| Project/Area Number |
04454107
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
畜産学(含草地学)
|
|---|
| Research Institution | KYOTO UNIVERSITY |
|---|
Principal Investigator |
SAKAI Hiroshi (1993-1994) Kyoto Univ.・Faculty of Agri., Associate Professor, 農学部, 助教授 (60089117)
内海 恭三 (1992) 京都大学, 農学部, 教授 (90033266)
|
|---|
| 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)
酒井 裕 京都大学, 農学部, 助教授 (60089117)
|
|---|
| Project Period (FY) |
1992 – 1994
|
| Project Status |
Completed (Fiscal Year 1994)
|
| Budget Amount *help |
¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1993: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1992: ¥2,500,000 (Direct Cost: ¥2,500,000)
|
|---|
| Keywords | Male specific DNA / Bovine SRY gene / Male sex differentiation / bSRY fusion proteins / 性決定遺伝子 / SRY / 胚の性判別 / ウシ / PCR法 / デオキシ鎖反応 / 性分化 / BOV97M |
|---|
| 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.
|
