Ribosomal Preparations Research Articles

Preparation of ribosomes loaded with truncated nascent proteins to study ribosome binding to mammalian mitochondria

Supporting a co-translational model of protein import into mitochondria, we have previously shown that ribosome–nascent chain complexes (RNCs) specifically bind to mitochondria. When producing RNCs using the rabbit reticulocyte lysate in vitro translation system, it was necessary to maximize ribosome loading with truncated nascent proteins because it had a direct impact on RNC binding. We describe here the optimal conditions for preparing RNCs. We show that translation temperature and reaction time are two critical factors, with 30 °C and 15 min being optimal, respectively. We also show that transcription reactions can be used directly in the translation reaction to create RNCs.

Methyltransferase Erm(37) Slips on rRNA to Confer Atypical Resistance in Mycobacterium tuberculosis

Members of the Mycobacterium tuberculosis complex possess a resistance determinant, erm(37) (also termed ermMT), which is a truncated homologue of the erm genes found in a diverse range of drug-producing and pathogenic bacteria. All erm genes examined thus far encode N(6)-monomethyltransferases or N(6),N(6)-dimethyltransferases that show absolute specificity for nucleotide A2058 in 23 S rRNA. Monomethylation at A2058 confers resistance to a subset of the macrolide, lincosamide, and streptogramin B (MLS(B)) group of antibiotics and no resistance to the latest macrolide derivatives, the ketolides. Dimethylation at A2058 confers high resistance to all MLS(B) and ketolide drugs. The erm(37) phenotype fits into neither category. We show here by tandem mass spectrometry that Erm(37) initially adds a single methyl group to its primary target at A2058 but then proceeds to attach additional methyl groups to the neighboring nucleotides A2057 and A2059. Other methyltransferases, Erm(E) and Erm(O), maintain their specificity for A2058 on mycobacterial rRNA. Erm(E) and Erm(O) have a full-length C-terminal domain, which appears to be important for stabilizing the methyltransferases at their rRNA target, and this domain is truncated in Erm(37). The lax interaction of the M. tuberculosis Erm(37) with its rRNA produces a unique methylation pattern and confers resistance to the ketolide telithromycin.

Reproducible growth of well diffracting ribosomal crystals

The crystallization of ribosomal particles is associated with extraordinary challenging demands. This originates mainly from the ribosome's natural tendency to deteriorate and from its multi-conformational heterogeneity, both of which stem from its functional flexibility. To increase the level of homogeneity of ribosomal preparations, systematic searches for conditions yielding populations of fully defined chemical compositions were employed and the variables essential for high functional activity were analyzed and optimized. These include temperature, cell-growth duration and media, the cell-harvesting stage, ribosomal purification and storage. The functional state that is most suitable to yield quality crystals was identified as that of the polysome and it was found that this fraction reproducibly yielded crystals of superior properties.

Exploiting the complementary nature of LC/MALDI/MS/MS and LC/ESI/MS/MS for increased proteome coverage

The goal of this work was to evaluate the improvement in proteome coverage of complex protein mixtures gained by analyzing samples using both LC/ESI/MS/MS and LC/MALDI/MS/MS. Parallel analyses of a single sample were accomplished by interfacing a Probot fractionation system with a nanoscale LC system. The Probot was configured to perform a post-column split such that a fraction (20%) of the column effluent was sent for on-line LC/ESI/MS/MS data acquisition, and the majority of the sample (80%) was mixed with a matrix solution and deposited onto the MALDI target plate. The split-flow approach takes advantage of the concentration sensitive nature of ESI and provides sufficient quantity of sample for MALDI/MS/MS. Hybrid quadrupole time-of-flight mass spectrometers were used to acquire LC/ESI/MS/MS data and LC/MALDI/MS/MS data from a tryptic digest of a preparation of mammalian mitochondrial ribosomes. The mass spectrometers were configured to operate in a data dependent acquisition mode in which precursor ions observed in MS survey scans are automatically selected for interrogation by MS/MS. This type of acquisition scheme maximizes the number of peptide fragmentation spectra obtained and is commonly referred to as shotgun analysis. While a significant degree of overlap (63%) was observed between the proteins identified in the LC/ESI/MS/MS and LC/MALDI/MS/MS data sets, both unique peptides and unique proteins were observed by each method. These results demonstrate that improved proteome coverage can be obtained using a combination of these ionization techniques.

