Cloning and characterization of a Xenopus poly(A) polymerase.
During oocyte maturation and early embryogenesis in Xenopus laevis, the translation of several mRNAs is regulated by cytoplasmic poly(A) elongation, a reaction catalyzed by poly(A) polymerase (PAP). We have cloned, sequenced, and examined several biochemical properties of a Xenopus PAP. This protein is 87% identical to the amino-terminal portion of bovine PAP, which catalyzes the nuclear polyadenylation reaction, but lacks a large region of the corresponding carboxy terminus, which contains the nuclear localization signal. When injected into oocytes, the Xenopus PAP remains concentrated in the cytoplasm, suggesting that it is a specifically cytoplasmic enzyme. Oocytes contain several PAP mRNA-related transcripts, and the levels of at least the one encoding the putative cytoplasmic enzyme are relatively constant in oocytes and early embryos but decline after blastulation. When expressed in bacteria and purified by affinity and MonoQ-Sepharose chromatography, the protein has enzymatic activity and adds poly(A) to a model substrate. Importantly, affinity-purified antibodies directed against Xenopus PAP inhibit cytoplasmic polyadenylation in egg extracts. These data suggest that the PAP described here could participate in cytoplasmic polyadenylation during Xenopus oocyte maturation.
Gebauer
F
F
Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Richter
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004625:Embryo, Nonmammalian / Q000196:embryology; D004926:Escherichia coli; D005260:Female; D015723:Gene Library; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009865:Oocytes / Q000201:enzymology; D016133:Polymerase Chain Reaction; D011062:Polynucleotide Adenylyltransferase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014982:Xenopus laevis
D017931:DNA Primers; D011994:Recombinant Proteins; D011062:Polynucleotide Adenylyltransferase