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 cell (PGC) determination in teleosts are unclear. Vasa is a member of the DEAD protein family and plays an indispensable role in germ cell determination in Drosophila and Xenopus species. In this study, we isolated and characterized a rainbow trout vasa cDNA as a first step towards understanding the molecular mechanisms of PGC determination and development and to develop a molecular marker to identify the PGCs in rainbow trout. Cloning of vasa cDNA was performed by degenerate- and RACE-PCR. The predicted amino acid sequence of rainbow trout Vasa contained eight consensus sequences for the DEAD protein family and five arginine-glycine-glycine repeats, a common character of known Vasa homologues. Overall amino acid similarity to the Vasa of Drosophila 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 large amounts of vasa gene transcripts. A reverse transcription-polymerase chain reaction analysis of unfertilized eggs proved that trout vasa is a maternal factor. Although we have not determined whether rainbow trout vasa functions as a germ cell determinant, its limited expression in the germ cell lineage 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 morphological characteristics.
Yoshizaki
G
G
Department of Aquatic Biosciences, Tokyo University of Fisheries, Tokyo, Japan. goro@tokyo-u-fish.ac.jp
Sakatani
S
S
Tominaga
H
H
Takeuchi
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D005854:Germ Cells / Q000378:metabolism*; D017403:In Situ Hybridization; D008297:Male; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss; D010053:Ovary / Q000378:metabolism; D012313:RNA / Q000032:analysis; D020365:RNA Helicases / Q000302:isolation & purification / Q000378:metabolism*; D016372:RNA, Antisense / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013737:Testis / Q000378:metabolism
D016372:RNA, Antisense; D012313:RNA; D020365:RNA Helicases