Related Experiment Videos
Evidence for circular permutation of the prophage genome of Bacillus subtilis bacteriophage phi 105
Abstract:
Analysis of DNA extracted from Bacillus subtilis lysogenic for bacteriophage phi 105 was performed by restriction endonuclease digestion and Southern hybridization using mature phi 105 DNA as a probe. The data revealed that the phi 105 prophage is circularly permuted. Digests using the enzymes EcoRI, SmaI, PstI, and HindIII localized the bacteriophage attachment site (att) to a region 63.4 to 65.7% from the left end of the mature bacteriophage genome. The phi 105 att site-containing SmaI C, PstI J, and HindIII L fragments were not present in digests of phi 105 prophage DNA. phi 105-homologous "junction" fragments were visualized by probing digests of prophage DNA with the purified PstI J fragment isolated from the mature bacteriophage genome. The excision of the phi 105 prophage was detected by observing the appearance of the mature PstI J fragment and the concomitant disappearance of a junction fragment during the course of prophage induction.
Insights
Researchers analyzed Bacillus subtilis DNA lysogenic for bacteriophage phi 105, revealing the prophage is circularly permuted. The bacteriophage attachment site was precisely mapped on the mature genome.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Bacillus subtilis can be lysogenic, harboring bacteriophage phi 105 DNA integrated into its genome.
- Understanding the structure and integration site of temperate bacteriophages is crucial for molecular biology and genetic engineering.
Purpose of the Study:
- To analyze the structure of the bacteriophage phi 105 prophage within Bacillus subtilis.
- To precisely map the bacteriophage phi 105 attachment site (att) on the mature phage genome.
- To investigate the excision process of the phi 105 prophage during induction.
Main Methods:
- Restriction endonuclease digestion of Bacillus subtilis DNA lysogenic for bacteriophage phi 105.
- Southern hybridization using mature phi 105 DNA as a probe.
- Analysis of specific DNA fragments (SmaI C, PstI J, HindIII L) and 'junction' fragments.
Main Results:
- The phi 105 prophage integrated into the Bacillus subtilis genome is circularly permuted.
- The bacteriophage attachment site (att) was localized to 63.4-65.7% from the left end of the mature phi 105 genome.
- Specific fragments containing the att site were absent in prophage DNA, but 'junction' fragments were detected, indicating integration.
Conclusions:
- The study successfully mapped the bacteriophage phi 105 attachment site within the Bacillus subtilis genome.
- The observed changes in DNA fragments during prophage induction confirm the excision mechanism of the phi 105 prophage.