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1992 Fiscal Year Final Research Report Summary

Expression cloning of the gene for complementing xeroderma pigmentosum group D.

Research Project

Project/Area Number 03454552
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 放射線5生物学
Research InstitutionKobe University

Principal Investigator

FUJIWARA Yoshisada  Kobe University School of Medicine, Department of Radiation Biophysics and Genetics, Professor., 医学部, 教授 (70030848)

Co-Investigator(Kenkyū-buntansha) MATSUMOTO Akira  Kobe University School of Medicine, Department of Radiation Biophysics and Genet, 医学部, 講師 (80181759)
Project Period (FY) 1991 – 1992
Keywordsxeroderma pigmentosum / DNA excision repair / repair defect / XPD-complementing cDNA transformation / Expression cloning / cDNA
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.

  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] Fujiwara.Y.,: "Amechanism for relief of replication block by activation of unused origins and age-dependent change in the caffeine susceptibility in xeroderma pigmentosum variant." Mutation Research. 254. 79-87 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kataoka,H.: "UV damago specfic DNA binding protein in xeroderma pigmentosum complementation group E." Biochem.Biophys.Res.Commun.175. 1139-1143 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Nakamura,T.: "Malignant Schwannoma associated with xeroderma pigmentosum in a patient belonging to complementation group D." Journal of American Academy of Dermatology. 25. 349-35.3 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Jimbo,O.,: "Role of excision repair in UVB-induced depletion and recovery of human epidermal Langerhans cells" Archieves pf Dermaotology. 128. 61-67 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Fujiwara.T.,: "A new anticancer platinum compornd,DWA2114R:DNA interstrand cross-linking,repair and lethal effects in normal human,Fanconi's anenia and xeroderma pigmentosum cells." British Journal of Cancer.

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Yamanoto,Y.,: "Roles of uracil-DNA glycosylase and apyrimidinic endonucleases in the molecular 5-bromo-2^1-deoxyuribine photosensitization in Escherichia coli K-12." Photochemistry and Photobiology.

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Fujiwara,Y., Ichihahsi,M., Matsumoto.A., and Kataoka,H.: "A mechanism for relief of replication block by activation of unused origins and age-dependent change in the caffeine susceptibility in xeroderma pigmentosum variant." Mutation Research. 254. 79-87 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kataoka,H. and Fujiwara,Y.: "UV damage specific DNA binding protein in xeroderma pigmentosum complementation group E." Biochem. Biophys. Res. Commun.175. 1139-1143 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [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
  • [Publications] Jimbo,P., Ichihashi,M., Mishima,Y. and Fujiwara,Y.: "Role of excision repair in UVB-induced depletion and recovery of human epidermal Langerhans cells." Archives of Dermal.128. 61-67 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Fujiwara,Y., Nakamura,M. and Yokoo,S.: "A new anticancer platinum compound, DWA2114R: DNA interstrand cross-linking, repair and lethal effects in normal human, Fanconi's anemia and xeroderma pigmento-sum cells." British J.Cancer.

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yamamoto,Y. and Fujiwara,Y.: "Roles of uracil-DNA glycosylase and apyrimidinic endonucleases in the molecular 5-bromo-2'-deoxyuridine photosensitization in Escherichia coli K-12." Photochem.Photobiol.

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Nakura,J., Miki,T., Kihara,K., Fujiwara,Y., et al: "Close linkage of the gene for Werner's syndrome to ANK1 and D8S87 in short arm of chromosome 8." Gerontology.

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Nakamura,M., Yamamoto,M., and Fujiwara,Y.: "Leber hereditary optic neuropathy in the Japanese pedigrees: Genetic control by the holoplasmic ND4 mutation and an abnormal X-linked recessive gene." Human Genetics.

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1994-03-24  

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