Electron microscopy and computerized evaluation of some partially denatured group P resistance plasmids
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Abstract:
DNA of the R plasmids RP1, RP4 and RP8 was isolated from various hosts. The lengths of these plasmid molecules were determined by electron microscopy: RP1 and RP4 were about 19 micron long, RP8 measured 31 micron. An RP4 plasmid mutant, designated RP4a, was isolated from Escherichai coli; it was about 1 micron shorter than normal RP4 DNA. To investigate the molecular relationship between RP4, RP4a and RP8 DNAs of these plasmids were partially denatured and examined in the electron microscope. Measurements of the length and denaturation pattern of the DNA molecules were used to construct physical maps. A new computer program was devised for the alignment of the circular molecules, and the effect of variations of different parameters on the reliability of the program was tested. A comparison of the denaturation pattern of RP4 and RP8 indicated that RP8 was composed of total RP4 plus an additional DNA fragment. The RP4a mutant plasmid could be defined as a deletion mutant with loss of 1 micron DNA.
Insights
Researchers mapped R plasmids RP1, RP4, and RP8 using electron microscopy. RP8 contains RP4 DNA plus an extra fragment, while RP4a is a deletion mutant lacking 1 micron of DNA.
Area of Science:
- Molecular biology
- Genetics
- Microbiology
Background:
- R plasmids are crucial for antibiotic resistance transfer.
- Understanding plasmid structure and evolution is vital for combating resistance.
- Previous studies have characterized various R plasmids, but detailed molecular relationships remain to be elucidated.
Purpose of the Study:
- To determine the molecular relationship between R plasmids RP4, RP4a, and RP8.
- To construct physical maps of these plasmids using DNA denaturation patterns.
- To analyze the genetic basis of the RP4a deletion mutant.
Main Methods:
- Isolation of R plasmid DNA (RP1, RP4, RP8) from various bacterial hosts.
- Electron microscopy for determining DNA molecule lengths and partial denaturation patterns.
- Development and application of a novel computer program for aligning circular DNA molecules.
- Comparative analysis of denaturation patterns to infer DNA sequence homology and structural variations.
Main Results:
- Plasmid lengths were determined: RP1 and RP4 (approx. 19 microns), RP8 (31 microns).
- RP4a, a mutant of RP4, was identified as a deletion mutant, approximately 1 micron shorter than RP4.
- Electron microscopy and denaturation mapping revealed that RP8 comprises the entire RP4 sequence plus an additional DNA fragment.
- The developed computer program facilitated accurate alignment and comparison of plasmid DNA structures.
Conclusions:
- RP8 is a composite plasmid, containing the full RP4 sequence and an additional DNA segment.
- RP4a represents a specific deletion within the RP4 plasmid.
- The study provides detailed physical maps and clarifies the molecular relationships between these R plasmids.
- The developed computational approach enhances the analysis of circular DNA molecule relationships.