DNA sequence analysis of spontaneous hprt mutations arising in vivo in cynomolgus monkey T-lymphocytes

✍ Scribed by P. R. Harbach; S. S. Mattano; D. M. Zimmer; Y. Wang; C. S. Aaron

• Publisher: John Wiley and Sons
• Year: 1995
• Tongue: English
• Weight: 683 KB
• Volume: 26
• Category: Article
• ISSN: 0893-6692
• DOI: 10.1002/em.2850260306

✦ Synopsis

To study the mechanisms of mutagenesis in vivo, we analyzed mutations at the hypoxanthine phosphoribosyl transferase (hprf) locus using cDNA from cynomolgus monkey T-lymphocytes. In the present study, the spectrum of spontaneous hprt mutations arising in vivo in wildcaught cynomolgus monkey peripheral T-lymphocytes is described. Cells were isolated from peripheral blood, and mutant clones were selected in bthioguanine, propagated, and stored frozen. cDNA was copied from hprt mRNA from a lysate of 7,000 to 20,000 cells. A 780base-pairs (bp) region including the coding region was amplified by polymerase chain reaction and directly sequenced. W e sequenced 40 spontane ous mutants from 11 monkeys. Of these 40 clones, 23 (57%) had base-pair substitutions, 1 1 (28%) had small (<20 bp) deletions and/or insertions, and 6 (1 5%) had large (>20 bp) deletions and/or insertions. Of the 23 base substitutions, 13 were transitions (1 1 G:C + A:T, 1 A:T + G:C, and 1 tandem l l + 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 61 7 were apparent substitution hotspots, which have also been observed as hotspots in human hprt. In 2 clones with large insertions, the inserted bases were of intronic origin. One of these lost 272 bp from exons 2-3 and contained a 93bp insertion from the middle of intron 3. Two clones with small deletions and 5 clones with large dele tions or insertions (7/40 or 17.5%) could be splice mutants.