DETERMINATION OF HPRT MUTANT AND MUTATION FREQUENCIES AND THE MOLECULAR CHARACTERIZATION OF HUMAN-DERIVED IN-VIVO T-LYMPHOCYTE MUTANTS

Using a T-lymphocyte clonal assay, 73 6-thioguanine resistant T-lympho cytes were isolated from two blood samples obtained 4 months apart fro m a 50-year-old male subject. Sixty-six of these mutants were characte rized at the DNA sequence level using cDNA. One particular single base substitution was recovered a total of 23 times. The majority of T-cel l receptors (TCR) of these mutants all share a common gamma-TCR rearra ngement, and thus likely represent a single mutational event that unde rwent clonal expansion in vivo. Siblings of this clone were recovered in both collections. Three other single base substitutions were also r ecovered more than once. In two of the three cases, the mutants were a lso found to be clonally related, while in one case they were not. A n umber of identical exon loss events were also recovered, yet none of t hese were clonally related. This probably reflects the multiple pathwa ys by which these mutations can arise. The TCR data was used to correc t the observed mutant frequency to produce an estimate of the actual m utation frequency. The two mutant frequencies, 18 x 10(-6) and 19 x 10 (-6), obtained from the first and second sampling periods, respectivel y, can thus be corrected to yield true mutation frequency's of 12 x 10 (-6) each. (C) 1995 Wiley-Liss, Inc.