An Analysis Of Extrachromosomal DNA Isolated From Bartonella henselae

Abstract

An unknown sequence of DNA discovered in Bartonella henselae was studied in order to determine rts conformation, size, and origin. The DNA was isolated from B. henselae cells using a standard miniprep method, commonly used to isolate small extrachromosomal DNA from cells. This DNA was subjected to a series of experiments including digestion with restriction endonucleases, SI, and Mung Bean nucleases, and hybridization to B. henselae genomic DNA. in order to determine whether there are any sequences in common. DNA fragments produced by digestion were separated using standard gel as well as Pulse-Field Gel Electrophoresis, and viewed after being stained with ethidium bromide. Fragments used for hybridization experiments were labeled with 32P. The results indicated that the unknown DNA contained many restriction sites for a majority of the restriction enzymes used in the experiment. Pulse Field Gel Electrophoresis of the unknown DNA digested with the SI and Mung Bean nucleases suggested that the DNA may be linear since both enzymes, which cut strictly circular DNA, did not seem to attack the unknown DNA. The results of the hybridization experiment indicated that the possibility of the unknown DNA sharing sequences with genomic DNA is unlikely. Finally, Pulse Field Electrophoresis of undigested B. henselae DNA suggested that the undigested DNA itself consists of a number of fragments of various sizes which were most likely compressed into a smgle band on the standard electrophoresis gel. This result ultimately disproves the t hypothesis that the unknown DNA was produced by a plasmid, bacteriophage, or a transposon, since such DNA would be expected to consist of fragments of uniform size.