Ribosomal immunotherapy for recurrent respiratory tract infections in children.

Recurrent respiratory tract infections are common in children. They reflect the immaturity of the immune system in its encounter with environmental antigens. Little or no specific protective immune response has yet been established. These infections represent an important public health problem in terms of both treatment (anti-inflammatory or antibacterial drugs for children) and economy. Immunotherapy has been proposed as a means of preventing these recurrent infections by providing children with small doses of inactive bacterial antigens liable to trigger specific and protective immune responses. Among such drugs, ribosomal preparations (to which this review is limited) appear to be not only well tolerated, but also ideally targeted to induce mucosal responses. One preparation of ribosomal mucosal vaccine is commercially available in several countries. Numerous clinical trials in the world have confirmed the positive role of this mucosal ribosomal bacterial vaccine in significantly reducing the number of infections, courses of antibacterials, and absenteeism. In vitro and ex vivo investigations have confirmed that such vaccines indeed trigger protective specific immune responses.

Altered Hepatic Mitochondrial Ribosome Structure Following Chronic Ethanol Consumption

Chronic ethanol consumption decreases the synthesis of all 13 polypeptides encoded by the hepatic mitochondrial genome. This alteration in mitochondrial protein synthesis is due to modifications in mitochondrial ribosomes. In the current study, the nature of these alterations was investigated by determining some of the hydrodynamic properties, namely sedimentation coefficient, shape, and mass of mitochondrial ribosomes. The effect of ethanol consumption on the capacity for mitochondrial ribosomes to translate proteins was also determined using an in vitro Poly (U) assay system. Rats were fed the Lieber–DeCarli diet for 31 days with ethanol as 36% of total calories. The sedimentation coefficient, measured by sedimentation velocity analyses, was slightly, but significantly lower in ethanol mitochondrial ribosomes (53.2 ± 0.5S) when compared with pair-fed controls (54.1 ± 0.5S) (P = 0.04). Mitochondrial ribosomes from ethanol-fed animals also had a greater tendency to dissociate into subunits. The diffusion coefficient, determined by dynamic light scattering, was lower in mitochondrial ribosomes from ethanol-fed rats than pair-fed controls and this indicated a significantly greater diameter for ethanol ribosomes (42.1 ± 0.2 nm) than for preparations from pair-fed controls (39.1 ± 0.5 nm; P = 0.008). These alterations to ethanol mitochondrial ribosomes occurred despite no change in molecular mass, which suggested a significant ethanol-related shape change in the ribosomes. The translation capacity of mitochondrial ribosome preparations from ethanol-fed animals was markedly reduced due to dissociation of the monosome into light and heavy subunits. In summary, these observations demonstrate that chronic ethanol consumption causes significant structural and functional alterations to mitochondrial ribosomes. The loss in ribosome function leads to impaired mitochondrial polypeptide synthesis and is an example of a pathology giving rise to an alteration in the mitochondrial ribosome structure.

A dynamic competition between release factor 2 and the tRNASec decoding UGA at the recoding site of Escherichia coli formate dehydrogenase H

Factors affecting competition between termination and elongation in vivo during translation of the fdhF selenocysteine recoding site (UGA) were studied with wild-type and modified fdhF sequences. Altering sequences surrounding the recoding site UGA without affecting RNA secondary structure indicated that the kinetics of stop signal decoding have a significant influence on selenocysteine incorporation efficiency. The UGA in the wild-type fdhF sequence remains 'visible' to the factor and forms a site-directed cross-link when mRNA stem-loop secondary structure is absent, but not when it is present. The timing of the secondary structure unfolding during translation may be a critical feature of competition between release factor 2 and tRNA(Sec) for decoding UGA. Increasing the cellular concentration of either of these decoding molecules for termination or selenocysteine incorporation showed that they were able to compete for UGA by a kinetic competition that is dynamic and dependent on the Escherichia coli growth rate. The tRNA(Sec)-mediated decoding can compete more effectively for the UGA recoding site at lower growth rates, consistent with anaerobic induction of fdhF expression.

