The starfish egg mRNA responsible for meiosis reinitiation encodes cyclin.
During meiotic maturation and early embryonic cycles, the activity of maturation-promoting factor (MPF) cycles in exact correspondence with the mitotic cycles. For the appearance of MPF activity in starfish, protein synthesis is required except in the first meiotic cycle. In order to identify newly synthesized proteins involved in the regulation of MPF activity, we extracted poly(A)+ RNA from starfish eggs, and found that the egg poly(A)+ RNA induced germinal vesicle breakdown (GVBD) upon injection into immature oocytes of starfish and Xenopus. The molecular size of the poly(A)+ RNA responsible for GVBD was estimated to be approximately 22S by sucrose density gradient centrifugation. Since these characteristics of the starfish egg poly(A)+ RNA are similar to those of cyclin mRNAs from sea urchin and surf clam eggs, we synthesized a 50-mer antisense-cyclin oligonucleotide probe coding for a part of the sea urchin cyclin cDNA and used this to screen starfish RNA. The Northern blot analysis showed that the starfish egg RNA contained cyclin homologous transcripts. Incubation of the starfish egg poly(A)+ RNA and the antisense-cyclin oligonucleotide with RNase H completely destroyed its GVBD-inducing activity. These results indicated that starfish cyclin mRNA was the only poly(A)+ RNA responsible for GVBD. We constructed a starfish egg cDNA library to clone starfish cyclin cDNA. The longest cDNA clone containing 2190 base pairs was sequenced. The longest open reading frame consisted of 395 amino acid residues, and the predicted molecular size was 48 kDa. Comparison of the deduced amino acid sequences of starfish cyclin with known cyclins indicated that the starfish cyclin belongs to the B-type. Injection of synthetic mRNA of starfish cyclin caused GVBD in immature oocytes of starfish and Xenopus, while injection of synthetic mRNA of human CDC2 had no effect. The Northern blot analysis of starfish RNA extracted at various stages of the meiotic cycles suggested that the starfish cyclin transcript was stored in its polyadenylated form even in immature oocytes and was further polyadenylated at maturation.
Biological Laboratory, Faculty of Science, Tokyo Institute of Technology, Japan.
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Tachibana
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Ishiura
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Uchida
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Kishimoto
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016213:Cyclins; D005260:Female; D015723:Gene Library; D007447:Invertebrate Hormones / Q000235:genetics*; D008540:Meiosis; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D009865:Oocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013215:Starfish; D014158:Transcription, Genetic
D016213:Cyclins; D007447:Invertebrate Hormones; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011061:Poly A; C008407:1-methyladenine; D012313:RNA; D000225:Adenine