Searching cytoplasmic factors involved in sex determination of vertebrate

性是一种主要的机制，主要参与生物多样性的产生和生物体的进化。到目前为止，人们的研究主要集中在性染色体上的性别决定基因上。最近，我们获得了一些数据，支持在卵子中存在参与青蛙性别决定的细胞质因子。本研究主要集中在以下三个方面：1.沃尔巴克氏菌为了鉴定蛙卵中的沃尔巴克氏菌，我们从蛙卵基因组DNA和cDNA中扩增出沃尔巴克氏菌FtsZ基因的特异性基因。结果从石原和广岛种群的卵子中扩增出扩增产物。然而，它们的核苷酸序列与FtsZ基因没有任何相似之处。2.线粒体为了在线粒体基因组中寻找与性别决定有关的新基因，我们确定了两个地方群体Isehara和Hiroshima的线粒体基因组结构，因为它们的杂交后代根据细胞质表现出显著的性别比偏差。结果，我们在Isehara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10个氨基酸组成，来自两条链的每三个框架。母性因素利用WW和ZZ卵的RNA差减和WW和ZZ胚胎的差异显示，我们分离到了一些只在两种卵或胚胎中高表达的基因。在WW卵中，半乳糖凝集素和线粒体COX I高表达，而在ZZ卵中，同源结构域DBX I和蛋白激酶Lyk-5高表达。从WW胚胎中分离到一个新的W连锁糖蛋白基因，该基因在性别决定过程中在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.