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WO2000015775A1 - Polypeptides rnase p - Google Patents

Polypeptides rnase p Download PDF

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Publication number
WO2000015775A1
WO2000015775A1 PCT/US1999/021644 US9921644W WO0015775A1 WO 2000015775 A1 WO2000015775 A1 WO 2000015775A1 US 9921644 W US9921644 W US 9921644W WO 0015775 A1 WO0015775 A1 WO 0015775A1
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WO
WIPO (PCT)
Prior art keywords
polynucleotide
rna
sequence
dna
polypeptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1999/021644
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English (en)
Inventor
Sabine Guth
Joanne Jennings
Catherine D. Prescott
Lisa A. Hegg
Hu Li
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SmithKline Beecham Ltd
SmithKline Beecham Corp
Original Assignee
SmithKline Beecham Ltd
SmithKline Beecham Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
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Priority to JP2000570302A priority Critical patent/JP2002525048A/ja
Priority to EP99948325A priority patent/EP1114149A1/fr
Publication of WO2000015775A1 publication Critical patent/WO2000015775A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by certain of these polynucleotides, molecular complexes of RNAs and polypeptides, the uses of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides and recombinant host cells transformed with the polynucleotides
  • the invention- relates particularly to such polynucleotides and polypeptides from Staphylococci. especially S aureus
  • This invention also relates to inhibiting the biosynthesis, assembly or action of such polynucleotides and/or polypeptides and to the use of such inhibitors m therapy
  • This invention relates to a novel bacterial ⁇ bonucleoprotem complex and the component parts thereof More specifically, this invention relates to RNase P, particularly
  • RNase P from Staphylococcus aureus. and the use of RNase P or components thereof in screens for the identification of antimicrobial compounds and to the use of such compounds in therapy
  • Staphylococci make up a medically important genera of microbes They are known to produce two types of disease, invasive and toxigenic Invasive infections are characterized generally by abscess formation effecting both skin surfaces and deep tissues S aureus is the second leading cause of bacteremia in cancer patients Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci The manifestation of these diseases result from the actions of exotoxms as opposed to tissue invasion and bacteremia These conditions include Staphylococcal food poisoning, scalded skm syndrome and toxic shock syndrome
  • This invention provides a novel ⁇ bonucleoprotein complex, particularly such complex from Staphylococcus aureus. and the separately isolated RNA and protein components thereof
  • polynucleotides which encode the protein and RNA components of such a complex
  • the invention provides polynucleotides having the DNA sequences given herein
  • the invention also relates to novel oligonucleotides derived from the sequences given herein which can act, for example, as antisense inhibitors of the expression of the RNA or protein components
  • the oligonucleotides or fragments or derivatives thereof can also be used to directly inhibit catalytic activity or indirectly inhibit activity by interference with RNA protein complex formation
  • the protein and the RNA components, either separately or in a complex, are also useful as targets in screens designed to identify antimicrobial compounds
  • a novel RNaseP protein from Staphylococcus aureus comprising the ammo acid sequence of Table 1 [SEQ ID NO 2]. or a variant thereof
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain contained in the deposited strain
  • a further aspect of the invention there are provided isolated nucleic acid molecules encoding RNaseP.
  • RNaseP particularly Staphylococcus aureus RNaseP, including mRNAs, cDNAs, genomic DNAs and catalytic RNAs
  • Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful vanants thereof, and- compositions compnsrng the same
  • a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization
  • particularly preferred embodiments of the invention are naturally occurring allelic vanants of RNaseP and polypeptides encoded thereby
  • RNaseP Staphylococcus aureus
  • compositions and methods for assessing RNaseP expression treatmg disease, for example, disease, such as. infections of the upper respiratory tract (e g , otitis media, bactenal tracheitis. acute epiglottitis, thyroiditis). lower respiratory (e g . empyema, lung abscess), cardiac (e g , infective endocarditis), gastrointestinal (e g , secretory diarrhoea, splenic abscess, retropentoneal abscess), CNS (e g , cerebral abscess), eye (e g , blephantis. conjunctivitis, keratitis.
  • infections of the upper respiratory tract e g , otitis media, bactenal tracheitis. acute epiglottitis, thyroiditis.
  • lower respiratory e g . empyema, lung abscess
  • cardiac e g , infective endocarditis
  • endophthalmitis preseptal and orbital cellulitis. darcryocystitis). kidney and urinary tract (e g , epi ⁇ dymitis, rntrarenal and penneplrnc absces. toxic shock syndrome), skin (e g , impetigo. folhcuhtis. cutaneous abscesses, cellulitis. wound infection, bactenal myositis) bone and joint (e g , septic arthntis. osteomyelitis), assaying genetic vanation.
