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Related Experiment Videos

Replication control genes of plasmid pE194.

R Villafane1, D H Bechhofer, C S Narayanan

  • 1Department of Microbiology, Public Health Research Institute, City of New York, Inc., New York 10016.

Journal of Bacteriology
|October 1, 1987
PubMed
Summary

The pE194 plasmid

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Area of Science:

  • Molecular Biology
  • Genetics
  • Antimicrobial Resistance

Background:

  • The pE194 plasmid confers resistance to macrolide, lincosamide, and streptogramin B antibiotics.
  • Understanding plasmid replication mechanisms is crucial for combating antibiotic resistance.

Purpose of the Study:

  • To precisely localize and characterize the replication genes (cop and repF) and origin of replication of the pE194 plasmid.
  • To correct previous sequence errors and elucidate the functional roles of identified genes.

Main Methods:

  • Molecular cloning and mutational analysis
  • DNA sequencing
  • Northern blot (RNA blot) analysis
  • In vitro transcription-translation assays

Main Results:

  • The CfoIB fragment of pE194 was confirmed to contain both cop and repF genes and support autonomous replication.
  • A single large open reading frame, proposed to encode the repF product, was identified in the corrected sequence.
  • Six transcripts were detected, all transcribed opposite to repF, with a 350-base transcript from the cop region.
  • A mutant repF product, when expressed from a different replicon, inhibited pE194 replication in trans.
  • The pE194 origin of replication was localized to a 200-base region.

Conclusions:

  • The study provides a corrected and detailed map of the pE194 replication region.
  • The repF gene product plays a role in plasmid replication and can exert a trans-acting inhibitory effect.
  • The findings contribute to understanding plasmid replication control mechanisms relevant to antibiotic resistance.

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