|Budget Amount *help
¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1991 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1990 : ¥1,500,000 (Direct Cost : ¥1,500,000)
(1) Isolation and sequence analysis of the human cAspAT gene : The human cAspAT gene is more than 30 kb long and contains 9 exons. When we compared the 5'-flanking sequences of the human and mouse cAspAT genes, we noted a high homology. Moreover, the sequences of binding sites for the transcription factor, CTF/NFI, previously identified in the mouse, are conserved between the human and mouse genes. The human cAspAT consists of the 413 amino acid residues, which showed high sequence homology with those of mouse, pig and chicken, in a range of 80% to 90%, and the amino acid residues essential for enzyme function are completely conserved among them.
(2) Regulation of the expression of the isozyme genes during cell differentiation : Upon appropriate stimulation, mouse preadipocyte 3T3-L1 cells undergo conversion into adipocytes. During this differentiation, the enzyme activities and mRNA levels for cMDH, mMDH and mAspAT but not cAspAT increased significantly.
(3) Effects of hormones on the expression of the isozyme genes : Using the rat hepatoma cell line, H4EII, we studied effects of dexamethasone (Dex) and CAMP on the expression of the isozyme genes, and found that cAspAT activity and mRNA are clearly increased after treatment of Dex and CAMP, but those of other isozymes (mAspAT, CMDII and mMDH) were not modified. Two sequences snowing homology to glueocorticoid-responsive element (GRE) have been found in the 5'-flanking region of the mouse cAspAT gene, one at the -581/-567 bp region (GRE-1) and the other at the -459/-441 bp region (GRE-2). Deletion analysis of the 5'-flanking region revealed that the GRE-2 sequence could be participate in the regulation of cAspAT by Dex.
(4) Site-directed inactivation of the mouse cAspAT gene by gene targeting : We constructed two classes of vectors, one for disruption of the gene by replacing endogenous dequences with exogenous sequences and another for partial inactivation by inserting a point mutation into the endogenous gene.