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-lymphocytes were isolated from two blood samples obtained 4 months apart from a 50-year-old male subject. Sixty-six of these mutants were characterized at the DNA sequence level using cDNA. One particular single base substitution was recovered a total of 23 times. The majority of T-cell receptors (TCR) of these mutants all share a common gamma-TCR rearrangement, and thus likely represent a single mutational event that underwent clonal expansion in vivo. Siblings of this clone were recovered in both collections. Three other single base substitutions were also recovered more than once. In two of the three cases, the mutants were also found to be clonally related, while in one case they were not. A number of identical exon loss events were also recovered, yet none of these were clonally related. This probably reflects the multiple pathways by which these mutations can arise. The TCR data was used to correct the observed mutant frequency to produce an estimate of the actual mutation frequency. The two mutant frequencies, 18 x 10(-6) and 19 x 10(-6), obtained from the first and second sampling periods, respectively, can thus be corrected to yield true mutation frequency's of 12 x 10(-6) each. (C) 1995 Wiley-Liss, Inc.