Analysis of high-molecular-weight free nuclear DNAs revealed in mammalian cells by pulsed-field gel electrophoresis

Pulsed-field electrophoresis revealed minor free DNA (fDNA) fractionsa andb in mammalian cells. The fractions differed in some properties from chromosomal DNA (chrDNA). Electron microscopy revealed rosette-like structures resistant to drastic deproteinization in fDNAs from Namalwa cells. Selective l...

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Bibliographic Details
Published in:Molecular biology (New York) 2000-05, Vol.34 (3), p.313-320
Main Authors: Budilov, A V, Domninskii, D A, Popenko, V I, Sukhova, T I, Botezatu, I V, Shelepov, V P, Serdyuk, O I, Alekhina, R P, Zakhariev, V M, Likhtenshtein, A V
Format: Article
Language:English
Subjects:
Bacteria
Cell differentiation
Chromatin
Deproteinization
Electron microscopy
Gel electrophoresis
Mammalian cells
Oligonucleotides
Pulsed-field gel electrophoresis
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Summary:Pulsed-field electrophoresis revealed minor free DNA (fDNA) fractionsa andb in mammalian cells. The fractions differed in some properties from chromosomal DNA (chrDNA). Electron microscopy revealed rosette-like structures resistant to drastic deproteinization in fDNAs from Namalwa cells. Selective labeling and immunochemical visualization located both fractions to the nucleus in terminally differentiated N1-E115 cells.Alu oligonucleotide-directed PCR showed that the distribution of unique and repetitive sequences in fDNAs differed from that in chrDNA. Representational difference analysis revealed four sequences probably located inb fDNA. Of these one proved homologous to the genes for human interleukin enhancer-binding factors (ILFs) and one, to the transcribed region neighboring the second internal transcribed spacer in the rDNA cluster. Theb fDNA was cleaved withBamHI,BglII, andBclI and cloned. The inserts ranging 120–7000 nt were completely or partly sequenced in 60 clones. The fDNAs were assumed to result from selective excision of some chromatin regions or to be extrachromosomal elements of unknown nature.
ISSN:0026-8933
1608-3245
DOI:10.1007/BF02759659