Translocation of Ribosomal Immunostimulant Through an In Vitro–Reconstituted Digestive Barrier Containing M‐Like Cells

Ribosomal preparations of pathogenic micro‐organisms of the upper respiratory tract can be delivered orally for the prevention of recurrent infectious episodes, because they induce mucosal and protective immune responses. The mechanism of mucosal barrier translocation is difficult to study in animal models, little is therefore known about this process. In order to circumvent these problems, we have examined the uptake of ribosomal preparations in three experimental systems that model human intestinal cells. We found that M‐like cells displayed a 8.7‐fold increase in the uptake of a ribosomal immunostimulant when compared to absorptive or crypt enterocyte‐like cells. The product was taken up, translocated, and delivered in the basolateral compartment by cultured M‐like cells. No translocation was observed across monolayers of T84 cells (model of crypt cells). Only minimal translocation occured through monolayers of Caco‐2 cells (model of absorptive enterocytes). This suggests that, in vivo, colyophilisat is delivered mainly through the M cells overlying lymphoid follicles (Peyer's patches) or nodules of the gut‐associated mucosal lymphoid tissue, which are the major inductor sites of mucosal responses. Use of the M‐like cell cultured model could be a key step for the development of even more efficient immunostimulators in animals and human.

Contrast analysis of the composition of ribosomes extracted with different purification procedures

The composition and hydration ofE. coliribosomes isolated with different purification protocols has been analysed by combining two experimental techniques: measurements of small-angle neutron scattering (SANS), for two different isotopic solvent compositions, and refractive index (RI) increments. From the contrast between the solvent and solute scattering densities and the molar polarizability, determined experimentally with SANS and RI measurements, three independent equations are obtained and three unknown quantities are determined: (i) the volume of the solute hydrated skeletonVs, (ii) the material contained in it, namely the biological components, intrinsic (rRNA and proteins) and extrinsic, such as aminoacylsynthetase and elongation factors, (iii) the number of water molecules structurally bound to the ribosome and non-exchangeable with the solvent. From the form factor at infinite contrast, a second definition of the solute volume is obtained, V_s^c, which represents the volume within the contour surface of the ribosome. This value is generally larger thanVsand can include a certain amount of water molecules,i.e.those inside the volume (V_s^c −Vs). Considering the molar volume of this water to be equal to that of the bulk water, it is possible to evaluate its amount. The particle density calculated from the ribosome components in V_s^c, including proteins, RNA, bound and unbound water molecules, corresponds to the buoyant density measured forE. coli70S particles. The two ribosomal preparations display different performances in protein synthesis; hence the results indicate that the optimal condition corresponds to a wider skeleton and contour volume but containing a smaller amount of segregated water molecules. It is believed that the method provides a reliable technique to determine the composition of ribosomes under various experimental conditions.

Two trans-acting rat-brain proteins, MARTA1 and MARTA2, interact specifically with the dendritic targeting element in MAP2 mRNAs

Different isoforms of the microtubule-associated protein 2 (MAP2) are somatodendritic components of neurons that seem to regulate the stability of the dendritic cytoskeleton. MAP2 localization into dendrites appears to be a complex multicausal mechanism that involves the specific recruitment of MAP2 mRNAs into dendritic compartments. Recently, we have functionally characterized a 640-nucleotide dendritic targeting element (DTE) in the 3′ untranslated region (3′ UTR) of MAP2 transcripts that mediates extrasomatic mRNA localization in primary neurons (Blichenberg et al., 1999). In analogy to molecular mechanisms regulating cytoplasmic RNA translocation in other cell systems, we propose that, in vivo, the cis-acting MAP2-DTE interacts with specific protein factors present in neurons. To identify putative trans-acting DTE-binding proteins, we performed in vitro ultraviolet crosslinking assays. Using this experimental system, two 90-kDa and 65-kDa MA P2- R NA t rans - a cting proteins, MARTA1 and MARTA2, were identified in rat-brain extracts. Both MARTAs bind with high affinity to the MAP2-DTE, but not to other investigated regions of MAP2 transcripts or the somatically restricted α-tubulin mRNA. Moreover, MARTA1 and MARTA2 do not bind significantly to other dendritically localized transcripts encoding vasopressin and arg3.1, nor to a dendritic trafficking element from the mRNA encoding the α-subunit of the Ca 2+/calmodulin-dependent protein kinase II. Binding of MARTA1 and MARTA2 to the MAP2-DTE occurs with an affinity in the nanomolar range. Whereas MARTA1 is clearly detectable in crude lysates, cytosolic and ribosomal salt-wash fractions, and in nuclear extracts, MARTA2 is preferentially found in the ribosomal salt-wash preparation. Neither MARTA is restricted to rat brain, and both are present in a number of other rat tissues. Thus, both proteins may be involved in a variety of nuclear and cytoplasmic events that regulate RNA metabolism in different cell types.

