Cloning and characterization of a vasa-like gene in rainbow trout and its expression in the germ cell lineage

The origin of germ cells and the molecular mechanisms of primordial germ ce ll (PGC) determination in teleosts are unclear. Vasa is a member of the DEA D protein family and plays an indispensable role in germ cell determination in Drosophila and Xenopus species. In this study, we isolated and characte rized a rainbow trout vasa cDNA as a first step towards understanding the m olecular mechanisms of PGC determination and development and to develop a m olecular marker to identify the PGCs in rainbow trout. Cloning of vasa cDNA was performed by degenerate- and RACE-PCR. The predicted amino acid sequen ce of rainbow trout Vasa contained eight consensus sequences for the DEAD p rotein family and five arginine-glycine-glycine repeats, a common character of known Vasa homologues. Overall amino acid similarity to the Vasa of Dro sophila was 79.2%. Whole-mount in situ hybridization of eyed stage embryos (eighty somite stage) revealed that signals were localized to the putative PGCs. In adult rainbow trout tissues, both ovaries and testes contained lar ge amounts of vasa gene transcripts. A reverse transcription-polymerase cha in reaction analysis of unfertilized eggs proved that trout vasa is a mater nal factor. Although we have not determined whether rainbow trout vasa func tions as a germ cell determinant, its limited expression in the germ cell l ineage proved that rainbow trout vasa can be used as a marker molecule for PGCs. This marker will make it possible to identify the PGCs or presumptive PGCs in early trout embryos whose germ cells can not be distinguished by m orphological characteristics. Mel. Reprod. Dev. 55:364-371, 2000. (C) 2000 Wiley-Liss, Inc.