  • kidney and urinary tract e g , epi ⁇ dymitis, rntrarenal and penneplrnc absces. toxic shock syndrome
  • skin e g , impetigo. folhcuhtis. cutaneous abscesses, cellulitis. wound infection, bactenal myositis
  • bone and joint e g , septic arthntis. osteomyelitis
  • RNaseP polypeptide or polynucleotide administered to an organism to raise an lmmunological response against a bactena, especially a Staphylococcus aureus bactena
  • polynucleotides that hybndize to RNaseP polynucleotide sequences, particularly under stringent conditions
  • antibodies against RNaseP polypeptides hi other embodutients of the invention there are provided methods for identifying compounds which b d to or otherwise interact with and inhibit or activate an activity of a ⁇ polypeptide or polynucleotide of the invention compnsrng contacting a polypeptide or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide or polynucleotide with the compound, and deternr-riing whether the compound binds to or otherwise interacts with and activates or inhibits an activity of the polypeptide or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction ofthe compound with the polypeptide or polynucleo
  • compositions compnsrng a RNaseP polynucleotide or a RNaseP polypeptide for adrnmistration to a cell or to a multicellular organism Vanous changes and modifications within the spmt and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descnptions and from reading the other parts ofthe present disclosure
  • Figure 1 illustrates the secondary structural modeling of RNase P RNAs based upon published sequence information
  • Figure 2 illustrates the sequence alignment of the Staphylococcal RNase P protein component ofthe invention compared to other protein components reported in the literature
  • Figure 3 illustrates the Staphylococcal DNA encoding a region of the RNase P gene [SEQ ID NO 14] encoding the RNA component used to clone the intact gene compared with the DNA sequence encoding the gene for the RNase P RNA component from B sub fib's
  • Figure 4 illustrates the predicted secondary structure for the RNA component of the RNase P of this invention
  • Figure 5 illustrates the ammo acid sequence of the protein component of the S aureus RNase P and the sequence of a DNA encoding same [SEQ ID NOS 1 and 2]
  • Figure 6 illustrates a schematic of a whole cell rescue assay to identify drug/RNA interaction
  • Figure 7 illustrates a schematic of a screen to identify compounds that disrupt
  • Figure 8 illustrates an example of a minimal RNase P substrate embodiment [SEQ -- ID NO 4]
  • Figure 9 shows a schematic diagram of a specific embodiment of the first amplification step of an RNase P gene
  • Figure 10 shows a schematic diagram of a specific embodiment of the second amplification step of an RNase P gene Glossary:
  • “Host cell” is a cell which has been transformed or transfected. or is capable of transformation or transfection by an exogenous polynucleotide sequence
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be. as determined by the match between strings of such sequences
  • Identity and similarity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk. A M . ed . Oxford University Press, New York. 1988. Biocomputing Informatics and Genome Projects. Smith, D W , ed . Academic Press, New York, 1993. Computer Analysis of Sequence Data. Part I.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested.
  • Methods to detenmne identity and similarity are codified m publicly available computer programs
  • Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J , et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN. and FASTA (Atschul, S F et al . J Molec Biol 215 403-410 (1990)
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul. S .
  • nucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO 1 it is intended that the nucleotide sequence of the polynucleotide is identical to the - ' reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO 1
  • up to 5% ofthe nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to
  • Polynucleot ⁇ de(s) generally refers to any polynbonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleot ⁇ de(s) include, without limitation, single- and double-stranded DNA, DNA that is a mixture of smgle- and double- stranded regions or smgle-.
  • polynucleotide refers to tnple-stranded regions compnsrng RNA or DNA or both RNA and DNA The strands in such regions may be from the same molecule or from different molecules The regions may include all of one or more ofthe molecules, but more typically involve only a region of some of the molecules One of the molecules of a tnple-he cal region often is an ohgonucleotide As used herem, the term "polynucleot ⁇ de(s)" also mcludes DNAs or RNAs as desc
  • DNAs or RNAs compnsrng unusual bases such as inos ne. or modified bases, such as tntylated bases, to name just two examples, are polynucleotides as the term is used herem
  • polynucleot ⁇ de(s) as it is employed herein embraces such chemically, enzymatically or metabohcally modified forms of polynucleotides, as well as the chemical forms of DNA and RNA charactenstic of viruses and cells, lncludmg, for example, simple and complex cells
  • Polynucleot ⁇ de(s) also embraces short polynucleotides often referred to as ol ⁇ gonucleot ⁇ de(s)
  • Polypept ⁇ de(s) refers to any peptide or protem compnsrng two or more amm
  • Polypeptides may contam ammo acids other than the 20 gene encoded ammo acids "Polypept ⁇ de(s)" include those modified either by natural processes, such as processmg and otlier post-translational modifications, but also by chemical modification techniques Such modifications are well descnbed m basic texts and m more detailed monographs.
  • a given polypeptide may contam many types of modifications Modifications can occur anywhere m a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini Modifications include, for example, acetylation, acylation, ADP-nbosylation. amtdation. covalent attachment of flav n.
  • covalent attachment of a heme moiety covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a lipid or lipid denvative, covalent attachment of phosphotidylrnositol, cross-linking, cyclization, disulfide bond formation, demethylation.
  • formation of covalent cross- links formation of cysteine.
  • formation of pyroglutamate formylation, gamma-carboxylation, glycosylation. GPI anchor formation, hydroxylation. lodination, methylation. mynstoylation, oxidation, proteolytic processmg, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation.
