THE EFFECTS OF DIFFERENTIAL POLYADENYLATION ON EXPRESSION OF THE DIHYDROFOLATE REDUCTASE-ENCODING GENE IN CHINESE-HAMSTER LUNG-CELLS

Three differently sized mRNAs are expressed from each of two DHFR (enc oding dihydrofolate reductase) alleles present in the Chinese hamster lung (CHL) cell line, DC-3F. The relative abundancy of the transcripts produced from each allele differs dramatically as a result of differe ntial utilization of the multiple poly(A) sites present in the DHFR ge ne and a genetic polymorphism located within the third poly(A) signal of one allele. We sought to determine whether such differences in poly adenylation affect the steady-state levels of DHFR mRNAs expressed fro m either allele and, in a more general sense, to ask whether differenc es in 3' end RNA processing in a gene containing multiple poly(A) site s affects the final level of gene expression. An SV40 promoter-based t ransient expression system producing chimeric cat::DHFR transcripts wa s developed to regenerate the in vivo mRNA polyadenylation patterns as sociated with each of the two DHFR alleles. The results demonstrate th at the total amount of polyadenylated RNA expressed from each of these constructs in vitro is the same regardless of the differential utiliz ation of the poly(A) signals that occurs between them. Moreover, measu rement of the individual turnover rates of the DHFR mRNAs expressed in vivo from each allele, as determined by pulse-chase labeling and acti nomycin D inhibition studies, revealed no significant allele-specific differences in transcript half-lives. Finally, measuring the steady-st ate levels of DHFR poly(A)(+) mRNA in parental DC-3F cells demonstrate d that both alleles are expressed to the same extent during normal gro wth, Thus, even though dramatic allele-specific differences in 3' end processing of DHFR transcripts occur in vivo, such differences do not appear to influence the steady-state levels of DHFR gene expression.