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Halobacterial megaplasmids are negatively supercoiled

P López-García1, J Antón, J P Abad

  • 1Centro de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, Spain.

Molecular Microbiology
|February 1, 1994
PubMed
Summary
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Researchers resolved large circular halobacterial megaplasmids using orthogonal-field alternating gel electrophoresis. Novobiocin treatment revealed these megaplasmids are negatively supercoiled and sensitive to topoisomerase II inhibition.

Area of Science:

  • Extremophile Biology
  • Molecular Genetics
  • Biochemistry

Background:

  • Haloferax mediterranei harbors large, covalently closed circular megaplasmids.
  • Understanding the in vivo topology of these megaplasmids is crucial for their functional characterization.

Purpose of the Study:

  • To resolve and characterize halobacterial megaplasmids using advanced electrophoresis techniques.
  • To investigate the native supercoiling state and topological properties of these large DNA molecules.

Main Methods:

  • Orthogonal-field alternating gel electrophoresis (OFAGE) was employed to resolve megaplasmids.
  • Analysis of electrophoretic mobility changes upon treatment with intercalating agents and novobiocin (a topoisomerase II inhibitor).

Main Results:

Related Experiment Videos

  • Successfully resolved multiple megaplasmids (up to >500 kb) from Haloferax mediterranei strains.
  • Intercalating agents altered mobility, suggesting negative supercoiling in vivo.
  • Novobiocin disrupted the native topological state, significantly impeding OFAGE migration.

Conclusions:

  • Halobacterial megaplasmids exist as negatively supercoiled DNA molecules in vivo.
  • Their topology is sensitive to topoisomerase II activity, influencing their electrophoretic behavior.