Amplification versus mutation as a mechanism for reversion of an HGPRT mutation.
We have used a cloned cDNA for hypoxanthine-guanine phosphoribosyltransferase (HGPRT) to analyze the HGPRT gene and mRNA in an HGPRT-deficient mutant of Chinese hamster cells (RJK10) and its HGPRT-positive revertants. By Southern blot analysis, no DNA rearrangements were detected within the genes from any of the cell lines examined. However, four of five spontaneous revertants each contained 10- to 20-fold more copies of the HGPRT gene than did RJK10 or wild-type cells. In contrast, the gene was not amplified in four mutagen-induced revertants. The RJK10 mutation did not alter the size or concentration of HGPRT mRNA and representatives of the revertants contained the mRNA in amounts proportional to the number of genes they carried. Examples of clones with either stable or unstable gene amplification were identified and their HGPRT-positive phenotypes were shown to be dependent on the gene amplification. In a stably amplified revertant, the extra genes were found to be syntenic with the X chromosome marker glucose-6-phosphate dehydrogenase. In an unstable revertant only one of the 10 to 20 copies of the gene could be shown to be X linked. Thus, we found that RJK10 can revert by at least two distinct mechanisms: amplification of the HGPRT gene, which occurred spontaneously, or point mutation, which predominated after exposure to mutagens.
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000378:metabolism*; D005784:Gene Amplification; D005796:Genes; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008168:Lung; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics
D012333:RNA, Messenger; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase