DNA sequence analysis of spontaneous and n‐ethyl‐n‐nitrosourea‐lnduced hprt mutations arising in vivo in cynomolgus monkey t‐lymphocytes

Abstract

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.

Analysis of in vivo mutation induced by N‐ethyl‐N‐nitrosourea in the hprt gene of rat lymphocytes

“…These observations suggest that our analysis method produces a mutational profile that is representative of the types of mutations induced by ENU in the entire hprr coding sequence. The pattern of ENU-induced mutations in rat lymphocytes also generally describes the ENU-induced mutations in exon 3 of TG' mouse lymphocytes reported by Skopek et al [1992], the ENU-induced h p n cDNA mutations found by Jansen et al [1994] for T G skin fibroblasts from the granuloma pouch assay, and the hprt cDNA mutations from T G monkey lymphocytes reported by Harbach et al [1992]. The differences that do exist between the mutational profiles may be due to the relatively small numbers of sequenced mutations.…”

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

“…They occur predominantly in A-T base pairs (70%), indicating that this lacZ surrogate mutation model detects a high proportion of ENU-induced mutations similar to, but at a somewhat lower frequency than, those observed in some endogenous mammalian genes-for example, offspring in the specific locus test [86% (28)] and in the somatic hprt mutations in the mouse Genetics: Douglas et al [90% (29)] and monkey [94% (30)], although any differences may simply be the result of different experimental conditions including dose (31). The proportion of mutations in AT vs. G-C base pairs in the present study is nearly identical to that reported for the lacI transgenic model [i.e., 68% (5)].…”

DNA damage, DNA repair, cell proliferation, and DNA replication: How do gene mutations result?

“…Is the persistent adduct O 2 -alkyl T and will the mutations then be A:T3T:A transversions? Will these mutations show the strand bias toward the nontranscribed strand found for these transversions at A:T base pairs induced both in vivo and in vitro in other systems including lacI transgenic mice by ENU (8,9,11,14,18)? Perhaps not, if this bias is caused by transcription-coupled repair, because the lacI gene is not transcribed.…”