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Base composition of Mycoplasma DNA and the formation of specific DNA-DNA hybrids

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Title:Base composition of Mycoplasma DNA and the formation of specific DNA-DNA hybrids
Authors:Fox, Richard Howard
Date Issued:1967
Abstract:Native deoxyribonucleic acid (DNA) was extracted and purified from eight parasitic Mycoplasma species and two saprophytic strains of Mycoplasma laidlawii. Triplicate samples of each of the DNA types were subjected to thermal denaturation (melting) to estimate the mean mole percent guanine plus cytosine (G+C%) composition. The DNA melting transitions were followed spectrophotometrically, where the relative absorbance was plotted as a function of temperature. From the midpoints of each transition the Tm (midpoint of the hyperchromic shift) values were estimated and the G+C% calculated from the following relationship: Tm = 69.3 + 0.41 (G+C). These experiments showed that the base composition of both the saprophytic and the parasitic Mycoplasma DNAs were characterized by a narrow G+C range of 32% to 35%. with the exception, of two species: Mycoplasma gallinarum, an avian parasite, 28% G+C; and the murine parasite Mycoplasma arthritidis, 29% G+C. Despite the narrow compositional range, the regions of greatest inflection of the melting transitions, from 17% to 85% of the hyperchromic shifts, were variable and indicated a compositional heterogeneity. A mathematical analysis of the transitions estimated the extent of the compositional heterogeneity in the different DNA types. Experiments were designed to evaluate the kinetics of immobilization of DNA to nitrocellulose membrane filters. Filtration of high molecular weight, denatured DNA, onto membranes resulted in the irreversible immobilization of the DNA to the membranes. DNA which was shear degraded by sonication for varying periods of time, then denatured, was bound irreversibly to the membranes. Incubation of blank membranes with an albumin solution and labeled sheared-denatured DNA, showed that the DNA was nonspecifically adsorbed to the membranes at about 10% of the input DNA concentration. The adsorption decreased slightly as the duration of exposure to shearing increased. Washing the membranes after incubation reduced this adsorption to about 2% of the input DNA. When sheared and denatured DNA was incubated with membranes in the absence of the albumin solution, the nonspecific adsorption was about twice that detected than in its presence. After washing, a similar 2% of the input DNA remained permanently adsorbed to the membranes. E. coli DNA immobilized on membranes was incubated with homologous labeled DNA to determine saturation of hybridizing sites as a function of the ratio of labeled to total DNA (symbolized as T.). When the value of T was about 0.5, nearly all of the labeled DNA was bound to the immobilized DNA. When the value of T was greater than 0.5, retention of DNA to the membranes decreased. This implied that all the hybridization sites of the immobilized DNA were saturated according to the expectation of the binomial distribution. Hybridization of homologous and heterologous Mycoplasma DNA by a modification of the nitrocellulose membrane filter technique was employed to study genetic relatedness among the Mycoplasma. Radiophosphorus labeled DNA from saprophytic Mycoplasma was bound to immobilized parasitic Mycoplasma DNA in all cases except for M. arthritidis. Labeled DNAs from E. coli K-12 and s. marcescens did not hybridize with Mycoplasma DNA, but did cross react with each other. Relative DNA-DNA homologies indicated that about 20 to 130 common cistrons were shared by the saprophytic and the parasitic Mycoplasma DNAs. Labeled DNA from B. circulans in similar hybridizations with Mycoplasma DNA indicated about 170 to 250 mutual cistrons. Hybridizations between B. circulans and Mycoplasma DNAs occurred only when the DNA from the Mycoplasma was immobilized to membranes. In reciprocal hybridizations no detectable homologies were observed. The possibility that the DNA extracted from B. circulans was contaminated by and complexed with teichoic acid was discussed as an explanation for the absence of reciprocity. This argument proposed that the retention of B. circulans DNA to DNA-membranes was not genuine, but rather a reflection of spurious adsorption of the teichoic acid. Based on the agreement between the relative amount of binding in reciprocal combinations of the saprophytic strain A and B DNA, it was concluded that these Mycoplasma strains are closely related. Genetic homology of some common cistrons also exist between the saprophytic and most of the parasitic Mycoplasma DNAs examined. Genetic relatedness between the DNAs from B. circulans and from Mycoplasma was concluded not to be definitive.
Thesis (Ph. D.)--University of Hawaii, 1967.
Bibliography: leaves 77-86.
ix, 86 l graphs, tables
Rights:All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Appears in Collections: Ph.D. - Microbiology

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