Characterization of the cis and trans elements essential for rat insulin II gene expression.
We have constructed 14 different linker-scanner (LS) mutants throughout the enhancer and promoter of a rat insulin II-CAT fusion gene. These LS mutants were transiently transfected into an insulin-producing (HIT) cell line and three mutants (LS-261/252, LS-102/91, LS-54/45) displayed drastically reduced levels of CAT activity. Therefore, at least three regions are essential for the in vivo expression of an insulin-CAT fusion gene. To identify the trans-acting factors which interact with this cell-specific promoter, we performed band-shifting assays with either HIT or HeLa nuclear extracts and an end-labelled insulin fragment (-100 to +49). Three binding activities were common to both extracts, and another one was unique to HIT cells. DNase I protection studies localized one binding activity to -60 to -40 bp 5' to the cap site. We show that this factor is common to both cell lines and is identical to a previously characterized transcription factor (COUP) which binds to the chicken ovalbumin upstream promoter.
Crowe
D T
DT
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.
Hwung
Y P
YP
Tsai
S Y
SY
Tsai
M J
MJ
D000818:Animals; D001483:Base Sequence; D005786:Gene Expression Regulation; D005796:Genes; D007328:Insulin / Q000235:genetics*; D009154:Mutation; D011487:Protein Conformation; D051381:Rats
D007328:Insulin