Mapping of intrinsic bent DNA sites in the upstream region of DNA puff BhC4-1 amplified gene.

We have identified bent DNA sites in the distal and proximal DNA puff BhC4-1 amplified gene promoter region of Bradysia hygida. The 2D modeling of the 3D DNA path and the ENDS ratio values calculated in this promoter region resulted in the identification of ten pronounced bent sites named BhC4B - 9 to + 1. The 1847 bp fragment (- 3697 to - 1850) in relation to the transcription start site shows multiple bending sites, BhC4B - 9 to BhC4B - 4, with periodicity approximately 300 bp. The analysis of the other identified bent region, starting at position - 957, reveals that the BhC4B + 1 bent site colocalizes with the putative BhC4-1 minimal promoter. The sequence analysis of bent site BhC4B - 4 shows a distribution of dA*dT at approximately 10 bp intervals between the middle of each tract, but intervals with more than one turn, approximately 20 bp, two helix turns, were detected in the other bent sites described here. The bent sites BhC4B - 6 and BhC4B - 4, contain two consensus sequences, with 60 bp each. The apparent molecular weight of fragments in the BhC4-1 promoter region were estimated in agarose gels and compared with the data obtained in polyacrylamide gels without and with ethidium bromide. The mobility reduction ratios (R-values) were determined, and a high R-value, 1.80, for a 1215 bp fragment in the distal promoter region and a 1.23 significant R-value for a 662 bp fragment in the proximal segment were found. To further analyze the predicted bent DNA sites in these fragments, the 2D trajectories of the 3D DNA path and other parameters, AT percentage, roll angle, ENDS ratio and DeltaG, were determined. The role of these bent sites in the BhC4-1 transcription regulation is discussed. Copyright 2001 Wiley-Liss, Inc.