Molecular Cytogenetical Studies on the Avian Sex Chromosomes

本联合研究的目的是从鸡W和Z染色体特异基因组文库中分离克隆，测定它们的核苷酸序列，并用荧光原位杂交(FISH)检测它们在灯刷Z-W二价染色体上的定位。主要研究结果如下：1)Z染色体：利用Ar~+激光烧毁鸡Z染色体末端区域，从剩余染色体区域微量提取DNA，用单一唯一的随机引物进行随机PCR扩增，获得了鸡Z染色体末端的Lambda gt10文库。随机选择100个克隆，混合后用作FISH对有丝分裂染色体的探针。这些探针与鸡Z染色体的异染色端区域杂交。用混合探针筛选母鸡的lambda GEM12基因组文库，得到克隆pCHZTH8。该克隆与Z染色体的短环区和异染色端的末端弓形环(TBL)杂交。PCHZTH8的部分序列在雀形目中是保守的，但其中大部分是Galus属所特有的。2)W染色体：用同样的技术获得W染色体特异的基因组文库。克隆了鸡W染色体上的CW01和CW50两个区域，每个区域包含约25kb的非重复序列。CW50区包含一个推测的CpG岛，CW01区包含一个序列，该序列在许多鸟类中广泛以W连锁的形式保守，因此是鉴定鸟类性别的有用的通用探针。

The aim of this joint research was to isolate clones from the chicken W and Z chromosome-specific genomic libraries, determine their nucleotide sequences, and to examine their localizations on the lampbrush Z-W bivalent chromosomes by fluorescence in situ hybridization (FISH). The major outcome is as follows.1) Z chromosome : Lambda gt10 libraries were prepared from a terminal region of the chicken Z chromosome by utilizing argon ion laser micro-irradiation to burn down all other chromosomes, micro-extraction of DNA from the remaining chromosomal region and random PCR amplification using a single unique primer. One hundred clones were selected randomly, mixed and used as probes in the FISH to mitotic chromosomes. Those probes hybridized to the heterochromatic end region of the chicken Z chromosome. A lambda GEM12 genomic library of the female chicken was screened with those mixed probes and a clone, pCHZTH8 was obtained. This clone hybridized to the short loops region and the terminal bow-like loop (TBL) at the heterochromatic end of the Z chromosome. A part of the pCHZTH8 sequence is conserved among the order Galliformes but most of them is unique to the genus Gallus.2) W chromosome : The same technique as above was applied to obtain W chromosome-specific genomic libraries. Two regions on the chicken W chromosome, CW01 and CW50 each consisting of about 25-kb non-repetitive sequences, were cloned. The CW50 region contains a putative CpG island and the CW01 region contains a sequnce that is conserved widely as W-linked among many avian species, and thus is a useful general probe for sexing avian species.