A FISH HOMOLOG OF THE CDC2-RELATED PROTEIN P40(MO15) - ITS CDNA CLONING AND EXPRESSION IN OOCYTES

We have previously shown that threonine (probably Thr-161) phosphoryla tion of cdc2 kinase (p34(cdc2)), the catalytic subunit of maturation-p romoting factor (MPF), is a crucial step for the 17 alpha,20 beta-dihy droxy-4-pregnen-3-one (17 alpha,20 beta-DP, a maturation-inducing horm one)induced activation of MPF in goldfish (Carassius auratus) oocytes. In this study, we have cloned a cDNA that encodes a goldfish homolog of p40(M015), the catalytic subunit of a protein kinase which has been shown to activate cdc2 kinase through phosphorylation of Thr-161, fro m a goldfish oocyte cDNA library. Reverse transcription PCR using olig onucleotide primers corresponding to highly conserved sequences in cdc 2-related genes was used to generate a 144 bp cDNA PCR product from go ldfish oocyte RNA. This probe was then used to screen the cDNA library for a full-length cDNA encoding a homolog of p40(M015) in goldfish. T he isolated clone of approximately 1.3 kbp contained an open reading f rame encoding a 344 amino acid protein that: was 83% and 55% identical to Xenopus p40(M015) and a homolog of p40(M015) in rice, respectively . Using Northern blot techniques, a 1.3 kb M015 mRNA was detected in b oth full-grown immature oocytes and mature oocytes of ginbuna (Carassi us auratus langsdorfii), a species closely related to goldfish. These data suggest that p40(M015) homolog is produced in fish oocytes and pl ays an important role in the 17 alpha,20 beta-DP-induced activation of MPF through threonine phosphorylation of cdc2 kinase.