Selective visualization of gene structure with ultraviolet light.
The ability of the ultraviolet (UV) "footprinting" technique to detect chromatin has been investigated in vitro. Two basic types of chromatin, a phased nucleosome and a phased nucleosome containing a phased H1 protein, have been reconstituted onto a cloned 5S ribosomal RNA gene from sea urchin. The histone-DNA interactions in each complex have been probed with exonuclease III, DNase I, dimethyl sulfate, and UV light. Whereas DNase I and exonuclease III readily detect interactions between histones and DNA, UV light and dimethyl sulfate do not. In contrast to histone-DNA interactions, we demonstrate that intimate sequence-specific contacts between the same sea urchin 5S DNA and the Xenopus laevis transcription factor IIIA (TFIIIA) are readily detected with UV light. Since the sensitivity of UV light for TFIIIA contacts is similar to its sensitivity for other regulatory protein-DNA contacts, these studies demonstrate the feasibility of using UV light to selectively visualize regulatory protein-DNA interactions in vivo with little or no interference from histone-DNA interactions.
Wang
Z
Z
Department of Biological Sciences, University of Pittsburgh, PA 15260.
Becker
M M
MM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002843:Chromatin / Q000528:radiation effects / Q000648:ultrastructure*; D004249:DNA Damage; D004275:DNA, Ribosomal / Q000378:metabolism / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005796:Genes; D006657:Histones / Q000378:metabolism; D008969:Molecular Sequence Data; D009710:Nucleotide Mapping / Q000379:methods*; D011485:Protein Binding; D012341:RNA, Ribosomal, 5S / Q000235:genetics; D012617:Sea Urchins; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000378:metabolism; D014466:Ultraviolet Rays
D002843:Chromatin; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D006657:Histones; D012341:RNA, Ribosomal, 5S; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors