Rapid assay for detection ofEschenchia colixanthine-guanine phosphoribosyltransferase activity in transduced cells
Open Access
- 1 January 1985
- journal article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 13 (8) , 2921-2930
- https://doi.org/10.1093/nar/13.8.2921
Abstract
Cultured mammalian cells transduced with the Escherichia coli gene, Ecogpt, synthesize the bacterial enzyme xanthine-guanine phosphoribosyl transferase (XGPT) (1). This paper describes a method for measuring XGPT activity in crude cell extracts by following the conversion of 14C-xanthine (X) to 14C-xanthine monophosphate (XMP) and 14C-xanthosine (XR) by thin layer chromatography. The method is rapid, easy to use, sensitive and linear over a wide range of XGPT activity and has been useful for detecting XGPT in cells that were transiently transfected or stably transformed with Ecogpt. During our studies, we have found that a human cell line (XP2OS) converts xanthine to XMP. This activity is probably catalyzed by a variant hypoxanthine-guanine phosphoribosyltransferase (HGPT) since the low activity is readily inhibited by hypoxanthine. A low level of conversion of X to XMP may explain why some cell lines are not killed in a medium containing mycophenolic acid and X.Keywords
This publication has 14 references indexed in Scilit:
- Purification and characterization of Escherichia coli guanine-xanthine phosphoribosyltransferase produced by a high efficiency expression plasmid utilizing a lambda PL promoter and CI857 temperature-sensitive repressor.Journal of Biological Chemistry, 1983
- Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase.Proceedings of the National Academy of Sciences, 1981
- Expression of a Bacterial Gene in Mammalian CellsScience, 1980
- Mapping temperature-sensitive and host-range mutations of adenovirus type 5 by marker rescueVirology, 1978
- Comparative studies of host-cell reactivation, colony forming ability and excision repair after UV irradiation of xeroderma pigmentosum, normal human and some other mammalian cellsMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, 1974
- A new technique for the assay of infectivity of human adenovirus 5 DNAVirology, 1973
- Guanine phosphoribosyltransferase from Escherichia coli. Specificity and propertiesBiochemistry, 1972
- Lesch-Nyhan Syndrome: Altered Kinetic Properties of Mutant EnzymeScience, 1971
- The inhibition of nucleic acid synthesis by mycophenolic acidBiochemical Journal, 1969
- A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells.Proceedings of the National Academy of Sciences, 1967