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 spectrum of spontaneous hprt mutations arising in vivo in wild-caught cynomolgus monkey peripheral T-lymphocytes is described. Cells were isolated from peripheral blood, and mutant clones were selected in 6-thioguanine, propagated, and stored frozen. cDNA was copied from hprt mRNA from a lysate of 7,000 to 20,000 cells. A 780-base-pairs (bp) region including the coding region was amplified by polymerase chain reaction and directly sequenced. We sequenced 40 spontaneous mutants from 11 monkeys. 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, 13 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 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 93-bp insertion 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.
Harbach
P R
PR
Investigative Toxicology Research, Upjohn Company, Kalamazoo, Michigan 49001, USA.
Mattano
S S
SS
Zimmer
D M
DM
Wang
Y
Y
Aaron
C S
CS
D000818:Animals; D000835:Animals, Wild; D016477:Artifacts; D001482:Base Composition; D001483:Base Sequence; D002999:Clone Cells; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D004251:DNA Transposable Elements; D005091:Exons; D005260:Female; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000096:biosynthesis / Q000235:genetics*; D007438:Introns; D008252:Macaca fascicularis / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D017384:Sequence Deletion; D013601:T-Lymphocytes / Q000201:enzymology*
D017931:DNA Primers; D004251:DNA Transposable Elements; D012333:RNA, Messenger; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase