Preparation and characterization of cell-free protein synthesis systems from oocytes and eggs of Xenopus laevis.
During the maturation of the oocytes of the frog Xenopus laevis, the rate of protein synthesis shows a twofold increase. Studies of the mechanisms involved in this stimulation have been seriously limited by the lack of an active cell-free translation system. We have now prepared such systems from oocytes, progesterone-matured oocytes and eggs of Xenopus laevis by induction of lysis by centrifugation of whole cells. The extracts are highly active in incorporation of labelled amino acids and, in the progesterone-matured and egg extracts, a substantial proportion of this is due to reinitiation on endogenous mRNA, as shown by the use of inhibitors. The increased rate of protein synthesis previously observed in intact oocytes following progesterone-induced maturation is reflected in the relative activities of the extracts. The difference in activity is not due to the presence of a dominant inhibitor of translation in the extracts from unstimulated oocytes. Labelling studies with initiator tRNA ([35S]Met-tRNAf) indicate a higher concentration of 43S preinitiation complexes in the extracts from unstimulated oocytes, suggesting an impairment of initiation of translation at or after the mRNA-binding step. Extracts from both oocytes and progesterone-matured oocytes translated endogenous mRNAs to give products ranging over a wide spectrum of molecular weight. However, significant translation of exogenous (globin) mRNA required the presence of reticulocyte postribosomal supernatant, suggesting that one or more factors required for mRNA recruitment is limiting in these extracts.
Patrick
T D
TD
Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Brighton, UK.
Lewer
C E
CE
Pain
V M
VM
D000818:Animals; D002474:Cell-Free System; D002498:Centrifugation; D005260:Female; D007700:Kinetics; D009865:Oocytes / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D010063:Ovum / Q000378:metabolism*; D011374:Progesterone / Q000494:pharmacology; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012358:RNA, Transfer, Met; D014982:Xenopus laevis
D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D011374:Progesterone