Functions and regulation of genes on animal sex chromosomes

1.采用激光微束切割和随机PCR扩增的方法，从鸡Z染色体异染色质末端构建了基因组文库，经DNA测序和荧光原位杂交（FISH）鉴定，克隆由一个大卫星家族组成，其中约24kb的NheI片段重复约830次。这一大卫星序列是鸡属特有的，表明其在进化时间尺度上相对较新的扩增.采用与上述相似的方法构建了来自单个鸡W染色体的基因组文库。从该文库中，获得了来自25kb长的非重复区域的克隆。经Southern印迹杂交证实，该区域的一个0.6kb EcoR Ⅰ片段（EE0.6）在鸟类W染色体上广泛保守，可作为鸟类性别鉴定的探针。鸡EE0.6包含一个推定的外显子，但所有三个翻译阅读框架包含终止密码子，因此该序列似乎已经失去了它的基因功能在进化过程中.克隆了东方白色鹳W和Z染色体上的EE 0.6相关序列，并测定了其核苷酸序列。虽然这两个序列在该物种中高度相似，但使用适当选择的引物序列，仅能通过PCR扩增出W染色体上的EE0.6相关序列.编码免疫球蛋白超家族蛋白的ZOV3基因位于鸡Z染色体短臂的中间。ZOV3 cDNA在大肠杆菌中表达为多肽，并在小鼠中产生针对其的多克隆抗体。免疫荧光研究表明，ZOV3主要作为膜糖蛋白存在于产生性类固醇激素的卵巢细胞中。

1. A genomic library was constructed from the heterochromatic end of the chicken Z chromosome by dissection with laser microbeam and amplification of DNA by random PCR.Characterization of clones by DNA sequencing and fluorescence in situ hybridization (FISH) demonstrated that the DNA of the terminal heterochromatin consisted of a macrosatellite family in which about 24kb Nhel fragment was repeated about 830 times. This macrosatellite sequence is specific to the genus Gallus, suggesting its relatively recent amplification on the evolutionary time scale.2. A genomic library from a single chicken W chromosome was constructed using the similar method as above. From this library, clones derived from a 25kb-long non-repetitive region were obtained. A 0.6kb EcoRl fragment (EE0.6) in this region was proved to be widely conserved in the W chromosome of birds and was shown to be a useful probe for sexing birds by Southern blot hybridization. The chicken EE0.6 contains a putative exon but all three translational reading frames contain stop codons and thus the sequence seems to have lost its gene function during evolution.3. The EE0.6-related sequences on the W and Z chromosome of Oriental white stork were cloned and their nucleotide sequences were determined. Although the two sequences are highly similar in this species, only the EE0.6-related sequence on the W chromosome could be amplified by PCR using properly selected primer sequences.4. A ZOV3 gene encoding an immunoglobulin superfamily protein was located at the middle of the short arm of the chicken Z chromosome. The ZOV3 cDNA was expressed into a polypeptide in Escherichia coli and a polyclonal antibody to it was raised in mice. lmmunofluorescence study demonstrated that ZOV3 is present as a membrane glycoprotein predominantly in ovarian cells producing sex steroid hormones.