Characterization of Escherichia coli 50S ribosomal protein L31

The published C-terminal sequence of Escherichia coli 50S ribosomal protein L31, …RFNK (Brosius, J. (1978) Biochemistry 17, 501–508), differs from that predicted by the gene sequence, …RFNKRFNIPGSK (GenBank accession no. X78541). This discrepancy might be due to post-translational processing of the protein. To examine this possibility, we have isolated L31 from E. coli strain MRE600 and sequenced the C-terminal tryptic peptide. We find the sequence to be FBIPGSK. Size comparisons of L31 from several E. coli strains demonstrate that all are identical in size to the protein isolated from MRE600 and larger than the previously described protein, indicating that …RFNKRFNIPGSK represents the true C-terminus of L31. In addition, we show that the failure to identify L31 in many ribosome preparations is probably due to the protein’s loose association with the ribosome and its ability to form various intramolecular disulfide bonds, leading to L31 forms with distinct mobilities in gels.

Characterization ofEscherichia coli50S ribosomal protein L31

The published C-terminal sequence of Escherichia coli 50S ribosomal protein L31, ellipsisRFNK (Brosius, J. (1978) Biochemistry 17, 501-508), differs from that predicted by the gene sequence, ellipsisRFNKRFNIPGSK (GenBank accession no. X78541). This discrepancy might be due to post-translational processing of the protein. To examine this possibility, we have isolated L31 from E. coli strain MRE600 and sequenced the C-terminal tryptic peptide. We find the sequence to be FBIPGSK. Size comparisons of L31 from several E. coli strains demonstrate that all are identical in size to the protein isolated from MRE600 and larger than the previously described protein, indicating that ellipsisRFNKRFNIPGSK represents the true C-terminus of L31. In addition, we show that the failure to identify L31 in many ribosome preparations is probably due to the protein's loose association with the ribosome and its ability to form various intramolecular disulfide bonds, leading to L31 forms with distinct mobilities in gels.

Inhibition of RNA polymerase III transcription by a ribosome-associated kinase activity.

Ribosomes prepared from somatic tissue of Xenopus laevis inhibit transcription by RNA polymerase III. This observation parallels an earlier report that a high speed fraction from activated egg extract, which is enrichedin ribosomes, inhibits RNA polymerase III activityand destabilizes putative transcription complexes assembled on oocyte 5S rRNA genes. Transcription of somatic- and oocyte-type 5S rRNA genes and a tRNA gene are all repressed in the present experiments. We find that 5S rRNA genes incubated in S150 extract prepared from immature oocytes exhibit an extensive DNase I protection pattern that is nearly identical to that of the ternary complex of TFIIIA and TFIIIC bound to a somatic 5S rRNA gene. The complexes formed in this extract are stable at concentrations of ribosomes that completely repress transcription, indicating that formation of the TFIII(A+C) complex is not the target of inhibition. Ribosomes taken through a high salt treatment no longer repress transcription of class III genes, establishing that the inhibition is due to an associated factor and not the particle itself. The inhibitory activity released from ribosomes is inactivated by treatment with proteinase K, but not micrococcal nuclease. Preincubation of ribosomes with a general protein kinase inhibitor, 6-dimethylaminopurine, eliminates repression of transcription. Western blot analysis demonstrates that p34(cdc2), which is known to mediate repression of transcription by RNA polymerase III, is present in these preparations of ribosomes and can be released from the particles upon extraction with high salt. These results establish that a kinase activity, possibly p34(cdc2), is the actual agent responsible for the observed inhibition of transcription by ribosomes.

Ribosomes from an oxazolidinone-resistant mutant confer resistance to eperezolid in a Staphylococcus aureus cell-free transcription-translation assay.

Oxazolidinone-resistant mutants of Staphylococcus aureus, isolated with a spiral plating technique, had a 16-fold higher MIC (2 versus 32 microg/ml) of eperezolid when compared to the parental sensitive strain. Eperezolid inhibited in vitro protein translation with 50% inhibitory concentrations of 30 microM for the oxazolidinone-sensitive S30 extract and 75 microM for the resistant extract. Experiments mixing various combinations of S100 and crude ribosome preparations from oxazolidinone-sensitive and -resistant S. aureus strains in a transcription-translation assay demonstrated that the resistant determinant resided within the ribosomal fraction. Ribosomes from the oxazolidinone-resistant strain bound less drug than ribosomes from the sensitive strain, indicating that the ribosome is the site of action for the oxazolidinones. These experiments demonstrate that an alteration of the ribosome is responsible for some or all of the oxazolidinone resistance observed in the S. aureus mutant.

