TRANSLATIONAL FRAMESHIFT BASED DRUG SCREENING ASSAY

Abstract: This invention relates to newly developed methods for discovering antimicrobial compounds using a ribosome-based assay system. This invention also relates to compositions of matter useful in carraying out the methods of the invention as well as antimicrobial compounds developed using such methods.

Description: TRANSLATIONAL FRAMESHIFT BASED DRUG SCREENING ASSAY FIELD OF THE INVENTION This invention relates to newly developed methods for discovering antimicrobial compounds using a ribosome- or a ribosomal subunit-based assay system. This invention also relates to compositions of matter useful in carrying out the methods of the invention as well as antimicrobial compounds developed using such methods. BACKGROUND OF THE INVENTION The accuracy of protein synthesis is critical to cell viability. Translational infidelity at the level of the ribosome can arise as a result of misincorporation of an aminoacylated tRNA during the codon-anticodon interaction at the acceptor site (A site) or as a result of a frameshift such that the ribosome moves relative to the translation reading frame of the messenger RNA. As a consequence, protein synthesis continues in a new and incorrect reading frame and an aberrant protein is synthesized. Proof-reading of the aminoacyl-tRNAs is mediated by various interactions that include those between the 16S ribosomal RNA, mRNA and/or tRNA, elongation factor Tu (EF-Tu) and/or the ribosome, the release of the deacylated tRNA from the exit site (E site) on the ribosome and/or the rate of peptidyl transferase. These events offer a plethora of steps for intervention, binding, inhibition, or activation by compounds, particularly small molecular weight compounds. Selectivity of such compounds is believed to be attributable to the known differences between the prokaryote and eukaryote ribosomes and translation factors (See for example, review by H.G. Wittmann (1986) Structure of Ribosomes, p.1-27. In B. Hardesty and G. Kramer (ed.), Structure, Function and Genetics of Ribosomes. Springer- Verlag, New York). There is a need for methods of screening for novel antimicrobial compounds, such as the screening methods of the invention, that have a present benefit of being useful to screen compounds for antibiotic activity. Such compounds can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases, such as any disease caused by or related to infection by a bacteria, including, particularly caused by an organism described elsewhere herein. SUMMARY OF THE INVENTION An embodiment of the invention is a method of screening for antimicrobial drugs comprising the steps of: providing a composition comprising at least one ribosome or at least one ribosomal subunit, at least one messenger-like RNA and at least one aminoacyl- tRNA or at least one labeled tRNA or tRNA-analog with a label; contacting such composition with at least one candidate compound; and determining whether at least one frameshift occurs, by monitoring either abberrant polypeptide synthesis or retention of the labeled tRNA or tRNA analog complexed to the ribosome or ribosomal subunit. Preferred methods use label that is radioactive, chromogenic or fluorescent. A preferred embodiment provides a method wherein said polypeptide is a nascent polypeptide chain or a substantially purified polypeptide. A further preferred embodiment provides a method wherein the binding of said tRNA or tRNA analog to a ribosome, ribosome subunit or mRNA is detected. A further preferred embodiment provides a method wherein at least one ribosome or at least one ribosomal subunit is in substantially purified form. Yet another preferred embodiment provides a method wherein said ribosome or ribosomal subunit is selected from the group consisting of a eubacterial ribosome or ribosomal subunit, an archaebacterial ribosome or ribosomal subunit, a fungal ribosome or ribosomal subunit, and a protozoan ribosome or ribosomal subunit. Still another preferred embodiment provides a method wherein said ribosome or ribosomal subunit is isolated from an organism selected from the group consisting of: a (i) prokaryote, including but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinotnycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further including, but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostήdium perfringens, Clostridium tetani, Clostridium botulinum, Treponema pallidum, Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon, including but not limited to Archaebacter, and (iii) a unicellular or filamentous eukaryote, including but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces ceriviseae, Kluveromyces lactis, or Candida albicans. Herein this group of organisms is also referred to as "organism(s)." Another embodiment of the invention provides a method of screening for antimicrobial drugs comprising the steps of: providing a composition comprising at least one ribosome, or at least one ribosomal subunit, at least one messenger-like RNA, at least one aminoacyl-tRNA, or at least one labeled tRNA or at least one labeled tRNA analog and at least one candidate compound; and determining whether there is at least one frameshift in the polypeptide synthesized or acceptance of a tRNA in a site that overlaps the A site, structurally or functionally, but is distinct from, the A site. Another embodiment of the invention provides a method for screening for at least one antimicrobial agent comprising the steps of: providing a composition comprising at least one purified ribosome or at least one purified ribosomal subunit, programmed with at least one defined messenger-like RNA and at least one aminoacyl-tRNAs or at least one labeled tRNA or at least one labeled tRNA analog; non-enzymatically positioning a cognate tRNA in the ribosome P-site on the ribosome; either enzymatically or non-enzymatically positioning at least one cognate aminoacyl-tRNAs or at least one labeled tRNA analog in a site that overlaps the A site, structurally or functionally, but is distinct from, the A site; adding a candidate compound to the composition; and determining binding of the candidate labeled tRNA or tRNA-analog. A further embodiment of the invention provides a composition comprising at least one substantially purified ribosome or at least one substantially purified ribosomal subunit programmed with a defined messenger-like RNA and at least one cognate aminoacyl- tRNAs, or at least one labeled tRNA or at least one labeled tRNA analog enzymatically or non-enzymatically positioned in the ribosome P-site on the ribosome, and an enzymatically or non-enzymatically positioned cognate aminoacyl-tRNA or labeled tRNA or labeled tRNA analog in a site that overlaps the A site, structurally or functionally, but is distinct from, the A site. