Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase.
cDNA clones encoding mammalian poly(A) polymerase were isolated with probes generated by the polymerase chain reaction based on amino acid sequences derived from the purified enzyme. A bovine cDNA clone was obtained encoding a protein of 82 kDa. Expression in Escherichia coli resulted in the appearance of a poly(A) polymerase activity that was dependent on the addition of the purified specificity factor CPF and the presence of the polyadenylation signal AAUAAA in the RNA substrate. The activity copurified with a polypeptide of the expected size. A second class of cDNAs encoded a polypeptide of 43 kDa which was closely related to the N-terminal half of the 82 kDa protein. Northern blots showed two mRNAs of 4.2 and 2.4 kb that probably correspond to the two classes of cDNAs, as well as a third band of 1.3 kb. The sequence of the N-terminal half of bovine poly(A) polymerase is 47% identical with the amino acid sequence of the corresponding part of yeast poly(A) polymerase. Homologies to other proteins are of uncertain significance.
Wahle
E
E
Universität Basel, Abteilung Zellbiologie, Switzerland.
Martin
G
G
Schiltz
E
E
Keller
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002417:Cattle; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011062:Polynucleotide Adenylyltransferase / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism
D012333:RNA, Messenger; D004247:DNA; D011062:Polynucleotide Adenylyltransferase