Amplification of mRNA of the hprt gene from lysates of mutant human cells and direct DNA sequencing to determine the spectrum of mutations induced by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha, epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.
Strong evidence points to mutation induction as one mechanism by which changes are introduced into normal cells to convert them into cancer cells. To understand the mechanisms by which mutations are induced in human cells by carcinogens, we are determining the kinds and spectra of mutations induced in the coding region of the hypoxanthine(guanine)phosphoribosyltransferase (hprt) gene. This region, composed of 654 bp, represents nine exons from a 44 kbp gene. To be able to analyze a large number of independent mutants rapidly and economically, we have optimized the conditions for copying mRNA directly from lysates of a small number of cells (e.g., 100) from a 6-thioguanine-resistant clone using reverse transcriptase and oligo(dT)12-18 primers. Then two 20-mer primers, specific for the cDNA of the hprt gene, are used to amplify the first and second strand cDNA 5 x 10(7)-fold during 30 cycles of polymerase chain reaction (PCR). The product (2 to 10 ng) is then purified by ultrafiltration, diluted 1:10, and subjected to an additional 30 cycles of PCR, using two 20-mer primers located just interior to the first set. The amplification product, 5 to 10 ug, is sequenced directly using three other end-labeled primers and Sequenase. To date, we have analyzed 26 mutants induced by (+-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha,epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene and found that 24/26 involved base substitutions. 97% of these involved G.C, predominantly G.C----T.A, distributed over seven exons, with many of the substitutions located in exon 3.
Maher
V M
VM
Department of Microbiology, Michigan State University, East Lansing 48824.
Yang
J L
JL
McCormick
J J
JJ
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000494:pharmacology*; D001483:Base Sequence; D002999:Clone Cells / Q000032:analysis; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D004274:DNA, Recombinant / Q000032:analysis; D004101:Dihydroxydihydrobenzopyrenes / Q000494:pharmacology*; D005347:Fibroblasts / Q000032:analysis / Q000187:drug effects; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics*
D004274:DNA, Recombinant; D004101:Dihydroxydihydrobenzopyrenes; D012333:RNA, Messenger; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase