| Co-Investigator(Kenkyū-buntansha) |
OGIHARA Jun Nihon University, College of Bioresource Sciences, Department of Agricultural and Biological Chemistry, Lecturer, 生物資源科学部, 講師 (50256830)
KATO Jun Nihon University, College of Bioresource Sciences, Department of Agricultural and Biological Chemistry, Lecturer, 生物資源科学部, 講師 (80130467)
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| Research Abstract |
(1) cDNA clones of a novel W chromosome-linked gene, PKCI-W, and its counterpart gene on the Z chromosome, PKCI-Z, were obtained from the female-minus-male subtraction cDNA library from day-S chicken embryos. Homology of PKCI-W and PKCI-Z at the amino acid sequence level was about 60%. The mRNA-level expression of both genes was high in genital ridge and neural tissues in early female embryos. PKCI-Z was a chicken ortholog of mammalian PKCI/HINT. PKCI-W lacked a HIT (histidine triad) motif which is essential for the function of PKCI/HINT but regions flanking the HIT motif which are involved in the homodimer formation of PKCI/HINT were well conserved. Both PKCI-W and PKCI-Z were expressed as fusion proteins with glutathione S-transferase or maltose-binding protein in E. coli and purified. PKCI-Z showed high affinity binding to adenosine and AMP-amidate hydrolyzing activity as demonstrated for mammalian PKCI/HINT, whereas PKCI-W did not show these activities. These activities of PKCI-Z w
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ere inhibited in a dominant-negative manner by forming a heterodimer with PKCI-W in vitro. These results supported the model in which PKCI-W might participate in the female sex determination.
(2) Genomic clones for spindlin gene (SPIN-W) and a novel SspI-family repetitive sequence were obtained from the chicken W chromosome-specific genomic library which was prepared by laser microdissection of a metaphase chromosome set followed by random PCR amplification. A clone for the counterpart gene, SPIN-Z, on the Z chromosome was subsequently obtained. Northern blot analysis using the 3'-untranslated region probe revealed that SPIN-Z was expressed ubiquitously but SPIN-W was expressed predominantly in the ovary. The SspI-family consisted of 11,300 tandem repeats of a 508-bp unit and localized at the euchromatin/ heterochromatin boundary on the short arm of the chicken W chromosome. Each repeating unit contained a 120-bp polypurine/polypyrimidine sequenece which might form a triple-stranded DNA. The SspI-family formed a diffuse chromatin structure in nuclei of female chicken embryonic fibroblasts.
(3) A clone for a specific region on the Z chromosome, designated MIIM (male-hypermethylated) region, was obtained from the chicken oocyte cDNA library. In females, MIL-IM region was transcribed actively into about 9.5-kb non-coding RNA (MHM-RNA) which accumulated at or in the close vicinity of the MHM region in a nucleus. In males, MHM region was highly methylated and its transcription was totally inactive. MHM-RNA accumulated very close to the DMRTI gene in a nucleus. The DMRT1 gene, which has been suggested to be essential for testis differentiation, is not expressed in female chickens after hatching and MHM-RNA was speculated to be involved in this transcriptional inactivation. Less
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