ABSTRACT. Molecular combing technology is an important new tool for the functional and physical mapping of genome segments. It is designed to identify amplifications, microdeletions, and rearrangements in a DNA sequence and to study the process of DNA replication. This technique has recently been used to identify and analyze the dynamics of replication in amplified domains. In Bradysia hygida, multiple amplification initiation sites are predicted to exist upstream of the BhC4-1 gene. However, it has been impossible to identify them using the available standard techniques. The aim of this study was to optimize molecular combing technology to obtain DNA fibers from the polytene nuclei of the salivary glands of B. hygida to study the dynamics of DNA replication in this organism. Our results suggest that combing this DNA without prior purification of the polytene nuclei is possible. The density, integrity, and linearity of the DNA fibers were analyzed, fibers 50 to 300 kb in length were detected, and a 9-kb fragment within the amplified region was visualized using 2061 ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 11 (3): 2060-2070(2012 Molecular combing analysis in Bradysia hygida biotin detected by Alexa Fluor 488-conjugated streptavidin technique. The feasibility of physically mapping these fibers demonstrated in this study suggests that molecular combing may be used to identify the replication origin of the BhC4-1 amplicon.

ABSTRACT. HMGB-like proteins are architectural chromatin factors, and their function is heavily dependent on their ability to interact with DNA (especially non-canonical DNA structures). HMGB1 is involved in many DNA processes, and dysregulation of HMGB protein expression has profound effects on cellular transcription, resulting in severe developmental defects as well as cancer. During DNA replication, elements that form the origin are still not well defined in metazoans. Sites with A (adenine) or T (thymine) repeats cause intrinsic curvatures in the DNA and are described to be involved in the replication machinery by providing binding sites to replication proteins. As a result, the DNA molecule shows intrinsically bent DNA sites, caused by periodic repeats of 2 or more As/Ts (dA/dT) as well as intrinsically non-bent DNA sites (INBDs), due to a succession of curvatures that cancel each other. In the present study, we mapped 11 INBDSs present in the AMPD2 gene that are related to each replication origin (oriGNAI3, oriC, oriB, and oriA). Following characterization of INBDSs, we tested the ability of HMGB1 to bind to the bent (b1, b2, b4a, b4b, b5, b6, b7, and b8) and non-bent DNA fragments (nb7, nb11, nb1, nb2, nb4, and nb5) via electrophoretic mobility shift assays. All fragments showed efficient binding to HMGB1. However, the non-bent DNA fragments nb2, nb4, and nb5 showed slightly reduced binding efficiency.