  • Polypeptides may be branched or cyclic, with or without branclimg Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well
  • 'Na ⁇ ant(s) 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 alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below
  • a typical variant of a polypeptide differs in ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, m many regions, identical
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, addition
  • RNase P plays a key role m the biosynthesis of transfer RNA (tRNA), itself a key intermediate in protein biosynthesis RNase P functions to process precursor RNAs into mature tRNAs by endo ⁇ bonucleolytic action
  • tRNA transfer RNA
  • the complex in prokaryotes is composed of two subumts a catalytic RNA and protein co-factor
  • Recent reviews of certain RNase P molecules exist See for example, L A Kirsebom, Molecular Microbiology 17(3), 41 1-420 (1995) or N R Pace and J W Brown, J Bacte ⁇ ol 177 (8). 1919-1928 (1995)
  • the mvention relates to novel RNaseP polypeptides and polynucleotides as descnbed m greater detail below
  • the mvention relates to polypeptides and polynucleotides of a novel RNaseP of Staphylococcus aureus, which is related by ammo acid sequence homology to RNase P polypeptide set forth in Figures 2 and 5 and SEQ ID NO 2
  • the mvention relates especially to RNaseP having the nucleotide and ammo acid sequences set out in Table 1, SEQ ID NO 1 and SEQ ID NO 2 respectively, and to the RNaseP nucleotide sequences ofthe DNA m the deposited stram and ammo acid sequences encoded thereby
  • the mvention also relates to the RNase P RNA component, particularly m its catalytic for, and sequences from which such component is transcnbed RNase P RNA Component
  • the full length sequence encoding the intact RNase P protein component can be obtained by probing a genomic library by for example in situ colony hybridization detailed m Mamatis et al (infra) using a probe(s) generated based on the sequences given in Figure 5 [SEQ ID NOS 1 and 2]
  • polypeptides of the mvention include the polypeptide of Table 1 [SEQ ID NO 2] (m particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of RNaseP, and also those which have at least 70% identity to the polypeptide of Table 1 [SEQ ID NO 2] or the relevant portion, preferably at least 80% identity to the polypeptide of Table 1 [SEQ ID NO 2], and more preferably at least 90% sinnlanty (more preferably at least 90% identity) to the polypeptide of Table 1 [SEQ ID NO 2] and still more preferably at least 95% srmilanty (still more preferably at least 95% identity) to the polypeptide of Table 1 [SEQ ID NO 2] and also mclude portions of such polypeptides with such portion of the polypeptide generally containmg at least 30 ammo acids and more preferably at least 50 ammo acids
  • the mvention also includes polypeptides ofthe formula set forth in Table 1 (D) [SEQ ID NO 2] wherein, at the ammo terminus, X is hydrogen, and at the carboxyl terminus. Y is hydrogen or a metal.
  • R j and R2 is any ammo acid residue, and n is an integer between 1 and 1000 Any stretch of ammo acid residues denoted by either R group, where R is greater than 1, may be either a heteropolymer or a homopolymer. preferably a heteropolymer
  • a fragment is a vanant polypeptide having an am o acid sequence that entirely is the same as part but not all of the ammo acid sequence ofthe aforementioned polypeptides
  • fragments may be "free-standing.” or compnsed withm a larger polypeptide of which they form a part or region, most preferably as a smgle contmuous region, a smgle larger polypeptide
  • Preferred fragments m include, for example, truncation polypeptides havmg a portion ofthe amino acid sequence of Table 1 [SEQ ID NO 2]. or of vanants thereof, such as a contmuous senes of residues that includes the ammo terminus, or a contmuous senes of residues that mcludes the carboxyl terminus Degradation forms of the polypeptides of the mvention m a host cell, particularly a Staphylococcus aureus.
  • fragments charactenzed by structural or functional attributes such as fragments that compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-formmg regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphrpathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic mdex regions
  • biologically active fragments which are those fragments that mediate activities of RNaseP including those with a similar activity or an improved activity, or with a decreased undesirable activity
  • fragments compnsrng receptors or domains of enzymes that confer a function essential for viability of Staphylococcus " aureus or the ability to initiate, or maintain cause disease m an individual, particularly a human
  • Vanants that are fragments of the polypeptides of the mvention may be employed for producmg the correspondmg full-length polypeptide by peptide synthesis, therefore, these vanants may be employed as intermediates for producmg the full-length polypeptides ofthe mvention Polynucleotides ofthe mvention
  • Another aspect ofthe mvention relates to isolated polynucleotides, including the full length gene, that encode the RNaseP polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and vanants thereof
  • a polynucleotide of the mvention encoding RNaseP polypeptide or RNA (such as that transcribed from SEQ ID NO 3) may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bactena usmg Staphylococcus aureus WCUH 29 cells as startmg matenal, followed by obtaining a full length clone
  • a polynucleotide sequence ofthe invention such as a sequence given m SEQ ID NOS 1, 3.
  • a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E cob or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, derived from a partial sequence
  • a radiolabeled ohgonucleotide preferably a 17-mer or longer
  • Clones carrying DNA identical to that of the probe can then be distinguished using stringent conditions
  • sequencing primers designed from the original sequence it is then possible to extend the sequence in both directions to determine the full gene sequence Conveniently, such sequencing is performed using denatured double stranded DNA prepared from a plasmid clone Suitable techniques are described by Maniatis. T .
  • RNaseP ofthe mvention is structurally related to other protems ofthe RNase P family, as shown by the results of sequencmg the DNA encoding RNaseP ofthe deposited strain
  • the protem exhibits greatest homology to B subtibs protem among known protems RNaseP of Table 1 [SEQ ID NO 2] has signficant identity and s milanty over its entire length with the ammo acid sequence of B subtibs RNase P polypeptide
  • the mvention provides a polynucleotide sequence identical over its entire length to the codmg sequence m Table 1 [SEQ ID NO 1] Also provided by the mvention is the codmg sequence for the mature polypeptide or a fragment thereof, by itself as well as the codmg sequence for the mature polypeptide or a fragment m reading frame with other codmg sequence, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protem sequence
  • the polynucleotide may also contain non-codmg sequences, mcluding for example, but not limited to non-codmg 5' and 3' sequences, such as the transcnbed, non-translated sequences, termination signals, nbosome binding sites, sequences that stabilize mRNA, mtrons, polyadenylation signals, and additional codmg sequence which encode additional am o acids
  • a marker sequence that facilitates punfication of the fused polypeptide can be encoded In
  • the marker sequence is a hexa-histidine peptide, as provided m the pQE vector (Qiagen. Inc ) and descnbed in Gentz et al , Proc Natl Acad Sci , USA 86 821-824 (1989). or an HA tag (Wilson et al . Cell 37 767 (1984)
  • Polynucleotides of the mvention also mclude. but are not limited to, polynucleotides compnsrng a structural gene and its naturally associated sequences that control gene expression
  • a preferred embodiment ofthe mvention is a polynucleotide of compnsrng nucleotide 1 to 351 or 354 set forth in SEQ ID NO 1 of Table 1 which encode the RNaseP polypeptide
  • the mvention also mcludes polynucleotides ofthe formula set forth in Table 1 (C)[SEQ ID NO 1 and (F)[SEQ ID NO 3] wherein, at the 5' end ofthe molecule, X is hydrogen, and at the 3' end ofthe molecule.
