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. 1991 Oct 1;88(19):8515–8519. doi: 10.1073/pnas.88.19.8515

Precise localization of the alpha-globin gene cluster within one of the 20- to 300-kilobase DNA fragments released by cleavage of chicken chromosomal DNA at topoisomerase II sites in vivo: evidence that the fragments are DNA loops or domains.

S V Razin 1, P Petrov 1, R Hancock 1
PMCID: PMC52539  PMID: 1656447

Abstract

We have mapped the position of the alpha-globin gene cluster in the 20- to 300-kilobase fragments of chromosomal DNA isolated from growing chicken HD3 erythroblastoid cells exposed to 4'-demethylepipodophyllotoxinthenylidene beta-D-glucoside. This epipodophyllotoxin traps functioning topoisomerase II molecules, the denaturation of which cleaves DNA and reveals their reaction sites. The DNA fragments, prepared by centrifugation in sucrose gradients, bind selectively to glass-fiber filters and are protected from lambda 5'-exonuclease, properties compatible with the presence of a topoisomerase II subunit bound to their 5' ends. Restriction enzyme cleavage of the fragments and hybridization with cloned alpha-globin-region probes reveal additional distinctive bands not seen in control DNA, allowing the localization of fragment ends near this gene cluster. The terminal regions of fragments from sucrose gradients or from field-inversion electrophoresis gels were also used to probe cloned regions of the gene cluster. Both approaches show that this cluster of three genes, which is not expressed in these cells, is located at a specific position in a approximately 20-kilobase DNA fragment. The upstream end of this fragment lies in a region that contains a site of DNA attachment to the nuclear matrix mapped by both in vivo and in vitro methods, and its downstream end is flanked by approximately 80% A + T sequences characteristic of matrix-attachment regions. These observations suggest that the DNA fragments are formed because topoisomerase II molecules can specifically and readily integrate into DNA at matrix-attachment regions and that the fragments represent entire DNA loops or domains.

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