Study of activity of promoter from mouse alpha 2(I) procollagen gene

Objective To clarify the segment and sequence in mouse alpha 2(I) procollag en gene which are responsible for high transcriptional activity during fibr ogenesis. Methods This study was focused on further fractional analysis of 2 kb-lengt h mouse alpha 2(I) procollagen gene promoter activity. Six chimeric genes w ere constructed in which various lengths of sequences between 2000 bp upstr eam of the start of transcription of the mouse alpha 2 (I) procollagen gene and 54 bp downstream of this site were fused to chloramphenicol acetyltran sferase (CAT) reporter gene. These recombinant plasmids were transfected tr ansiently 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 tr ansfected cells. Results The highest and partial cell specific CAT expression was observed i n construction driven by -780 to +54 bp fragment. The construction containi ng sequence deleted the proximal 500 bp from the transcription start site a nd part of exon I, and resulted in the lowest CAT expression. Conclusions Some essential elements might exist in the 500 bp fraction prox imal to transcription start site and part of exon I in mouse alpha 2(I) pro collagen gene. The high potential promoter sequence between -780 bp from th e start of transcription site and +54 bp from this site is of great signifi cance in our following study of searching for specific DNA-binding proteins in activated collagen-producing cells.