We have previously characterized an extracellular nuclease from Pseudomonas BAL 31 which, in addition to other activities, displays a double-strand exonuclease activity which progressively shortens both strands of linear duplex DNA molecules from both termini. This degradation is accomplished without the introduction of detectable scissions away from the ends of the duplexes. When this nuclease is used to produce a series of progressively shortened samples from a linear duplex DNA, subsequent digestion of these samples with a site-specific restriction endonuclease and analysis of the resulting fragments by gel electrophoresis permits the rapid establishment of the order of the restriction enzyme fragments through the entire genome. This is accomplished by noting from the electropherograms the order in which the various restriction enzyme fragments become noticeably shortened or disappear. Using this method, the five cleavage sites for the endonuclease Hpa I and the single cleavage sites for the nucleases Hpa II and Pst I have been mapped in PM2 bacteriophage DNA. In a more stringent test of the method, 18 of the 24 fragments produced by cleavage of coliphage lambdab2b5c DNA with the Pst I nuclease have been mapped, and five of the six remaining fragments have been assigned to small regions of the genome.

The culture medium of Pseudomonas BAL 31 contains endonuclease activities which are highly specific for single-stranged DNA and for the single-stranded or weakly hydrogen-bonded regions in supercoiled closed circular DNA. Exposure of nicked DNA to the culture medium results in cleavage of the strang opposite the sites of preexisting single-strand scissions. At least some of the linear duplex molecules derived by cleavage of supercoiled closed circular molecules contain short single-stranded ends. Single-strand scissions are not introduced into intact, linear duplex DNA or unsupercoiled covalently closed circular DNA. Under these same reaction conditions, 0X174 phage DNA is extensively degraded and PM2 form I DNA is quantitatively converted to PM2 form III linear duplexes. Prolonged exposure of this linear duplex DNA to the concentrated culture medium reveals the presence of a double-strand exonuclease activity that progressively reduces the average length of the linear duplex. These nuclease activities persist at ionic strengths up to 4 M and are not eliminated in the presence of 5% sodium dodecyl sulfate. Calcium and magnesium ion are both required for optimal activity. Although the absence of magnesium ion reduces the activities, the absence of calcium ion irreversibly eliminates all the activities.

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HeLa S3 cells were cloned, recloned, and then selected for growth in the presence of increasing concentrations of bromodeoxyuridine. Cultures of these cloned thymidine kinase minus (TK^-) cells were found to harbor mycoplasma which sedimented with mitochondria in sucrose density step gradients. Examination of mitochondrial DNA (mitDNA) components by restriction enzyme analysis and electron microscopy revealed no gross alterations in size, sequence arrangements, or replicative forms compared with mitDNA of HeLa S3 cells. Restriction enzyme cleavage sites for BamHl (one site), PstI (two sites) and HpaI (three sites) were mapped on this genome relative to the three cleavage sites for each of EcoRI and HindIII, respectively. Analysis of topological complexity revealed similar frequencies of catenated mitDNA molecules in both cloned TK^– (22.5 ± 1.5% of mass) and HeLa S3 cells (25.6 ± 1.5% of mass). Unicircular dimers comprise 6.7 ± 0.9% of the mitDNA mass in cloned TK^– cells but were not detected in HeLa S3 mitDNA. Examination of the mycoplasmal contaminant of mitochondrial DNA after digestion with various restriction enzymes and agarose gel electrophoresis revealed that most of the DNA was distributed in discretely sized fragments in patterns that can probably be used to unambiguously identify and classify the organism.