Molecular Cytogenetical Studies on the Avian Sex Chromosomes

本联合研究的目的是从鸡W和Z染色体特异性基因组文库中分离克隆，确定其核苷酸序列，并通过荧光原位杂交（FISH）检测其在灯刷Z-W二价染色体上的定位。主要结果如下。1) Z染色体：利用氩离子激光微照射灼烧鸡Z染色体末端区域的所有其他染色体，从剩余染色体区域微提取DNA，利用单个独特引物随机扩增，制备Lambda gt10文库。随机选择100个无性系，混合后作为FISH中有丝分裂染色体的探针。这些探针与鸡Z染色体的异色末端区杂交。利用这些混合探针筛选了一个母鸡lambda GEM12基因组文库，获得了一个克隆pCHZTH8。该克隆杂交于Z染色体异色末端的短环区和末端弓形环（TBL）。部分pCHZTH8序列在鸡形目中是保守的，但大部分是鸡属所特有的。2)W染色体：采用相同的技术获得W染色体特异性基因组文库。克隆了鸡W染色体上的两个区域CW01和CW50，各包含约25kb的非重复序列。CW50区域包含一个假定的CpG岛，CW01区域包含一个在许多鸟类中广泛保守的w连锁序列，因此是一个有用的鸟类性别鉴定的一般探针。

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.