Characterization of Homology Between Actinobacillus actinomycetemcomitans Chromosomal and Extrachromosomal DNA. Since there have been few reports in the literature of isolation and characterization of extrachromosomal DNA from Actinobacillus actinomycetemcomitans (Aa), a plasmid enrichment procedure was used to demonstrate the presence of extrachromosomal DNA in 2 of 39 strains of Aa. Dye buoyant density gradient analyses confirmed the covalently closed circular conformation of these molecules. One, pVT745, hybridized under stringent conditions to genomic DNA from 15 Aa isolates obtained from geographically diverse regions of the U.S. Southern blot analyses revealed 5 strain-specific patterns of hybridization. The purpose of the current study is to characterize this 24Kb plasmid and to locate the molecule sequences responsible for hybridization to Aa genomic DNA. pVT745 was digested with the restriction endonuclease enzymes, BamHl and Pst1, into 3 nonoverlapping fragments of approximately 7, 8 and 9Kb. The fragments were cloned in a E. col host on the low copy number vector, pGB2. Although these 3 fragments exhibited no cross hybridization, genomic DNA from isolates representing each of the strain-specific patterns hybridized with 2 or 3 of the cloned fragments. This suggests that 2 or more unique sequences within pVT745 may insert into Aa genomic DNA. It is possible that these sequences represent 2 or more insertion sequence elements and/or transposons. Upon insertion, these sequences may serve to either disrupt or activate genes, resulting in alteration of the phenotypic characteristics of the host strain. Current efforts are directed toward elucidating the nature of these sequences utilizing molecular biology techniques to accomplish the following specific aims: 1. Development of a restriction endonuclease map and determination (through Southern Blot analysis) of the specific region(s) on pVT745 that are responsible for hybridization to Aa genomic DNA. 2. Characterize the nature of the extrachromosomal molecule utilizing phage induction experiments and genetics transfer experiments (conjugation transformation, transduction). 3. Sequences regions of pVT745 responsible for hybridization to Aa genomic DNA. Compare these sequences with known gene banks in an attempt to elucidate their nature. 4. Due to the strain specific pattern of hybridization seen with pVT745, it is proposed that this molecule be evaluated as a tool in the classification of Aa clinical isolates.
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