JP2001514025A - New RNASEP - Google Patents

Abstract

  (57) [Summary] The present invention relates to a novel ribonucleoprotein complex and a component thereof. More particularly, the present invention relates to RNaseP isolated from S. pneumoniae and the use of RNaseP or a component thereof in screening for the identification of antimicrobial compounds and the use of such compounds in therapy.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention relates to newly identified polynucleotides and polypeptides, as well as catalytic RNAs encoded by certain of these polynucleotides, molecular complexes of RNA and polypeptides, such polynucleotides and polypeptides. It relates to the use of peptides, and to the production of such polynucleotides and polypeptides and to recombinant host cells transformed with the polynucleotides. The invention relates in particular to the genus Streptococcus, in particular to Streptococcus pneumoniae.
s pneumoniae, also referred to as "S pneumoniae"). The invention also relates to inhibiting the biosynthesis, assembly or action of such polynucleotides and / or polypeptides, and further relates to the use of such inhibitors in therapy.

BACKGROUND OF THE INVENTION The present invention relates to novel bacterial ribonucleoprotein complexes and their constituent parts. More particularly, the invention relates to RNaseP, in particular RNaseP from S. pneumoniae, and RnaseP in screening for the identification of antimicrobial compounds.
It relates to the use of NaseP or its components as well as the use of such compounds in therapy. Streptococci form a medically important microbial genus, and in humans, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, empyema and endocarditis, most particularly Are known to cause several types of diseases, including meningitis such as, for example, cerebrospinal fluid infection. Since more than 100 years have passed since the isolation of the genus Streptococcus, Streptococcus pneumoniae is one of the microorganisms that has been studied in more detail. For example, in fact, much of the early view that DNA is the genetic material was stated in Griffith's and Avery, Macleod and McCarty studies using this microorganism. Despite the vast amount of research on Streptococcus pneumoniae, many questions remain regarding the toxicity of this microorganism. It is particularly preferred to use Streptococcus genes and gene products as targets for the development of antibiotics.

[0003] The frequency of Streptococcus pneumoniae infections has increased dramatically over the past two decades. This has been attributed to the emergence of multiple antibiotic resistant strains and an increasing population of people with a compromised immune system. It is no longer uncommon to isolate Streptococcus pneumoniae strains that are resistant to some or all of the standard antibiotics. This phenomenon has created a need for new antimicrobial agents, vaccines and diagnostic tests for this organism.

[0004] Although certain Streptococcus factors associated with virulence have been identified, additional targets are always useful. This is because antimicrobial screening targets often bias the results. Thus, new targets, such as the RNase P of the present invention, will enable the discovery of a new class of antimicrobial agents.

[0005] BRIEF DESCRIPTION OF THE INVENTION The present invention, novel ribonucleoprotein complex, in particular, complex consuming from S. pneumoniae, and separately provides isolated the RNA and protein components. According to another aspect of the invention, there is provided a polynucleotide (DNA or RNA) encoding the protein and RNA components of such a complex. In particular, the present invention provides polynucleotides having the DNA and RNA sequences provided herein. The invention also relates to novel oligonucleotides derived from the sequences described herein, which can act, for example, as antisense inhibitors on the expression of RNA or protein components. Using an oligonucleotide or a fragment or derivative thereof,
Catalytic activity can be directly inhibited, or activity can be indirectly inhibited by interfering with RNA protein complex formation. The protein and RNA components, either separately or in a complex, are also useful as targets in screens designed to identify antimicrobial compounds. It is an object of the present invention to provide a polynucleotide that is transcribed to RNase P, especially a catalytic RNA. It is a further object of the present invention to provide a polynucleotide isolated from Pneumoniae using the nucleic acid sequence shown in SEQ ID NO: 1 or 5 as a probe or primer. It is a further object of the present invention to provide a polynucleotide isolated from Pneumoniae set forth in SEQ ID NO: 5, and variants thereof. According to another aspect of the present invention there is provided an isolated nucleic acid molecule transcribed into RNase P RNA by S. pneumoniae strain 0100993 contained in the deposited strain.

According to a further aspect of the present invention there is provided an isolated nucleic acid molecule encoding RNase P RNA, in particular RNase P of Streptococcus pneumoniae, including mRNA, cDNA, genomic DNA. You. Further embodiments of the present invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising them. According to another aspect of the present invention there is provided the use of a polynucleotide of the present invention for therapeutic or prophylactic purposes, particularly for genetic immunity. Particularly preferred embodiments of the present invention include naturally occurring allelic variants of RNase P and the polypeptides encoded thereby. In another aspect of the present invention, novel polypeptides of Streptococcus pneumoniae, referred to herein as RNaseP, and fragments, variants thereof that are useful biologically, diagnostically, prophylactically, clinically or therapeutically And derivatives, and variants and derivatives of the foregoing fragments and analogs, and compositions comprising them. Particularly preferred embodiments of the invention include variants of the RNaseP polypeptide encoded by naturally occurring alleles of the RNaseP gene. In a preferred embodiment of the present invention, there is a method for producing the RNaseP polypeptide. According to yet another aspect of the present invention, there are provided inhibitors of such polypeptides, including, for example, antibodies, which are useful as antibacterial agents. According to certain preferred embodiments of the present invention, assessing RNaseP expression, treating diseases such as otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, empyema and endocarditis, most particularly For example, treatment of meningitis, such as infection of the cerebrospinal fluid, assays for genetic variation, and the use of RNaseP polypeptides or polynucleotides to produce an immunological response against bacteria, especially Streptococcus pneumoniae bacteria. Products, compositions and methods for administration are provided. According to certain preferred embodiments of this and other aspects of the invention, there are provided polynucleotides that hybridize, particularly under stringent conditions, to RNaseP polynucleotide sequences. In certain preferred embodiments of the present invention, there are provided antibodies against RNaseP polypeptides.

In another embodiment of the present invention, there is provided a method of identifying a compound that binds or interacts with a polypeptide or polynucleotide of the present invention to inhibit or activate their activity, the method comprising: The polypeptide or polynucleotide of the present invention is contacted with the compound to be screened under conditions that allow binding or other interaction between the polypeptide or polynucleotide and binding or other interaction with the compound. (Such binding or interaction is associated with a second compound capable of providing a detectable signal in response to the binding or interaction of the polypeptide or polynucleotide with the compound), and ,
By detecting the presence or absence of a signal resulting from the binding or interaction of the compound with the polypeptide or polynucleotide, the compound binds or interacts with the polypeptide or polynucleotide to activate or inhibit their activity Deciding whether to do so. According to yet another aspect of the invention, there are provided RNase P agonists and antagonists, preferably bacteriostatic or bactericidal agonists and antagonists. In a further aspect of the invention, for administration to a single or multicellular organism,
Compositions comprising an RNaseP polynucleotide or RNaseP polypeptide are provided. 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 description and from reading the remainder of the specification.

Terminology The following description is provided to facilitate understanding of certain terms that are commonly used herein. Other specific definitions are set forth elsewhere herein. A “host cell” is a cell that has been transformed or transfected, or is capable of being transformed or transfected by an exogenous polynucleotide sequence. As is well known in the art, "identity" is a relationship between two or more polypeptide sequences or between two or more polynucleotide sequences, as determined by comparing the sequences. is there. In the art, "identity" also sometimes means the degree of relatedness between two polypeptide or polynucleotide sequences, as determined by the match between the two strands of such sequences. “Identity” can be easily calculated by a known method (Computational Molecular Biology, L
esk, AM, Oxford University Press, New York, 19
1988; Biocomputing: Informatics and Genome Projects, Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Da
ta, part I, edited by Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje
, G., Academic Press, 1987; and Sequence Analysis Primer, Gribs.
kov, M. and Devereux, J. eds., M Stockton Press, New York, 1991;
And Carillo, H., and Lipman, D., SIAM J., Applied Math., 48: 1073 (1988)). Preferred methods for determining identity are designed to give the best match between the two sequences tested. Methods for determining identity and similarity are codified in publicly available computer programs. A preferred computer program method to determine identity and similarity between two sequences is the GCG program package (Devereux, J. et al., Nucleic Acids Research 12).
(1): 387 (1984), BLASTP, BLASTN and FASTA (Atschul, SF et al., J. Molec. Biol.
.215: 403 (1990))). BLAST
The X program is publicly available from NCBI and other sources (BLAST Manual,
Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et
al., J. Mol. Biol. 215: 403-410 (1990)). Identity may be determined using the well-known Smith Waterman algorithm.

Parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970) Comparison matrix: Hentikoff and Hentikoff, Proc. Natl Acad. Sci. USA. 8
9: BLOSSUM62 from 10915-10919 (1992) Gap penalty: 12 Gap length penalty: 4 A useful program for these parameters is the Genetics Computer Grou
Publicly available as a "gap" program from p. Madison WI. The above parameters are default parameters for polypeptide comparison (without penalty for end gaps). Preferred parameters for polynucleotide comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970) Comparison matrix: match = + 10, mismatch = 0 gap penalty: 50 Gap length penalty: 3 A useful program for these parameters is the Genetics Computer Group
Publicly available as a "gap" program from p. Madison WI. The above parameters are default parameters for polynucleotide comparison.

[0010] Preferred meanings for "identity" for polynucleotides and polypeptides are set forth in (1) and (2) below. (1) Further, specific examples of the polynucleotide have at least 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the control sequence of SEQ ID NO: 1, 3, 4, or 7. The polynucleotide sequence of the invention may be identical to the control sequence of SEQ ID NO: 1, 3, 4, or 7, or may have up to a certain number of nucleotides compared to the control sequence. May be changed. Such changes may be due to deletions, substitutions (at least one nucleotide).
Transitions and transversions) or insertions, wherein the changes are individually at nucleotides in the control sequence at positions at the 5 'or 3' end of the control nucleotide sequence or between those terminal positions. Or they may occur interspersed or as one or more contiguous groups in a control sequence. Multiplying the total number of nucleotides in SEQ ID NO: 1, 3, 4 or 7 by the individual percent identity value (divided by 100), and multiplying the product by SEQ ID NO: 1, 3
The number of nucleotide changes is determined by subtracting from the total number of nucleotides in 4 or 7. This is described by the following _{equation: n n ≦ x n - (} x n · y) wherein, n _n is the number of nucleotide alterations, x _n is SEQ ID NO: 1, 3, 4 or 7
Where y is 0.70 for 70% and 0 for 80%, for example.
. 0.85 for 80, 85%, 0.90 for 90%, 0.95, 97 for 95%
% Is 0.97, 100% is 1.00, and is a product operator. The non-integer product of _xn and y is rounded down to the nearest integer and then subtracted from _xn . Changes in the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 or 6 can preferably cause nonsense, missense or frameshift mutations in the coding sequence, and are concomitant with such changes. The polypeptide encoded by the polynucleotide changes. For example, a polynucleotide sequence of the present invention may be identical to the control sequence of SEQ ID NO: 1, 3, 4, or 7, ie, may be 100% identical, or may have some number compared to the control sequence. (In which case the percent identity is less than 100%). Such alterations are selected from the group consisting of deletions, substitutions (including transitions and transversions) or insertions of at least one nucleic acid, wherein the alterations are at the 5 'or 3' end of a control polynucleotide sequence or at their position. At positions between the terminal positions, they may occur individually or interspersed in the nucleic acid in the control sequence, or as one or more contiguous groups in the control sequence. Subtracting the total number of nucleic acids in SEQ ID NO: 1 or 5 from the total number of nucleic acids in SEQ ID NO: 1 or 5, multiplying the total number of nucleic acids in SEQ ID NO: 1, 3 or 4 by an integer representing the identity of each individual divided by 100 To determine the number of nucleic acid changes for the% identity value.
Or is described by this formula below: _n n ≦ x n - in (x _n · y) equation, n _n is the number of nucleic acid variation, x _n is SEQ ID NO: 1, 3, 4 or 7 in Where y is 0.70 for 70%, 0.85 for 85%, etc.
Non-integer products of _xn and y are rounded down to the nearest integer and then subtracted from _xn .

