Molecular analysis of group A xeroderma pigmentosum

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Molecular analysis of group A xeroderma pigmentosum Kunimitsu

Iwai and Kiyoji Tanaka

Institute for Molecular and Cellular Biology, Osaka University, Suita, Osaka MS, Japan

We cloned a human DNA excision repair gene which complements the defect of group A XP(XPAC gene). Human XPAC cDNA encodes a hydrophilic protein of relative molecular mass 31K. The protein displays a distinct zinc-finger motif, indicating that it interacts directly with DNA. Human XPAC cDNA was expressed in E. coli to obtain a recombinant xpac protein. Microinjection of a recombinant xpac protein restored UV-induced unscheduled DNA synthesis in group A XP cells. It was also injected into rabbit to elicit a polyclonal anti xpac protein antibody. Immunoprecipitation of crude cell extract with this antibody detected about 40 K xpac protein on SDS-PAGE in normal human cells, but

not in group A XP cells. The molecular basis of group A XP was investigated. We found three different kinds of mutations of XPAC gene in Japanese group A XP patients. One was a G to C transversion at the 3’ splice acceptor site of intron 3, which altered to obligatory AG acceptor dinucleotides to AC, creating two abnormally spliced mRNA forms. Second one was C to T transition in exon 6 altering Arg codon(CGA) to a nonsense codon(TGA). The third one was a T to A transversion in exon 3, altering Tyr codon(TAT) to a nonsense codon(TAA). Frequency of alleles with these mutations in Japanese group A XP patients was 85x, 13% and 4%, respectively.

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Alterations of ras oncogenes in UV induced skin tumors Chikako Department

of Dermatology’

Nishigori ‘, Hiraku Takebe 2 and Sadao Imamura and Department of Experimental

Alterations of DNA sequences in oncogenes in UV-induced skin tumors in hairless mice were analyzed qualitatively and quantitatively. Skin tumors were produced by irradiation of the back of the mice with UVB light (Toshiba FS-20SE, emission peak, 305 nm, 3430 J/m*/exposure), 2-3 times a week. Cells originating from the tumors were grown in culture, and DNA was extracted from the cells and transfected to golden hamster embryo cells by the calcium phosphate

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Radiology’, Faculty of Medicine. Kyoto University, Kyoto

coprecipitation. Transformants were identified by the focus formation. Presence of the mouse repetitive sequence and ras oncogenes in DNA of the transformants were detected by the Southern blotting using ~7014, v-H, K- and N-ras as probes. Among 17 transformant clones with high transforming ability, 15 had the mouse repetitive sequence. Presence of the mouse H-, K- and N-ras oncogenes was detected in 9 transformants and three base transversions (one G +T in