1992 Fiscal Year Final Research Report Summary
Expression cloning of the gene for complementing xeroderma pigmentosum group D.
| Project/Area Number |
03454552
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
放射線5生物学
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| Research Institution | Kobe University |
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Principal Investigator |
FUJIWARA Yoshisada Kobe University School of Medicine, Department of Radiation Biophysics and Genetics, Professor., 医学部, 教授 (70030848)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUMOTO Akira Kobe University School of Medicine, Department of Radiation Biophysics and Genet, 医学部, 講師 (80181759)
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| Project Period (FY) |
1991 – 1992
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| Keywords | xeroderma pigmentosum / DNA excision repair / repair defect / XPD-complementing cDNA transformation / Expression cloning / cDNA |
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| Research Abstract |
Xeroderma pigmentosum (XP) group D (XPD) exhibits a severe defect in excision repair of UV-induced pyrimidine dimers and (6-4) photoproducts in the DNA and a high frequency of sunlight-induced skin cancers. We have so far assigned 13 definite and 6 possible XPD patients in Japan (-10% of all assigned XPs) by the cell fusion-complementation test. We established an SV40 origin-defective T antigen DNA-immortalized cell line, XP59TOpSV, from fibroblasts of Japanese XPD patient XP59TO. XP59TOpSV cells were transfected by large scale repetitions with the three types of normal human fibroblast cDNA libraries constructed in pcD, pcD2 (with the neo gene), and lambdapCEV mammalian expression vector (with unidirectionally inserted cDNA and the neo gene). After the 3 X UV selections for pcD-cDNA transfected cells and the G418 - 2 X UV selections for pcD2-cDNA introduction, the acquired 10 and 21 clones respectively revealed a very slight UV resistance without obvious recovery in the DNA repair, suggesting no full-length complementing cDNA. Then we introduced the unidirectional normal-cDNA constructed in lambdapCEV expression vector into 5 x 10^7 XP59TOpSV cells, and obtained the two final, significantly UV-resistant clones revealing the 3-fold recovery compared with the parental cell line. The genomic DNA of one more resistant clone contained the insertion of -2.5 kb cDNA, which showed the same Not I site in the middle and the same PCR product as in ERCC2, indicating that the present cDNA obtained by the expression cloning method complements the repair defect in XPD. RT-PCR of mRNA from three XPD strains indicated an abnormally low, unapmlifiable level of XPD mRNA in a case. Now we are recloning from the libraries for base sequencing.
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Research Products
(14 results)
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[Publications] Nakamura,T., Ono,T., Yoshimura,K., Arao,T., Ichihashi,M., Matsumoto,A. and Fujiwara,Y.: "Malignant Schwannoma associated with xeroderma pigmentosum in patient belonging to complementation group D." Journal of American Academy of Dermatol.25. 349-353 (1991)
Description
「研究成果報告書概要(欧文)」より
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