Searching cytoplasmic factors involved in sex determination of vertebrate

性是生物体产生生物多样性和进化的主要机制。迄今为止，研究主要集中在性染色体上的性别决定主基因。近年来，我们获得了一些资料，支持卵细胞质因子参与青蛙性别决定。本研究主要集中在以下三个方面：1. Wolbachia为了鉴定蛙卵中的Wolbachia细菌，我们从蛙卵的基因组DNA和cDNA中扩增了Wolbachia细菌FtsZ基因的特异性基因。结果从伊势原和广岛种群的卵中均获得了扩增产物。然而，它们的核苷酸序列与FtsZ基因没有任何相似之处。2.线粒体为了鉴定线粒体基因组中任何参与性别决定的新基因，我们测定了两个当地种群，伊势原和广岛的线粒体基因组结构，因为它们的杂交后代显示出明显的细胞质性比偏差。结果表明，在伊势原种群的D-loop区域内发现了一个独特的序列。它可以编码60-100个氨基酸，由两条链每三个框架的十个氨基酸的重复组成。3.母体因子利用WW和ZZ两种卵的RNA差减技术和WW和ZZ两种胚胎的差异显示技术，分离出了仅在两种卵或胚胎中表达的基因。WW卵高表达galectin和线粒体考克斯Ⅰ，ZZ卵高表达同源结构域dbx Ⅰ和蛋白激酶LYK-5。我们从WW胚胎中分离到一个新的W-连接糖蛋白基因，该基因在ZW雌性性腺中特异表达。

Sex is a major mechanism to be essentially involved in the production of biological diversities and evolution in the organisms. So far, researches have been focused on a master sex determining gene on sex chromosomes. Recently, we have got some data that support existence of cytoplasmic factors in eggs involved in sex determination of frogs. The present study focused on the following three points.1.WolbachiaTo identify Wolbachia bacteria in the eggs of frog, Rana rugosa, we amplified a gene specific to the FtsZ gene of Wolbachia from genomic DNA and cDNA of the frog eggs. As the results, amplified products were got from eggs of Isehara and Hiroshima populations. However, their nucleotide sequences did not show any similalities to the FtsZ gene.2.MitochondriaTo identify any new genes involved in sex determination within the mitochondiral genome, we determined mitochondrial genome organization of the two local populations, Isehara and Hiroshima, because their hybrids show striking bias of sex ratio according to the cytoplasm. As the results, we found one unique sequence within the D-loop region of the Isehara population. That can encode 60-100 amino acids, comprising of repeats of ten amino acids, from every three frame of both strands. The ten amino acids are similar to unknown protein of the protozoan pathogen Trypanosoma cruzi.3.Maternal factorUsing RNA subtraction between WW and ZZ eggs and differential display between WW and ZZ embryos, we isolated some genes highly expressed only in each of the two kinds of eggs or embryos. In WW eggs, one galectin and mitochondrial cox I were highly expressed, while in ZZ eggs, homeodomain dbx I and protein kinase LYK-5 were highly expressed. From the WW embryos, we isolated a new W-linked glycoprotein gene, which was expressed specifically in ZW female gonads during sex determinination.