| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1999: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
To understand the mechanisms of smooth muscle (SM) cell differentiation, we are analyzing the transcriptional regulation system of human smooth muscle a-actin (SmaA) gene. We made several transgenic mice having the upstream region from the transcription initiation site and the first intron region following a CAT gene. The transgenic mice having the both regions (EA4.7-CAT and 123int-CAT) showed very strong CAT expressions in aorta, heart and intestine, but weak in liver, skeletal muscle. Using histochemical stains by CAT-activity and CAT-antibody, CAT is detected only in tunica media in artery and SM layer in gastrointestinal tract, where intrinsic SmaA are expressed. Whereas, the transgenic mice deleting the 137-bp region in intron 1 (D#0-CAT) which is highly conserved among several species (human, mouse and chick) showed weak CAT expression in all tissues we examined. Therefore, it is showed that the 137-bp sequence is necessary for SM tissue-specific expression in adult mice. In the 137-bp region, one CArG box element (#0) and A/TBF binding element whose motif is ATTA sequence locate overlaply and nuclear factors, SRF and A/TBF, can bind to each element independently. To test whether these two elements are important for SM tissue-specific in vivo expression, point mutants (-1M and 4M) are introduced into each element and measured promoter activities in mouse embryos. At 11.5 dpc embryos, the wild-type SmaA promoter (hSmaA-lacZ) worked in aorta, heart and diencephalon whereas the mutated promoters in both elements worked in heart and diencephalon but not in aorta. From these results, CArG box and A/TBF binding elements in the intron 1 of the SmaA gene are very important for SM tissue-specific in vivo expression.
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