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

(1)从鸡胚雌-雄减法cDNA文库中获得了新的W染色体连锁基因PKCI-W及其在Z染色体上的对应基因PKCI-Z的cDNA克隆。PKCI-W和PKCI-Z在氨基酸序列水平上的同源性约为60%。这两个基因在早期雌性胚胎的生殖嵴和神经组织中表达水平较高。PKCI- z与哺乳动物PKCI/HINT同源。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同型二聚体形成的区域保存良好。PKCI-W和PKCI-Z在大肠杆菌中以谷胱甘肽s转移酶或麦芽糖结合蛋白的融合蛋白表达并纯化。PKCI- z在哺乳动物PKCI/HINT中表现出与腺苷的高亲和力结合和amp -酰胺水解活性，而PKCI- w则没有表现出这些活性。pkci - zw…More在体外与PKCI-W形成异源二聚体，以显性负向方式抑制这些活性。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染色体特异性基因组文库中获得了spindlin基因（SPIN-W）的基因组克隆和一个新的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。利用3'-非翻译区探针进行Northern blot分析发现，SPIN-Z在卵巢中普遍表达，而SPIN-W在卵巢中主要表达。sspi家族由11,300个508 bp的串联重复序列组成，定位于鸡W染色体短臂上的常染色质/异染色质边界。每个重复单元包含一个120 bp的多嘌呤/多嘧啶序列，可能形成一个三链DNA。sspi家族在鸡胚成纤维细胞细胞核中形成弥漫性染色质结构。(3)从鸡卵母细胞cDNA文库中获得了Z染色体上一个名为MIIM （male-hypermethylated）区的克隆。在雌性中，MIL-IM区被主动转录成约9.5 kb的非编码RNA (MHM-RNA)，这些非编码RNA聚集在细胞核中MHM区或其附近。在雄性中，MHM区域高度甲基化，其转录完全失活。MHM-RNA在细胞核中非常靠近DMRTI基因聚集。DMRT1基因一直被认为是睾丸分化所必需的，但在母鸡孵化后不表达，推测MHM-RNA参与了这种转录失活。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