MUTATIONAL SPECTRUM OF ICR-191 AT THE HPRT LOCUS IN HUMAN LYMPHOBLASTOID-CELLS

Human TK6 lymphoblasts were treated with the acridine derivative ICR-1 91, and mutants at the hprt locus were isolated. Mutant hprt cDNA was reverse-transcribed from mRNA, amplified by polymerase chain reaction (PCR), and sequenced. Additions of single G:C base pairs (+1 frameshif t mutations) in repetitive G:C sequences were found in 82% (32/39) of the mutants. Sixteen of the +1 frameshifts analyzed were located in a single sequence of six consecutive guanine bases in exon 3. The remain ing +1 frameshifts occurred at six different GGG sequences (14 mutants ) and a single GGGG sequence (2 mutants) in other hprt exons. The repe titive guanine sequences that underwent frameshift mutagenesis were lo cated in both the transcribed and nontranscribed strands of hprt. No s ingle base deletions (-1 frameshift mutations) were observed. Base sub stitutions were observed in 13% (5/39) of the clones analyzed and occu rred at both G:C and A:T bases. Loss of exon 4 from the cDNA was also observed in 5% (2/39) of the mutants. Hprt mutants containing seven co nsecutive guanines (produced from a +1 frameshift in a GGGGGG sequence ) were treated with ICR-191 and wild-type revertants selected in CHAT medium. Revertants were recovered at a frequency of approximately 10(- 7) and contained the wild-type sequence (GGGGGG) in all clones analyze d. The observed frequency of ICR-191-induced -1 frameshift reversion i n the GGGGGGG sequence was approximately 500-fold lower than the estim ated frequency of +1 frameshifts observed in the wild-type GGGGGG sequ ence following the same ICR-191 treatment. These results suggest that ICR-191 produces predominantly +1 frameshift mutations at the hprt loc us in human cells. (C) 1994 Wiley-Liss, Inc.