Multiple sequence-specific DNA binding activities are eluted from chicken nuclei at low ionic strengths.

DNA sequence-specific binding proteins eluted from chicken erythrocyte and thymus nuclei, and fractionated as described by Emerson and Felsenfeld (19), have been investigated by filter binding and footprint analyses. The erythrocyte nuclear protein fraction specifically binds to at least two sites within the 5' flanking chromatin hypersensitive site of the chicken beta A-globin gene, and to a site 5' to the human beta-globin gene. The major chicken beta A globin gene binding site [G)18CGGGTGG) and the human beta-globin gene binding site [TA)6(T)8C(T)4) occur at or near sequences which are hypersensitive to S1 nuclease cleavage in supercoiled plasmids. Downstream, the second chicken beta A-globin gene binding site includes the beta-globin gene CACCC consensus sequence. Filter binding studies also show other sequence specific binding activities to human N-ras and human (but not chicken) c-myc gene sequences.     

Nuclease hypersensitivity in the beta-globin gene region of K562 cells

We have investigated chromatin structure in the beta-globin gene region of the K562 human erythroleukemic cell line by using S1 and DNase I nuclease sensitivity assays. Despite the lack of beta-globin gene expression in these cells, we find nuclease-hypersensitive sites to these enzymes in its 5' and 3' flanking regions in K562 chromatin. This result is in contrast to previous reports in which no hypersensitive sites were found in the immediate vicinity of this gene. In the 3' region, one major hypersensitive site at 0.9 kpb 3' and three minor hypersensitive sites at 0.7 kbp, 0.5 kbp 3' and 0.2 kbp 5' of the polyadenylation site were observed; these sites are very similar to those found in fetal liver and adult bone marrow cells in which the beta-globin gene is expressed. We find hypersensitive sites to both enzymes in the 5' region of the beta-globin gene: a major site 0.8 kbp 5' to the cap site, and two minor sites 1.2 and 1.5 kbp 5' to the cap site. The -0.8 kbp site is also present in plasmids containing the beta-globin gene. Our results suggest that the lack of beta-globin gene expression may be related to the lack of hypersensitivity sites in the immediate (150 bp) 5' flanking region of the beta-globin gene, as occurs in other active globin genes.     

Possible structures of homopurine-homopyrimidine S1-hypersensitive sites.

S1 nuclease hypersensitive sites in the 5' flanking regions of eukaryotic genes, present in small artificial supercoiled DNA circles, reside in homopurine-homopyrimidine stretches. The hierarchical behavior which these sites exhibit is consistent with the notion that they act as sinks of torsional free energy. By employing DMS as a single-strand-specific reagent, we show that these sites (despite their S1 sensitivity) are regions of duplex DNA. A simple thermodynamic treatment indicates that the high torsional stress in the small DNA circles is almost certain to be relieved by the formation of alternate DNA structures. The same treatment places some constraints on the types and sizes of the regions with alternate conformation. While no definitive structural conclusions can be drawn, left-handed helices seem most consistent with the extent and the pattern of sensitivity to S1 nuclease.     

Structure and characterization of a murine chromosomal fragment containing the interferon beta gene

In this paper we report on the cloning and characterization of the murine interferon (IFN) beta gene. We have isolated and sequenced a 2.8 kb genomic fragment containing the murine IFN beta gene flanked by 1.2 kb 5' and 1 kb 3' untranslated regions (1 kb = 10(3) base-pairs). The mRNA cap site has been defined. An extensive analysis of the flanking sequence is provided and points out striking features such as: the presence of A + T-rich motifs characteristic of transiently expressed mRNAs, and homologies to repetitive R-type element flanks and to hormone-responsive elements. Comparison of the MuIFN beta 5' flanking region with those from other species reveals similarities in the sequences required for the regulated expression of such inducible genes. Computer analysis of the 130 base-pairs preceding the cap site has revealed TGAAAG motifs and shows that the presence of such elements and their permutants have biological significance, according to statistical calculations. Thus, the comparison between the mouse promoter reported here and the promoters from other species highlights the region containing the hexanucleotide blocks, which is strongly conserved.     

A cruciform in the direct repeats of the yeast 2 micron DNA: Selective S1 nuclease cleavage at one of the three homologous palindromes

An S1-hypersensitive site was found at the 60 bp direct repeats of the cis-acting, stability and/or copy number control region of the yeast 2 micron DNA in the supercoiled hybrid plasmid pDB248'. It was retained in a different plasmid, pYK2121, consisting of pBR322 and the 300 bp long repeated DNA. Analyses of 5'-end-labeled fragments and nucleotide sequence determination showed that the S1-cleavage site was at the central part of an AT-rich 19 bp palindrome present in the repeats. Two other homologous palindromes (21 and 15 bp) containing the 12 bp consensus sequences were not cleaved. The nucleotide sequences at the base of the stem and/or loop may determine the efficiency of the cruciform extrusion.