| Co-Investigator(Kenkyū-buntansha) |
MINAMI Naojiro Kyoto Univ.・Faculty of Agri., Researcher, 農学部, 助手 (30212236)
HOSOI Yoshihiko Kinki Univ.・Res.Inst.of Bio-Oriented Sci.and Tech., Instructor, 生物理工学部, 講師 (70192739)
YAMADA Masayasu Kyoto Univ.・Faculty of Agri., Associate Professor, 農学部, 助教授 (10243073)
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| Research Abstract |
Cloning of bovine sex determining region of Y gene (bSRY), which is supposed to induce male differentiation of gonad during bovine embryogenesis, and characterization of bSRY-coding proteins were designed. The SRY was first identified by positional cloning within the region of the human Y chromosome and expressed at high levels in the testes where no function has been determined. A sequence has been found that is conserved on SRY of all mammals tested. We therefore attempted to isolate a cDNA clone of bSRY using RT-PCR with human SRY sequence as primers and total RNA extracted by AGPC method from bovine testes. Sequence analysis of this clone, designated as bSRYcDNA-l revealed that it consisted of a total 317 nucleotides, which contained conserved region of SRY.We next attempted to determine the upstream region of bSRYcDNA using 5'RACE.As a result, we could further determine the 101 bp upstream sequence of bSRYcDNA-1, the cDNA cloned by 5'RACE was designated as bSRYcDNA-2. Surprisingly, when the nucleotide sequences of bSRYcDNA-1 and bSRYcDNA-2 were compared, it was found that the sequences in 5'direction from the 108 bp upstream of the conserve region were different, at which point the characteristic of a potential splice acceptor was found. Therefore, the difference of the sequence may be attributed to the circular structure of bSRY mRNA,as has been described in muse SRY.
Next to characterize a fusion proteins of bSRY-1 encoding polypeptides and beta-galactosidase, pETSRYlacZ plasmid was transfected into BL21 cells. The fusion proteins were highly expressed in the cells with IPTG induction. We are now trying to isolate the fusion proteins using beta-galactosidase affinity column.
Moreover, we could find that the primers derived from bSRYcDNA-1 were useful for sexing of bovine embryos at the blastocyst stage using PCR method.
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