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1(A, B, C) shows a schematic diagram of an embodiment of the invention showing site A binding, no site A binding and competition for the site A binding due to a tRNA or tRNA analog that binds to a site that overlaps the A site, structurally or functionally, but is distinct from, the A site. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for screening for compounds alter the accuracy of translation, particularly those that do not inhibit protein synthesis per se. Applicants believed that such compounds will have an enormous impact on 'global' protein synthesis, and are therefore predicted to be bactericidal. An embodiment of the invention is a method of screening for antimicrobial drugs or drug precursors. A preferred embodiment in this regard comprises the steps of: providing a composition comprising at least one ribosome or ribosomal subunit, at least one messenger-like RNA and at least one aminoacyl-tRNA or labeled tRNA or labeled tRNA analog. Preferred messenger-like RNAs are prepared which serve as a template for translation. Any RNA sequence that may be translated to yield a polypeptide may be used in the invention, however, preferred RNAs are characterized by the presence of a Shine- Dalgarno sequence usually positioned 7-12 nucleotides upstream of a start codon, such as that which encodes methionine (AUG). Additional defined codons will be selected to measure frameshifting, particularly frameshifting in either the -1, -2, +1 or +2 positions. Natural messenger RNAs, preferably isolated ones, may also be used in the method of the invention. The next step comprises contacting this composition with at least one candidate compound. A "candidate compound," as used herein, may be any chemical element, compound or composition, particularly those suspected of being capable of binding to or interfering with any component of the translational machinery. Once the composition is contacted by a candidate compound, it is determined whether there is at least one frameshift such that a tRNA or tRNA analog is accepted other than that cognate to the A site and/or alters the amino acid sequence in a polypeptide synthesized. One may detect frameshifting directly, such as at the RNA level, or indirectly, such as by detecting alterations of the polypeptide sequence as compared to the wild type protein. If it is determined that there is a frameshift, the candidate compound may be useful as an antimicrobial compound. This may be readily determined using any of the many well-known methods for testing antimicrobial activity, such as, for example, by disk diffusion assay followed by an MIC determination. As used herein "polypeptide(s)" or "protein(s)" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. The term "polypeptide(s)" or "protein(s)" refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids. The term "polypeptide(s)" or "protein(s)" include, for example, those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, sulfation, transfer- RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993) and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth. Enz mol. 182:626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well. The determining step of the invention may be carried out in many ways in view of the teachings of the present invention. For example, binding is monitored by the introduction of a label into the amino acid or directly on to the tRNA or tRNA analog and retention of the ribosome-tRNA or ribosomal subunit-tRNA complex by filtration on to filter paper, any other filtration device or any other support. In this example, the free aminoacyl-tRNAs are washed off the filters. The peptidyl-tRNA does not bind the A site (Figure 1A). Cognate aminoacyl-tRNA will bind the A site once the P site is occupied (Figure IB). Compounds that promote a frameshift (for example in the +1 direction) can be monitored by the introduction of a second aminoacyl-tRNA that is cognate to the +1 position (Figure 1C). The aminoacyl-tRNA cognate to the A site can be labeled with for example, a radioisotope (e.g., l ^C) that is distinct from that used to label (^H) the aminoacyl-tRNA cognate to the A +1 codon. In this way, if both aminoacyl-tRNAs are present, compounds that cause a +1 frameshift will result in an altered ratio of ^C:^H, than in the absence of the drug. Frameshifts at +2, - 1 and -2 can also be monitored using the appropriate cognate aminoacyl-tRNAs or tRNA analog. A preferred embodiment provides a method wherein the polypeptide translated in the screening assay is a nascent polypeptide chain or a substantially purified polypeptide. In any case, the polypeptide in the assay may be labeled using any of the well-known polypeptide labeling methods, such as, radiolabeling or chromogenic labeling, or detected using an appropriate antibody and immunopreciptation reaction. It is preferred that the label used be readily observable or detectable, such as by being luminescent, radiolabeled, colored or fluorescent. Other preferred methods of the invention comprise labeled tRNA and/or tRNA analogue. Such labeling may be performed using any of the well-known RNA labeling methods, such as, radiolabeling or chromogenic labeling. It is preferred that the label used