Harbach P R, Mattano S S, Zimmer D M, Wang Y, Aaron C S
Investigative Toxicology Research, Upjohn Company, Kalamazoo, Michigan 49001, USA.
Environ Mol Mutagen. 1995;26(3):218-25. doi: 10.1002/em.2850260306.
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.
为了研究体内诱变机制，我们使用食蟹猴T淋巴细胞的cDNA分析了次黄嘌呤磷酸核糖转移酶（hprt）基因座的突变。在本研究中，描述了野生捕获的食蟹猴外周T淋巴细胞体内自发产生的hprt突变谱。从外周血中分离细胞，在6-硫鸟嘌呤中选择突变克隆，进行扩增并冷冻保存。从7000至20000个细胞的裂解物中的hprt mRNA复制cDNA。通过聚合酶链反应扩增包括编码区在内的780个碱基对（bp）区域并直接测序。我们对11只猴子的40个自发突变体进行了测序。在这40个克隆中，23个（57%）发生了碱基对替换，11个（28%）有小（<20 bp）缺失和/或插入，6个（15%）有大（>20 bp）缺失和/或插入。在23个碱基替换中，13个是转换（11个G:C→A:T，1个A:T→G:C，1个串联TT→CC），10个是颠换（3个G:C→T:A，3个G:C→C:G，2个A:T→T:A，2个A:T→C:G）。碱基209和617是明显的替换热点，在人类hprt中也观察到是热点。在2个有大插入的克隆中，插入的碱基来自内含子。其中一个从外显子2 - 3缺失了272 bp，并包含来自内含子3中间的93 bp插入。2个有小缺失的克隆和5个有大缺失或插入的克隆（7/40或17.5%）可能是剪接突变体。