  • Y is hydrogen or a metal
  • Rj and R2 is any nucleic acid residue
  • n is an mteger between 1 and 3000 Any stretch of nucleic acid residues denoted by either R group, where R is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer
  • a preferred embodiment for the sequence set forth m Table 1 (F) [SEQ ID NO 3] has R j or R2 bemg between 1 and 10 or 1 and 20, and especially being 1.
  • polynucleotide encodmg a polypeptide encompasses pol nucleotides that mclude a sequence encoding a polypeptide of the mvention, particularly a ⁇ bactenal polypeptide and more particularly a polypeptide of the Staphylococcus aureus RNaseP havmg the ammo acid sequence set out in Table 1 [SEQ ID NO 2]
  • the term also encompasses polynucleotides that mclude a smgle contmuous region or discontmuous regions encodmg the polypeptide (for example, interrupted by mtegrated phage or an insertion sequence or editmg) together with additional regions, that also may contam codmg and/or non-codmg sequences
  • the mvention further relates to vanants of the polynucleotides descnbed herem that encode for vanants of the polypeptide havmg the deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Vanants that are fragments of the polynucleotides of the mvention may be used to synthesize full-length polynucleotides ofthe mvention
  • N means that any of the four DNA or RNA bases may appear at such a designated position in the DNA or RNA sequence, except that, in preferred embodiments. N can not be a base that when taken in combination with adjacent nucleotide positions, when read in the correct reading frame, would have the effect of generating a premature termination codon in such reading frame
  • RNaseP vanants that have the ammo acid sequence of RNaseP polypeptide of Table 1 [SEQ ID NO 2] m which several, a few, 5 to 10, 1 to 5. 1 to 3, 2, 1 or no ammo acid residues are substituted, deleted or added, m any combination Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of RNaseP
  • polynucleotides that are at least 50%. 60%) or 70% identical over their entire length to a polynucleotide encodmg RNaseP polypeptide havmg an ammo acid sequence set out m Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides
  • polynucleotides that compose a region that is at least 80% identical over its entire length to a polynucleotide encoding RNaseP polypeptide of the deposited stram and polynucleotides complementary thereto
  • polynucleotides at least 90*% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95%o are especially preferred
  • those with at least 97%o are highly preferred among those with at least 95%, and among these those with at least 98%) and at least 99% are particularly highly preferred,
  • Preferred embodiments of the mvention are polynucleotides that are at least 50%. 60% or 70% identical over their entire length to an RNaseP polynucleotide havmg a nucleotide sequence set out m SEQ ID NO 3.
  • polynucleotides that are complementary to such polynucleotides are most highly preferred. polynucleotides that compnse a region that is at least 80%> identical over its entire length to an RNaseP polynucleotide of the deposited stram and polynucleotides complementary thereto
  • polynucleotides at least 90%) identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred
  • those with at least 97% are highly preferred among those with at least 95%. and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99%o bemg the more preferred It is especially preferred that these polynucleotides be RNAs, especially catalytic RNAs
  • Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by the DNA of Table 1 [SEQ ID NO 1] or as the RNase P RNA component transcnbed by the DNA of SEQ ID NO 3. 4 or 14
  • the mvention further relates to polynucleotides that hybndize to the herem above- descnbed sequences
  • the mvention especially relates to polynucleotides that hybndize under strmgent conditions to the herem above-descnbed polynucleotides
  • strmgent conditions and “strmgent hybndization conditions” mean hybndization will occur only if there is at least 95% and preferably at least 97% identity between the sequences
  • An example of stringent hybridization conditions is overnight incubation at 42°C m a solution comprising 50% formamide, 5x SSC (150mM NaCl.
  • the invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO 1 or SEQ ID NO 3 or SEQ ID NO 4 or SEQ ID NO 14 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth m SEQ ID NO 1 or SEQ ID NO 3 or SEQ ID NO 4 or SEQ ID NO 14 respectively or a fragment thereof, and isolating said DNA sequence Fragments useful for obtaining such a polynucleotide include, for- example, probes and primers described elsewhere herem
  • polynucleotide assays of the mvention may be used as a hybndization probe for RNA.
  • cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encodmg RNaseP and to isolate cDNA and genomic clones of other genes that have a high sequence similanty to the RNaseP gene
  • Such probes generally will compnse at least 15 bases
  • such probes will have at least 30 bases and may have at least 50 bases
  • Particularly preferred probes will have at least 30 bases and will have 50 bases or less
  • Polynucleotides of the invention that are oligonucleotides derived from the sequences of SEQ ID NOS 1 and or 2 and/or 3 and/or 4 and/or 14 may be used in the processes herein as described, but preferably for PCR, to detennme whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue It is recognized that such sequences will also have utility m diagnosis of the stage of infection and type of infection the pathogen has attained
  • the mvention also provides polynucleotides that may encode a polypeptide that is the mature protem plus additional ammo or carboxyl-terrninal ammo acids, or amino acids mtenor to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance) Such sequences may play a role m processmg of a protem from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half-life or may facilitate manipulation of a protem for assay or production, among other things As generally is the case in vivo, the additional ammo acids may be processed away from the mature protem by cellular enzymes
  • a precursor protem. havmg the mature form of the polypeptide fused to one or more prosequences may be an mactive form of the polypeptide When prosequences are removed such mactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proprotems In sum.
