Functions and regulation of genes on animal sex chromosomes

动物性染色体基因的功能和调控

基本信息

批准号：
07456042
负责人：
MIZUNO Shigeki
金额：
$ 4.35万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07456042/
关键词：
chicken Oriental white stork W chromosome Z chromosome heterochromatin sexing by DNA probe sex chromosome-specific genomic library immunoglobulin superfamily ゲノムライブラリー PCR法性染色体マクロサテライト配列性判別DNAプローブ

项目摘要

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.

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次。该宏观阳性层序列特定于Gallus属，这表明其在进化时间尺度上相对较新的扩增。2。使用与上述类似的方法构建了来自单个鸡W染色体的基因组文库。从这个库中获得了源自25kb长的非重复区域的克隆。事实证明，该区域的0.6kb Ecorl片段（EE0.6）在鸟类的W染色体中被广泛保守，并被证明是通过Southern印迹杂交对鸟类进行性爱的有用探针。 Chicken EE0.6包含一个推定的外显子，但所有三个翻译读物都包含终止密码子，因此该序列似乎在进化过程中失去了基因功能。3。克隆东方白鹳W和Z染色体上的EE0.6相关序列，并确定其核苷酸序列。尽管这两个序列在该物种中高度相似，但只能使用正确选择的引物序列来通过PCR扩增W染色体上的EE0.6相关序列。4。编码免疫球蛋白超家族蛋白的ZOV3基因位于鸡Z染色体的短臂的中间。 ZOV3 cDNA被表达为大肠杆菌中的多肽，并在小鼠中提出了多克隆抗体。 lmmunof荧光研究表明，在产生性类固醇激素的卵巢细胞中，ZOV3主要是膜糖蛋白。