Investigation on functions of genes on the Z and W sex chromosomes of birds with respect to sex determination and sex differentiation

(1)从S鸡胚雌-负-雄消减文库中克隆了一个新的W染色体连锁基因PKCI-W和Z染色体上的相应基因PKCI-Z。PKCI-W与PKCI-Z在氨基酸序列水平上的同源性约为60%。在早期雌性胚胎中，这两个基因在生殖器脊和神经组织中的表达水平较高。PKCI-Z是哺乳动物PKCI/HINT的鸡同源基因。PKCI-W缺乏对PKCI/HINT功能至关重要的HIT(组氨酸三联体)基序，但HIT基序两侧参与PKCI/HINT同源二聚体形成的区域被很好地保存下来。将PKCI-W和PKCI-Z分别与谷胱甘肽S转移酶或麦芽糖结合蛋白在大肠杆菌中进行融合表达和纯化。与哺乳动物的PKCI/HINT一样，PKCI-Z具有与腺苷和AMP-酰胺的高亲和力结合活性，而PKCI-W则不显示这些活性。PKCI-Z w…的这些活动在体外，通过与PKCI-W形成异二聚体，以显性-负性方式抑制更多的ERE。这些结果支持PKCI-W可能参与雌性性别决定的模型。(2)从鸡W染色体特异的基因组文库中获得Spindlin基因的基因组克隆(Spin-W)和一个新的SspI家族重复序列。随后获得了Z染色体上对应基因Spin-Z的克隆。用3‘-非翻译区探针进行Northern杂交分析表明，Spin-Z普遍表达，而Spin-W主要在卵巢中表达。SspI家族由11300个508bp的串联重复序列组成，定位于鸡W染色体短臂上常染色质/异染色质交界处。每个重复单元包含一个120bp的多嘌呤/多嘧啶序列，可能形成三链DNA。SspI家族在雌性鸡胚胎成纤维细胞的细胞核内形成弥漫的染色质结构。(3)从鸡卵母细胞cDNA文库中获得了Z染色体上特定区域的克隆，命名为MIIM(雄性高甲基化)区。在雌性中，MIL-IM区被主动转录成约9.5kb的非编码RNA(MHM-RNA)，并聚集在细胞核中MHM区或其附近。在男性中，MHM区域高度甲基化，其转录完全不活跃。MHM-RNA在细胞核中积聚在与DMRTI基因非常接近的位置。DMRT1基因被认为是睾丸分化所必需的，但在母鸡孵化后不表达，MHM-RNA被推测参与了这种转录失活。较少

(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 … More 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