C Gao1, H Wang, C Huang, X Kong. 1. Clinical Immunology Center, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
Abstract
OBJECTIVE: To clarify the segment and sequence in mouse alpha 2(I) procollagen gene which are responsible for high transcriptional activity during fibrogenesis. METHODS: This study was focused on further fractional analysis of 2 kb-length mouse alpha 2(I) procollagen gene promoter activity. Six chimeric genes were constructed in which various lengths of sequences between 2000 bp upstream of the start of transcription of the mouse alpha 2 (I) procollagen gene and 54 bp downstream of this site were fused to chloramphenicol acetyltransferase (CAT) reporter gene. These recombinant plasmids were transfected transiently to collagen-producing cells (NIH/3T3) and non-collagen-producing cells (COS7) with liposomal transfection method. The activities of putative promoters were observed and compared by means of CAT measurement in the transfected cells. RESULTS: The highest and partial cell specific CAT expression was observed in construction driven by -780 to bp fragment. The construction containing sequence deleted the proximal 500 bp from the transcription start site and part of exon I, and resulted in the lowest CAT expression. CONCLUSIONS: Some essential elements might exist in the 500 bp fraction proximal to transcription start site and part of exon I in mouse alpha 2(I) procollagen gene. The high potential promoter sequence between -780 bp from the start of transcription site and bp from this site is of great significance in our following study of searching for specific DNA-binding proteins in activated collagen-producing cells.
OBJECTIVE: To clarify the segment and sequence in mouse alpha 2(I) procollagen gene which are responsible for high transcriptional activity during fibrogenesis. METHODS: This study was focused on further fractional analysis of 2 kb-length mouse alpha 2(I) procollagen gene promoter activity. Six chimeric genes were constructed in which various lengths of sequences between 2000 bp upstream of the start of transcription of the mouse alpha 2 (I) procollagen gene and 54 bp downstream of this site were fused to chloramphenicol acetyltransferase (CAT) reporter gene. These recombinant plasmids were transfected transiently to collagen-producing cells (NIH/3T3) and non-collagen-producing cells (COS7) with liposomal transfection method. The activities of putative promoters were observed and compared by means of CAT measurement in the transfected cells. RESULTS: The highest and partial cell specific CAT expression was observed in construction driven by -780 to bp fragment. The construction containing sequence deleted the proximal 500 bp from the transcription start site and part of exon I, and resulted in the lowest CAT expression. CONCLUSIONS: Some essential elements might exist in the 500 bp fraction proximal to transcription start site and part of exon I in mouse alpha 2(I) procollagen gene. The high potential promoter sequence between -780 bp from the start of transcription site and bp from this site is of great significance in our following study of searching for specific DNA-binding proteins in activated collagen-producing cells.