(2) Further, specific examples of the polypeptide have at least 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the polypeptide control sequence of SEQ ID NO: 2 or 6. An isolated polypeptide comprising a polypeptide having
The polypeptide sequence may be identical to the control sequence of SEQ ID NO: 2 or 6,
Alternatively, it may have up to a certain number of amino acid changes compared to a control sequence. Such alterations are selected from the group consisting of deletions, substitutions (including conservative and non-conservative substitutions) or insertions of at least one amino acid, wherein the alterations are at the amino or carboxy terminal position of the control polypeptide sequence or at those positions. May occur individually or interspersed with amino acids in the control sequence, or as one or more contiguous groups in the control sequence. SEQ ID NO: 2
Or multiplying the total number of amino acids in 6 by an integer indicating the identity divided by 100;
The number of amino acid changes is determined by subtracting the product from the total number of amino acids in SEQ ID NO: 2 or 6. This is described by the following equation: n _a ≦ x _a - in (x _a · y) formula, n _a is the number of amino acid alterations, x _a is SEQ ID NO: is the total number of amino acids in two or 6, y Is, for example, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97%, 1.71
If 100% 1.00, • is the symbol for the multiplication operator, and wherein any non-integer product of x _a and y is the rounded down to the nearest integer prior to subtracting it from x _a. For example, a polypeptide sequence of the invention may be identical to the control sequence of SEQ ID NO: 2 or 6, ie, may be 100% identical, or may contain up to a certain number of amino acids as compared to the control sequence. May have a change (in which case the percent identity is less than 100%). Such alterations are selected from the group consisting of deletions, substitutions (including conservative or non-conservative substitutions) or insertions of at least one amino acid, wherein the alterations are at the amino or carboxy terminal position of the control polypeptide sequence or at those positions. May occur individually or interspersed with amino acids in the control sequence, or as one or more contiguous groups in the control sequence. % Identity by multiplying the total number of amino acids in SEQ ID NO: 2 or 6 by the individual percent identity value (divided by 100) and subtracting the product from the total number of amino acids in SEQ ID NO: 2 or 6 Determine the number of amino acid changes for the value.
This is described by the following _{equation: n a ≦ x a - (} x a · y) wherein, n _a is the number of amino acid alterations, x _a is SEQ ID NO: is the total number of amino acids in 2 or 6, y is, for example, 0.70 for 70%, 0.85 for 85%, and the like.
Non-integer products of x _a and y are rounded down to the nearest integer and then subtracted from x _a . "Isolated" means altered "by the hand of man" from its natural state, ie, in the case of natural products, altered or removed from its original environment or both. For example, a polynucleotide or polypeptide naturally occurring in an organism is not `` isolated, '' but the same polynucleotide or polypeptide separated from its coexisting material in its natural state is the term used herein. As "isolated".

“Bacteria” include (i) Streptococcus, Staphylococcus, Bordetella, Corynebac
terium, Mycobacterium, Neisseia, Haemophilus, Actinomyces, Streptomyces, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Pasteurella, Moraxella, A
cinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacill
us, Listeria, Calymmatobacterium, Brucella, Bacillus, Closterdium, Trepo
nema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Bo
rrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseu
members of the genus (but not limited to) domonas, Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma;
ococcus, Streptococcus of group G, Streptococcus pneumoniae, Streptococ
cus pyrogenes, Streptococcus agalactiae, Streptococcus faecalis, Strepto
coccus faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria me
ningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebac
terium diptheriae, Garnella vaginalis, Mycobacterium tuberculosis, Mycob
acterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, Actinomyce
s israelli, Listeria monocytogenes, Bordetella pretusis, Bordetella para
pretusis, Bordetella bronchiseptica, Esherichia coli, Shigella dysenteri
ae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainflu
enzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter fr
eundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis, Klebsiella
pneumoniae, Serratia marcessens, Vibrio cholera, Shigella dysenterii, S
higella flexneri, Pseudomonas aeruginosa, Franscisella tularensis, Bruce
lla abortis, Bacillus anthracis, Bacillus cereus, Clostridium perfringen
s, Clostridium tetani, Clostridium butulinum, Treponema pallidum, Ricket
tsia rickettsii and Chlamydia trachomitis, prokaryotes, including, but not limited to, members of a species or group; (ii) archaebacteria, including, but not limited to, Archaebacter; and (iii) protozoa, fungi, Saccharomyce
s, members of, but not limited to, the genus Kluveromyces or Candida, and Sacc
A unicellular or filamentous eukaryote, including but not limited to members of the species haromyces cerevisiae, Kluveromyces lactis or Candida albicans.

“Polynucleotide (s)” generally refers to either polyribonucleotides or polydeoxyribonucleotides, which may be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotide" refers to single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, single-stranded and Hybrids comprising RNA which is a mixture of double-stranded regions and DNA and RNA which may be single-stranded or more typically a mixture of double- or triple-stranded regions or single- and double-stranded regions Molecules, including but not limited to molecules. further,
As used herein, "polynucleotide" refers to a triple-stranded region consisting of RNA or DNA, or both RNA and DNA. The chains in these regions may be from the same molecule or from different molecules. The regions may include all one or more of these molecules, but more typically include only some regions of the molecule. One of the molecules of the triple helix region is often an oligonucleotide. As used herein, "polynucleotide (s)"
The term "DNA" also includes one or more modified bases.
Or RNA. That is, DNA or RNA having a backbone that has been modified for stability or for other reasons is also "polynucleotide (s)" as that term is intended herein. In addition, DNA or RNA (only two examples are shown) that include unusual bases such as inosine or modified bases such as tritylated bases are also polynucleotides as that term is used herein.
It will be appreciated that a variety of modifications have been made to DNA and RNA and have been used for many useful purposes, as will be known to those skilled in the art. As used herein, the term "polynucleotide" refers to such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as viruses and cells, such as, for example, simple and complex cells. Includes characteristic DNA and RNA chemical forms. "Polynucleotide (s)" also encompasses relatively short polynucleotides, often referred to as oligonucleotide (s).

“Polypeptide (s)” refers to any peptide or protein comprising two or more amino acids joined together by peptide bonds or modified peptide bonds. "Polypeptide (s)" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 amino acids encoded by the gene. "Polypeptide (s)" have been modified by either natural processes, such as processing and other post-translational modifications, or by chemical modification techniques. Such modifications are described in detail in the basic text and in more detailed research articles, as well as in the extensive research literature, which are well known to those skilled in the art. It will be apparent that the same type of modification may be present at the same or varying degrees at several sites in a given polypeptide. Also, a given peptide may have many types of modifications. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, phosphotidylinositol Bond, crosslink, cyclization, disulfide bond formation, demethylation, covalent crosslink formation, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, sugar chain formation, GPI anchor formation, hydroxylation Iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipidation, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosyl , Selenoylation, sulfation, arginylation Addition of amino acids to proteins mediated transfer RNA, and encompasses ubiquitination. For example, PROTEINS
−STRUCTURE AND MOLECULAR PROPERTIES, 2nd edition, TE Creighton, WHF
reeman and Company, New York (1993) and Wold, F., POSTTRANSLATIONAL
COVALENT MODIFICATION OF PROTEINS, Posttranslational Protein Modificatio
ns: Perspectives and Prospects, pgs. 1-12, edited by BC Johnson, Academic Press
, New York (1983); Seifter et al., Meth Enzymol. (1990) 182: 626-646, and
Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging,
See Ann NY Acad Sci (1992) 663: 48-62. Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides are the result of natural post-translational processing and can likewise be synthesized in entirely synthetic ways.

The term “variant” as used herein is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not change the amino acid sequence of the polypeptide encoded by the control polynucleotide. As discussed below, nucleotide changes result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the control polypeptide and the variant are closely similar overall and, in many regions, identical. Variant and control polypeptides can have an altered amino acid sequence by one or more substitutions, additions, deletions occurring in any combination. The substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be naturally occurring, for example, an allelic variant, or may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis techniques or direct synthesis, and other recombinant methods known to those of skill in the art. "Plasmids" are generally designed according to standard nomenclature familiar to those skilled in the art, preceded by a lowercase p and / or followed by uppercase letters and / or numbers. The starting plasmids described herein are commercially available, commonly used, or can be constructed from available plasmids by versatile well-known procedures. Furthermore, plasmids equivalent to those described are well known,
It will be apparent to those skilled in the art.

“Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used in the present invention are commercially available and their reaction conditions, cofactors and other essential factors will be known to those skilled in the art. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Suitable buffers and substrate amounts for individual restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at about 37 ° C. are typically used, but may vary as described by the supplier. After digestion, the reaction is electrophoresed directly on an agarose gel to isolate the desired fragment. An "oligonucleotide" is a single-stranded polydeoxynucleotide or two complementary polydeoxynucleotide chains, which may be chemically synthesized. Because such synthetic oligonucleotides lack a 5 'phosphate, they will not ligate to another oligonucleotide without adding a phosphate with ATP in the presence of a kinase. The synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated. "Ligation" refers to the process of forming a phosphodiester bond between two double-stranded nucleic acid fragments (Maniatis, T. et al., Supra). Unless otherwise specified, ligation may be performed using 10 units of T4 DNA ligase (referred to as ligase) per 0.5 μg of approximately equimolar DNA fragment to be ligated under known buffers and conditions. Preferably, the polypeptides and polynucleotides of the present invention are in isolated form, and preferably are purified to homogeneity.

A “replicon” is a genetic element (eg, a plasmid, chromosome, virus) that functions as an autonomous unit of DNA in vivo, ie, can replicate under its own control. A "vector" is a replicon, such as a plasmid, phage or cosmid, that is capable of binding another DNA segment and causing replication of the linked segment. "Double-stranded DNA molecule" refers to deoxyribonucleotides in a double helix polymer form (both relaxed and supercoiled). This term is used only for the primary and secondary structure of the molecule and is not limited to individual tertiary forms. Thus, the term includes double-stranded DNA, especially linear DNA molecules (eg, restriction fragments), viruses, plasmids, and chromosomes. When referring to the structure of a particular double-stranded DNA molecule, the untranscribed strand of DNA (ie, mRNA
Strands having a sequence homologous to) can be shown in the 5 'to 3' direction. The DNA "coding sequence" or "encoding nucleotide sequence" of a particular protein is a DNA sequence that is transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences. A “promoter sequence” binds to RNA polymerase in a cell and binds downstream (3 ′
Direction) is a DNA regulatory region capable of initiating transcription of the coding sequence. For purposes of defining the present invention, a promoter sequence may include a translation initiation codon (eg, A
TG) attached to the 3 'end of the coding sequence, extending upstream (5' direction) and containing the minimum number of bases or elements necessary to initiate detectable levels of transcription above background. I have. In the promoter sequence are found a transcription initiation site (conveniently determined by mapping with nuclease S1) as well as a protein binding domain (consensus sequence) that can respond to the binding of RNA polymerase. Eukaryotic promoters are often, but not always, “TATA”
Box and a "CAT" box. Prokaryotic promoters are -10 and-
Includes 35 consensus sequences. DNA "control sequences" collectively refer to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, etc., which all direct the expression of coding sequences (ie, transcription and translation) in host cells. Is what you do. RNA polymerase binds to the promoter sequence and replaces the coding sequence with mRN
When transcribed to A, the control sequence "directs expression" of the coding sequence in the cell.
The mRNA is then translated into the polypeptide encoded by the coding sequence.

A “host cell” is a cell that has been transformed or transfected, or that can be transformed or transfected, by a foreign DNA sequence. When such exogenous DNA is introduced into the cell membrane, the cells are "
"Transformed". The foreign DNA may or may not be integrated (covalently linked) into the chromosomal DNA to form the genome of the cell. In prokaryotes and yeast, for example, foreign DNA may be maintained on episomal elements, such as plasmids. For eukaryotic cells, a stably transformed or transfected cell line is a cell in which foreign DNA has become integrated into a chromosome so that it is inherited by chromosome duplication in daughter cells. This stability is due to roasting DN
Indicated by the ability of eukaryotic cells to establish cell lines or clones containing a population of daughter cells containing A. A "clone" is a population of cells derived from a single cell or from a common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations. A "heterologous" region of a DNA construct is an identifiable DNA segment present in or associated with another DNA molecule that is not found in association with another molecule in nature.

Further Description and Definitions The coding regions of the RNase P RNA structural gene and the protein gene are described , for example, in the National Collection of Industrial and Marine.
S. pneumoniae deposited at Bacteria Limited (referred to as NCIMB), 23 Street, McDorough Drive, Aberdeen AB21RY, Scotland on April 11, 1996 and assigned NCIMB accession number 40794.
It may be isolated by screening using a deposit containing the 0993 strain.
After the deposit, the deposited strain is called S. pneumoniae 0100993 strain. In the present specification, the S. pneumoniae strain deposit is referred to as “deposited strain” or “DNA of the deposited strain”.
" The deposited strain contains the full length RNaseP catalytic RNA structural gene as well as the RNaseP protein gene. The polynucleotide sequence contained in the deposited strain, as well as the amino acid sequence of the polypeptide encoded thereby, will dominate in events that conflict with any description of the sequences herein. Deposits of the deposited strains have been made under the terms of the Budapest Treaty on the International Recognition of Microbial Deposits for Patent Procedure. Once the patent is issued, without any restrictions or conditions,
Eventually, the shares will be sold. The deposit is provided solely for the convenience of those skilled in the art and does not acknowledge that the deposit is a feasible requirement, as required under 35 USC 112. A license is required to make, use or sell the deposit, but such a license has not been granted here.

