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Amplification of DNA at a prophage attachment site in Haemophilus influenzae
1Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6076.
Abstract:
The Escherichia coli plasmids pBR322 and pBR327 can be taken up by Haemophilus influenzae but do not replicate in this organism; however, integration of pBR into the H. influenzae chromosome was achieved by ligation to a fragment of the Haemophilus phage S2 that carried a phage attachment site (attP). Once these sequences were integrated, they could serve as sites of recombination and amplification for homologous (pBR or phage) DNA. Amplification appeared to occur in one of two prophage sites (attB) present in the H. influenzae chromosome. The extent of amplification was different in different cells and reflected the ability of these sequences to undergo rearrangement leading to the formation of a DNA ladder. The ladder was obtained by treatment of DNA with restriction enzymes that cut outside of the inserted DNA, i.e., did not cut in the repeat sequence, and represented different numbers of repeat elements. Reversed-field gel electrophoresis was instrumental in resolving amplified structures. Inasmuch as single-cell isolates gave rise to the same ladder structure, it was assumed that amplification was under regulatory control and that it reproduced the same equilibrium of repeat structures. Transformation of E. coli with the amplified H. influenzae DNA resulted in precise excision and replication of the original monomeric plasmids. This excision was independent of the recA and recBC genes.
Insights
Escherichia coli plasmids were integrated into Haemophilus influenzae chromosomes via phage DNA. This integration enabled DNA recombination and amplification, which could be precisely excised later.
Area of Science:
- Molecular Biology
- Microbial Genetics
- Bacteriophage Biology
Background:
- Escherichia coli plasmids pBR322 and pBR327 do not replicate in Haemophilus influenzae.
- Integration of foreign DNA into bacterial chromosomes is a key genetic manipulation technique.
Purpose of the Study:
- To achieve integration and replication of E. coli plasmids in H. influenzae.
- To investigate DNA recombination and amplification mechanisms in H. influenzae.
- To analyze the regulatory control of DNA amplification and excision.
Main Methods:
- Ligation of pBR plasmids to Haemophilus phage S2 DNA containing a phage attachment site (attP).
- Integration into H. influenzae chromosome at prophage attachment sites (attB).
- Reversed-field gel electrophoresis to analyze DNA amplification and rearrangement.
- Transformation of E. coli with amplified H. influenzae DNA.
Main Results:
- Successful integration of pBR sequences into the H. influenzae chromosome.
- Demonstration of homologous DNA recombination and amplification at prophage sites.
- Formation of a DNA ladder representing amplified repeat elements, resolved by electrophoresis.
- Evidence of regulatory control over amplification, with consistent ladder structures from single-cell isolates.
Conclusions:
- Integration of phage DNA facilitates stable maintenance and amplification of foreign DNA in H. influenzae.
- Amplification is a regulated process leading to reproducible DNA structures.
- Precise excision and replication of original plasmids from amplified H. influenzae DNA in E. coli is possible and recA/recBC independent.