A genetic polymorphism within the third poly(A) signal of the DHFR gene alters the polyadenylation pattern of DHFR transcripts in CHL cells.
Two polymorphic dihydrofolate reductase (DHFR) alleles, termed 20 K and 21 K, exist in Chinese hamster lung cells. Three major transcripts of different lengths are transcribed from each allele, and the expression of these transcripts differs dramatically between the alleles as a result of differential utilization of three poly(A) sites. Transcripts from the 20 K allele are preferentially polyadenylated at the first poly(A) site, while those from the 21 K allele are preferentially polyadenylated at the third site. In this study, transient expression experiments were used to demonstrate that a 2.1 kb genomic fragment containing the three DHFR poly(A) sites is sufficient to reproduce the allele-specific polyadenylation pattern on transiently expressed CAT-DHFR transcripts in COS cells. Site-directed mutagenesis allowed identification of the sequence elements which are responsible for this allele-specific polyadenylation. These studies indicate that a single-base change in the third poly(A) signal sequence, which alters the consensus AAUAAA signal in the 21 K allele to a weak AAUAAU signal in the 20 K allele, is primarily responsible for the dramatic difference in polyadenylation between the two alleles. Thus, as a result of this single-base change in the third poly(A) signal sequence, utilization of the first poly(A) site, located 1.2 kb upstream, changes dramatically.
Yang
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Graduate Program in Molecular and Cell Biology, University of Maryland, Baltimore 21201.
Melera
P W
PW
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA; D008168:Lung / Q000166:cytology / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011061:Poly A / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D011061:Poly A; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D015500:Chloramphenicol O-Acetyltransferase