From Peyer's patches to tonsils. Specific stimulation with ribosomal immunotherapy.

Ribosomal immunotherapy has been successfully used since the 1960s to boost the immune system and provide protection against microbial infections. We have investigated both whether and how these immunostimulants behave as natural immunogens in the mucosa-associated immune system. According to current understanding of the physiology of the mucosal immune response, intestinal Peyer's patches and the related solitary nodules are the primary inductive sites involved in the immune protection of all mucosal surfaces. Sensitised lymphocytes generated at these sites reach the general circulation through lymphatic drainage and relocate in mucosal areas by means of specialised 'high endothelial venules'. We hypothesised that orally administered ribosomal preparations would yield sensitised B cells specific for bacterial antigens from the parent strains. These cells should then be detectable in the peripheral blood after ribosomal intake, and identifiable as plasma cells in mucosae-associated tissues after completing their terminal differentiation. Ultimately, specific IgA should appear in secretions. To this end, we studied the immune responses generated in children and adults after 'Ribomunyl' administration, according to various consecutive protocols. The initial hypothesis was confirmed by the identification of specific B cells in the peripheral blood, plasma cells in the tonsillar tissue and specific IgA in the saliva. An animal model involving the use of twin sheep enabled detection of the specific cells in mesenteric and cervical lymph nodes. Analysis of these data indicates that ribosomal preparations trigger the production of lymphocytes specific for both ribosomes themselves and whole bacterial antigens. This supports the fact that small antigenic motifs are carried as partly synthesised peptides on the ribosomal particles. Therefore, ribosomes boost an array of B cells that are specific for many antigenic determinants of the bacteria from which they are extracted. We were also able to show that the stimulation provided was specific, since no response to other bacteria could be detected. Finally, analysis of the kinetics of this stimulation confirmed that oral immunisation generates rapid and transient secretory responses, building increased numbers of memory cells that are readily available to respond to further challenges by either more ribosomal preparations or potential pathogens.

Use of Bacterial Ribosomal Immunostimulators in Respiratory Tract Infections

Bacterial ribosomes, composed of proteins and RNA, have been used for nearly 30 years as immunostimulators. Preparations from about 30 different species of bacteria, as well as from fungi or parasites, have been used. Ribosomes have been tested in a number of animal models, and demonstrated to induce both specific immunity and protection. Paradoxically, although only a small part of these studies have dealt with micro-organisms involved in respiratory infections, most of the clinical trials involving ribosomes have used preparations obtained from such bacteria. Ribosomal preparations have been shown to reduce and prevent recurrent infections of the upper respiratory tract in humans.

Insights into erythromycin action from studies of its activity as inducer of resistance

Appreciation of the inducibility of erythromycin resistance began as an observation in the clinical bacteriology laboratory during susceptibility testing of erythromycin-resistant clinical isolates of Staphylococcus aureus. It was noted that inhibition zones surrounding spiramycin, lincomycin, and pristinamycin I (streptogramin B family) test disks placed close to an erythromycin test disk deviated from the expected circular shape and assumed a distorted ‘‘D’’ shape instead. Such observations suggested a possible antagonistic interaction between erythromycin, on the one hand, and spiramycin, lincomycin, or pristinamycin I, on the other (5, 7). The interaction turned out to be a functional rather than a physical antagonism, and out of these observations grew the notion of erythromycin-inducible resistance toward erythromycin, initially (41, 55), and then, more generally, toward all the macrolide, lincosamide, and streptogramin type B (MLS) antibiotics (57). Vazquez (50) and Vazquez and Monro (52) showed that antibiotics belonging to each subclass of the MLS antibiotics competed with chloramphenicol for uptake by intact cells. Competition for binding to purified 50S ribosome subunits was shown only for macrolides and lincosamides but not for streptogramin type B antibiotics. Collectively, these observations suggested that an alteration of 50S subunit function was involved in resistant cells. In a study of the time and concentration dependence of induction, Weisblum et al. (58) showed that (i) the optimal erythromycin concentration for induction was between 10 and 100 ng/ml, the threshold of its inhibitory action, (ii) at the optimal inducing concentration of erythromycin cells became phenotypically resistant within 40 min, and (iii) ribosomes from induced cells apparently bound labeled erythromycin and lincomycin with a reduced affinity. By mixing ribosome preparations from susceptible and resistant cells and noting no loss of expected antibiotic binding activity, it was possible to exclude the alteration of the antibiotic by modifying enzymes present as contaminants in the ribosome preparation. Consistent with this picture, Allen (1) showed that cell extracts of resistant S. aureus carried out erythromycin-resistant protein synthesis in vitro, suggesting that a component of the protein-synthesizing machinery had been altered. A posttranscriptional methylation of a single adenine residue in 23S rRNA, comprising the induced biochemical alteration (30, 31), was located at Escherichia coli coordinate 2058 (A-2058) (47), and translational attenuation (16, 24), the mechanism for its regulation, was proposed. As discussed below, this unusual mechanism of gene regulation requires no repressor proteins but, instead, is based on the conformational isomerization of the ermC message to a translationally active form. How might this be achieved?

