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日龄鸡胚的雌性-雄性差减cDNA文库中获得了一个新的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在氨基酸水平上的同源性约为60%。这两个基因的mRNA水平的表达是高的生殖嵴和神经组织在早期女性胚胎。PKCI-Z是哺乳动物PKCI/HINT的鸡直系同源物。PKCI-W缺乏HIT（组氨酸三联体）基序，该基序对于PKCI/HINT的功能是必不可少的，但是参与PKCI/HINT的同源二聚体形成的HIT基序侧翼区域是非常保守的。PKCI-W和PKCI-Z在大肠杆菌中表达为谷胱甘肽S-转移酶或麦芽糖结合蛋白融合蛋白。coli中进行纯化。如哺乳动物PKCI/HINT所示，PKCI-Z显示出与腺苷的高亲和力结合和AMP-酰胺水解活性，而PKCI-W没有显示出这些活性。PKCI-Z的这些活性与 关于我们 在体外通过与PKCI-W形成异源二聚体而以显性负性方式抑制。这些结果支持了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克隆了spindlin基因（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。利用3 '-非翻译区探针进行的北方印迹分析表明，SPIN-Z在卵巢中广泛表达，而SPIN-W主要在卵巢中表达。SspI家族由11，300个508 bp的串联重复序列组成，位于鸡W染色体短臂上的常染色质/异染色质边界。每个重复单元含有一个120 bp的聚嘌呤/聚嘧啶序列，可能形成一个三链DNA。SspI家族在雌性鸡胚胎成纤维细胞的细胞核中形成弥散的染色质结构。(3)从鸡卵母细胞cDNA文库中获得了Z染色体上一个特定区域的克隆，命名为MIIM（雄性高甲基化）区域。在雌性中，MIL-IM区域被积极地转录成约9.5-kb的非编码RNA（MHM-RNA），其积累在核中的MHM区域处或其附近。在男性中，MHM区域高度甲基化，其转录完全失活。MHM-RNA在细胞核中积累得非常接近DMRTI基因。DMRT 1基因，这已被认为是睾丸分化所必需的，是不表达的母鸡孵化后，MHM-RNA被推测参与这种转录失活。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