be readily observable or detectable, such as by being luminescent, radiolabeled, colored or fluorescent. A further preferred embodiment provides a method wherein a ribosome or ribosomal subunit is in substantially purified form. As used herein the terms "Isolated" and "substantially purified" each mean altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Ribosomes and or ribosomal subunits may be isolated using methods well known in the art, including, for example, density centrifugation. Yet another preferred embodiment provides a method wherein said ribosome or ribosomal subunit is selected from the group consisting of a eubacterial or ribosomal subunit ribosome, an archaebacterial ribosome or ribosomal subunit, a fungal ribosome or ribosomal subunit, and a protozoan ribosome or ribosomal subunit. Other organisms from which ribosomes and ribosomal subunits may be isolated from streptococci, staphylococci, enterococci E. coli, streptomyces and Bacillus subtilis cells, yeast cells, and Aspergillus cells. Ribosomes and ribosomal subunits from higher eukaryotes may be used in assays to show selectivity of compounds putatively identified as antimicrobial compounds using the method of the invention. Such ribosomes and ribosomal subunit useful in the method of the invention include, for example, CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells. Selectivity of a candidate compound as an antimicrobial may be shown by demonstrating that such compound is associated with frameshifting in an assay using a ribosome or ribosomal subunit from a microbe, but no, or substantially less framshifting is associated in an assay using a ribosome or ribosomal subunit from a higher eukaryote, particularly a human. Another embodiment of the invention provides a method for screening for antimicrobial agents comprising the steps of: providing a composition comprising purified ribosomes programmed with a defined messenger-like RNA and at least one aminoacyl- tRNA or tRNA analog. Cell-free assays using purified ribosomes programmed with a defined messenger-like RNA and aminoacyl-tRNAs are known . Ribosomes may be also be programmed by non-enzymatic positioning of appropriate cognate tRNA (e.g., N- acetylated-Phe tRNAP he on UUU or formyl-Met-tRNA met to AUG, among others) in the peptidyl-tRNA binding site (P-site) on the ribosome can be followed by the enzymatic positioning of cognate aminoacyl-tRNAs (in a ternary complex with elongation factor Tu (EF-Tu) and GTP) in the acceptor site (A site). Non-enzymatically positioning a cognate tRNA in the ribosome P-site on the ribosome is achieved, for example, as detailed by Ofengang, J., Liou, R., Kohut, J., Schwartz, I. and Zimmerman, R.A. (1979) Biochemistry 18, 4322-4332. Enzymatically positioning at least one cognate aminoacyl-tRNAs in the A site is achieved, for example, as detailed by Cunningham, P.R., Nurse, K, Bakin, A., Weitz ann, C, Pflumm, M. and Ofengand, J. (1992) Biochemistry 31, 12012-12022. Once the composition comprising the programmed ribosomes and cognate tRNAs treated as described in the forgoing is prepared, one or more candidate compounds are added to the composition to test whether any of the candidate compounds is associated with translational frameshifting. If a mixture of candidate compounds is associated with frameshifting, the mixture may be deconvoluted to determine which compound or compounds is active. One method to achieve this is to test each component of the mixture separately in the assay. Deconvolution may also be performed using any of the known deconvolution methods. It is preferred that the method of the invention is formatted for high throughput screening (herein "HTS"). Skilled artisans can readily adapt the method of the invention for HTS. A particularly preferred embodiment of the screening methods of the invention is a high throughput screen for compounds that interfere with the proper functioning of the ribosome, such as compounds that are associated with framshifting during translation. A further embodiment of the invention provides a composition comprising substantially purified ribosomes programmed with a defined messenger-like RNA. Such compositions may also comprise at least one aminoacyl-tRNAs (or tRNA analog) and/or a cognate tRNA non-enzymatically positioned in the ribosome P-site on the ribosome. These compositions may still further comprise a cognate aminoacyl-tRNA enzymatically positioned in the A site. Potential antimicrobial compounds identified using the method of the invention include, among other things, small organic molecules, polynucleotides, peptides, polypeptides and antibodies that bind the ribosome, ribosomal subunit or message, or mimic the activity of a tRNA. The Applicants believe that certain aminoglycosides will cause frameshifting and are thus preferred candidate compounds for screening. The term "Antibody(ies)" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library. Potential antagonists include a small molecule that binds to and occupies the a ribosomal site thereby preventing binding of natural factors, such as, for example, tRNAs, such that normal biological activity is prevented. Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules. The Applicants believe that that certain compounds screened using the methods of the invention could cause or be associated with the rapid release of tRNA by the translation factor thereby enhancing misincorporation and/or frameshifting. This mechanism of action is provided as a non-limiting example to allow skilled artisans to better understand the invention. Moreover, this type of frameshift error, as well as others, may be detected using the methods of the invention and compounds that cause or are associated with such errors are expected to be effective antimicrobial compounds. The invention further provides assay packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. Each reference disclosed herein is incorporated by reference herein in its entirety. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety.

