A homolog of the serinehhreonine protein kinase (~3 4~~~' ) , encoded by the cdcZf gene of the fission yeast (Schizosaccharomyces pombe), is a catalytic subunit of maturation-promoting factor and a key regulator of the cell cycle. We have raised a monoclonal antibody against the most conserved amino acid sequence, the PSTAIR sequence (EGVPSTAIREISLLKE) of ~3 4~~~' This antibody recognizes 31 -34 kDa proteins by immunoblotting in all species examined so far. The proteins recognized by the anti-PSTAIR antibody are probably either ~3 4~~~' itself or proteins highly homologous to ~3 4~~~' in the given species, since, in all species studies to date, they are all precipitated with p13suc1, the fission yeast S U C~+ gene product, which binds to ~3 4~~~' with high specificity. The anti-PSTAIR imrnunoprecipitate had no histone H1 kinase activity and did not contain cyclin 6, suggesting that the PSTAIR region is masked when ~34'~'' forms a complex with cyclin B as an active kinase. lmmunoblotting with the anti-PSTAIR antibody demonstrated that the fastest-migrating form of ~3 4~~~' homologues becomes abundant, when oocytes mature or the cell enters M phase. The possible significance of this observation is discussed in relation to the phosphorylation and activity state of p34cdc2 The observed broad cross-reactivity of the anti-PSTAIR antibody against ~34'~'' homologues in various species should permit us to examine the role of ~34'~'' homologues in the regulation of the cell cycle in a variety of organisms.

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We isolated the structural gene encoding cytochrome P-450 aromatase (P-450arom), for the first time from a nonmammalian vertebrate, the medaka (a teleost fish, Oryzias latipes), using the rainbow trout P-450arom cDNA as a probe. The structure of the entire P-450arom gene, the nucleotide sequence of its 5' flanking region, and the transcriptional initiation sites were determined. The medaka P-450arom gene consists of nine exons, but spans only 2.6 kb, being much smaller than the human P-450arom gene (at least 70 kb), as a result of extremely small introns (medaka, 73-213 bp vs. human, 1.3-10 kbp). The splicing junctions are located at exactly the same positions as those found in the human P-450arom. The deduced amino acid sequence is 51-52% identical to those of mammals and chicken, and 75% identical to the rainbow trout amino acid sequence. Genomic Southern blots revealed the presence of a single medaka gene. Promoter analyses indicated two major transcription initiation sites 60 and 61 bp upstream from a putative initiation codon. The promoter region of medaka P-450arom gene also contains potential Ad4BP sites and estrogen responsive element (ERE) half-sites. These results suggest that the basic structural organization of P-450arom genes and the regulatory mechanisms of expression are well conserved throughout the vertebrates.

The enzyme aromatase P-450 (P450arom) catalyses the conversion of androgen to oestrogen. A cDNA insert encoding P450arom was isolated from a rainbow trout (Oncorhynchus mykiss) ovary cDNA library. The insert was sequenced and found to contain an open-reading frame predicted to encode a protein of 522 amino acid residues. The deduced polypeptide is 52% homologous with human, mouse and rat P450arom and 53% homologous with that of chicken. The insert was confirmed to encode P450arom by introducing it into COS-1 monkey kidney tumour cells (COS-1 cells) and detecting the conversion of testosterone to oestradiol-17 beta by radioimmunoassay. The N-terminal region of the deduced polypeptide was 19 amino acids longer than that of the other four species, and was found by hydropathy plotting to be very hydrophobic. Northern blot analysis revealed 2.6 kb RNA transcripts which were present in the trout ovary during vitellogenesis and hybridized to the cDNA insert. In preparations from subsequent stages of ovarian development, no RNA transcripts hybridized to the probe. Since the RNA transcripts are present only during the stage of oestradiol-beta production by the ovarian follicles, oestradiol-17 beta production may be regulated, in part, by the amount of P450arom mRNA present.

We demonstrate, for the first time in fish, that a Ca' +-independent and cyclic-nucleotide-independent histone H1 kinase activity oscillates according to the cell cycle of the oocyte, peaking at the first and the second meiotic metaphase with a transient drop between them. The kinase, M-phase-specific histone H1 kinase (M-HlK), was purified from mature carp oocytes by using two exogenous substrates for assaying its activity: histone H1 and a synthetic peptide (SP peptide, KKAAKSPK KAKK) containing the sequence KSPKK, which includes the consensus sequence of the site phosphorylated by a serinelthreonine-specific protein kinase encoded by the fission yeast cdc2' gene (cdc2 kinase). The M-H1K and maturation-promoting factor (MPF) activities coincided closely throughout four steps of purification, strongly suggesting the identity of M-HIK and MPF. The final preparation was purified 5000-fold with a recovery of 4%, when histone H1 was used for the kinase assay, and 10000-fold with a recovery of 7% when SP peptide was used. The purified molecular mass of the kinase was estimated to be 100 kDa by gel filtration and contained four proteins of 33,34,46 and 48 kDa. Anti-PSTAIR antibody recognizing cdc2 kinase cross-reacted with the 33-kDa and 34-kDa proteins, while the 46-kDa and 48-kDa bands cross-reacted with monoclonal antibodies raised against cyclin B. The 33-kDa protein was also recognized by an antibody against a goldfish cdk2 (Egl) kinase, a cdc2-related kinase which has the PSTAIR sequence and binds to p13""'1 but does not form a complex with cyclin B. M-H1K activity corresponded well to the 34-kDa, 46-kDa and 48-kDa proteins but not to the 33-kDa protein. These results strongly suggest that M-HIK consists of cdc2 kinase forming a complex with cyclin B, and that cdk2 kinase is not a component of M-HlK, although it is found in the highly purified M-H1K. The purified M-H1K utilized Mg2+, Mn2+, ATP and GTP, and had a wide pH optimum ranging over 8.0-10.5. The kinase was thermolabile and sensitive to freezing/ thawing.Fully grown oocytes can be fertilized only after undergoing oocyte maturation, which is triggered by maturation-inducing hormone (MIH) synthesized in and secreted from follicle cells surrounding the oocytes, under the influence of gonadotropin [l]. Oocyte maturation involves the progression of the cell cycle from G2 to M, and includes morphological changes such as germinal vesiclc breakdown (GVBD), condensation of chromosomes and formation of the first meiotic spindle. MIH has been fully identified only for two animal groups so far: one is 1 -methyladenine in starfish [2], and the other is 17a,20p-dihydroxy-4-pregnen-3-one in salmonid fishes [3]. The site of action of MIH is the oocyte surface, since injection of MIH into the oocytes has no effect on inducing oocyte maturation, while its external application is fully active [4]. It is therefore clear that MIH reception at the oocyte surface must trigger the activation of a cytoplasmic mediator, which leads the