The nucleotide sequences described herein can be obtained by synthetic chemistry methods known in the art, and probes for DNA prepared using probes constructed from the individual sequences disclosed herein. Thus, it can also be obtained from S. pneumoniae 0100993. Alternatively, oligonucleotides from the disclosed sequences can act as PCR primers in a PCR cloning sequence from bacterial genomic sources. It will be appreciated that such sequences are also useful in diagnosing the type of infection achieved by the pathogen. The polynucleotide of the present invention may be in the form of RNA or DNA, which DNA includes cDNA, genomic DNA and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding (antisense) strand. The coding sequence that encodes the polypeptide may be identical to the coding sequence shown, or may encode the same polypeptide but have a different coding sequence as a result of the redundancy or degeneracy of the genetic code. Thus, the term "polynucleotide encoding a polypeptide" encompasses polynucleotides that include only the coding sequence of the polypeptide as well as polynucleotides that include additional coding and / or non-coding sequences. Therefore, the present invention relates to a polynucleotide sequence wherein the coding sequence of a mature polypeptide in a polynucleotide aids in the same reading frame in assisting expression and secretion of the polypeptide from the host cell, e.g., transport of the polypeptide from the cell. Includes polynucleotides that may be fused to a leader sequence that functions as a controlling secretory sequence. A polypeptide having a leader sequence is a preprotein and may have a leader sequence that is cleaved by the host to yield a mature form of the polypeptide.
The polynucleotide may encode a proprotein with an additional 5 ′ amino acid residue added to the mature protein. A mature protein having a prosequence is a proprotein, which is an inactive form of the protein. When the prosequence is cleaved, the active mature protein remains. Thus, for example, the polynucleotide of the present invention may encode a mature protein, or may encode a protein having both a prosequence and a presequence (leader sequence). Furthermore, the amino acid sequence shown herein NH ₂ - and ends methionine residue is shown. However, during the post-translational modification of the peptide, this residue may be deleted. Thus, the present invention contemplates the use of both methionine-containing and methionine-deleted amino-terminal variants of each of the proteins disclosed herein.

The polynucleotide of the invention has a coding sequence fused in frame to a marker sequence at the 5 ′ or 3 ′ end of the gene, which allows for purification of the polypeptide of the invention. You may. The marker sequence may be a hexa-histidine tag provided by a pQE series of vectors (commercially available from Quiagen Inc.) to allow purification of the polypeptide fused to the marker sequence in the case of a bacterial host. Good. Alternatively, maltose binding protein (MBP)
A fusion system may be used. In this system, the gene of interest is ma, which encodes MBP.
Fused to the IE gene (provided by New England BioLabs). The fusion product is purified in one step with MBP affinity for maltose. The pre-treated Xa cleavage site allows for efficient removal of MBP components from the gene product of interest.

[0022] RNase P is a detailed description ribonucleoprotein of the invention shows an important role in the metastasis of RNA (tRNA) in the biosynthesis is an important intermediate of its own protein biosynthesis. R
Nase P has a function of processing a precursor tRNA into a mature tRNA by an endoribonucleolytic activity. The complex in the prokaryotic cells studied is composed of two subunits. They are catalytic RNA and protein cofactors. There are recent reviews on certain RNase P molecules. See, for example, LA Kirsebom, Molecular Microbiology 17 (3), 411-420 (1995) or NR Pace and JW Brown, J. Bacteriol. 177 (8), 1919-1928 (1995). The present invention relates to novel RNase P polynucleotides described in more detail below. In particular, the present invention relates to novel S. pneumoniae RNase P polynucleotides related by structure and nucleotide sequence homology to the RNase P gene shown in FIGS. In particular, the present invention relates to RNase P having the nucleotide sequence shown in Table 1 [SEQ ID NOs: 3 or 7, each being a minor functional variant] and the RNase P nucleotide sequence of the DNA of the deposited strain and the RNA transcribed therefrom. About. The present invention also provides an RNase
It relates to the RNA component of P, in particular this catalytic form as well as the sequence to which such component is transcribed.

The RNA component phylogenetic comparison of RNase P facilitates secondary structure modeling and identification of minimal consensus structures. Data on the RNA structure of RNase P is available, for example, the RNase P database on www (http://jwbrown.mbio.ncsu.edu/RNaseP/home.html). The polynucleotides of the present invention to which the RNA component is transcribed are shown in Table 1 [SEQ ID NOs: 3 and 7]. Generally, a small number of nucleotides are conserved, but correct base changes in the hydrogen bonding region indicate that the overall structure is conserved in eubacteria.
The universal conservation of primary sequences (E. coli: 61-74, 353-360), together with other conserved or semi-conserved nucleotides, indicates functional significance, but their significance is unknown. To date, all RNase P RNA molecules are able to fold and conform to a consensus “cage-like” structure, and beyond this domain, between prokaryotic and eukaryotic RNase P RNAs. Has no persuasive structural similarity.

The exact functional role of the RNase P protein component protein is unknown. Although it is possible to establish experimental conditions in vitro where the protein component is not required for catalytic activity, it appears that the protein component is required in vivo. In the present invention, other RNases
Among the P components, RNase P protein components derived from S. aureus, B. subtilis, E. coli and other bacteria can be used together with the RNase P RNA component of the present invention. These components will complement the activity of this RNA when expressed in vivo in bacteria, especially in bacteria other than S. pneumoniae. The S. pneumoniae RNase P protein component may be used with the RNase P RNA of the invention (see coding sequences SEQ ID NO: 5 and SEQ ID NO: 6). Combining an RNase P protein derived from Staphylococcus aureus, such as a protein having the sequence shown in SEQ ID NO: 2, with an RNAse of RNase P derived from Streptococcus pneumoniae as shown in SEQ ID NO: 3 or 7 May be used. Other bacterial RNase P proteins may be used in this manner. By searching a genomic library using degenerate primers prepared from the amino acid and polynucleotide sequences of S. aureus or S. pneumoniae RNase P shown in Table 1 below, the full length encoding the intact RNase P protein component is obtained. An array may be obtained.

Table 1 RNase P polynucleotide and polypeptide sequences (A) Sequence derived from S. aureus RNase P polynucleotide sequence [SEQ ID NO: 1] 5′-ATG TTA TTG GAA AAA GCT TAC CGA ATT AAA AAG AAT GCA GAT TTT CAG AGA ATA TAT AAA AAA GGT CAT TCT GTA GCC AAC AGA CAA TTT GTT GTA TAC ACT TGT AAT AAT AAA GAA ATA GAC CAT TTT CGC TTA GGT ATT AGT GTT TCT AAA AAA CTA GGT AAT GCA GTG TTA AGA AAC AAG ATT AAA AGA GCA ATA CGT GAA AAT TTC AAA GTA CAT AAG TCG CAT ATA TTG GCC AAA GAT ATT ATT GTA ATA GCA AGA CAG CCA GCT AAA GAT ATG ACG ACT TTA CAA ATA CAG AAT AGT CTT GAG CAC GTA CTT AAA ATT GCC AAA GTT TTT AAT AAA AAG ATT AAG TAA-3 '

(B) RN of S. aureus deduced from the polynucleotide sequences in this table
se P polypeptide sequence [SEQ ID NO: 2] NH ₂ -MLLEK VYRIK KNADF GRIYK KGHSV ANRQF VVYTC NNKEI DHFRL GISVS KKLGN AVLRN KIKRA IRENF KVHKS HILAK DIIVI ARQPA KDMTT LQIQN SLEHV LKIAK VFNKK IKCOOH

(C) Specific Example of Polynucleotide Sequence [SEQ ID NO: 3] X- (R1) n-GATGTGCAATTTTTGGATAATCGAGTGAAGGGAATTGCTTCTCATGAGGAAAGTCCATGATAGCACAGGCTG
TGATGCCTGTAGTATTTGTGATAGGCGAAACCATAAGCCTAGGGACGAGAATTCGTTACGGCAGTTGAAATG
GATAAGTCCTTGGATAAGCCAGAGTAGGCTTGAAAGTGCCACAGTGACGGAGTCTTTCTGGAAACAGAGAGA
GTGGAACGCGGTAAACCCCTCAAGCTAGCAACCCAAATTTTGGTCGGGGCATGGAGTACGCGGAAACGGACG
TAGTATTCTGACTGGTATCAGCTAGAGCTGTTAGTGGTAGACAGATGATTATCGAAGGAAGTGGTCCTAGTC
ACTTTTGGAACAAAACATGGCTTATAGAAAATTGCATATAGG- (R2) nY

(D) RNAse sequence of RNase P [SEQ ID NO: 3] 5′-GATGTGCAATTTTTGGATAATCGAGTGAAGGGAATTGCTTCTCATGAGGAAAGTCCATGATAGCACAGGCTG
TGATGCCTGTAGTATTTGTGATAGGCGAAACCATAAGCCTAGGGACGAGAATTCGTTACGGCAGTTGAAATG
GATAAGTCCTTGGATAAGCCAGAGTAGGCTTGAAAGTGCCACAGTGACGGAGTCTTTCTGGAAACAGAGAGA
GTGGAACGCGGTAAACCCCTCAAGCTAGCAACCCAAATTTTGGTCGGGGCATGGAGTACGCGGAAACGGACG
TAGTATTCTGACTGGTATCAGCTAGAGCTGTTAGTGGTAGACAGATGATTATCGAAGGAAGTGGTCCTAGTC
ACTTTTGGAACAAAACATGGCTTATAGAAAATTGCATATAGG-3 '

(E) RNA gene of RNase P (5 ′ side) [SEQ ID NO: 4] 5′-GATGTGCAATTTTTGGATAATCGCGTGAGGAGAATTGCTTCTCATGAGGAAAGTCCATGCTAGCACAGGCTG
TGATGCCTGTAGTGTCTGTGCTACGCGAAACCATAAGCCTATGGACGAGAAATCGTTACGGCAGTTGAAATG
GCTAATTCCTTGGATAAGCCACAGTATGCTTGAAATTGCCACAGTGACCGAGTCTTTCTGGAAACACATAGA
TTGGAACGCCGTAAACCCCTCAAGCTAGCAACCCAAATTTTGGTCGGGGCA-3 '

(F) Specific example of RNA gene of RNase P (5 ′ side) [SEQ ID NO: 4] X- (R1) n-GATGTGCAATTTTTGGATAATCGCGTGAGGAGAATTGCTTCTCATGAGGAAAGTCCATGCTAGCACAGGCTG
TGATGCCTGTAGTGTCTGTGCTACGCGAAACCATAAGCCTATGGACGAGAAATCGTTACGGCAGTTGAAATG
GCTAATTCCTTGGATAAGCCACAGTATGCTTGAAATTGCCACAGTGACCGAGTCTTTCTGGAAACACATAGA
TTGGAACGCCGTAAACCCCTCAAGCTAGCAACCCAAATTTTGGTCGGGGCA- (R2) nY

(G) RNase P protein coding sequence [SEQ ID NO: 5] 5′-TTGAAGAAAAACTTTCGTGTAAAAAGAGAGAAAGATTTTAAGGCGATTTTCAAGGAGGGGACAAGTTTTGCT
AATCGCAAATTTGTGGTCTACCAATTAGAAAACCAGAAAAACCATTTTCGAGTAGGTCTATCAGTTAGCAAA
AAACTGGGGAATGCCGTCACTAGAAATCAAATTAAGCGACGGATTCGGCATATTATCCAGAATGCAAAAGGG
AGTCTGGTAGAAGATGTCGACTTTGTTGTCATTGCTCGAAAAGGAGTCGAAACCTTGGGATACGCAGAGATG
GAGAAAAATCTACTCCATGTATTAAAATTATCAAAGATTTACCGGGAA-3 '

(H) RNase P protein [SEQ ID NO: 6] NH ₂ -LKKNFRVKREKDFKAIFKEGTSFANRKFVVYQLENQKNHFRVGLSVSKKLGNAVTRNQIKRRIRHIIQNAKG
SLVEDVDFVVIARKGVETLGYAEMEKNLLHVLKLSKIYRE-COOH

(I) RNase P protein [SEQ ID NO: 6] X- (R1) n-TTGAAGAAAAACTTTCGTGTAAAAAGAGAGAAAGATTTTAAGGCGATTTTCAAGGAGGGGACAAGTTTTGCT
AATCGCAAATTTGTGGTCTACCAATTAGAAAACCAGAAAAACCATTTTCGAGTAGGTCTATCAGTTAGCAAA
AAACTGGGGAATGCCGTCACTAGAAATCAAATTAAGCGACGGATTCGGCATATTATCCAGAATGCAAAAGGG
AGTCTGGTAGAAGATGTCGACTTTGTTGTCATTGCTCGAAAAGGAGTCGAAACCTTGGGATACGCAGAGATG
GAGAAAAATCTACTCCATGTATTAAAATTATCAAAGATTTACCGGGAA- (R2) nY

(J) RNase P protein [SEQ ID NO: 6] X- (R1) n-LKKNFRVKREKDFKAIFKEGTSFANRKFVVYQLENQKNHFRVGLSVSKKLGNAVTRNQIKRRIRHIIQNAKG
SLVEDVDFVVIARKGVETLGYAEMEKNLLHVLKLSKIYRE- (R2) nY

(K) Full length RNase PRNA gene [SEQ ID NO: 7] 5′-GAT GTG CAA TTT TTG GAT AAT CGC GTG AGG AGA ATT GCT TCT CAT GAG GAA AGT CCA TGC TAG CAC AGG CTG TGA TGC CTG TAG TGT TTG TGC TAG GCG AAA CCA TAA GCC TAG GGA CGA GAA ATC GTT ACG GCA GTT GAA ATG GCT AAG TCC TTG GAT AAG CCA GAG TAG GCT TGA AAG TGC CAC AGT GAC GGA GTC TTT CTG GAA ACA GAG AGA GTG GAA CGC GGT AAA CCC CTC ATA GCA ACC CAA ATT TTG GTC GGG GCA TGG AGT ACG CGG AAA CGA ACG TAG TAT TCT GAC TGC TAT CAG CTA GAG CTG TTA GTG GTA GAC AGA TGA TTA TCG AAG GAA GTG GTC CTA GTC ACT TCT GGA ACA AAA CAT GGC TTA TAG AAAATT GCA TAT AGG-3 '

