Cloning and characterization of the gene for the somatic form of DNA topoisomerase I from Xenopus laevis.
Two distinct tissue-specific forms of DNA topoisomerase I with M(r) of 165 and 110 kDa have been purified from oocytes and somatic cells respectively of the African frog Xenopus laevis. In this paper, cDNAs encoding a Xenopus topoisomerase I were cloned using PCR primers derived from sequences of yeast and human topoisomerase I. A polypeptide expressed from a portion of the coding sequence was recognized by an antiserum directed against the somatic topoisomerase I that had previously been shown to be unable to cross-react with the oocyte enzyme. Thus, the clone encodes the somatic cell topoisomerase I. An antiserum raised against a synthetic peptide containing the sequence surrounding the active site tyrosine of the somatic topoisomerase I reacts with the enzymes purified from both oocytes and somatic cells, indicating that the two enzymes share some limited sequence homology. RNA blot hybridization showed that oocytes contain an abundant store of somatic topoisomerase I mRNA that is not efficiently polyadenylated in oocytes. This stored RNA contains a consensus cytoplasmic polyadenylation element that is found in a variety of mRNAs that are translationally repressed in oocytes. Microinjection into oocytes of in vitro transcribed mRNA prepared from a Myc-tagged construct of the somatic topoisomerase I sequence is translated to yield a 110 kDa product. This suggests that the oocyte-specific 165 kDa topoisomerase I is not produced by tissue-specific post-translational modification of the somatic topoisomerase I. The oocyte enzyme appears to be produced from a minor mRNA species in oocytes that has not yet been identified.
Pandit
S D
SD
Department of Biochemistry, State University of New York at Stony Brook, USA.
Richard
R E
RE
Sternglanz
R
R
Bogenhagen
D F
DF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry; D004331:Drosophila melanogaster; D000939:Epitopes; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009865:Oocytes / Q000201:enzymology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D012568:Schizosaccharomyces; D014982:Xenopus laevis
D017931:DNA Primers; D018076:DNA, Complementary; D000939:Epitopes; D012333:RNA, Messenger; D004264:DNA Topoisomerases, Type I