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 (encoding 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 differential utilization of the multiple poly(A) sites present in the DHFR DHFR gene and a genetic polymorphism located within the third poly(A) signal of one allele. We sought to determine whether such differences in polyadenylation affect the steady-state levels of DHFR and mRNAs expressed from either allele and, in a more general sense, to ask whether differences in 3' end RNA processing in a gene containing multiple poly(A) sites affects the final level of gene expression. An SV40 promoter-based transient expression system producing chimeric cat::DHFR transcripts was developed to regenerate the in vivo mRNA polyadenylation patterns associated with each of the two DHFR alleles. The results demonstrate that the total amount of polyadenylated RNA expressed from each of these constructs in vitro is the same regardless of the differential utilization of the poly(A) signals that occurs between them. Moreover, measurement of the individual turnover rates of the DHFR mRNAs expressed in vivo from each allele, as determined by pulse-chase labeling and actinomycin D inhibition studies, revealed no significant allele-specific differences in transcript half-lives. Finally, measuring the steady-state levels of DHFR poly(A)+ mRNA in parental DC-3F cells demonstrated that both alleles are expressed to the same extent during normal growth. 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.
Yang
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H
Graduate Program in Molecular and Cell Biology, University of Maryland, Baltimore, MD 21201, USA.
Hussain
A
A
Melera
P W
PW
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D015971:Gene Expression Regulation, Enzymologic; D008168:Lung / Q000201:enzymology*; D008969:Molecular Sequence Data; D011061:Poly A / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011061:Poly A; D013762:Tetrahydrofolate Dehydrogenase