(L) Specific Example of Sequence of RNase P RNA Gene [SEQ ID NO: 7] X- (R1) n-GAT GTG CAA TTT TTG GAT AAT CGC GTG AGG AGA ATT GCT TCT CAT GAG GAA AGT CCA TGC TAG CAC AGG CTG TGA TGC CTG TAG TGT TTG TGC TAG GCG AAA CCA TAA GCC TAG GGA CGA GAA ATC GTT ACG GCA GTT GAA ATG GCT AAG TCC TTG GAT AAG CCA GAG TAG GCT TGA AAG TGC CAC AGT GAC GGA GTC TTT CTG GAA ACA GAG AGA GAA CGC GGT AAA CCC CTC AAG CTA GCA ACC CAA ATT TTG GTC GGG GCA TGG AGT ACG CGG AAA CGA ACG TAG TAT TCT GAC TGC TAT CAG CTA GAG CTG TTA GTG GTA GAC AGA TGA TTA TCG AAG GAA GTG GTC CTA GTC ACT TGA ACA AAA CAT GGC TTA TAG AAA ATT GCA TAT AGG- (R2) nY

Polypeptide of the Present Invention The polypeptide of the present invention has the sequence shown in Table 1 [SEQ ID NO: 6]
S. aureus, such as the sequence of S. aureus shown in [SEQ ID NOs: 1 and 2], particularly includes polypeptides isolated from the deposited strain. Specifically, it encompasses mature polypeptides and polypeptides and fragments, particularly those having the biological activity of RNase P, and further comprises at least 50% of the polypeptide of Table 1 [SEQ ID NO: 2] or a significant portion thereof. %, 60% or 70% identity, more preferably Table 1 [SEQ ID NO: 2]
At least 80%, even more preferably at least 90% similarity to the polypeptide of Table 1 [SEQ ID NO: 2 or 6] (even more preferably With at least 90% identity), and more preferably at least 9 against the polypeptide of Table 1 [SEQ ID NO: 2 or 6].
Polypeptides and fragments having 5% similarity (even more preferably at least 95% identity), and more usually portions of such polypeptides comprising at least 30, more preferably at least 50 amino acids Include.

Fragments are variant polypeptides that have an amino acid sequence that is entirely identical to some, but not all, of the amino acid sequences of the aforementioned polypeptides. RNas
For eP polypeptides, the fragments may be "free standing," or may be included within a larger polypeptide by forming a portion or region, most preferably a single polypeptide. As a large single polypeptide. Preferred fragments include, for example, truncated polypeptides having a portion of the amino acid sequence of RNaseP of SEQ ID NO: 6. Table 1 [SEQ ID NO: 1, 2,
5 or 6] or a variant thereof, such as a series of residues including the amino terminus or a series of residues including the carboxyl terminus. Also preferred are degradation forms of the polypeptides of the present invention in host cells or bacteria, especially S. pneumoniae. Also, fragments characterized by structural or functional attributes, such as alpha helices and alpha helix forming regions, beta sheets and beta sheet forming regions, turns and turn forming regions, coils and coil forming regions, hydrophilic regions, hydrophobic Also preferred are a region, an alpha-amphiphilic region, a beta amphipathic region, a variable region, a surface-forming region, a substrate-binding region, and a fragment containing a high antigenic index region. Also preferred are biologically active fragments which are those that mediate or improve the activity of RNase P and have reduced undesirable activity. Also included are fragments that are antigenic or immunogenic in animals, especially humans. Fragments containing a receptor or domain of an enzyme that confers a function essential to the survival of S. pneumoniae in an individual, especially a human, or the ability to initiate or maintain disease pathogenesis are particularly preferred. Variants that are fragments of the polypeptides of the present invention may be used for the production of corresponding full-length polypeptides by peptide synthesis, and thus these variants may be used as intermediates for producing full-length polypeptides. A variant that is a fragment of a polynucleotide of the invention may be used to synthesize a full-length polynucleotide of the invention. The polypeptides of the invention may be used, for example, to evaluate the binding of small molecule substrates and ligands in cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands can be natural substrates and ligands, and can be structural or functional mimetics. For example, Coligan et al., Cur
See rent Protocols in Immunology 1 (2): Chapter 5 (1991).

Polynucleotides of the Invention Another aspect of the invention relates to isolated polynucleotides, as described in Table 1
Full length gene encoding the RNaseP polypeptide of [SEQ ID NO: 6], for example, a gene isolated using the sequence of Table 1 [SEQ ID NO: 1, 2, 5, or 6], for example, prepared from these sequences Included are sequences obtained by amplification with degenerate primers, as well as closely related polynucleotides and variants thereof. Using the information provided herein, for example, using the polynucleotide sequence shown in SEQ ID NO: 1, 2, 3, 4, 5 or 7, to clone and clone a chromosomal DNA fragment from the starting material Streptococcus pneumoniae 0100993 cells Using standard cloning and screening, such as those used for sequencing, a polynucleotide of the invention encoding an RNaseP polypeptide or RNA (such as that transcribed from SEQ ID NO: 3 or 7) is obtained, Then, a full-length clone may be obtained. For example, Streptococcus pneumoniae in E. coli or some other suitable host to obtain a polynucleotide sequence of the invention, such as the sequence set forth in SEQ ID NO: 3 or 7, or the RNaseP structural protein gene. A typical library of chromosomal DNA clones is derived from a partial sequence, for example from the nucleotide sequence of SEQ ID NO: 1, 3, 4, 5 or 7 as appropriate, preferably a 17-mer or more. Probe with a length of radiolabeled oligonucleotide. Clones carrying DNA identical to the probe DNA can be distinguished using stringent conditions. By sequencing the individual clones thus identified using sequencing primers designed from the original sequence, the sequence can be extended in both directions and the entire gene sequence can be determined. Such sequencing is conveniently performed using denatured double-stranded DNA prepared from a plasmid clone. For appropriate techniques, see Maniat
is, T., Fitsch, FF and Sambrook et al., Molecular Cloning, A Laboratory Manu.
al, 2nd edition; described in Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) (Screenin
g By Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Te
mplates 13.70). In a typical example of the invention, the polynucleotides shown in SEQ ID NOs: 3 and 7 are the DNs from Streptococcus pneumoniae 0100993.
Found in the A library.

The present invention relates to a coding sequence for a mature polypeptide or a fragment thereof, as well as a coding sequence for a mature polypeptide or a fragment thereof in an open reading frame having other coding sequences, such as a leader or secretory sequence, pre, pro, prepro. A sequence encoding a protein sequence is provided. The polynucleotide is
For example, transcribed untranslated sequences, termination signals, ribosome binding sites, mRNA
Transcribed non-translated sequences such as stabilizing sequences, introns, polyadenylation signals,
And non-coding sequences such as non-coding 5 'and 3' sequences, such as additional coding sequences encoding additional amino acids, but are not limited thereto. For example, it may encode a marker sequence that facilitates purification of the fusion polypeptide. In one preferred embodiment of the invention, the marker sequence is a hexa-histidine peptide (Gent) as provided in a pQE vector (Qiagen, Inc.).
Natl. Acad. Sci., USA, 86: 821-824 (1989)) or HA tag (Wilson et al., Cell, 37: 767 (1984)). The polynucleotides of the present invention also include, but are not limited to, structural genes and native sequences that regulate gene expression.

The present invention also encompasses polynucleotides of the formula shown in Table 1 (C) [SEQ ID NO: 3] and (K) [SEQ ID NO: 7], wherein X is hydrogen at the 5 'end. Yes,
At the 3 ′ end, Y is hydrogen or metal, R ₁ and R ₂ are nucleic acid residues, n is 1
To an integer of 3000. The extended portion of the nucleic acid residue represented by any of the R groups (R is greater than one) may be a heteropolymer or a homopolymer, and is preferably a heteropolymer. Table 1 (C) [SEQ ID NO: 3] or (
K) A preferred specific example of the sequence represented by [SEQ ID NO: 7] is that R ₁ or R ₂ has 1 to 10 or 1 to 20, especially 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. The present invention also provides RNA transcribed from such a polynucleotide, particularly catalytic RNA.

The term “polynucleotide encoding a polypeptide” as described herein above.
Encompasses a polynucleotide comprising a sequence encoding the polypeptide sequence of the present invention, in particular, a bacterial polypeptide, more specifically a Streptococcus pneumoniae having the amino acid sequence shown in Table 1 [SEQ ID NO: 2]. Includes a polynucleotide comprising a sequence encoding a RNase P polypeptide. The term refers to a single contiguous or discontinuous region encoding a polypeptide (eg, an integrated phage or sequence of phage or sequence), with additional regions that may include coding and / or non-coding sequences. (Interrupted by insertion or editing of the sequence). The invention further relates to variants of the polynucleotides described herein that encode variants of the polypeptides of the invention. A variant that is a fragment of a polynucleotide of the invention may be used to synthesize a full-length polynucleotide of the invention.

A further particularly preferred embodiment is a polynucleotide encoding an RNase P variant having the amino acid sequence of the RNase P polypeptide of Table 1 [SEQ ID NO: 2], wherein some, a few It has an amino acid sequence in which 5 to 10, 1 to 5, 1 to 3, 2, 1 or 0 amino acid residues are substituted, deleted or added in any combination. Among them, particularly preferred is RNas
Silent substitutions, additions and deletions that do not alter the properties and activities of eP.

In a further preferred embodiment of the invention, there is at least 50%, 60% or 70% identity over the entire length of the polynucleotide encoding the RNase P polypeptide [SEQ ID NO: 6]. Polynucleotides, particularly polynucleotides obtained using the amino acid or polynucleotide sequences shown in Table 1 [SEQ ID NOs: 1-7], and polynucleotides complementary to such polynucleotides. Alternatively, most highly preferred are polynucleotides comprising a region that is at least 80% identical over its entire length to the polynucleotide encoding the RNase P polypeptide of the deposited strain, and polynucleotides complementary thereto. In this regard, polynucleotides that are at least 90% identical over their entire lengths are particularly preferred, particularly those with at least 95% identity, and more preferably at least 97% among those with at least 95% identity. It is more preferred, especially at least 98% and at least 99% identity, and even more preferably at least 99% identity. Preferred embodiments of the present invention are directed to RNaseP polynucleotides having the nucleotide sequence set forth in SEQ ID NOs: 3, 4 or 7, and to polynucleotides complementary to such polynucleotides at least 50%, 60% or 70% over their entire length. % Polynucleotides. Alternatively, most preferred is a polynucleotide comprising a region having at least 80% identity over its entire length to a deposited strain RNaseP polynucleotide and a polynucleotide complementary thereto. In this regard, among these particularly preferred polynucleotides, those having at least 90% identity to the above polynucleotides are preferred, and those having at least 95% identity are particularly preferred. Furthermore, those with at least 97% identity are highly preferred among those with at least 95% identity, and those with at least 98% identity, and even at least 99% identity are particularly highly preferred. preferable. It is particularly preferred that these polynucleotides are RNA, especially catalytic RNA. Preferred embodiments are RNase P RNA transcribed by the mature polypeptide encoded by the DNA of the present invention or the DNA of SEQ ID NO: 3, 4 or 7.
A polynucleotide that encodes a polypeptide that retains substantially the same biological function or activity as the component.

The invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the present invention particularly relates to polynucleotides that hybridize under stringent conditions to the herein above-described polynucleotides. As used herein, the terms "stringent conditions" and "stringent hybridization conditions" refer to hybridization that occurs only when the identity between the sequences is at least 95%, preferably at least 97%. .
Examples of stringent hybridization conditions include 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate),
Incubation overnight at 42 ° C. in a solution containing 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / ml of denatured, sheared salmon sperm DNA, followed by The filter is washed in 0.1 × SSC at about 65 ° C. Hybridization and washing conditions are well known and are illustrated in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition; Cold Spring Harbor, New York (1989), especially Chapter 11 thereof.

The present invention also provides for the use of a suitable library containing the complete gene for a polynucleotide sequence of the present invention under stringent hybridization conditions with a probe having the sequence of the above polynucleotide sequence shown in Table 1 or a fragment thereof. Also provided are polynucleotides that essentially comprise the polynucleotide sequence obtainable by screening and isolating the DNA sequence. Fragments useful for obtaining such polynucleotides include, for example, probes and primers described elsewhere herein.

As discussed further herein with respect to the polynucleotide assays of the present invention, for example, the polynucleotides of the present invention described above may be used to isolate full length and genomic clones of cDNA encoding RNase P, and to enhance the RNase P gene. It can be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNA and genomic clones of other genes with great sequence similarity. Such probes usually have at least 15
Including base. Preferably such probes have at least 30 bases and may have at least 50 bases. Particularly preferred probes are at least 3
It has 0 bases and 50 bases or less. The polynucleotides of the present invention which are oligonucleotides derived from the sequences of SEQ ID NO: 1 and / or 2 and / or 3 and / or 4 and / or 5 and / or 6 and / or 7 are used in the methods described herein. Although preferably used for PCR to determine whether all or a portion of the polynucleotides identified herein are transcribed into infected tissues. It is understood that such sequences are also useful in diagnosing the stage and type of infection achieved by the pathogen.

