Partial purification of native rRNA and tRNA cistrons from mycoplasma sp. (Kid)

Abstract

Precise optical melting profiles of purified DNA from Mycoplasma sp. (Kid) show a secondary hyperchromic rise, corresponding to 1.4 per cent of the total DNA, occurring at 88 degrees C, while the bulk of the DNA melts at 79.5 degrees C, indicating an average base composition of 24.9 per cent guanine-cytosine (G-C). A method is presented, using sonication followed by hydroxyapatite column chromatography, for the partial purification of regions of a genome which contain significantly higher G-C than the average value for the genome. The procedure does not involve denaturation and renaturation of the high G-C material so that purified DNA is in its native, double-stranded state and has a normal melting profile. When applied to Mycoplasma sp. (Kid), the method yielded a fraction of native DNA enriched 40 times with respect to those regions coding for rRNA and tRNA. This enriched DNA has a saturation hybridization value of 15.9 per cent with Kid rRNA plus tRNA. The saturation hybridization values of the bulk DNA with rRNA and tRNA are 0.26 per cent and 0.16 per cent, respectively. Based on a genome size of 6.84 x 10(8), obtained by electron microscopy, this indicates that Mycoplasma sp. (Kid) contains only enough ribosomal DNA to code for one set of 23S plus 16S rRNA and only enough DNA complementary to tRNA to code for 44 different tRNA molecules.