SPLICING MUTANTS AND THEIR 2ND-SITE SUPPRESSORS AT THE DIHYDROFOLATE-REDUCTASE LOCUS IN CHINESE-HAMSTER OVARY CELLS

Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were scree ned for aberrantly spliced dhfr mRNA by RNase protection and/or revers e transcriptase coupled with cDNA amplification by the polymerase chai n reaction (PCR). Of 115 mutants screened, 28 were found to be affecte d in splicing. All exhibited less than 1% correct splicing, probably b ecause the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at t he sites flanking the first and last exons resulted in the efficient r ecruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many c ases, multiple exons were skipped, suggesting the clustering of adjace nt exons prior to actual splicing. Six mutations fell outside the well -conserved GU and AG dinucleotides. All but one were donor site single -base substitutions that decreased the agreement with the consensus an d resulted in little or no correct splicing. Starting with five of the se donor site mutants, we isolated 31 DHFR+ revertants. Most revertant s carried a single-base substitution at a site other than that of the original mutation, and most had only partially regained the ability to splice correctly. The second-site suppression occurred through a vari ety of mechanisms: (i) a second change within the consensus sequence t hat produced a better agreement with the consensus; (ii) a change clos e to but beyond the consensus boundaries, as far as 8 bases upstream i n the e''n or 28 bases downstream in the intron; (iii) mutations in an apparent pseudo 5' site in the intron, 84 and 88 bases downstream of a donor site; and (iv) mutations that improved the upstream acceptor s ite of the affected exon. Taken together, these second-site suppressor mutations extend the definition of a splice site beyond the consensus sequence.