The invention also provides polynucleotides that can encode a polypeptide that is a mature protein with additional amino or carboxyl terminal amino acids, or amino acids present in the mature polypeptide (eg, the mature form of one or more polypeptides). Having a peptide chain). Such sequences play a role in the processing of the protein from the precursor to the mature form, allowing for the transport of the protein, extending or shortening the half-life of the protein, or otherwise manipulating the protein for assay or production. Can be easier. Generally, in vivo, additional amino acids are processed by cellular enzymes and removed from the mature protein. A precursor protein having the mature form of the polypeptide fused to one or more prosequences can be an inactive form of the polypeptide. Removal of the prosequence usually activates such inactive precursors. Some or all of the prosequence can be removed prior to activation. Usually such precursors are called proproteins. In short, the polynucleotide of the present invention is a mature protein, a mature protein to which a leader sequence is added (also referred to as a preprotein), a precursor of a mature protein having one or more prosequences which are not the leader sequence of the preprotein, Alternatively, it may encode a preproprotein that is a precursor of a proprotein having a leader sequence and one or more prosequences, which are usually removed during a processing step that produces an active and mature form of the polypeptide. .

Cloning of the RNA Structural Gene of S. pneumoniae RNase P The present invention also relates to polynucleotides or vectors containing the polynucleotides of the present invention, host cells genetically engineered with the vectors of the present invention, and recombinant vectors of the present invention. It also relates to the production of polypeptides. Therefore, according to a further aspect of the present invention, there is provided a method for producing the polypeptide of the present invention by a recombinant method by expressing a polynucleotide encoding the polypeptide in a host and recovering the expression product. You. Alternatively, the polypeptides of the invention can be produced synthetically on a conventional peptide synthesizer. A host cell is genetically engineered (transduction or transformation or transfection) with a vector of the invention (eg, which may be a cloning vector or an expression vector). The vector may be in the form of a plasmid, cosmid, phage, etc. Activating the genetically engineered host cells,
The cells are cultured in a conventional nutrient medium modified so as to be suitable for selection of transformants or amplification of genes. Culture conditions, such as temperature, pH, etc., have already been used in the host cell for expression and will be apparent to those skilled in the art. Suitable expression vectors include those derived from chromosomal, non-chromosomal and synthetic DNA sequences such as bacterial plasmids, phage DNA, baculovirus, yeast plasmids, combinations of plasmids and phage DNA. However, other vectors may be used as long as they are replicable and viable in the host. Expression systems or portions thereof, or polynucleotides of the invention, can be incorporated. Introduction of a polynucleotide into a host cell is described, for example, in Davis et al., Basic.
Methods in Molecular Biology (1986); Sambrook et al., Molecular Cloning; A
Laboratory Manual, 2nd edition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989),
It can be performed by methods described in many standard laboratory manuals, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid mediated transfection, electroporation, transduction, There are scrape loading, ballistic introduction and infection.

Representative of suitable hosts include bacterial cells, such as Streptococcus, Staphylococci, Enterococci, E. coli, Streptococcus. Mrs. (Streptomyce
s) and Bacillus subtiis cells; fungal cells, eg, yeast cells and Aspergillus cells; insect cells, eg, Drosophila S2 and Spodoptera Sf9 cells; animal cells, eg, CHO, COS, HeLa, C127, 3T3, BHK
, 293 and Bows melanoma cells; and plant cells.

Numerous expression systems can be used to produce the polypeptides of the present invention. Such vectors include chromosomal, episomal and viral derived vectors, such as bacterial plasmid derived, bacteriophage derived, transposon derived, yeast episomal derived, insertion element derived, yeast chromosomal element derived such as baculovirus, papovavirus such as SV40, Vectors derived from viruses such as vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus, and vectors derived from combinations thereof, such as vectors derived from genetic elements of plasmids and bacteriophages, such as cosmids And phagemids. The expression system constructs may contain regulatory regions that control and cause expression. In this regard, in general,
Any system or vector suitable for retaining, extending or expressing a polynucleotide in a host and / or expressing a polypeptide can be used for expression.
Appropriate DNA sequences may be inserted into the expression system by any of a variety of well-known and conventional techniques, for example, as described in Sambrook et al., Molecular Cloning, A Laboratory Manual (
Above).

The appropriate DNA sequence can be inserted into the vector by various methods. Generally, the DNA sequence is inserted into an appropriate restriction endonuclease site by methods known in the art. The DNA sequence in the expression vector may be linked to a suitable expression control sequence (promoter) to direct mRNA synthesis. Typical examples of such promoters include the LTR or SV40 promoter, E. coli lac or trp.
, The PL promoter of lambda phage and other promoters known to control the expression of genes in eukaryotic or prokaryotic cells or their viruses. The expression vector may include a ribosome binding site for translation initiation and / or a transcription terminator. The vector may include a sequence suitable for enhancing expression.

Further, preferably, the expression vector comprises one or more selectable markers that confer a phenotype for selection of transformed host cells, such as dihydrofolate for eukaryotic cell culture. Resistant to reductase or neomycin,
E. coli is resistant to tetracycline or ampicillin. The gene can be placed under the control of a promoter, a ribosome binding site (for bacterial expression), and optionally an operator (collectively referred to herein as "control elements"), so that the expression construct is In a host cell transformed with the containing vector, a DNA sequence encoding the desired protein is transcribed into RNA. A coding sequence may or may not include a signal or leader sequence. For example, the polypeptide of the present invention can be expressed using an E. coli tac promoter or a protein A gene (spa) promoter and a signal sequence. The leader sequence may be removed by the bacterial host during translation processing. For example, U.S. Patent Nos. 4,431,739 and 4,425,437
No. 4,338,397. The promoter region can be selected from the desired gene using a CAT (chloramphenicol transferase) vector or other vector having a selectable marker. Two suitable vectors are pKK
232-8 and pCM7. lacI, lacZ, T3, T7, gpt,
Lambdas P _R , P _L and trp are mentioned in particular. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is within the level of ordinary skill in the art. In addition to regulatory sequences, it may be desirable to add regulatory sequences that can regulate the expression of the protein sequence in relation to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and include those that turn gene expression on or off in response to a chemical or physical stimulus, such as the presence of a regulatory compound. Other types of regulatory elements, such as enhancer sequences, may be present in the vector. The expression vector is constructed such that the particular coding sequence is present in the vector along with the appropriate regulatory sequences, and the position and orientation of the coding sequence relative to the control sequences is such that the coding sequence is transcribed under the `` control '' of the control sequences. (
That is, the RNA polymerase that binds to the DNA molecule at the control sequence transcribes the coding sequence). Modification of the coding sequence may be desirable to achieve this purpose. For example, in some cases, it may be necessary to modify the sequence so that it is linked to the control sequence in the proper orientation, ie, to maintain the reading frame. Control and other regulatory sequences may be ligated to the coding sequence before insertion into the vector, such as the cloning vectors described above. Alternatively, the coding sequence may be cloned directly into an expression vector which already has the control sequences and appropriate restriction sites.

In general, the recombinant expression vectors contain an origin of replication and a selectable marker that allows transformation of the host cell, such as the ampicillin resistance gene of E. coli and the TRP1 gene of S. cerevisiae and It will include a promoter from a highly expressed gene for direct transcription of the downstream structural gene. Heterologous structural sequences are assembled together with translation initiation and termination sequences, and preferably a leader sequence capable of directly secreting the translated protein in the periplasmic space or extracellular medium, to form an appropriate arrangement. If desired, the heterologous sequence may encode a fusion protein containing an N-terminal identification peptide that confers the desired property, eg, stabilizes, or facilitates purification of the expressed recombinant product. An appropriate host may be transformed with a vector containing the appropriate DNA sequence and an appropriate promoter or control sequence to express the protein in the host. More particularly, the present invention also encompasses a recombinant construct comprising one or more of the sequences outlined above. A construct comprises a vector, such as a plasmid or viral vector, into which the sequences of the invention have been inserted in a forward or reverse orientation. In a preferred embodiment of this embodiment, the construct further comprises a regulatory sequence, including, for example, a promoter operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.
For example, there are the following vectors. Bacterial vector: pET3 vector (Stratagene)
, PQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, pharges
cript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16
a, pNH18A, pNH46A, (Stratagene), ptrc99a, pKK2
33-3, pDR540, pRIT5 (Pharmacia). Eukaryotic cell vector: pBlue
BacIII (Invitrogen), pWLNEO, pSV2CAT, pOG44, pXT1
, PSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharma
cia). However, other plasmids or vectors may be used as long as they are replicable and viable in the host.

Examples of recombinant DNA vectors for cloning and host cells which can be transformed are bacteriophage λ (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (Gram negative bacteria) ), PG
V1106 (Gram negative bacteria), pLAFR1 (Gram negative bacteria), pME29
0 (non-E. Coli gram negative bacteria), pHV14 (E. coli and Bacillus
Subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pU
C6 (Streptomyces), YIp5 (Saccharomyces), Baculovirus insect cell line, YCp19 (Saccharomyces). Generally, "DNA Cloning": Vols.
I & II, Glover et al. Ed. IRL Press Oxford (1985) (1987) and T. Maniati
s et al. "Molecular Cloning" Cold Spring Harbor Laboratory (1982).

In some cases, it may be preferable to add sequences that cause secretion of the polypeptide from the host cell, followed by cleavage of the secretory signal. The polypeptide can be expressed in a host cell under the control of a suitable promoter. Such a protein may be obtained using a cell-free translation system and RNA derived from the DNA construct of the present invention. Suitable cloning and expression vectors for use in prokaryotic and eukaryotic cells are described in Sambrook, et al., Molecular Cloning: A Laboratory Manual.
, Second Edition, Cold Spring Harbor, NY, (1989), which is hereby incorporated by reference. After transformation of the appropriate host strain and growth of the host cell to an appropriate cell density, the selection promoter is induced by appropriate means (eg, temperature shift or chemical induction) and the cells are cultured for an additional period. Typically, the cells are collected by centrifugation, the cells are disrupted by physical or chemical means, and the resulting crude extract is saved for further purification. Microbial cells used for protein expression can be disrupted by conventional methods, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents, and such methods are well known to those skilled in the art. ing. Depending on the selected expression system and host, the polypeptide of the present invention may be produced by growing host cells transformed with the above expression vector under conditions that allow the expression of the target polypeptide. Then, isolating the polypeptide from the host cell,
Purify. If the expression system secretes the polypeptide into the growth medium, the polypeptide can be purified directly from the medium. If the polypeptide is not secreted, the polypeptide is isolated from the cell lysate or recovered from the cell membrane fraction. Where the polypeptide is localized on the cell surface, whole cells or isolated cell membranes can be used as an assayable source of the desired gene product. Polypeptides expressed in bacterial hosts, such as E. coli, may require isolation and regeneration from inclusion bodies. If the mature protein has a highly hydrophobic region that leads to overexpression of the insoluble product, it may be desirable to express a truncated protein in which the hydrophobic region has been deleted. Selection of appropriate growth conditions and recovery methods are within the skill of the art. Recombinant polypeptides by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography It can be recovered from cell culture and purified.
In completing the configuration of the natural protein, a protein regeneration step can be used if necessary. Finally, high quality liquid chromatography (HPLC) may be used for the final purification step. Depending on the host used in the recombinant production method, the polypeptide of the present invention may or may not be glycosylated. The polypeptide of the present invention may include the first methionine amino acid residue.

Preparation of the RNA Component of RNase P The RNA molecule of RNase P was prepared using the run-off of in vitro transcription using T7 RNA polymerase under standard conditions (recommended by the supplier, eg, Promega).
-off). The plasmid is linearized using an appropriate restriction enzyme to obtain linear dsDNA containing a full-length gene encoding RNase P RNA. RNA is purified from preparative denaturing acrylamide gels or precipitated before use in in vitro cleavage assays. Substrates for RNase P RNA and RNA complexed with its protein (RNase P protein) can be obtained by in vitro transcription of cloned genes. Useful substrates are pre-tRN
A. ^Met or E. coli pre-4.5S or B. subtilis pre-sc RNA molecules, including but not limited to, using an in vitro transcription system directed by a T7 RNA polymerase as described above. It may be expressed.