Claims: What is claimed is: 1. A method of screening for antimicrobial drugs comprising the steps of: providing a composition comprising at least one ribosome or ribosomal subunit, at least one messenger-like RNA and at least one aminoacyl-tRNA; contacting such composition with at least one candidate compound; and determining whether there is at least one frameshift in a gene product synthesized. 2. The method of claim 1 wherein said gene product is a nascent polypeptide chain or a substantially purified polypeptide. 3. The method of claim 1 wherein said ribosome or ribosomal subunit is in substantially purified form. 4. The method of claim 1 wherein said ribosome or ribosomal subunit is selected from the group consisting of a eubacterial ribosome or ribosomal subunit, an archaebacterial ribosome or ribosomal subunit, a fungal ribosome or ribosomal subunit, and a protozoan ribosome or ribosomal subunit. 5. A method of screening for antimicrobial drugs comprising the steps of: providing a composition comprising at least one ribosome or ribosomal subunit, at least one messenger-like RNA, at least one aminoacyl-tRNA, and at least one candidate compound; and determining whether there is at least one frameshift in the gene product synthesized. 6. The method of claim 5 wherein said gene product is a nascent polypeptide chain or a substantially purified polypeptide. 7. The method of claim 5 wherein said ribosome or ribosomal subunit is in substantially purified form. 8. The method of claim 5 wherein said ribosome or ribosomal subunit is selected from the group consisting of a eubacterial ribosome or ribosomal subunit, an archaebacterial ribosome or ribosomal subunit, a fungal ribosome or ribosomal subunit, and a protozoan ribosome or ribosomal subunit. 9. The method of claim 1 wherein said determining step comprises detecting a detectable marker. 10. The method of claim 5 wherein said determining step comprises detecting a detectable marker. 11. The method of claim 1 wherein said determining step comprises detecting a detectable marker on said gene product. 12. The method of claim 5 wherein said determining step comprises detecting a detectable marker on said gene product. 13. A method for screening for antimicrobial agents comprising the steps of: providing a composition comprising purified ribosomes programmed with a defined messenger-like RNA and at least one aminoacyl-tRNAs; non-enzymatically positioning a cognate tRNA in the ribosome P-site on the ribosome; enzymatically positioning at least one cognate aminoacyl-tRNAs in the A site; adding a candidate compound to the composition; and determining binding of the candidate compound. 14. A composition comprising substantially purified ribosomes programmed with a defined messenger-like RNA and at least one aminoacyl-tRNAs, a non- enzymatically positioned cognate tRNA in the ribosome P-site on the ribosome, and an enzymatically positioned cognate aminoacyl-tRNA in the A site. 15. The method of claim 1 wherein said ribosome or ribosomal subunit is isolated from an organism selected from the group consisting of: a (i) prokaryote, including but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further including, but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainβuenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens. Vibrio cholera, Shigella dysenterii, Shigella flexneri, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostridium perfringens, Clostridium tetani, Clostridium botulinum, Treponema pallidum, Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon, including but not limited to Archaebacter, and (iii) a unicellular or filamentous eukaryote, including but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces ceriviseae, Kluveromyces lactis, or Candida albicans. 16. The method of claim 5 wherein said ribosome or ribosomal subunit is isolated from an organism selected from the group consisting of: a (i) prokaryote, including but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma, and further including, but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, Actinomvctes israelii, Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostridium perfringens, Clostridium tetani, Clostridium botulinum, Treponema pallidum, Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon, including but not limited to Archaebacter, and (iii) a unicellular or filamentous eukaryote, including but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces ceriviseae, Kluveromyces lactis, or Candida albicans.

Inventors: NURSE KELVIN CHRISTOPHER, PRESCOTT CATHERINE DENISE, NURSE, KELVIN, CHRISTOPHER, PRESCOTT, CATHERINE, DENISE
Applicants: NURSE KELVIN CHRISTOPHER, PRESCOTT CATHERINE DENISE, SMITHKLINE BEECHAM CORP, NURSE, KELVIN, CHRISTOPHER, PRESCOTT, CATHERINE, DENISE, SMITHKLINE BEECHAM CORPORATION
Application year: 1998
Main classification: C12Q1
IPC codes: A61K45, C12N15, C12P21, C12Q1