Immunogenic Mycobacterial Ribosomal and Ribonucleic Acid Preparations: Chemical and Physical Characteristics

Five to 20% linear sucrose gradients were used to obtain sedimentation patterns of mycobacterial ribosomes, ribosomal subfractions, and ribonucleic acid (RNA) preparations. Classical 70 S ribosomes were obtained when 10 −1 m magnesium chloride was used. These, when dialyzed against 10 −4 m MgCl 2 , yielded typical 50 S , 30 S , and smaller ribosomal subunits. The 30 S subunits were the most immunogenic under these conditions. A ribosomal preparation containing subunits which varied from 2.5 to 40 S was fractionated by collecting five fractions from a sucrose gradient; based upon the amount of nucleic acid present, the fraction containing the 40 S particles was most immunogenic. Physical and chemical evidence suggested that mycobacterial RNA preparations extracted with 65% ethyl alcohol from the ribosomes and diluted in distilled water, were either double-stranded, or mostly double-helical, or had a highly organized secondary structure. This was based on the following observations. (i) Native RNA was resistant to trace amounts of ribonuclease. (ii) The approximate T m value in SSC buffer (0.15 m NaCl plus 0.015 m sodium citrate) was greater than 85 C and in 0.1 SSC buffer was 55 C; the RNA diluted in SSC buffer produced a hypochromic effect on cooling at room temperature. (iii) Formaldehyde, in the presence of SSC buffer, decreased the T m of the RNA to approximately 55 C, and there was no hypochromic effect on cooling. (iv) Formaldehyde did not increase the wavelength of maximal adsorption of the RNA. (v) The purine/pyrimidine ratio was close to one. (vi) The major peak of the RNA sedimented in the more dense zones of the sucrose gradients. There was a relationship between the sedimentation pattern obtained with the RNA-protein subunits on sucrose gradients and immunogenicity; several examples are given. RNA-protein complexes of approximately 14 to 20 S , and occasionally 23 S in the major peak, appeared to produce the highest immune response. Smaller RNA-protein complexes such as 6 S , which were obtained when the RNA preparation was diluted in certain buffers, were much less immunogenic. This was confirmed by collecting five fractions from sucrose gradients and finding the third fraction (containing RNA-protein complexes approximately 15 to 16 S ) the most immunogenic. Immunogenic activity was apparently related to the structure of the RNA since it was maximal when the RNA appeared to be either double stranded, double helical, or had a highly organized structure.