Antagonists and Agonists-Assays and Molecules The present invention provides methods for screening for agents that interfere with the RNA portion, protein portion, and / or intact RNA / protein complex of RNase P described herein. The method comprises measuring the inhibition of protein and / or RNA activity by the test agent. For example, because the selected RNA moiety has catalytic activity, after appropriate purification and formulation, the activity of the RNA can be tracked by its ability to convert natural or synthetic RNA substrates. Different chemically synthesized test compounds or natural products can be used in such enzyme activity assays to detect additives that compete with natural or synthetic substrates or inhibit enzyme activity. The present invention also provides a compound screening method for identifying a compound that enhances (agonist) or blocks (antagonist) the action of an RNaseP polypeptide or polynucleotide, specifically a bacteriostatic and / or bactericidal compound. Also provide. The screening method is a high throughput method. For example, to screen for agonists or antagonists, synthetic reaction mixtures, membranes, cell compartments, such as cell surface membranes or cell walls, or RNase polypeptides or polynucleotides comprising such substrates, or ligands, comprising RNase polypeptides or polynucleotides. Either preparation is incubated in the presence or absence of candidate molecules that can be agonists or antagonists of the RNaseP polypeptide or polynucleotide. The ability of a candidate molecule to enhance or block the action of an RNaseP polypeptide and / or polynucleotide is reflected in reduced binding of a labeled ligand or reduced production of a product from such a substrate. Molecules that bind and have no effect, ie, do not induce the effects of an RNaseP polypeptide or polynucleotide, may be the best antagonists. Molecules that bind well and increase the rate of product formation from the substrate are agonists. The production rate or level of product from the substrate can be enhanced by using a reporter system. Reporter systems useful in this regard include labeled substrates for colorimetry, RNaseP, which are converted to products.
A reporter gene responsive to a change in polynucleotide or polypeptide activity,
And binding assays well known in the art, but are not limited thereto. Another example of an assay for antagonists to RNaseP is a competition assay, in which RNaseP and a potential antagonist are converted to a polypeptide binding molecule, a recombinant polypeptide binding molecule, a natural substrate or ligand, under conditions suitable for a competition inhibition assay. Or mixed with a substrate or ligand mimetic. RNaseP can be labeled, for example, with a radioactive or colorimetric compound, and the number of RNaseP molecules bound to the binding molecule or converted to product can be accurately determined to assess the effect of the potential antagonist. Potential antagonists include small organic molecules, peptides, polypeptides, and antibodies that bind to a polynucleotide or polypeptide of the present invention, thereby inhibiting or eliminating its activity. Also, potential antagonists are small organic molecules, peptides, polypeptides, such as closely related proteins or antibodies,
Potential antagonists bind to the same site on the binding molecule, but do not induce the activity of RNaseP, and thus eliminate RNaseP from binding by eliminating RNaseP from binding.
It hinders the action of Potential antagonists include polypeptides and / or polynucleotides,
In particular, there are small molecules that bind to and occupy the binding site of RNase P and thereby interfere with binding to cellular binding molecules, thereby disrupting normal biological activity. Examples of small molecules include, but are not limited to, small organic molecules, peptides, peptide-like molecules, and the like. Other potential antagonists include antisense molecules (see Okano, J., Neuroche for a description of these molecules).
m.56: 560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene
Expression, CRC Press, Boca Raton, FL (1988)). Preferred potential antagonists include RNaseP related compounds and RNaseP variants.

[0059] Each of the DNA sequences provided herein may be used in the discovery and development of antibacterial compounds. The encoded protein, when expressed, can be used as a target for screening for antibacterial agents. Furthermore, an antisense sequence is constructed using a DNA sequence encoding the amino-terminal region of the encoded protein or the Shine-Dalgarno sequence or other translation-enhancing sequence of each mRNA to control the expression of the target coding sequence. You can also. The antagonists and agonists of the present invention may be used, for example, in upper respiratory tract diseases (eg, otitis media, bacterial tracheitis, acute pharyngitis, thyroiditis), lower respiratory tract diseases (eg, pyometra, pulmonary abscess), heart diseases (eg, infectious Endocarditis), gastrointestinal disorders (eg, secretory diarrhea, spleen abscess, retroperitoneal abscess), CNS disorders (eg, cerebral abscess), eye disorders (eg, blepharitis, conjunctivitis, keratitis, endophthalmitis, anterior Septal and orbital cellulitis, lacrimal cystitis), renal and ureteral diseases (eg, epididymitis, intrarenal and perirenal abscess, toxic shock syndrome), skin diseases (eg, impetigo, folliculitis, skin abscess, It may be used for controlling and treating diseases such as cellulitis, wound infections, bacterial myositis, and bone and joint diseases (eg, septic arthritis, osteomyelitis).

Assays for detecting compounds that inhibit the cleavage of RNA substrates by the HTP screening method RNase P can be developed. Several possible assay formats are suitable for HTP screening based on the ability to incorporate labels into RNA site-specifically by chemical synthesis. Conventional radioactivity-based formats are preferred, but homogeneous fluorescence-based formats are useful for subsequent lead compound follow-up. The use of either format is contemplated by the present invention.

Functional RNase P Assay Biotin is introduced at the appropriate position in the RNA substrate and the 5 'end is labeled with ³² P.
The substrate is bound to the 96-well plate via streptavidin. Substrate R
After Nase P-dependent hydrolysis, the radiolabeled 5 'leader cleavage product is released into the bulk solution phase and subjected to scintillation counting. Alternatively,
The RNA substrate is bound to a streptavidin-coated flash plate and the signal is reduced by release of the radioactive hexamer into the solution phase. This has the advantage that no further steps are required after the start of the assay, except for the filtration step in certain embodiments, which is a homogeneous and continuous assay format. Either format is useful in the practice of the present invention. This is because both use the same RNA substrate.

[0062] Efficient approaches to identifying compounds that interact with RNA fragment library rescue RNA are contemplated. The concept is based on overexpression of a drug binding site regenerated on the RNA fragment, which will sequester the drug and continue the functioning of the intact ribozyme. This approach has recently been described because of the search for ligands that bind to ribosomal RNA (Howard, BA, et al., Biochem. Cell Bio. 73
(11/12): 1161-1166 (1995)). After selection, any RNA fragments that clearly show the minimal target structure for drug recognition are incorporated into the protocol for rational drug design. Thus, a random fragment library based on RNase P RNA is obtained and used for HTP screening to identify compounds that disrupt RNA / protein interactions.

Incorporating the constraints on the cyclic peptide phage library conformation into a flexible lead compound is a powerful strategy for increasing the potential of the lead compound and is particularly useful in the field of peptidomimetic design. (Al-Obeidi, F. et al., J. Med. Chem . 32
: 2555-2561 (1989); Barker, PL, et al., J. Med.Chem . 35: 2040-2048 (
1992)). In this case, a cyclic peptide library is constructed that includes adjacent cysteine residues to allow for efficient disulfide bond formation and cyclization in the phage assembly. The streptavidin-bonded crystal structures of the two disulfide-bridged peptides were both shown to be in beta-turn conformation (Ka
hn, M. (Guest, Ed., 1993) Tetrahedron 49 , Symp. 50, 3433-3677). Beta-turns are important recognition elements in many biological interactions, and efforts have therefore been directed at the design of small, constrained beta-turn mimics (Kahn, M. (Guest Ed. ., 1993) Tetrahedron 49 , Symp. 50, 3433-3677).
This approach, when applied to RNase P, can identify suitable cyclic peptides for the synthesis of peptide mimetics as inhibitor molecules. A cyclic octapeptide phage display library can be constructed and used to identify peptides that interact with certain RNA domains.

Secondary Evaluation SELEX: Systematic Evolution of Ligands by Exponential Enrichment As an aid to the rational drug design and the secondary evaluation of compounds identified by HTP screening, RNase for structural analysis was P RNA (M1
This approach may be used to identify the RNA recognition motif to which the (RNA) protein binds. The method is based on repeated selections and amplification of RNA fragments that specifically bind proteins with high affinity (Szostak, JW, TIB S 17: 89-93 (1992)). Fragment libraries based on S. pneumoniae and E. coli RNase P RNA can be constructed to perform in vitro synthesis of RNA and subsequent selection of molecules that bind to their individual proteins. Interaction sites may be mapped using methods to search for chemical and enzyme structures in conjunction with protein / RNA protection studies. The resulting RNA
Fragment-based SELEX may be further utilized to determine the minimum structural requirements for RNA recognition.

The identification of RNase P assembly disruption protein / RNA-fragment pairs allows the development of screens for compounds that disrupt those assemblies. Drug-induced destruction of labeled RNA (biotin / streptavidin) bound to immobilized protein is accompanied by a reduction / loss of the signal caused by the presence of the RNA.

RNA / Drug Interactions RNase P RNA fragments conferring drug resistance (RNA fragment rescue library described above) were sequenced to search for chemical and enzyme structures in the presence and absence of drug. To do so, the binding sites may be mapped by expression in vitro. SELEX may be applied to the lead compound to identify minimum structural requirements for drug binding.

The minimal RNA substrate including RNase P substrate pre-tRNA and pre-scRNA derivatives is H
It may be chemically synthesized for TP screening. RNA-ligand interactions involving the 2'-hydroxyl group of a particular nucleotide may be explored by chemically synthesizing appropriately modified RNA fragments. Characteristic C ^{3 ribonucleotides'} - to hold the endo configuration, 2'-methoxy and 2'full Oro can be used ribonucleotide analogs, sterically latter is preferred.
C ² nucleotides undesirable lacking 2'substituent ^'- take endo configuration. Further, the present invention relates to inhibitors identified by any of the methods described herein. It is understood that, due to the enzymatic nature of the RNase P action, inhibitors can be identified that act as mimetics of the transition state, inhibitors of product release or inhibitors of substrate binding.

Diagnostic Assays The present invention also relates to the use of RNase P polynucleotides for use as diagnostic reagents. Detection of RNase P in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease. Eukaryotes (also referred to herein as "individuals"), particularly mammals, and especially humans, infected with an organism containing the RNase P gene can be detected at the DNA level by various methods. Diagnostic nucleic acids can be obtained from cells and tissues of infected individuals, such as bone, blood, muscle, cartilage and skin. Genomic DNA may be used directly for detection, or may be amplified enzymatically by using PCR or other amplification methods prior to analysis. RNA or cDNA can also be used in the same way. Using amplification methods, prokaryotic strains present in eukaryotes, particularly mammals, and especially humans, can be characterized by genotyping prokaryotic genes. Deletions and insertions can be detected by a change in size of the amplification product when compared to the genotype of the control sequence. Point mutations can be identified by hybridizing the amplified DNA to a labeled RNase P polynucleotide sequence. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. By detecting changes in electrophoretic mobility of DNA fragments in gels with or without denaturing agents, or by direct DNA sequencing,
Sequence differences may be detected. See, for example, Meyers et al., Science, 230: 1242 (1985). Sequence changes at specific locations can also be determined by nuclease protection assays, such as R
It may be revealed by Nase and S1 protection or chemical cleavage methods. See, e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985). Cells carrying mutations or polymorphisms in the gene of the invention may be detected at the DNA level by various techniques, for example by serotyping. For example, mutations can be detected using RT-PCR. It is particularly preferred to use RT-PCR in combination with an automatic detection system, such as GeneScan. RN
A or cDNA may be used for PCR or RT-PCR for the same purpose. By way of example, PCR primers complementary to a nucleic acid encoding RNase P can be used to identify and analyze mutations. Using these primers, RNase P DNA isolated from infected individuals was amplified and
The gene may be subjected to various techniques for examining the A sequence. In this way,
Mutations in the DNA sequence can be detected and used to diagnose infection and serotype and / or classify infectious agents.

The invention also relates to diseases, preferably bacterial infections, more preferably infections by Streptococcus pneumoniae, most preferably otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, empyema and endocardium. The present invention provides a method for diagnosing a disease such as meningitis, such as, for example, infection of cerebrospinal fluid, which is described in Table 1 [SEQ ID NO: 1, 3, 4, 5 or 7]. Any of the above) is detected from a sample derived from an individual. Increasing or decreasing RNase P polynucleotide expression can be determined by any method known in the art for quantifying polynucleotides, such as amplification, PCR, RT-PCR.
, RNase protection, Northern blotting and other hybridization methods. In addition, a diagnostic assay of the invention for detecting overexpression of RNase P protein compared to normal control tissue samples may be used, for example, to detect the presence of an infection. Assay techniques that can be used to determine levels of a RNase P protein, in a sample derived from a host are well-known to those of skill in the art. Such assays include:
There are radioimmunoassay, competitive binding assay, western blot analysis and ELISA assay.

Antibodies Antibodies immunospecific for such polypeptides can be obtained using the polypeptides of the present invention or variants thereof, or cells expressing them, as an immunogen. As used herein, “antibody” includes monoclonal and polyclonal antibodies, chimeras, single chains, monkey and humanized antibodies, and Fab fragments, as well as Fab fragments, such as the products of an immunoglobulin expression library. Included. Fab fragments may be prepared from the parent monoclonal antibody by enzymatic treatment, for example, by cleaving the Fab portion from the Fc portion using papain. Antibodies raised against the polypeptides of the invention can be obtained by administering fragments, analogs or cells bearing the polypeptides or epitopes, preferably to non-human animals, using conventional laboratory methods. The antibodies so obtained will bind to the polypeptide itself. In this way, even sequences encoding only fragments of the polypeptide can be used to obtain antibodies that bind to the whole raw polypeptide. Using such an antibody, a target polypeptide can be isolated from a tissue that expresses the target polypeptide. Monoclonal antibodies can be prepared using techniques well known to those skilled in the art, which provide antibodies produced by continuous cell line cultures. Examples include Kohler, G. and Milst
ein, C., Nature, 256: 495-497 (1975); Kozbor et al., Immunology Today, 4:
72 (1983); Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R Li.
SS, Inc., pages 77-96 (1985). One or more of the original polypeptides and / or fusion proteins may be used to screen hybridomas to select for cell lines that have high affinity and favor cross-reactivity with other Staphylococcus species. . Techniques described for the production of single-chain antibodies (US Pat. No. 4,946,778) can be applied to obtain single-chain antibodies to the polypeptides of the invention. Also,
Transgenic mice or other organisms, such as other mammals, may be used to express antibodies such as humanized antibodies.

