| Project/Area Number |
61860007
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
応用生物化学・栄養化学
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| Research Institution | TOHOKU UNIVERSITY |
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Principal Investigator |
MIZUNO Shigeki Faculty of Agriculture, Tohoku University, 農学部, 教授 (90112903)
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| Co-Investigator(Kenkyū-buntansha) |
NAGATA Yoshiho Vice Di Research Institute, Nichirei Corporation, 次長 (60013322)
MIZUMA Yutaka Professo Faculty of Agriculture, Tohoku University, 農学部, 教授 (50005584)
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 1987: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1986: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Keywords | Chicken / Turkey / Fpheasant / Sexing of early emvryos / W chgomosome / Repetitive DNA / Dot blot hybridization / ビオチン標識プローブ / 性染色体 / Y染色体 / 初期胚の性判定 |
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| Research Abstract |
Two repeating units, 0.7kb and 1.1kb, of the XhoI family repetitive DNA, which is specific to the W chtomosome of chicken were cloned. These repeating units consist of tandem repeat of about 21-bp sequence and migrate unusually slow in 4% ployacrylamide gel electrophoresis, suggesting that these units form curved DNA configuration. Undef the non-stringent hybridization conditions allowing the presence of about 60% base pair mismatches in the resulting hybrids and using the_<32>p-labeled 0.7-kb repeating unit as a probe, Xhoi family-related and W chromosome-specific repeating units were selected and cloned from the female genomic DNAs of turky and pheasant: PUMG0401 carrying a PstI-0.4-kb fragment from the turkey and PUPV0501 carrying a TaqI-0.5-kb fragment from the pheasant.
Feasibility of employing these sex chromosome-specific repetitive DNA clones in sexing of early embryos was tested using the cloned 0.7-kb unit of the chicken. It was demonstrated that a cleat female-specific hybredization signal could be obtained when a crude DNA preparation isolated from a part of the indivisual 6 day chicken embryo was subjected to dot blot hybrisization with the_<32>p-labeled probe. When the XhoI 0.7-kb unit was biotinylated by nick translation with biotin-11-dUTP and used as a probe in the dot blot thbridization and the hybrid formation was deterced by an avidin-alkalinephosphatase method, ad little as 100ng DNA was proven to be sufficient to give clear demale-specific color development. Similar method was also shown to be applicable to in sity hybridization with the nuclei from the 72h chiecken embryos.
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