| Project/Area Number |
07456042
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Tohoku University |
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Principal Investigator |
MIZUNO Shigeki Tohoku University, Department of Applied Biological Chemistry, Professor, 農学部, 教授 (90112903)
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| Co-Investigator(Kenkyū-buntansha) |
HARATA Masahiko Tohoku University, Department of Applied Biological Chemistry, Assistant, 農学部, 助手 (70218642)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 1996: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1995: ¥4,200,000 (Direct Cost: ¥4,200,000)
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| Keywords | chicken / Oriental white stork / W chromosome / Z chromosome / heterochromatin / sexing by DNA probe / sex chromosome-specific genomic library / immunoglobulin superfamily / ゲノムライブラリー / PCR法 / 性染色体 / マクロサテライト配列 / 性判別DNAプローブ |
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| Research Abstract |
1. A genomic library was constructed from the heterochromatic end of the chicken Z chromosome by dissection with laser microbeam and amplification of DNA by random PCR.Characterization of clones by DNA sequencing and fluorescence in situ hybridization (FISH) demonstrated that the DNA of the terminal heterochromatin consisted of a macrosatellite family in which about 24kb Nhel fragment was repeated about 830 times. This macrosatellite sequence is specific to the genus Gallus, suggesting its relatively recent amplification on the evolutionary time scale.
2. A genomic library from a single chicken W chromosome was constructed using the similar method as above. From this library, clones derived from a 25kb-long non-repetitive region were obtained. A 0.6kb EcoRl fragment (EE0.6) in this region was proved to be widely conserved in the W chromosome of birds and was shown to be a useful probe for sexing birds by Southern blot hybridization. The chicken EE0.6 contains a putative exon but all three translational reading frames contain stop codons and thus the sequence seems to have lost its gene function during evolution.
3. The EE0.6-related sequences on the W and Z chromosome of Oriental white stork were cloned and their nucleotide sequences were determined. Although the two sequences are highly similar in this species, only the EE0.6-related sequence on the W chromosome could be amplified by PCR using properly selected primer sequences.
4. A ZOV3 gene encoding an immunoglobulin superfamily protein was located at the middle of the short arm of the chicken Z chromosome. The ZOV3 cDNA was expressed into a polypeptide in Escherichia coli and a polyclonal antibody to it was raised in mice. lmmunofluorescence study demonstrated that ZOV3 is present as a membrane glycoprotein predominantly in ovarian cells producing sex steroid hormones.
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