Alternatively, a phage display technique is used to screen for the presence of anti-RNaseP from a repertoire of PCR amplified v-genes of human lymphocytes screened for having an anti-polypeptide. Antibody genes having binding activity to polypeptides can be selected from humans or from untreated libraries (McCafferty, J. et al., Nature 3).
48: 552-554 (1990); Marks, J. et al., Biotechnology 10: 779-783 (1992)).
The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., Nature 352: 624-628 (1991)). Antibodies should be screened again for high affinity for the polypeptide and / or fusion protein. As described above, a fragment of the final antibody may be prepared. The antibody is an intact antibody having a molecular weight of about 150,000 or a derivative thereof, for example, Skerra
, A and Pluckthum, A., Science 240: 1038-1040 (1988). If there are two antigen binding domains, each domain is directed against a different epitope, termed a "bispecific" antibody. In particular, derivatives that are slightly longer or slightly shorter than the intact protein or polypeptide fragment of the invention may be used. In addition, polypeptides in which one or more amino acid residues have been modified may be used. Such peptides may be prepared, for example, by amino acid substitution, addition or transfer, or by chemical modification. All such substitutions and modifications are widely known to those skilled in peptide chemistry.

Using the above antibodies, clones expressing the polypeptide can be isolated and identified, and the polypeptide can be purified by affinity chromatography. Thus, in particular, RNaseP polypeptides or, for example, RNAs of RNaseP
Infections, especially bacterial infections, especially otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pyothorax and endocarditis, using antibodies against RNaseP polynucleotides, including Diseases such as meningitis, such as cerebrospinal fluid infections, may be treated. Preferably, an antibody is prepared by expressing a DNA polymer encoding the antibody in an appropriate expression system as described above for expression of a polypeptide of the invention. Selection of the vector for the expression system will depend in part on the host, for example, prokaryotic cells such as E. coli (preferably strain B) or Streptomyces species, mouse C127, mouse It may be myeloma, human HeLa, Chinese hamster ovary, filamentous fungus or unicellular fungus or insect cells. The host may be a transgenic animal or a transgenic plant (eg, as described in Hiatt, A. et al., Nature 340: 76-78 (1989)). Suitable vectors include plasmids, bacteriophages, cosmids, and recombinant viruses, for example, from baculovirus and vaccinia.

[0073] Polypeptide variants include antigenically, epitopically or immunologically equivalent variants and are particular embodiments of the present invention. "Antigenically equivalent derivative" as used herein
The term refers to a polypeptide or polypeptide thereof specifically produced by a particular antibody that, when produced against a protein or polypeptide according to the present invention, interferes with the immediate physical interaction between the pathogen and the mammalian host. Includes equivalents. As used herein, the term "immunologically equivalent derivative" refers to an antibody that, when used in a formulation suitable for producing antibodies in vertebrates, causes immediate physical contact between the pathogen and the mammalian host. Includes peptides or equivalents that act to interfere with the interaction. Polypeptides, such as antigenically, immunologically equivalent derivatives, or fusion proteins thereof, can be used as antigens to immunize mice or other animals, such as rats or chickens. Fusion proteins can confer stability on a polypeptide. The antigen can bind to an immunogenic carrier protein, such as, for example, by conjugation, to bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH). Alternatively, multi-antigen peptides containing multiple copies of the protein or polypeptide, or polypeptides that are antigenically or immunologically equivalent thereto, have sufficient antigenicity to improve immunogenicity. No need to use carrier. Preferably, the antibodies or variants thereof are modified to reduce immunogenicity in the individual. For example, if the individual is a human, most preferably the antibody is "humanized"; in this case, the complementarity determining region of the antibody from the hybridoma is human.
Monoclonal antibodies have been transplanted, e.g., Jones, P. et al., Nature 321: 522-525.
(1986) or Tempest et al., Biotechnology 9: 266-273 (1991). Humanized monoclonal antibodies or fragments thereof having binding activity form a particular aspect of the invention. The modifications are not limited to those "humanized", and other primate sequences may be used. When the polynucleotide of the present invention is used in genetic immunization, for example, direct injection of plasmid DNA into muscle (Wolff et al., Hum. Mol. Genet. 1: 363 (1992); Ma)
nthorpe et al., Hum. Gene Ther. 4: 419 (1963)), delivery of a complex of a specific protein carrier and DNA (Wu et al., J. Biol. Chem. 264: 16985 (1989)), calcium phosphate Co-precipitation with DNA (Benvenisty & Reshef, PNAS 83: 9551 (1986)), encapsulation of DNA in various forms of liposomes (Kaneda et al., Science 243: 375 (1989)), particle bombardment (Tang et al., Nature 356). : 152 (1992); Eisenbraun et al., DNA Cell Bio.
l.12: 791 (1993)) and in vivo infection with cloned retroviral vectors (Seeger et al., PNAS 81: 5849 (1984)).

Vaccine Another aspect of the present invention relates to a method of inducing an immunological response in an individual, particularly a mammal, comprising the step of producing an individual from an infection, particularly a bacterial infection, most particularly a S. pneumoniae infection. Injecting an individual with sufficient antibody to protect and / or RNaseP or a fragment or variant thereof to generate a T cell immune response. Also provided is a method wherein such an immunological response slows bacterial replication. Yet another aspect of the present invention relates to a method of inducing an immunological response in an individual, the method comprising the step of in vivo RNaseP, or a fragment or variant thereof, whether or not the disease has been established in the individual. A nucleic acid vector that directs the expression of RNaseP or a fragment or variant thereof for expression is delivered to such an individual, for example, to generate an antibody and / or a T cell immune response (eg, a cytokine producing T cell or a cytotoxic T cell). Inducing an immunological response to produce antibodies that protect the individual from disease. As a method for rapidly administering a gene into a desired cell, there is a method such as coding on a particle. Such nucleic acid vectors may include DNA, RNA, modified nucleic acids, or DNA / RNA hybrids.

A further aspect of the invention is that an immunological response can be induced in a host or, when introduced into a host in which it has been induced, an immunological response to RNaseP or a protein encoded thereby can be induced in the host. For an immunological composition to induce, the composition comprises recombinant RNaseP or a protein encoded thereby,
D encoding and expressing an antigen for eP or the protein encoded thereby
NA. The immunological response may be used therapeutically or prophylactically, and the immunological response may be in the form of antibody immunity or cellular immunity, such as that arising from CTLs or CD4 ⁺ T cells. RNaseP polypeptides or fragments thereof can be combined with a co-protein capable of producing a fusion protein that does not itself produce antibodies but stabilizes the first protein and has immunogenic and protective properties. They may be fused. Preferably, such a fusion recombinant protein solubilizes an antigen co-protein, eg, a coexisting protein such as lipoprotein D from Hemophilus influenzae, glutathione-S-transferase (GST) or beta-galactosidase. And relatively large coexisting proteins that promote their production and purification. Furthermore, co-proteins provide a universal stimulus in the immune system,
It can act as an adjuvant. The coexisting protein may be bound to either the amino or carboxy terminus of the first protein. An RNaseP polynucleotide, particularly RNaseP RNA or a fragment thereof, is fused to RNA, DNA or PNA (herein such a fusion is referred to as "co-polynucleotide"), and the coexisting polynucleotide is itself an antibody. , But can stabilize the first polynucleotide, resulting in a fusion polynucleotide having immunogenic and protective properties. The recombinant polynucleotide thus fused preferably further comprises a copolynucleotide or polynucleotide that facilitates purification. Moreover, copolynucleotides can serve as adjuvants in the sense that they cause systemic stimulation of the immune system. The copolynucleotide is 5 ′ of the first polynucleotide.
Or it may be attached to any of the 3 'ends.

The present invention relates to polypeptides or polynucleotides of the invention and Sato Y. et al., S
cience 273: 352 (1966), including vaccine compositions and methods, including immunostimulatory DNA sequences. The present invention has also been described, in an animal model infected with Streptococcus pneumoniae, as having been described which encodes a constant region of a bacterial cell surface protein in a DNA construct used for such genetic immunization experiments. Or, inter alia, methods of using fragments thereof are provided, which are particularly useful for identifying protein epitopes capable of stimulating a prophylactic or therapeutic immune response. This study is particularly valuable from the essential organs of animals that have succeeded in resisting and clearing bacterial infections in mammals, especially humans, for the development of preventive or therapeutic treatments for bacterial infections, especially Streptococcus pneumoniae infections. It will be possible to prepare monoclonal antibodies successfully. The polypeptide may be used as an antigen for host inoculation to obtain a specific antibody that protects against bacterial invasion, for example by blocking bacterial attachment to damaged tissue. Examples of tissue damage include, for example, mechanical, chemical or thermal damage,
Or wounds of the skin or connective tissue, or mucous membranes such as those of the mouth, mammary gland, urethra or vagina, caused by implantation of an indwelling device.

The invention also includes a vaccine formulation that includes an immunogenic recombinant protein together with a suitable carrier. Since the protein can be broken down in the stomach, it can be parenterally, for example, subcutaneously,
It is preferably administered intramuscularly, intravenously or intradermally. Formulations suitable for parenteral administration include aqueous or non-aqueous sterilization which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the body fluids of the individual, preferably blood. Injectable solutions and sterile aqueous or non-aqueous suspensions which may contain suspending or thickening agents, and the like. The formulations may be presented in single-dose or multi-dose containers, for example, sealed ampules and vials, and stored in a lyophilized condition requiring only the addition of a sterile liquid carrier immediately before use. Vaccine formulations may also contain adjuvant systems that increase the immunogenicity of the formulation, such as oil-in-water or other systems well known in the art. The dosage depends on the specific activity of the vaccine and can be readily determined by routine experimentation. Although the invention has been described with respect to particular RNase P proteins, the invention relates to naturally occurring proteins and fragments of similar proteins with additions, deletions or substitutions that do not substantially affect the immunogenic properties of the recombinant protein. It will be understood that it includes.

Compositions, Kits and Administration The present invention also relates to compositions comprising the polynucleotides or polypeptides described above, or agonists or antagonists thereof. The polypeptides of the present invention can be used in admixture with non-sterile or sterile carriers used for cells, tissues or organisms, for example, with a pharmaceutical carrier suitable for administration to a subject.
Such compositions comprise, for example, a solvent additive or a therapeutically effective amount of a polypeptide of the invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration. The present invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the above-described compositions of the present invention.

The polypeptides of the present invention and other compounds may be used alone or in combination with other compounds such as therapeutic compounds. The pharmaceutical composition may be administered by any effective and convenient method, for example, topically, orally, vaginally, intravenously, intraperitoneally, intramuscularly, subcutaneously, intranasally, or intradermally. . In therapy or as a prophylactic, the active substance can be administered to the individual as an injectable composition, for example, preferably as an isotonic sterile aqueous dispersion. Alternatively, the compositions may be formulated for topical application, for example, in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwashes, impregnated bandages and suture threads, and aerosols. Ointments and creams may contain suitable conventional additives such as preservatives, solvents to aid penetration of the drug, and ointments and creams. Such topical formulations may contain compatible conventional carriers, such as cream or ointment bases and, for lotions, ethanol or oleyl alcohol. Such carriers may represent from about 1% to about 98% by weight of the formulation, more usually up to about 80% by weight of the formulation. For administration to mammals, especially humans, the daily dose of active substance is:
It is from 0.01 mg / kg to 10 mg / kg, typically about 1 mg / kg. The physician will always determine the actual dosage which will be most suitable for the individual and will vary according to age, weight and especially the individual's responsiveness. The above dosages are typical of the average case. Of course, there are individual examples where the high and low dose ranges fit, and such examples are within the scope of the present invention.

Indwelling devices include surgical implants, prosthetic devices, catheters, and the like, that is, those that are introduced into the body of an individual and remain in place for an extended period of time. Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (cont
There is an infinite ambulatory peritoneal dialysis (CAPD) catheter. The compositions of the present invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of the indwelling device. After surgery, treatment may continue for as long as the device is present in the body. In addition, it can be used on covers that spread during surgical procedures to protect against bacterial wound infections, especially those of Streptococcus pneumoniae. Many orthopedic surgeons believe that humans with prosthetic joints should be considered for antibiotic prophylaxis before dental treatments that can result in bacteremia. Delayed severe infection is a serious complication that sometimes leads to loss of prosthetic joints, with significant morbidity and mortality. It is therefore possible in this context to extend the use of active substances as an alternative to prophylactic antibiotics.

