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  • INPECFION AND IMMUNITY, Nov. 1970, p. 659-668 Vol. 2, No. 5Copyright 1970 American Society for Microbiology Printed In U.S.A.

    Immunogenic Mycobacterial Ribosomal andRibonucleic Acid Preparations: Chemical

    and Physical CharacteristicsANNE S. YOUMANS AND GUY P. YOUMANS

    Department of Microbiology, Northwestern University Medical School, Chicago, Illinois 60611

    Received for publication 2 March 1970

    Five to 20% linear sucrose gradients were used to obtain sedimentation patternsof mycobacterial ribosomes, ribosomal subfractions, and ribonucleic acid (RNA)preparations. Classical 705 ribosomes were obtained when 10-' M magnesiumchloride was used. These, when dialyzed against 104 M MgCl2, yielded typical 50S,30S, and smaller ribosomal subunits. The 30S subunits were the most immunogenicunder these conditions. A ribosomal preparation containing subunits which variedfrom 2.5 to 40S was fractionated by collecting five fractions from a sucrose gradient;based upon the amount of nucleic acid present, the fraction containing the 40Sparticles was most immunogenic. Physical and chemical evidence suggested thatmycobacterial RNA preparations extracted with 65% ethyl alcohol from the ribo-somes 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 follow-ing observations. (i) Native RNA was resistant to trace amounts of ribonuclease.(ii) The approximate Tm value in SSC buffer (0.15 M NaCl plus 0.015 M sodiumcitrate) was greater than 85 C and in 0.1 SSC buffer was 55 C; the RNA diluted inSSC buffer produced a hypochromic effect on cooling at room temperature. (iii)Formaldehyde, in the presence of SSC buffer, decreased the Tm of the RNA toapproximately 55 C, and there was no hypochromic effect on cooling. (iv) Formalde-hyde did not increase the wavelength of maximal adsorption of the RNA. (v) Thepurine/pyrimidine ratio was close to one. (vi) The major peak of the RNA sedi-mented in the more dense zones of the sucrose gradients. There was a relationshipbetween the sedimentation pattern obtained with the RNA-protein subunits onsucrose gradients and immunogenicity; several examples are given. RNA-proteincomplexes of approximately 14 to 20S, and occasionally 23S in the major peak,appeared to produce the highest immune response. Smaller RNA-protein complexessuch as 6S, which were obtained when the RNA preparation was diluted in certainbuffers, were much less immunogenic. This was confirmed by collecting five frac-tions from sucrose gradients and finding the third fraction (containing RNA-proteincomplexes approximately 15 to 16S) the most immunogenic. Immunogenic activitywas apparently related to the structure of the RNA since it was maximal when theRNA appeared to be either double stranded, double helical, or had a highly or-ganized structure.

    We have reported recently (41) that carefully of these to immunogenic activity. We will alsoprepared mycobacterial ribosomal and ribo- present evidence which indicates that highlynucleic acid (RNA) fractions can be obtained immunogenic mycobacterial RNA preparationswhich are as immunogenic, on a weight basis, contain nucleic acid which exhibits characteris-as the viable cells from which they were prepared. tics of double-stranded RNA, or, highly organizedA high correlation also was found between the double-helical secondary structures.quality of the RNA and immunogenic activity. In addition, we have included data showingIt is the purpose of the present paper to present that 70S particles can readily be isolated frommore of the chemical and physical characteristics mycobacterial particulate fractions, providedof these preparations and to try to relate certain adequate concentrations of magnesium chloride

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  • YOUMANS AND YOUMANS

    are employed. It will also be shown that, whenthe concentration of magnesium chloride islowered, these 70S particles break up intothe classical 50 and 30S as well as smaller sub-units. Finally, we wish to stress that our concernin this paper with the characteristics of prepara-tions containing ribosomal subunits, rather thanwith 70S particles, is solely because the prepara-tions containing subunits have appeared moreimmunogenic (38). Our primary interest is incharacterizing immunogenic material, not inpreparing and characterizing such classical bac-terial structures as ribosomes.

    MATERIALS AND METHODS

    Mycobacterial cultures. The attenuated H37Rastrain of Mycobacteriwn tuberculosis was used as thesource of the ribosomal and RNA preparations.The highly virulent H37Rv strain of M. tuberculosiswas used to challenge mice. Both strains were main-tained, and the suspensions made, as described previ-ously (41).

