DNase I and nuclease S1 sensitivity of the rabbit β1 globin gene in nuclei and in supercoiled plasmids

Jean B. Margot, Ross C. Hardison

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Abstract

We have examined the nuclease sensitivity of the 5′ flanking region of the rabbit β1 globin gene in bone marrow nuclei and in supercoiled plasmids. A DNase I hypersensitive site was found about 100 base-pairs 5′ to the cap site in bone marrow nuclei. S1 nuclease can introduce a specific double-strand cut in the DNA in the same region. The presence of the nuclease-hypersensitive region correlates with the active transcription of gene β1 in bone marrow. Treatment with nuclease S1 of a supercoiled plasmid containing 1400 base-pairs of 5′ flanking sequences as well as part of the β1 gene reveals a major double-strand cut 400 base-pairs 5′ to the cap site. This cut maps within a stretch of repeating dinucleotides (C-T)12 and does not correspond to the in vivo site. Introduction of an RsaI fragment containing the nuclease S1-hypersensitive site into plasmid pBR322 shows that this fragment alone is sufficient to generate the hypersensitive site. Deletion of that RsaI fragment from the β1 plasmid reveals another site 1300 base-pairs upstream. Further deletion of this secondary site uncovers numerous other sites, none of which corresponds to the site in nuclei. Chromatin reconstitution with plasmids carrying the 5′ flanking region of β1 and histones is capable of suppressing the in vitro nuclease-S1-hypersensitive site at -400 but is incapable of generating the in vivo site at -100. Fine analysis at the nucleotide level of the early events in the digestion with nuclease S1 shows that the enzyme attacks preferentially the sequence (G-A)12 on the message complementary strand. The region of DNA containing the supercoil-dependent S1 site adopts at least three different conformations that can be resolved electrophoretically. These different conformations are detected in linear restriction fragments and may represent non-B DNA or unusual B-form DNA.

Original languageEnglish (US)
Pages (from-to)195-210
Number of pages16
JournalJournal of Molecular Biology
Volume184
Issue number2
DOIs
StatePublished - Jul 20 1985

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All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

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