YAMAUCHI Masatake NATIONAL INSTITUTE OF RADIOLOGICAL SCIENCES,THE 2ND RESEARCH GROUP,SENIOR RESEAR, 第二研究グループ, 主任研究官 (00260240)
TSUJI Hideo NATIONAL INSTITUTE OF RADIOLOGICAL SCIENCES,THE 5TH RESEARCH GROUP,SUBGROVP READ, 第五研究グループ・サブグループ, リーダ (40163795)
|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1996 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1995 : ¥1,300,000 (Direct Cost : ¥1,300,000)
In order to clarify defense mechanisms against active oxygens in mammalian cells, we isolated many mutants sensitive to active oxygen generating agents, plumbagin (PG). They were classified into 4 groups according to sensitivity to PG and methylviologen (MV). Most of them were also sensitive to mitomycin C (MMC). It was concluded they were classified into 4 genetic complemen-tation groups. In order to identify responsible genes for MMC sensitivity in these mutants, the following experiments were carried out by using complementation group I (Pa13) and II (Pb4).
(1) Human chromosome #9 on which Fanconi's anaemia C group gene was isolated was introduced into Pa13 and Pb4 cells by minrocell fusion methods. MMC-sensitivity of these mutants was not complemented by chromosome #9.
(2) Pb4 and human normal diploid (TIG7) cells were fused. Two hybrid clones, resistant to MMC,containing human chroosome, 4H1 and 4H2, were selected by Alu-PCR method. 4H1 was resistant to MMC almost like wild type and 4H2 was partially resistant. Each clone have about 10 human chromosomes. Subclones of 4H1 partially lost human chromosome were isolated by colcemid treatment. 7 subclones were tested and found to contain several common chromosomal regions. Only 15q. 20q. 22q regions were fond also in 4H2 and its subclones. The gene related to active oxygen scavenging activity were identified as Cu/Zn-SOD (21q22.1) , Mn-SOD (6q21) , exo-SOD (4p16.3-q21) , catalse (11p13) , glutathione peroxidase (3q11-q12) . These results suggest that defective gene in Pb4 mutant is different from them.
(3) mRNA levels of Cu, Zu-SOD,Mu-SOD,catalase in the mutants, complementation groups 1 to 4, were almost same with that of wild-type cells.
From these results, it was suggested that new gene was related to active oxygen scavenging gene.