1996 Fiscal Year Final Research Report Summary
Analysis of gene expressing during sex differentiation.
| Project/Area Number |
06454076
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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 | KYOTO UNIVERSITY |
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Principal Investigator |
OHYAMA Kanji Kyoto Univ.Fac.Agric.Prof., 農学部, 教授 (40135546)
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| Co-Investigator(Kenkyū-buntansha) |
FUKUZAWA Hideya Kyoto Univ.Fac.Agric.Assoc.Prof., 農学部, 助教授 (30183924)
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| Project Period (FY) |
1994 – 1996
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| Keywords | liverwort / PAC library / FISH / sex chromosome / CDPK / alternative splicing / subtraction / finger printing |
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| Research Abstract |
We obtained 23 clones with 50-750 kb insert DNAs using genomic DNA from suspension cultured cells of a female liverwort in an attempt to construct a yeast artificial chromosome (YAC) library. Two of the clones had approximately 50 kb and 500 kb insert DNAs, which were shown to hybridize to chromosomes of a female liverwort by fluorescent in situ hybridizatioin (FISH). Because of low cloning efficiency and complicated manipulation of YAC clones, P1-derived artificial chromosome (PAC) has been adopted as a cloning vector. We have cloned about 5000 PAC clones with 50-250 kb insert DNAs from male and female thalli. The signals were shown by FISH using one of them with 50 kb insert DNA as a probe. Thus, it is possible to isolate the clones containing the sex chromosomal DNAs by this method.
Taking advantage of differences of genomic DNAs between male and female thalli, random amplified polymorphic DNAs (RAPD) were cloned and sequenced, one of which was a homologue of a calcium-dependent protein kinase (CDPK). We cloned the CDPK gene from suspension cultured cells of a female liverwort and showed that the 6th and 7th exons (Exon 6A and Exon 6B) of the CDPK gene were almost identical. RT-PCR (PCR in conjunction with reverse transcription) analysis revealed that two species of mature mRNA were actually generated from a single CDPK gene by alternative splicing mutually exclusively Exon 6A and Exon 6B in male and female suspension cultured cells, thalli and sexual organs.
We have isolated 3 novel cDNA fragments by cDNA subtraction between female sexual organs and female thalli and many cDNAs by RNA finger printing.
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