DNA sequence analysis of spontaneous and N-ethyl-N-nitrosourea-induced hprt mutations arising in vivo in cynomolgus monkey T-lymphocytes.
The study of hprt mutations in cynomolgus monkey T-lymphocytes is part of our effort to understand the mechanisms of mutagenesis in vivo. This primate model allows us to study mutations and their kinetics at the molecular level under well-controlled conditions using recently developed techniques for selection of mutant T-cells and polymerase chain reaction (PCR) amplification of hprt cDNA, which is directly sequenced. This is the first report of the sequence of the coding region of the cynomolgus monkey hprt gene and PCR/DNA sequence analysis of seven spontaneous mutant T-cell clones, as well as 23 mutant clones isolated 63 and 601 days after treatment with ethylnitrosourea (ENU, 77 mg/kg, intraperitoneal). cDNA was reverse transcribed from hprt mRNA directly from a lysate of about 2-4 x 10(3) cells, and a 700 bp fragment including the coding region was amplified by PCR and sequenced. Of the seven spontaneous mutants, only one point mutation (GC----AT transition) was detected, and the other six failed to amplify by PCR, possibly due to functional deletions. Of the 14 mutant clones isolated 63 days after ENU treatment, nine base substitutions were detected in ten clones: four transitions (three AT----GC and one GC----AT) and five transversions (four AT----TA and one AT----CG). Of the nine mutants isolated 601 days after ENU treatment, six single base substitutions were detected in six clones (five AT----TA and one AT----CG transversions), and one mutant had a large deletion or insertion. No changes were detected in three clones (one Day 63 and two Day 601 clones). In summary, only one of 15 single base substitutions isolated after ENU treatment was a GC----AT transition mutation and the rest were transitions and transversions at AT sites.