were isolated that survived on medium toxic to cells expressing thymidylate synthase (TS). The subline exhibiting the lowest TS activity, designated as C18, was characterized. Extracts from C18 cells were mixed with extracts from parental C cells to determine whether the TS-deficient phenotype is trans-acting. No effect was observed on the activity of TS in parental extracts. The levels of functional TS in C18 cells were analyzed by the binding of the mechanism-based inhibitor 5-fluoro-2'-deoxyuridylate (FdUMP) under conditions that allowed for the detection of 10 fmol of TS. Only a low level of FdUMP-TS complexes was detected in C18 extracts. The level of TS expression in C18 cells was similar to that in parental C cells, as indicated by immunoblot and RNA analyses. DNA sequence analysis of TS cDNA from C18 cells revealed the existence of a point mutation (C-->T) at nucleotide 647 that predicts the replacement of Ser216 by a leucine residue. That the C18 cell line was homozygous for this mutation was indicated by restriction fragment-length polymorphism analysis and by primer extension analysis. To provide additional evidence that substitution of Ser216 by a leucine residue created a defective protein, a TS-deficient bacterial strain was transformed with an expression vector containing the mutated human TS cDNA. The transformed strain exhibited thymidine auxotrophy, indicating that the mutant TS (Leu216) is nonfunctional.
Databáze: MEDLINE
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