  • a polynucleotide ofthe mvention may encode a mature protem, a mature protem plus a leader sequence (which may be referred to as a preprotem), a precursor of a mature protem havmg one or more prosequences that are not the leader sequences of a preprotem.
  • preproprotem which is a precursor to a proprotem, havmg a leader sequence and one or more prosequences, which generally are removed during processmg steps that produce active and mature fonns ofthe polypeptide Cloning of S aureus RNase P RNA structural gene ⁇
  • the coding region ofthe RNaseP gene may be isolated, for example, by screening usmg a deposit containmg a Staphylococcus aureus WCUH 29 stram which has been deposited with the National Collections of Industnal and Marine Bactena Ltd (herem "NCIMB"), 23 St Machar Dnve, Aberdeen AB2 1RY.
  • NCIMB National Collections of Industnal and Marine Bactena Ltd
  • Staphylococcus aureus WCUH29 On deposit
  • the Staphylococcus aureus stram deposit is referred to herem as "the deposited stram” or as "the DNA ofthe deposited stram "
  • the deposited stram contains the full length RNaseP gene
  • the sequence of the polynucleotides contained m the deposited stram, as well as the ammo acid sequence of the polypeptide encoded thereby, are controlling m the event of any conflict with any descnption of- sequences herem
  • the deposit ofthe deposited stram has been made under the terms ofthe Budapest Treaty on the International Recogmtion of the Deposit of Micro-organisms for Purposes of Patent Procedure
  • the stram will be irrevocably and without restnction or condition released to the public upon the issuance of a patent
  • the deposited stram is provided merely as convemence to those of skill m the art and is not an admission that a deposit is required for enablement. such as that required under 35 U S C ⁇ 112
  • a license may be required to make, use or sell the deposited stram, and compounds denved therefrom, and no such license is hereby granted
  • nucleotide sequences disclosed herein can also be obtained by synthetic chemical techniques known in the art or can be obtained from S aureus WCUH 29 by probing a DNA preparation with probes constructed from the particular sequences disclosed herein Alternatively, oligonucleotides derived from a disclosed sequence can act as PCR primers in a process of PCR-based cloning of the sequence from a bacterial genomic source It is recognized that such sequences will also have utility m diagnosis of the type of infection the pathogen has attained
  • a polynucleotide of the present invention may be in the form of RNA or in the form of DNA which DNA includes cDNA, genomic DNA. and synthetic DNA
  • the DNA may be double-stranded or single-stranded, and if smgle stranded may be the coding strand or non- coding (anti-sense) strand
  • the coding sequence which encodes the polypeptide may be identical to the coding sequence shown or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encoding the same polypeptide
  • polynucleotide encodmg a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-codmg sequence
  • the present invention therefore includes polynucleotides, wherein the coding sequence for the mature polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids m expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell
  • the polypeptide having a leader sequence is a preprotem and may have the leader sequence cleaved by the host cell to form the mature form ofthe polypeptide
  • the polynucleotides may also encode for a proprotem which is the mature ⁇ protein plus additional 5' ammo acid residues A mature protem having a prosequence
  • the polynucleotide of the present invention may encode for a mature protem, or for a protem having a prosequence or for a protem having both a prosequence and a presequence (leader sequence)
  • leader sequence the ammo acid sequences provided herein show a methionine residue at the NH2-termmus It is appreciated, however, that during post-translational modification of the peptide, this residue may be deleted Accordingly, this invention contemplates the use of both the methionme-containmg and the methionineless ammo terminal variants of each protein disclosed herein
  • the polynucleotides ofthe present invention may also have the coding sequence fused in frame to a marker sequence at either the 5' or 3' terminus of the gene which allows for purification ofthe polypeptide ofthe present invention
  • the marker sequence may be a hexa- histidme tag supplied by the pQE series of vectors (supplied commercially by Quiagen Inc ) to provide for purification of the polypeptide fused to the marker m the case of a bacterial host
  • the maltose binding protein (MBP) fusion system may be employed In this system the gene of interest is fused the malE gene encoding the MBP (supplied by New England BioLabs)
  • the fusion product is purified in a one step procedure based on the MBP affinity for maltose
  • a pre-engineered Xa cleavage site allows for efficient removal of the MBP component from the gene product of interest
  • Plasmids are designated by a lower case p preceded and/or followed by capital letters and/or numbers
  • the startmg plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures
  • equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan
  • “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 use T- herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan
  • typically 1 ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution
  • typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme m a larger volume
  • Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer
  • Incubation times of about 1 hour at 37°C are ordinarily used, but may vary in accordance with the supplier's instructions
  • After digestion the reaction is electrophoresed directly on an agarose gel to isolate the desired fragment Size separation of the cleaved fragments is generally performed using a 1 % percent agarose gel
  • Oligonucleotides refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another ohgonucleotide without adding a phosphate with an ATP in the presence of a kinase A synthetic ohgonucleotide will ligate to a fragment that has not been dephosphorylated
  • “Ligation” refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Mamatis, T . et al . supra . p 146) Unless otherwise provided, hgation may be accomplished using known buffers and conditions with 10 units to T4 DNA hgase ("hgase") per 0 5 ⁇ g of approximately equimolar amounts of the DNA fragments to be hgated
  • a "rephcon” is any genetic element (e g , plasmid. chromosome, virus) that functions as an autonomous unit of DNA replication in v vo, 1 e , capable of replication under its own control
  • a "vector” is a rephcon, such as a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication ofthe attached segment
  • double-stranded DNA molecule refers to the polymeric form of deoxy ⁇ bonucleotides (bases adenine, guanine, thymme, or cytosine) in a double-stranded helix, both relaxed and supercoiled
  • This tenn refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms
  • this term includes double-stranded DNA found, inter aba, in linear DNA molecules (e g , — restriction fragments), viruses, plasmids, and chromosomes