In addition to the treatments described above, the compositions of the present invention are generally used as therapeutic agents for wounds,
Bacteria may be prevented from adhering to the exposed matrix proteins in the wound tissue, and may be used prophylactically in dental treatment instead of or in combination with antibiotic prophylaxis. Alternatively, the composition of the present invention may be used to bathe an indwelling device immediately prior to insertion. For immersion of wounds or indwelling devices, the active substance is from 1 μg / ml to 10 m
Preferably, the concentration is g / ml. The vaccine composition is conveniently in injectable form. Conventional immune adjuvants may be used to enhance the immune response. A unit dose suitable for vaccination is 0.5-5 μg / kg of antigen, and such dose is preferably administered once to three times at intervals of one to three weeks. For the compounds of the present invention, no adverse toxicological effects are observed that would prevent administration to appropriate individuals in the dosage ranges indicated. Each document disclosed herein is considered to be incorporated herein by reference in its entirety. Any patent application for which this application claims priority is deemed to be incorporated herein by reference in its entirety.

EXAMPLES The following examples, except as described in detail, are performed using standard techniques well known to those skilled in the art. The examples are given for illustrative purposes only and do not limit the invention. Example 1 An example of an assay for influencing and detecting the cleavage of Streptococcus pneumoniae RNaseP is shown below. The assay is to first perform a kinetic analysis to obtain the appropriate K _M and k _cat to screen. Thus, if formation of the holoenzyme is desirable for the assay, the holoenzyme may be formed using Streptococcus pneumoniae RNA and either E. coli or Staphylococcus aureus proteins. In order to easily determine the catalytic activity, preferably the holoenzyme is present in the assay system at a final concentration of 100 nM.
The ³² P-substrate analog was present in only trace amounts (eg, about 50,000 counts per minute per reaction (CPM) per reaction). RNA (including the ³² P-substrate analog) was added to dH ₂ O and denatured at 95 ° C. for 1.5 minutes. They were then cooled at room temperature for about 3-4 minutes. At this point, 2X
Buffer (200 mM Tris pH 7.0, 300 mM KCl, 20 mM
The MgCl _2, 10% PEG) was added to the RNA / dH ₂ O mixture, and incubated 4-5 minutes at room temperature. Then, the individual proteins were mixed gently and RNA / dH ₂ O / 2X buffer. Substrates do not incorporate proteins. All cocktails are incubated for an additional 10 minutes at room temperature. After this incubation, the reaction is started by adding the holoenzyme cocktail to the substrate cocktail and allowed to react for 30 minutes at room temperature. Volume ratio 1
The reaction was stopped by adding a 1: 1 inactivation buffer to the reaction (typically 10 microliters of reaction for screening). At this point, the reaction product was loaded on the gel, moved on the gel, and the amount of the reaction product was quantified. The E. coli C5 RNaseP protein may be used in the reactions described in this example, as well as in other screening assays and assays for activity described elsewhere herein. This protein can be found, for example, in Hansen F., E. Hansen, and T. Atlung.
1985.Physical mapping and nucleotide sequence of the rnpA gene that en
codes the protein of ribonuclease P in Escherichia coli. Gene 38: 85-93.

[0083]

[Sequence list]

 SEQUENCE LISTING <110> SmithKline Beecham Corporation <120> Novel Rnase P <130> GM50036 <140> PCT / US98 / 18291 <141> 1998-09-03 <150> US 60 / 057,520 <151> 1997-09-04 < 160> 7 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 354 <212> DNA <213> S. pneumoniae <400> 1 atgttattgg aaaaagctta ccgaattaaa aagaatgcag attttcagag aatatataaa 60 aaaggtcatt ctgtagcca cagacaattt gttgtata catttag gattgtatacattg aaaaaactag gtaatgcagt gttaagaaac 180 aagattaaaa gagcaatacg tgaaaatttc aaagtacata agtcgcatat attggccaaa 240 gatattattg taatagcaag acagccagct aaagatatga cgactttaca aatacagaat 300 agtcttgagc acgtacttaaattatta aaa attagatta 2 ag Glu Lys Val Tyr Arg Ile Lys Lys Asn Ala Asp Phe Gly 1 5 10 15 Arg Ile Tyr Lys Lys Gly His Ser Val Ala Asn Arg Gln Phe Val Val 20 25 30 Tyr Thr Cys Asn Asn Lys Glu Ile Asp His Phe Arg Leu Gly Ile Ser 35 40 45 Val Ser Lys Lys Leu Gly Asn Ala Val Leu Arg A sn Lys Ile Lys Arg 50 55 60 Ala Ile Arg Glu Asn Phe Lys Val His Lys Ser His Ile Leu Ala Lys 65 70 75 80 Asp Ile Ile Val Ile Ala Arg Gln Pro Ala Lys Asp Met Thr Thr Leu 85 90 95 Gln Ile Gln Asn Ser Leu Glu His Val Leu Lys Ile Ala Lys Val Phe 100 105 110 Asn Lys Lys Ile Lys 115 <210> 3 <211> 402 <212> DNA <213> S. pneumoniae <400> 3 gatgtgcaat ttttggataa tcgagtgaag ggaattgctt ctcatgagga agcat 60 tagcacaggc tgtgatgcct gtagtatttg tgataggcga aaccataagc ctagggacga 120 gaattcgtta cggcagttga aatggataag tccttggata agccagagta ggcttgaaag 180 tgccacagtg acggagtctt tctggaaaca gagagagtgg aacgcggtaa acccctcaag 240 ctagcaaccc aaattttggt cggggcatgg agtacgcgga aacggacgta gtattctgac 300 tggtatcagc tagagctgtt agtggtagac agatgattat cgaaggaagt ggtcctagtc 360 acttttggaa caaaacatgg cttatagaaa attgcatata gg 402 <210> 4 <211> 267 <212> RNA <213> S. pneumoniae <400> 4 gatgtgcaat ttttggataa tcgcgtgagg agaattgctt ctcatgagga aagtccatgc 60 tagcacaggc tgtgatgcct gtagtgtctg tgctacgcga aaccataagc ctatggacga 120 ga aatcgtta cggcagttga aatggctaat tccttggata agccacagta tgcttgaaat 180 tgccacagtg accgagtctt tctggaaaca catagattgg aacgccgtaa acccctcaag 240 ctagcaaccc aaattttggt cggggca 267 <210> 5 <211> 336 <212> DNA <213> S. pneumoniae <400> 5 ttgaagaaaa actttcgtgt aaaaagagag aaagatttta aggcgatttt caaggagggg 60 acaagttttg ctaatcgcaa atttgtggtc taccaattag aaaaccagaa aaaccatttt 120 cgagtaggtc tatcagttag caaaaaactg gggaatgccg tcactagaaa tcaaattaag 180 cgacggattc ggcatattat ccagaatgca aaagggagtc tggtagaaga tgtcgacttt 240 gttgtcattg ctcgaaaagg agtcgaaacc ttgggatacg cagagatgga gaaaaatcta 300 ctccatgtat taaaattatc aaagatttac cgggaa 336 <210> 6 <211> 112 <212> PRT <213> S. pneumoniae <400 > 6 Leu Lys Lys Asn Phe Arg Val Lys Arg Glu Lys Asp Phe Lys Ala Ile 1 5 10 15 Phe Lys Glu Gly Thr Ser Phe Ala Asn Arg Lys Phe Val Val Tyr Gln 20 25 30 Leu Glu Asn Gln Lys Asn His Phe Arg Val Gly Leu Ser Val Ser Lys 35 40 45 Lys Leu Gly Asn Ala Val Thr Arg Asn Gln Ile Lys Arg Arg Ile Arg 50 55 60 His Ile Ile Gln Asn Ala Lys Gl y Ser Leu Val Glu Asp Val Asp Phe 65 70 75 80 Val Val Ile Ala Arg Lys Gly Val Glu Thr Leu Gly Tyr Ala Glu Met 85 90 95 Glu Lys Asn Leu Leu His Val Leu Lys Leu Ser Lys Ile Tyr Arg Glu 100 105 110 <210> 7 <211> 402 <212> RNA <213> S. pneumoniae <400> 7 gatgtgcaat ttttggataa tcgcgtgagg agaattgctt ctcatgagga aagtccatgc 60 tagcacaggc tgtgatgcct gtagtgtttg tgctaggcga aaccataagc ctagggacga 120 gaaatcgtta cggcagttga aatggctaag tccttggata agccagagta ggcttgaaag 180 tgccacagtg acggagtctt tctggaaaca gagagagtgg aacgcggtaa acccctcaag 240 ctagcaaccc aaattttggt cggggcatgg agtacgcgga aacgaacgta gtattctgac 300 tgctatcagc tagagctgtt agtggtagac agatgattat cgaaggaagt ggtcctagtc 360 acttctggaa caaaacatgg cttatagaaa attgcatata gg 402

[Brief description of the drawings]

FIG. 1 is a side-by-side comparison of the two sequences in FIG. 1 with differences highlighted in gray. There are 20 differences in the first 280 nucleotides.

FIG. 2 is a prediction of the secondary structure of E. coli and B. subtilis RNaseP.
The minimal consensus RNase P RNA structure is also shown.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/395 A61P 29/00 4C086 45/00 31/04 48/00 43/00 105 A61P 29/00 C12N 1/14 31/04 1/20 43/00 105 C12Q 1/68 A C12N 1/14 C12N 15/00 ZNAA 1/20 A61K 37/02 5/10 37/54 C12Q 1/68 C12N 5/00 A ( 72) Inventor Lisa A. Hegg, United States 19333 Tanglewood Lane, 554, Devon, PA (72) Inventor Fri United States, 19426 Collegeville, PA, East Fifth Avenue 74, (72) Inventor Catherine・ Di Prescott UK, CB 1.1E1, Kembridge, Elden Str REIT No. 6 (72) Inventor Amy M. Chappell, United States 19555 Pennsylvania, Stone Maker Road, Stonehill Road No. 629 F-term (reference) 4B024 AA13 BA11 CA04 DA05 DA06 EA04 GA11 HA14 HA15 4B063 QA01 QA19 QQ06 QQ15 QQ44 QQ52 QR08 QR33 QR35 QR42 QR56 QS25 QS34 QX02 QX07 4B065 AA01X AA26X AA49X AA49Y AA57X AA72X AA90X AB01 BA02 CA24 CA31 CA46 4C084 AA07 AA13 AA17 CA04 NA14 ZB352 4C007AA AA ABA A03 A04 A04 A04

Claims (21)

[Claims] 1. A polynucleotide having at least 50% identity to a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 3, 4 or 7;
A polynucleotide having at least 50% identity to a polynucleotide comprising the same catalytic RNA as expressed by the RNA gene of P; (c) complementary to the polynucleotide of (a) or (b) And a polynucleotide comprising a polynucleotide sequence selected from the group consisting of: (d) a polynucleotide comprising at least 15 contiguous bases of the polynucleotide of (a), (b) or (c). 2. The polynucleotide according to claim 1, which is DNA. 3. The polynucleotide according to claim 1, which is RNA. (A) SEQ ID NO: 1, 3 as probe or primer
A polynucleotide having a length of at least 30 nucleotides obtained by hybridization to the nucleic acid sequence shown in 4, 4, or 7; and (b) the polynucleotide shown in SEQ ID NO: 1, 3, 4, 5 or 7. A) a polynucleotide isolated from Streptococcus pneumoniae selected from the group consisting of polynucleotides sufficiently complementary to the sequence of the polynucleotide of (a) or (b). 5. The method according to claim 2, which comprises the nucleotide shown in SEQ ID NO: 3, 4 or 7.
Polynucleotide. 6. A vector comprising the polynucleotide of claim 1. 7. A host cell comprising the vector of claim 6. 8. A method for producing RNA, comprising expressing the RNA transcribed by the DNA from the host of claim 7. 9. A method for producing RNaseP RNA or fragment, comprising culturing the host of claim 7 under conditions sufficient for the production of RNaseP RNA or fragment. 10. The polynucleotide according to claim 3, wherein said RNA is a catalytic RNA. 11. An antagonist that inhibits the activity or expression of the RNA of claim 3. 12. A method of treating an individual in need of inhibiting RNaseP RNA, comprising administering to the individual a therapeutically effective amount of the antagonist of claim 11. 13. A method for diagnosing a disease associated with the expression or activity of the RNA of claim 3 in an individual, comprising: (a) determining a nucleic acid sequence that transcribes the RNA; and / or (b) originating from the individual Analyzing the presence or amount of the RNA in the sample of the above. 14. A method for identifying a compound that interacts with the RNA of claim 10 to inhibit or activate its activity, wherein the RNA is capable of interacting with the RNA under conditions that allow the interaction between the compound and the RNA. And evaluating the interaction of the compound with the compound to be screened (the interaction is associated with a second component that can provide a detectable signal in response to the interaction of the RNA with the compound). And then determining whether the compound interacts with the RNA to activate or inhibit its activity by detecting the presence or absence of a signal resulting from the interaction of the compound with the RNA. . 15. An isolated RNase P from S. pneumoniae. 16. The RNase P according to claim 15, which has an RNA component having the sequence of SEQ ID NO: 3. 17. The isolated RNA component of S. pneumoniae RNase P. 18. An isolated protein component of S. pneumoniae RNase P. 19. An isolated DNA encoding the component of claim 18. 20. A polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, or 7. 21. A polynucleotide consisting of a polynucleotide sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, or 7.