Fundamental studies of a developing heteromorphic sex chromosome pair in hynobiid salamanders.

韩国济州岛的水蛭（Hynobius leechii）有一对雌性异型性染色体，其中W染色体根据其栖息地表现出大小和形状的多态性（Ikebe未发表的数据）。另一方面，日本关东地区的东京海参（Hynobius tokyoensis）也具有相同类型的性染色体多态性，其W染色体与leechii不同。这两个物种的异型性明显是由W染色体的异染色质增加引起的。相反，居住在leechii和h.t okoensis栖息地之间的大部分neburrosus没有异型性染色体对（Ikebe & Kohno 1979, Ikebe et al. 1987）。也就是说，可以很自然地理解，在这两个物种中，异型性染色体对应该是独立发育的。为了进一步了解两种植物性染色体对异型性的促进因素，首先，我们尝试检测水蛭W染色体中扩增的重复序列。对1998年收集的松panak和2000年收集的kyyorairi胚胎中期W染色体的异染色质区和着丝粒区进行了染色体显微解剖。采用微克隆、三步简并寡核苷酸引物聚合酶链反应（dopp - pcr）和Southern blot杂交技术，分离出一个起源于leechii基因组DNA的DNA片段。对该片段进行测序，长度为208个碱基对。在未来的研究中，我们需要分离出leechii h . W染色体上由异染色质组成的序列，并将其与东京血吸虫进行比较。

Hynobius leechii in Cheju Island (Korea) has a pair of female heteromorphic sex chromosomes in which the W chromosome shows polymorphism in its size and shape according to its habitat (Ikebe unpublished data). On the other hand, Hynobius tokyoensis in Kanto district (Japan) also has the same kind of sex chromosomes with polymorphism and the W chromosomes in this species are different from those of H.leechii. Heteromorphism in the two species are clearly caused by increase of heterochromatin in their W chromosomes. On the contrary, most of H.neburosus which inhabits between the habitats of H.leechii and H.tokyoensis, does not have a heteromorphic sex chromosome pair (Ikebe ＆ Kohno 1979, Ikebe et al. l987). Namely, it is natural to understand that the heteromorphic sex chromosome pairs should be developed independently in these two species.For obtaining the fundamental knowledge in promoting factors of heteromorphism in the sex chromosome pair of the two species, first, we tried to detect the repeat sequences which amplified in W chromosomes in H.leechii. Microdissection of the chromosomes was carried on the heterochromatin region and the centromeric region of the W chromosomes in metaphases of embryos from Songpanak (collected in 1998) and Kyorairi (collected in 2000). By using the microcloning, the three step degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR), and the Southern blot hybridization, one DNA fragment which is originated in genomic DNA of H.leechii was isolated. The fragment was seqenced and the size was 208 base pairs. It was not specific to the female genomic DNA of H.leechii..In near future, we have to isolate the sequence which consists of heterochromatin in W chromosomes of H.leechii and have to compare it to that of H.tokyoensis.