Inactivation of a transfected gene in human fibroblasts can occur by deletion, amplification, phenotypic switching, or methylation.
Plasmids containing the bacterial gpt gene under control of the simian virus 40 promoter were transfected into a simian virus 40-transformed human fibroblast line. Two transfectants, E2 and C10, which contain stably integrated single copies of the gpt gene, were isolated. These two lines produce Gpt- variants spontaneously with a frequency of about 10(-4). We carried out a detailed molecular analysis of the spectrum of alterations which gave rise to the Gpt- phenotype in these variants. DNA from 14 of 19 Gpt- derivatives of one of the cell lines (E2) contains deletions or rearrangements of gpt-containing sequences. In four of the remaining five lines, the Gpt- phenotype was correlated with reduced levels of expression rather than with changes in the gross structure of the gpt gene, and it was possible to reactivate the gpt gene. In one Gpt- line, gpt mRNA was present at normal levels, but no active enzyme was produced. Spontaneous Gpt- derivatives of the other cell line (C10) produced a completely different spectrum of alterations. Very few deletions were found, but several derivatives contained additional extrachromosomal gpt sequences, and, remarkably, in two other Gpt- lines, gpt-containing sequences were amplified more than 100-fold. The phenotypes of the majority of the Gpt- derivatives of C10 could be attributed to alterations in gene expression caused by methylation.
D002460:Cell Line; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005784:Gene Amplification; D005796:Genes; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase; D008745:Methylation; D010430:Pentosyltransferases / Q000235:genetics; D010641:Phenotype; D010957:Plasmids
D010430:Pentosyltransferases; D007041:Hypoxanthine Phosphoribosyltransferase