Characterization of HAT- and HAsT-resistant HPRT mutant clones of V79 Chinese hamster cells.
HPRT mutant clones of V79 Chinese hamster cells, isolated after 6-thioguanine (6TG) selection, normally exhibit sensitivity to growth in medium containing the folic acid inhibitor aminopterin or the glutamine analogue L-azaserine (e.g., HAT or HAsT medium). However, it has been shown that some HPRT- clones are resistant to both HAT and HAsT medium. The present study was undertaken to investigate whether any common structural gene alteration exists for such 6TGr-HATr-HAsTr clones. Four clones were studied, 1 of spontaneous origin, 2 induced by a low dose of MNU and 1 EMS-induced. In contrast to wild-type cells and a mutant clone carrying a complete deletion of the HPRT gene, these 4 investigated 6TGr-HATr-HAsTr clones all showed an enhanced incorporation of exogenous 3H-hypoxanthine in the presence of aminopterin and L-azaserine suggesting that these clones carry mutations in the structural part of the HPRT gene. Sequence analysis of PCR-amplified HPRT cDNA from these mutants showed that the spontaneous and the 2 MNU-induced mutant clones lacked exon 4, while the EMS-induced mutant had a GC to AT transition in exon 6. Southern blot analysis of genomic DNA after digestion with BglII, EcoRI and PstI showed no changes in fragment patterns as compared to the wild type. Further sequence analysis of PCR-amplified genomic DNA using exon 4-specific primers showed that all these 3 mutants had an AT to GC or GC to AT transition in exon 4, but had no alterations in the splice sites of exon 4. Based on their characteristics of hypoxanthine incorporation, the present mutant clones fit the model for the proposed functional domains of the HPRT protein.
Zhang
L H
LH
Department of Genetic and Cellular Toxicology, Wallenberg Laboratory, Stockholm University, Sweden.
Jenssen
D
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D000630:Aminopterin / Q000494:pharmacology; D000818:Animals; D001377:Azaserine / Q000494:pharmacology*; D015139:Blotting, Southern; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000302:isolation & purification; D004351:Drug Resistance / Q000235:genetics; D005796:Genes / Q000235:genetics*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification; D013866:Thioguanine
D012313:RNA; D001377:Azaserine; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine; D000630:Aminopterin