Abstract
A total of seven DNA bend sites were mapped in the 4.4-kilobase human beta-globin gene region by the circular permutation assay. The periodicity of these sites (except one) was about every 700 (average 685.5 +/- 267.7) base pairs. All of the sites contained the sequence feature of short poly(dA) tracts, which are typical of DNA bending. The relative positions of the sites to the cap site were identical to those in the epsilon-globin gene region, suggesting that the bend sites were conserved during molecular evolution of the two globin genes. To explain this periodicity and conservation of the sites within the evolutionary unstable noncoding regions, we focused upon the appearance of a potential bend core sequence, A2N8A2N8A2 (A/A/A), and its complement, T2N8T2N8T2 (T/T/T). These sequences appeared in or very close to most of the bend sites of the globin gene regions, whereas other A+T-rich sequences or candidates for DNA bending did not. The distances between any two of the core sequences in the entire beta-globin locus showed a strong bias to a length of about 700 base pairs and its multiples, suggesting that the periodicity exists throughout the locus. The data presented here strengthen the idea of sequence-directed nucleosome phasing.
Highlights
A total of seven DNA bend sites were mapped in the 4.4-kilobase human p-globin gene region by the circular permutation assay
The distances between any two of the core sequences in the entire p-globin locus showed a strong bias to a length of about 700 base pairs and its multiples, suggesting that the periodicity exists throughout the locus
The positions of the bend sites were detected by polyacrylamide gel electrophoresis at 4 °C as the fastest migrating band among the fragments created by various restriction enzymes, and the bend centers were likely to be located between the second and third nearest sites and close to the nearest site
Summary
Materials-Restriction enzymes were purchased from Takara (Kyoto) or New England Biolabs. Plasmid Construct-Plasmids were constructed by subcloninga fragment from the plasmid containing the region between the positions -2282 (Pst!) and 2161 (Pst!) in pUC8 vector. Tandem duplicates of each fragment were inserted into the multiple cloning site of pBluescript SK(-). About 1 ""g of plasmid DNAs that contained duplicates of the regions of interest were linearized with the restriction enzymes shown (see Fig. 1). Electrophoresis at 55 DC (3 V/cmfor 7 h) was performed with at least one clone for each bend site. A Defined as the region between the second and third nearest sites in the assays by the circular permutation method. Computer Analysis-DNA sequences were analyzed by the program supplied by Software Development Co. using the sequence of the human {3-globin gene locus (73,326 bp, entry name HSHBB) or other sequences from the GenBank data bases
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