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Mycobacterium smegmatis dnaA region and autonomous replication activity
M Rajagopalan1, M H Qin, D R Nash
1Department of Microbiology, University of Texas Health Center at Tyler 75710, USA.
Journal of Bacteriology
|November 1, 1995
Summary
Researchers identified a DNA fragment from Mycobacterium smegmatis containing the dnaA gene. This fragment enables autonomous replication in M. smegmatis, suggesting it harbors the origin of replication.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Replication initiation in eubacteria requires the DnaA protein and a replication origin.
- Understanding mycobacterial replication is crucial for this important genus.
Purpose of the Study:
- To investigate the replication initiation process in Mycobacterium smegmatis.
- To isolate and characterize DNA elements involved in replication initiation.
Main Methods:
- Isolation of a 4-kb chromosomal DNA fragment from Mycobacterium smegmatis containing the dnaA gene.
- Nucleotide sequence analysis to identify homologous genes.
- Cloning the DNA fragment into a non-replicative plasmid (pUC18) and testing for autonomous replication in M. smegmatis.
Main Results:
- The isolated 4-kb fragment contains the dnaA gene, homologous to rpmH and dnaN.
- The DNA fragment, when cloned into pUC18, conferred autonomous replication ability to the plasmid in M. smegmatis.
- This indicates the presence of an origin of replication within the fragment.
Conclusions:
- The 4-kb DNA fragment from M. smegmatis contains essential elements for replication initiation.
- The M. smegmatis chromosomal origin of replication is likely located within this isolated fragment.
- This finding is a significant first step in studying mycobacterial replication initiation.