Bacterial crude extracts or ribosomes are recognized similarly by peripheral and mucosal B cells

Bacterial ribosomes have been shown to induce effective humoral and cellular immunological responses to whole microorganisms. In this study, the numbers of specific antibody producing cells directed towards Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae ribosomes or whole bacteria sonicates were compared in the peripheral blood and tonsils of 7 children, and in the tonsils, mesenteric and cervical lymph nodes of 10 sheep. No significant difference was noted between the two types of antigens, confirming that ribosomal preparations are able to mimic the immunogenicity of whole bacteria in the mucosae-associated lymphoid tissue.

Drosophila ribosomal protein S3 contains an activity that cleaves DNA at apurinic/apyrimidinic sites.

A rat cDNA-encoding ribosomal protein S3 was used to clone the S3 homolog in Drosophila melanogaster. The Drosophila gene was in turn used to construct fusions between S3 and glutathione S-transferase that were overexpressed in Escherichia coli, purified on affinity columns, and subsequently used for antibody production and biochemical analysis. Antibody specific for S3 was originally tested to determine the subcellular location of S3 by Western (immunoblot) analysis. As expected, the S3 antigen was found associated with purified preparations of ribosomes, but notably the protein was also observed in the nucleus where it was found to be tightly associated with the nuclear matrix. This result, combined with the fact that S3 contains a nuclear localization signal and that the protein shares some homology to a yeast nuclease gene, suggested that S3 might possibly have a role in DNA metabolism. Tests were initially performed to see if S3 contained DNase activity, where it was subsequently determined that the protein specifically cleaved DNA containing an apurinic/apyrimidinic site via a beta-elimination reaction. The DNase activity was inactivated by antibody to S3, indicating that the apurinic/apyrimidinic lyase activity was associated with the Drosophila S3 protein. Taken together, these results suggest that S3 is among a growing class of multifunctional proteins with roles in transcription/translation and DNA repair.

Compartmentalization of specific B-cells in sheep mucosae associated lymphoid organs

Numerous studies have shown that Peyer's patches (PP) contribute to the seeding of other lymphoid organs in sheep. This was demonstrated by perfusing labeled lymphocytes in PP, and later investigating their presence in drainage lymph nodes, spleen, peripheral blood or bone marrow. These data showed that PP export considerable numbers of cells every day, but provided no information as to their specificity. In this work, we used the enzyme-linked immunosorbent assay (ELISA) spot method to investigate, in the peripheral blood, mesenteric and cervical lymph nodes and tonsils from ten sheep, the numbers of specific B-cells, directed to four common bacteria of the oro-pharyngeal area of mammals: Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae or Klebsiella pneumoniae. The data were obtained from five sets of monozygous sheep, one animal of each pair being previously fed ribosomal preparations of these bacteria. Both prior to and after oral challenge, specific B-cells could be found in all the tissues tested. They were mostly IgG-producing cells and preferentially located in oro-pharyngeal drainage lymph nodes and tonsils. Their numbers increased in these lymph nodes after stimulation, while they decreased in mesenteric lymph nodes. These observations are consistent with the current hypothesis suggesting intestinal sensitization, proliferation and fast emigration of specific B-cells after oral challenge.