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontransc ⁇ bed strand of DNA (l e , the strand having the sequence homologous to the mRNA)
  • a DNA "coding sequence of or a "nucleotide sequence encoding" a particular protem is a DNA sequence which is transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence
  • the promoter sequence is bound at the 3' terminus by a translation start codon (e g , ATG) of a coding sequence and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background
  • a transcription initiation site (conveniently defined by mapping with nuclease S I), as well as protem binding domains (consensus sequences) responsible for the binding of RNA polymerase
  • Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes
  • Prokaryotic promoters contain the -10 and -35 consensus sequences
  • control sequences refers collectively to promoter sequences, ⁇ bosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the expression (I e , the transcription and translation) of a coding sequence in a host cell
  • a control sequence "directs the expression" of a coding sequence m a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence
  • a "host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous DNA sequence
  • Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome ofthe cell
  • the exogenous DNA may be maintained on an episomal element, such as a plasmid
  • a stably transformed or transfected cell- is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cell containing the exogenous DNA
  • a “clone” is a population of cells derived from a single cell or 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 segment of DNA withm or attached to another DNA molecule that is not found in association with the other molecule in nature Preparation ofthe RNase P protein component
  • the present mvention also relates to vectors which include polynucleotides of the present mvention, host cells which are genetically engineered with vectors ofthe invention and the production of polypeptides ofthe invention by recombinant techniques
  • polypeptide of the invention by recombinant techniques by expressing a polynucleotide encoding said polypeptide m a host and recovering the expressed product
  • polypeptides of the invention can be synthetically produced by conventional peptide synthesizers
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be. for example, a cloning vector or an expression vector
  • the vector may be, for example, in the fonn of a plasmid, a cosmid, a phage, etc
  • the engineered host cells can be cultured m conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan
  • Suitable expression vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e g , bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA
  • any other vector may be used as long as it is rephcable and viable in the host For
  • appropnate hosts include bactenal cells, such as streptococci. staphylococci, enterococci E cob, streptomyces and Bacillus subtibs cells, fungal cells, such as yeast cells and Aspergillus cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127. 3T3. BHK. 293 and Bowes melanoma cells, and plant cells
  • vectors mclude. among others, chromosomal, episomal and virus-denved vectors. e g . vectors denved from bactenal plasmids, from bactenophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses. fowl pox viruses, pseudorabies viruses and retroviruses.
  • the expression system constructs may contam control regions that regulate as well as engender expression
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide m a host may be used for expression in this regard
  • the appropnate DNA sequence may be mserted mto the expression system by any of a vanety of well-known and routme techniques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, (supra)
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures In general, the DNA sequence is inserted into an appropriate restriction endonuclease s ⁇ te(s) by procedures known m the art
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis
  • promoter there may be mentioned LTR or SV40 promoter, the E cob lac or trp. the phage lambda P j ⁇ promoter and other promoters known to control expression- of genes in eukaryotic or prokaryotic cells or their viruses
  • the expression vector may also contains a ⁇ bosome binding site for translation initiation and/or a transcription terminator
  • the vector may also include appropriate sequences for amplifying expression
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracychne or ampicillm resistance m E cob
  • the gene can be placed under the control of a promoter, ⁇ bosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired protem is transcribed into RNA in the host cell transformed by a vector containing this expression construction
  • the coding sequence may or may not contain a signal peptide or leader sequence
  • the polypeptides of the present invention can be expressed using, for example, the E cob tac promoter or the protem A gene (spa) promoter and signal sequence Leader sequences can be removed by the bacterial host in post-translational processing See, e g , U S Patent Nos 4,
  • Promoter regions can be selected from any desired gene using CAT (chloramphemcol transferase) vectors or other vectors with selectable markers
  • CAT chloramphemcol transferase
  • Two appropriate vectors are PKK232-8 and PCM7
  • Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and trp
  • Eukaryotic promoters include CMV immediate early. HSV thymidme kmase.
  • regulatory sequences which allow for regulation ofthe expression ofthe protem sequences relative to the growth of the host cell Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound
  • Other types of regulatory elements may also be present in the vector, for example, enhancer sequences
  • An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" ofthe control sequences (1 e , RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence) Modification of- the coding sequences may be desirable to achieve this end For example, in some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation, I e , to maintain the reading frame
  • the control sequences and other regulatory sequences may be hgated to the coding sequence prior to insertion mto a vector, such as the cloning vectors described above Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transfonnation of the
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence
  • a promoter operably linked to the sequence
  • suitable vectors and promoters are known to those of skill in the art, and are commercially available
  • the following vectors are provided by way of example Bacterial pET-3 vectors (Stratagene), pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescnpt, ps ⁇ X174, pbluesc ⁇ pt SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptr
  • Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bactenophage ⁇ (E cob), pBR322 (E cob), pACYC177 (E cob), pKT230 (gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFRl (gram- negative bacteria), pME290 (non-i? cob gram-negative bacteria), pHV14 (E cob and Bacillus subtibs), pBD9 (Bacillus).
