Searching cytoplasmic factors involved in sex determination of vertebrate

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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.