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Molecular Biology

, Volume 34, Issue 3, pp 313–320 | Cite as

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

  • A. V. Budilov
  • D. A. Domninskii
  • V. I. Popenko
  • T. I. Sukhova
  • I. V. Botezatu
  • V. P. Shelepov
  • O. I. Serdyuk
  • R. P. Alekhina
  • V. M. Zakhariev
  • A. V. Likhtenshtein
Genomics. Proteomics. Bioinformatics
  • 38 Downloads

Abstract

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.

Key words

free DNA pulsed-field gel electrophoresis polymerase chain reaction electron microscopy cloning sequencing 

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Copyright information

© MAIK “Nauka/Interperiodica” 2000

Authors and Affiliations

  • A. V. Budilov
    • 1
  • D. A. Domninskii
    • 1
  • V. I. Popenko
    • 1
  • T. I. Sukhova
    • 2
  • I. V. Botezatu
    • 2
  • V. P. Shelepov
    • 2
  • O. I. Serdyuk
    • 2
  • R. P. Alekhina
    • 2
  • V. M. Zakhariev
    • 1
  • A. V. Likhtenshtein
    • 2
  1. 1.Engelhardt Institute of Molecular BiologyRussian Academy of SciencesMoscowRussia
  2. 2.Institute of Carcinogenesis, Cancer Research CenterRussian Academy of Medical SciencesMoscowRussia

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