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

鸟类Z和W性染色体基因在性别决定和性别分化中的功能研究

基本信息

批准号：
12460036
负责人：
MIZUNO Shigeki
金额：
$ 10.3万
依托单位：
Nihon University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2003
项目状态：
已结题

项目摘要

(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

（1）新型W染色体链接基因PKCI-W的cDNA克隆及其在Z染色体上的对应基因PKCI-Z是从Day-S鸡胚胎的雌性微米 - 毛线减法cDNA库中获得的。 PKCI-W和PKCI-Z在氨基酸序列水平上的同源性约为60％。早期雌性胚胎的生殖脊和神经组织中，这两个基因的mRNA水平表达均高。 PKCI-Z PKCI-W缺乏命中（组氨酸三合会）基序，这对于PKCI/提示的功能至关重要，但是构成了PKCI/提示的同二聚体形成涉及的命中基序的区域构成很好。 PKCI-W和PKCI-Z均表示为大肠杆菌中的谷胱甘肽S-转移酶或麦芽糖结合蛋白的融合蛋白并纯化。 PKCI-Z显示出高亲和力与腺苷和AMP降低水解活性，如哺乳动物PKCI/提示所示，而PKCI-W没有显示这些活性。 PKCI-Z W的这些活性通过在体外与PKCI-W形成异二聚体，以显性阴性的方式抑制。这些结果支持了PKCI-W可能参与女性性别确定的模型。（2）Spindlin Gene（Spin-W）的基因组克隆（Spin-W）和一种新型的SSPI型家庭重复序列，是从鸡W染色体特异性基因组库中获得的，该基因组特异性基因组库是由Rashase Microdection for Rystaphase Chromosome组制备的，由随机化合酶组合集合了PRCR PREFIFIFIAT。随后获得了Z染色体上的对应基因Spin-Z的克隆。使用3'-非翻译区域探针的北印迹分析表明，Spin-Z普遍存在，但Spin-W主要在卵巢中表达。 SSPI家庭由508 bp单元的11,300个串联重复序列组成，并位于鸡W染色体短臂上的白染色质/异染色质边界。每个重复单元都包含一个120 bp的息肉素/息肉酰胺序列，该序列可能形成三链DNA。 SSPI家庭在雌性鸡胚胎成纤维细胞的核染色质结构中形成了弥漫性染色质结构。（3）从Z染色体上的特定区域的克隆（指定为MIIM（雄性甲基化）区域），从鸡肉卵母细胞cDNA库中获得。在女性中，将MIL-IM区域积极转录为约9.5 kb的非编码RNA（MHM-RNA），该RNA（MHM-RNA）积聚在核中MHM区域的接近或附近。在男性，MHM区域高度甲基化，其转录完全不活跃。 MHM-RNA在核中非常接近DMRTI基因。 DMRT1基因被认为是睾丸分化至关重要的，在孵化和MHM-RNA后，在雌性鸡中不表达DMRT1基因，并未表达MHM-RNA参与这种转录失活。较少的