IDENTIFICATION OF 2 REGULATORY ELEMENTS WITHIN THE PROMOTER REGION OFTHE MOUSE CONNEXIN-43 GENE

To define the minimal sequences required for expression of the connexi n 43 gene (cx43) in myometrial cells, we generated 5' deletion constru cts of a fragment extending 1686 base pairs upstream and 162 base pair s downstream of the transcription start site and determined their abil ity to drive expression of the chloramphenicol acetyltransferase repor ter gene in transfected myometrial cell lines, Our investigation revea led two cis-acting regulatory elements within this fragment, Deletion of a region extending from -102 to -92 led to an increase of the promo ter activity by greater than 10-fold, indicating a presence of a repre ssor element, Deletion of a region extending from -72 to -62 caused a decrease of the promoter activity of a similar extent, implying the ex istence of a positive element, Electrophoretic mobility shift assays d emonstrated that synthetic oligonucleotides derived from these two sma ll regions can each bind with a nuclear protein(s) prepared from myome trial cells, and an introduction of three and two base substitutions i nto each of these oligomers was sufficient to abolish their protein bi nding capability, These same mutations, when incorporated in the chlor amphenicol acetyltransferase constructs, diminished regulatory functio ns of the negative and positive elements, and the protein(s) that bind to these functional elements was found in several tissues known to ex press cx43 gene,