DNA-SEQUENCE ANALYSIS OF SPONTANEOUS HPRT MUTATIONS ARISING IN-VIVO IN CYNOMOLGUS MONKEY T-LYMPHOCYTES

To study the mechanisms of mutagenesis in vivo, we analyzed mutations at the hypoxanthine phosphoribosyl transferase (hprt) locus using cDNA from cynomolgus monkey T-lymphocytes. In the present study, the spect rum of spontaneous hprt mutations arising in vivo in wild-caught cynom olgus monkey peripheral T-lymphocytes is described. Cells were isolate d from peripheral blood, and mutant clones were selected in 6-thioguan ine, propagated, and stored frozen. cDNA was copied from hprt mRNA fro m a lysate of 7,000 to 20,000 cells. A 780-base-pairs (bp) region incl uding the coding region was amplified by polymerase chain reaction and directly sequenced. We sequenced 40 spontaneous mutants from 11 monke ys. Of these 40 clones, 23 (57%) had base-pair substitutions, 11 (28%) had small (<20 bp) deletions and/or insertions, and 6 (15%) had large (>20 bp) deletions and/or insertions. Of the 23 base substitutions, 1 3 were transitions (11 G:C --> A:T, 1 A:T --> G:C, and 1 tandem TT +/- CC) and 10 were transversions (3 G:C --> T:A, 3 G:C --> C:G, 2 A:T -- > T:A, 2 A:T --> C:G). Bases 209 and 617 were apparent substitution ho tspots, which have also been observed as hotspots in human hprt. In 2 clones with large insertions, the inserted bases were of intronic orig in. One of these lost 272 bp from exons 2-3 and contained a 93-bp inse rtion from the middle of intron 3. Two clones with small deletions and 5 clones with large deletions or insertions (7/40 or 17.5%) could be splice mutants. (C) 1995 Wiley-liss, Inc.