    Preparation of ribosomal subunits and RNA.These were done as described in detail previously(41). Briefly, the particulate fraction was obtained bydifferential centrifugation of ruptured viable H37Racells. A given concentration (50 mg/ml, wet weight)was added to an equal volume of 0.5% sodium dode-cyl sulfate (SDS) at room temperature. This wasrecentrifuged at 144,700 X g for 3 hr to sedimentthe ribosomes. To obtain 70S ribosomes withoutsome dissociation into 50 or 30S subunits, it wasnecessary to use 10-1 M MgCl2 in the sucrose bufferin which the cells were ruptured instead of the 10" MMgCl2 used by other investigators (7, 8, 37). The70S ribosomes were dialyzed overnight against asolution containing 10-4 M MgCl2. After dialysis,the material was recentrifuged at 144,000 X g tosediment the 50 and 30S particles. RNA was pre-pared from the ribosomal fraction by ethanol pre-cipitation, a modification of the method of Crest-field et al. (6).

    Chemical and physical methods. The preparationswere standardized by measurement of absorbance at260 to 280 nm with a Beckman DU-2 spectrophotome-ter and by the orcinol method of Dische as discussedby Ashwell (1) by using mycobacterial RNA as thestandard. Protein was determined by the method ofLowry et al. (23) with crystalline bovine albumin asa standard. The quality of each preparation of RNAwas determined by the per cent increase in hyper-chromicity of a ribonuclease hydrolyzed portion, asdescribed earlier (41). The tests used to determinewhether the RNA was single or double stranded werebased primarily on those of Lampson et al. (19)and were as follows. (a) A small amount of ribo-nuclease (0.2 ,ug/20 ,ug of RNA) was used uponnative RNA and upon RNA which had been heatdenatured at 110 C for 7 to 8 min and then placedin ice to prevent reannealing of the strands. After theribonuclease was added to each of these two samples,and to single-stranded yeast RNA, absorption read-

    ings at 260 nm were taken at 5-min intervals at roomtemperature to observe increase in hyperchromicity.(b) Three per cent formaldehyde was added to anequal volume (40 ,g/ml) of mycobacterial RNAand to yeast RNA and incubated at 35 C for 4 hr;controls were included in which water was addedinstead of formaldehyde. The absorption of eachwas measured at 5-nm intervals from 220 through280 nm. (c) The effect of formaldehyde, in a concen-tration of 2.76%, upon the thermal transition point(Tm) of the RNA preparation was determined. (d)The approximate Tm was also measured in twoionic concentrations of 0.15 M NaCl plus 0.015 Msodium citrate (SSC), pH 7.0, and 0.1 SSC.The base composition of the RNA was determined

    by a modification of the paper chromatographymethod of Bendich, by using an isopropanol-hydro-chloride solvent system (3).

    Sucrose gradients. Linear 5 to 20% sucrose gra-dients were prepared approximately 18 hr before useand refrigerated.Volumes of 0.4 and 0.5 ml containing 2.0 and 2.5

    mg of ribosomal fraction and RNA, respectively,were layered upon the gradients. Alkaline phospha-tase, used as a point of reference, was contained in avolume of 0.1 ml and was layered on top of the ribo-somal and RNA preparations. Before use, the alka-line phosphatase was diluted 1:10 with 1.0 M this(hydroxymethyl)aminomethane (Tris) buffer, pH8.0, and dialyzed against Tris buffer for 24 hr toremove the ammonium sulfate present.

    It was necessary to use 10-l M MgCI2 in the 5 to20% sucrose gradients to obtain 70S ribosomeswithout some dissociation into 50 and 30S subunits.After dialysis and recentrifugation at 144,000 X gfor 3 hr, the 50 and 30S subunits were centrifuged in5 to 20% sucrose gradients containing 104 M MgC12.The centrifugations were done for 1.5 hr in a SW-39Spinco rotor at 39,000 rev/min. Two markers wereused, alkaline phosphatase, and an 80S mammalianbrain ribosomal marker (20) (kindly supplied byTerry C. Johnson).

    Since we had found that by using higher concen-trations of magnesium ions the particulate fractionswere not as immunogenic (38), smaller ribosomalsubunits were obtained by centrifugation of the ribo-somal preparations in sucrose gradients to which nomagnesium ions had been added.The ribosomal subunit preparations other than the

    50 and 30S were centrifuged at 39,000 rev/min in aSpinco SW-39 rotor for 2 hr, and the RNA prepara-tions were spun for 4 hr. At the end of this time, thegradients were collected manually from the bottomby gravity flow by using a very short 20-gauge needleto puncture the bottom of the tube. Each 2-dropfraction was collected in 3 ml of distilled water, andthe adsorption of each was measured at 260 nm.Alkaline phosphatase was measured by using freshlyprepared 0.001 M p-nitrophenyl phosphate in 1.0 MTris buffer, pH 8.0. In the assay, 0.1 ml of sucrosegradient fraction was added to 3.0 ml substratemixture and read at 410 nm, after 10 min of incuba-tion at room temperature.The approximate S values of the ribosomal and

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