  • pIJ61 Streptomyces
  • pUC6 Streptomyces).
  • YIp5 Sacharomyces
  • YCpl9 Sacharomyces
  • sequences which cause the secretion of the polypeptide from the host organism may be desirable to add sequences which cause the secretion of the polypeptide from the host organism, with subsequent cleavage ofthe secretory signal
  • Polypeptides can be expressed in host cells under the control of appropriate promoters Cell-free translation systems can also be employed to produce such protems using RNAs derived from the DNA constructs of the present invention
  • RNAs derived from the DNA constructs of the present invention Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al , Molecular Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor, N Y , (1989), the disclosure of which is hereby incorporated by reference
  • the selected promoter is induced by appropriate means (e g , temperature shift or chemical induction) and cells are cultured for an additional period
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification
  • Microbial cells employed in expression of protems can be disrupted by any convenient method, including freeze-thaw cycling, sonication. mechanical disruption, or use of cell lysing agents, such methods are well known to those skilled in the art
  • the polypeptide of the present invention may be produced by growing host cells transformed by an expression vector described above under conditions whereby the polypeptide of interest is expressed The polypeptide is then isolated from the host cells and purified If the expression system secretes the polypeptide mto growth media, the polypeptide can be purified directly from the media If the polypeptide is not secreted, it is isolated from cell lysates or recovered from the cell membrane fraction Where the polypeptide is localized to the cell surface, whole cells or isolated membranes can be used as an assayable source of the desired gene product Polypeptide expressed in bacterial hosts such as E cob may require isolation from inclusion- bodies and refolding Where the mature protein has a very hydrophobic region which leads to an insoluble product of overexpression, it may be desirable to express a truncated protein m which the hydrophobic region has been deleted The selection of the appropriate growth conditions and recovery methods are within the skill ofthe art
  • the polypeptide can be recovered and purified from recombinant cell cultures by methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Protein refolding steps can be used, as necessary, in completing configuration of the mature protem Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps
  • polypeptides of the present invention may be glycosylated or may be non-glycosylated
  • Polypeptides ofthe invention may also include an initial methionine ammo acid residue Preparation ofthe RNase P RNA Component
  • the RNase P RNA molecules are prepared by run-off in vitro transcription using T7 RNA polymerase as according to standard conditions - usually as recommended by the supplier, e g , Promega
  • the plasmid is linearized with an appropriate restriction enzyme generating a linear dsDNA comprising the full length gene encoding the RNase PRNA
  • the RNA is purified either from a preparative denaturing acrylamide gel or is precipitated prior to use in in vitro cleavage assays
  • the substrates for the RNase P RNA and the RNA complexed with its protem can be obtained by in vitro transcription of cloned genes Useful substrates included but are not limited to pre-tRNA ⁇ et or E cob or B subtibs pre-4 5S molecules and may be expressed using an n vitro transcription system directed by T7 RNA polymerase as described above
  • the RNA can also be prepared by automated synthesis Antagonists and agonists - assays and molecules
  • This invention provides a method of screening drugs to identify those which interfere with the RNA portion, the protem portion and/ or the mtact RNA/protein complex of the RNase P described herein, which method comprises measuring the interference of the activity of the protem and/or RNA by a test drug For example since the RNA portion selected has a catalytic activity, after suitable purification and formulation the activity of the RNA can be followed by its ability to convert its natural or synthetic RNA substrates By incorporating- different chemically synthesized test compounds or natural products into such an assay of enzymatic activity one is able to detect those additives which compete with the natural or synthetic substrate or otherwise inhibit enzymatic activity
  • Polypeptides of the mvention may also be used to assess the binding of small molecule substrates and hgands m. for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures These substrates and hgands may be natural substrates and hgands or may be structural or functional mimetics See, e g , Coligan et al . Current Protocols in Immunology 1(2) Chapter 5 (1991)
  • the mvention also provides a method of screening compounds to identify those which enhance (agomst) or block (antagonist) the action of RNaseP polypeptides or polynucleotides, particularly those compounds that are bactenostatic and/or bactenocidal
  • the method of screening may involve high-tliroughput techniques For example, to screen for agomsts or antagonists, a synthetic reaction mix.
  • a cellular compartment such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsrng RNaseP polypeptide and a labeled substrate or hgand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a RNaseP agomst or antagonist
  • the ability ofthe candidate molecule to agonize or antagonize the RNaseP polypeptide is reflected in decreased binding ofthe labeled hgand or decreased production of product from such substrate Molecules that bmd gratuitously, i e , without mducmg the effects of RNaseP polypeptide are most likely to be good antagonists
  • Molecules that bmd well and mcrease the rate of product production from substrate are agonists Detection ofthe rate or level of production of product from substrate may be enhanced by usmg a reporter system Reporter systems that may be useful m this regard mclude but are not limited to colonn etnc label
  • an assay for RNaseP antagonists is a competitive assay that combines RNaseP and a potential antagonist with RNaseP -binding molecules, recombinant RNaseP binding molecules, natural substrates or hgands, or substrate or hgand mimetics, under appropnate conditions for a competitive inhibition assay
  • RNaseP can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of RNaseP molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist
  • Potential antagomsts mclude small organic molecules, peptides, polypeptides and antibodies that bmd to a polynucleotide or polypeptide of the mvention and thereby inhibit or- extinguish its activity
  • Potential antagomsts also may be small organic molecules, a peptide, a polypeptide such as a closely related protem or antibody that bmds the same sites on a binding molecule, such as a binding molecule, without mducmg RNaseP -mduced activities, thereby preventmg the action of RNaseP by excluding RNaseP from binding
  • Potential antagomsts m include a small molecule that bmds to and occupies the binding site of the polypeptide thereby preventmg binding to cellular binding molecules, such that normal biological activity is prevented
  • small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules
  • Other potential antagomsts m clude antisense molecules (see Okano, J Neurochem 56 560 (1991), OL1GODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press. Boca Raton, FL (1988).
