SOUTHERN BLOT ANALYSIS OF T-CELL RECEPTOR GENE REARRANGEMENTS IN CYNOMOLGUS MONKEYS, AND IDENTIFICATION OF A PROGENITOR-CELL HPRT MUTATION

Increases in peripheral blood T-lymphocyte HPRT mutant frequency may r eflect either a number of independent HPRT gene mutational events or c lonal proliferation of a single HPRT mutant. Sequence analysis of HPRT mutations in conjunction with T-cell receptor (TCR) gene rearrangemen t pattern analysis can distinguish these possibilities. Our laboratory previously characterized a nonhuman primate model for in vivo mutatio n studies using the clonal HPRT mutation assay. In the present study w e report the use of probes for human TCR beta and gamma genes to chara cterize TCR rearrangements in cynomolgus monkeys. Together, these meth ods were used to examine a monkey which exhibited a mean spontaneous H PRT mutant frequency (MF) of 16.4 x 10(-6), compared to the normal mea n MF of 3.03 x 10(-6). The elevated MF resulted from the occurrence of a single HPRT mutation in a lymphocyte progenitor cell or stem cell, since T-cell clones isolated from the monkey exhibited a G to T transv ersion at bose pair 539 in the HPRT coding region, and had unique rear rangements of TCR gamma along with an apparent germline TCR beta confi guration. In a preliminary in vivo mutation study, the animal was trea ted with the investigational potent mutagen and antitumor agent adozel esin (U-73975). No increase in HPRT mutant frequency was observed. The HPRT mutant clones isolated after treatment showed rearrangement of b oth TCR gamma and beta genes. Possible explanations for these findings ore discussed. (C) 1995 Wiley-Liss, Inc.