Analysis Of The Dnase I-Hypersensitive Chromatin Structure 5' To A Human Hsp70 Gene

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Biochemistry and Molecular Biology


A DNase I-hypersensitive site occurred in minichromosomes containing a human hsp70 gene segment in COS 7 cells. This site was present in the absence of heat treatment and did not change in intensity or position upon heat shock. We have mapped this site to sequences in the 5' nontranscribed region (between -50 and -260, where +1 represents the site of hsp70 transcription initiation). By contrast, a barely detectable site was seen over Drosophila hsp70 5' flanking sequences in similar experiments. By analyzing deletion mutants, we have determined that a 280 base pair (bp) sequence coincident with the hypersensitive site is entirely responsible for this altered chromatin structure. Information about protein binding was obtained using exonuclease III, gel mobility retardation and DNase I footprinting. Barriers to exonuclease III digestion were mapped at two distinct locations: one located within the region that is hypersensitive and the other near the start of transcription. Both of these stop sites map near imperfect inverted repeat elements and may reflect proteins specifically bound at these locations. In addition, there are several potential binding sites for transcription factor Sp1 within the hypersensitive region and deletion mutants lacking some of these sites show reduced hypersensitivity. We believe that the hypersensitive site is caused by specific interaction of proteins with more than one site in this region since no single polypeptide chain would be expected to span a 280 bp segment of DNA.


Biology, Molecular; Chemistry, Biochemistry

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