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PMID: 7862135 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Cloning and characterization of a Xenopus poly(A) polymerase.

Molecular and cellular biology ·Vol. 15 ·No. 3 ·1995-03-00 ·Pages 1422-30

Gebauer F, Richter JD

Abstract

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.

MeSH Terms
Amino Acid Sequence Animals Base Sequence Cattle Chromatography, Affinity Chromatography, Ion Exchange Cloning, Molecular DNA Primers Electrophoresis, Polyacrylamide Gel Embryo, Nonmammalian/embryology Escherichia coli Female Gene Library Kinetics Molecular Sequence Data Molecular Weight Oocytes/enzymology Polymerase Chain Reaction Polynucleotide Adenylyltransferase/biosynthesis,isolation & purification,metabolism Recombinant Proteins/biosynthesis,isolation & purification,metabolism Sequence Homology, Amino Acid Sequence Homology, Nucleic Acid Xenopus laevis
Chemicals
DNA Primers Recombinant Proteins Polynucleotide Adenylyltransferase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Gebauer F
Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Richter J D
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.15.3.1422
Published
1995-03-00
Pages
1422-30
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC230366
Subset
IM
Grants
NIGMS NIH HHS · GM46779 · United States
Databases
GENBANK
U23456
Corrections
ErratumIn
PMID: 7760843
External Links
PubMed source DOI: 10.1128/MCB.15.3.1422 PMC Full Text View journal details
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