Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6 | JoVE Visualize

Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6

K N Timmis, F Cabello, S N Cohen

Molecular & General Genetics : MGG|June 14, 1978

View abstract on PubMed

Summary

Researchers mapped antibiotic resistance genes and replication origins on R6-5 plasmids using DNA cloning and heteroduplex analysis. This study precisely located key genetic elements, including replication functions and resistance determinants, on the plasmid genome.

Area of Science:

Molecular Biology
Genetics
Microbiology

Background:

Antibiotic resistance plasmids, such as R6 and R6-5, are crucial for understanding bacterial evolution and developing new therapeutic strategies.
Characterizing the genetic organization of these plasmids is essential for deciphering their replication mechanisms and gene expression.

Purpose of the Study:

To precisely map the EcoRI and HindIII restriction endonuclease cleavage sites on the R6 and R6-5 plasmid genomes.
To determine the location of genes conferring resistance to various antibiotics and mercury.
To identify and characterize regions responsible for plasmid replication, including those capable of autonomous replication and those supporting replication in specific bacterial strains.

Main Methods:

Cloning of EcoRI and HindIII generated DNA fragments from R6 and R6-5 plasmids into high copy number vectors (ColE1, pML21) using insertional inactivation.
Electron microscope heteroduplex analysis and agarose gel electrophoresis to determine the location of restriction sites on parent plasmids.
Ultracentrifugation in cesium chloride gradients to analyze fragment buoyant density and associate it with specific resistance genes.
Functional analysis of hybrid plasmids to map antibiotic/mercury resistance and replication functions.

Main Results:

Localized high buoyant density regions of R6-5 to fragments carrying streptomycin-spectinomycin, sulfonamide, and mercury resistance genes, and a low buoyant density region to the tetracycline resistance determinant.
Precisely mapped genes for chloramphenicol, kanamycin, fusidic acid, and tetracycline resistance using identified restriction sites.
Identified one region for autonomous replication (EcoRI fragment 2, HindIII fragment 1) and two additional replication activity regions (RepB, RepC) on the R6-5 plasmid.

Conclusions:

The study successfully mapped key functional regions of the R6-5 plasmid, including antibiotic resistance genes and replication origins.
The identified replication regions provide insights into plasmid maintenance and propagation mechanisms.
This detailed genetic map serves as a foundation for further studies on plasmid biology and the development of novel antimicrobial strategies.