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

Abstract

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.

Intrinsic bent DNA sites in the chromosomal replication origin of Xylella fastidiosa 9a5c

“…The CAnT motif is located either in the center or near the curvature and seems to be responsible for high bent peak formation (7). For the analysis of the nucleotide sequences in the bent DNA sites described in the present study, we confirmed the DNA helix turn phasing of two or more adenine/thymine tracts, which were located around the sites, and the presence of the CAnT motifs.…”

Replication origins oriGNAI3 and oriB of the mammalian AMPD2 locus nested in a region of straight DNA flanked by intrinsically bent DNA sites

“…The 2023 bp SacI/AvaI fragment, which contains the intrinsically bent DNA b1 and b2, migrated faster through the PA gel, yielding an R-value of approximately 0.90. Such electrophoretic behavior is indicative of non-centered bent DNA sites (5,18). Indeed, the b1 and b2 sites lie in the initial third part of the fragment.…”

“…3B, PA gel, lanes 1 and 2, white arrowhead; R-values 0.84 and 0.83). The observed increase in mobility indicates the presence of bent DNA sites in one or both extremities (5,18). Taking into account the position of restriction sites in the region, site b5 was difficult to centralize in a fragment and was instead assayed by circular permutation.…”

Molecular combing in the analysis of developmentally regulated amplified segments of Bradysia hygida

“…Studies of this DNA segment have identified a region with numerous sites of bent DNA and a significant site localized to approximately 2000 bp upstream of the BhC4-1 gene transcription start site (Fiorini et al, 2001). Sites of association with the nuclear matrix have also been identified in this segment (Mikami, 2000;Polinarski, 2005).…”