Isolation and Characterization of DNA from a Mycoplasmalike Organism. Barbara B. Sears. Department of Botany and Plant Pathology, Genetics Program, Center for Electron Optics and Pesticide Research Center, Michigan State University, East Lansing 48824 U.S.A. Pyung-Ok Lim(2), Neta Holland(1), Bruce C. Kirkpatrick(4), and Karen L. Klomparens(1,3). (1)Department of Botany and Plant Pathology, (2)Genetics Program, and (3)Department of Entomology, Center for Electron Optics and Pesticide Research Center, Michigan State University, East Lansing 48824 U.S.A. (4)Department of Plant Pathology, University of California, Davis 95616 U.S.A. MPMI 2:175-180. Accepted 8 March 1989. Copyright 1989 The American Phytopathological Society.

A method was developed for the purification of high molecular weight DNA from a representative of the plant-pathogenic mycoplasmalike organisms (MLOs). This procedure utilized leaf tip cultures of the evening primrose, Oenothera, in which MLOs have been maintained in the laboratory at a high titer. Through differential centrifugation, the MLOs were isolated along with the plant mitochondria. Contaminating plant chromosomal and chloroplast DNAs could be removed by a DNase treatment, followed by washes with buffer containing 20 mM EDTA, before lysing the mitochondria and MLOs for further purification of their DNAs. From this material, two DNA bands were recovered by equilibrium centrifugation in CsCI gradients, which contained the DNA-binding fluorochrome, bisbenzimide. The lower band corresponded to plant mitochondrial DNA; the upper DNA band was unique to the samples containing DNA extracted from the MLO-infected plant material, and this DNA was used to make a partial DNA library. One particular clone hybridized specifically to MLO DNA, and in particular to an endogenous plasmid of the MLO, with no crosshybridization to plant DNA. Characterizations of the purified MLO DNA indicated that its G+C content is less than 30%, and the DNA was shown to be resistant to digestion by most endonucleases having four or more GC pairs in their recognition sites. Other restriction enzymes produced discrete banding patterns, indicating that the DNA is of relatively low complexity. The stoichiometry of the bands suggests that the MLO plasmid is maintained at a high copy number.