Genomic sequences encoding two types of medaka hemopexin-like protein Wap65, and their gene expression profiles in embryos.
Medaka genomic BAC clones, which contained two types of medaka hemopexin-like protein gene (Wap65), mWap65-1 and mWap65-2, were screened and their genomic sequences were determined by the shotgun strategy. The exon-intron organizations were highly conserved between both mWap65s and human hemopexin genes. The 5'-flanking regions of mWap65-1 and mWap65-2 contained various putative transcription factor binding sites including elements for developmental regulation. The expression patterns of mWap65s during embryonic development were examined by quantitative real-time PCR, demonstrating that both mWap65 transcripts were observed in early embryonic stages, but their expression patterns were different. Interestingly, in situ hybridization revealed that mWap65-2 transcripts were restricted to liver, whereas mWap65-1 transcripts were detected along the edge of pectoral fin buds and the median fin fold of tail buds in embryos at stage 32. Furthermore, we generated transgenic medaka expressing GFP driven by mWap65-1 and mWap65-2 promoters and observed GFP expression patterns during ontogeny. Although localizations of GFP varied among individuals, embryos uniformly expressed GFP 1 day after injection of mWap65-1-hrGFP and mWap65-2-hrGFP constructs, suggesting that mWap65-1 and mWap65-2 promoters were activated in very early stages. The differences between mWap65-1 and mWap65-2 in their expression profiles indicate their distinct roles during ontogeny.
Nakaniwa
Makiko
M
Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.
Hirayama
Makoto
M
Shimizu
Atsushi
A
Sasaki
Takashi
T
Asakawa
Shuichi
S
Shimizu
Nobuyoshi
N
Watabe
Shugo
S
D000818:Animals; D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial; D017931:DNA Primers; D004625:Embryo, Nonmammalian / Q000378:metabolism; D040461:Gene Components; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D014644:Genetic Variation; D049452:Green Fluorescent Proteins; D006466:Hemopexin / Q000235:genetics* / Q000378:metabolism*; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D009990:Oryzias / Q000196:embryology* / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D049452:Green Fluorescent Proteins; D006466:Hemopexin