  • Preferred potential antagomsts mclude compounds related to and vanants of RNaseP
  • the encoded protem upon expression, can be used as a target for the screening of antibacterial drugs
  • the DNA sequences encodmg the amino terminal regions of the encoded protem or Shme-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression ofthe coding sequence of interest
  • the invention also provides the use of the polypeptide, polynucleotide or inhibitor of the invention to interfere with the initial physical interaction between a pathogen and mammalian host responsible for sequelae of infection
  • the molecules of the invention may be used in the prevention of adhesion of bacteria, in particular gram positive bacteria, to mammalian extracellular matrix proteins on m-dwelhng devices or to extracellular matrix proteins in wounds, to block RNaseP protein-mediated mammalian cell invasion by.
  • the antagomsts and agomsts of the mvention may be employed, for instance, to mhibit and treat disease, such as, infections of the upper respiratory tract (e g , otitis media, bactenal tracheitis. acute epiglottitis, thyroiditis), lower respiratory (e g .
  • Assays can be developed to detect compounds that inhibit RNase P directed cleavage of RNA substrates
  • Assay formats are suitable for HTP screening based upon the ability to incorporate labels withm the RNA in a site-specific fashion by chemical synthesis
  • the conventional radioactive-based format is preferred, while a homogeneous fluorescence-based format is useful for subsequent follow-up of lead compounds
  • the use of both formats is contemplated by this invention Functional RNase P Assay
  • Biotm is introduced in to an appropriate position withm the RNA substrate and the 5' terminus labeled with 32p,
  • the substrate is linked via streptavidin withm a 96-well plate Following RNase P dependent hydrolysis of the substrate, the radiolabelled 5' leader cleavage product is released mto the bulk solution phase, and subject to scintillation counting
  • the RNA substrate is bound to a streptavidin-coated flashplate such that the release of the radioactive 6-mer mto the solution phase results in a decrease in signal
  • cyclic peptide libraries were constructed with flanking cysteine residues to allow efficient disulfide bond formation and cychzation during phage assembly
  • the streptavidm bound crystal structures of two disulfide bridged cyclic peptides showed both peptides to be in beta-tum conformations (Kahn, M (Guest, Ed . 1993) Tetrahedron 49. Symp 50, 3433- 3677)
  • Beta-turns are key recognition elements in many biological interactions therefore effort has been focused on 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, could identify cyclic peptides suitable for peptide mimic synthesis as inhibitor molecules
  • a cyclic octapeptide phage display library may be constructed and used to identify peptides that interact with defined RNA domains Secondary Evaluation
  • RNA recognition motifs for RNase P RNA Ml RNA
  • the technology is based on the repeated selection and amplification of RNA fragments that specifically bmd to a protem with high affinities (Szostak. J W .
  • Fragment libraries based on the S aureus and E cob RNase P RNAs may be constructed for the in vitro synthesis of RNA fragments and the subsequent selection of molecules that bmd their respective protems
  • Chemical and enzymatic structure probing technologies may be employed in combination with protein/RNA protection studies to map the interactive sites SELEX based on the resulting RNA fragment(s) may be further exploited to determine the minimal structural requirements for RNA recogmtion Disruption of RNase P Assembly —
  • RNase P RNA fragments that confer drug resistance may be sequenced and expressed in vitro for chemical and enzymatic structure probing in the presence and absence of the drug in an attempt to map the binding site SELEX may be applied to lead compounds m an attempt to identify the minimal structural requirements for drug binding RNase P Substrates
  • Minimal RNA substrates may be chemically synthesized for HTP screening including both pre-tRNA and pre-4 5 S RNA derivatives (e g Figure 8) RNA-hgand interactions involving ⁇ bose 2'-hydroxyl groups of specific nucleotides may be probed via chemical synthesis of the appropriately modified RNA fragment
  • 2'-methoxy and 2'-fluoro ⁇ bonucleot ⁇ des analogues can be used, the latter being preferred on ste ⁇ c grounds Nucleotides lacking a 2'- substituent adopt the undesired C'-endo configuration
  • the invention also relates to inhibitors identified by any of the techniques described herem Because of the enzymatic nature of RNase P action, it is appreciated that inhibitors may be identified which act as transition state mimics, inhibitors of product release or inhibitors of substrate binding Diagnostic Assays
  • This mvention is also related to the use ofthe RNaseP polynucleotides ofthe mvention for use as diagnostic reagents Detection of RNaseP in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease Eukaryotes (herem also " ⁇ nd ⁇ v ⁇ dual(s)"), particularly mammals, and especially humans, infected with an organism compnsrng the RNaseP gene may be detected at the nucleic acid level by a vanety of techmques
  • Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skm Genomic DNA may be used directly for detection or may be amplified enzymatically by usmg PCR or other amplification technique pnor to analysis RNA or cDNA may also be used in tlie same ways Usmg amplification, characte ⁇ zation of the- species and stram of prokaryote present in an mdividual, may be made by an analysis of the genotype ofthe prokaryote gene Deletions and insertions can be detected by a change m size of tlie amplified product m companson to the genotype of a reference sequence Pomt mutations can be identified by hybndizmg amplified DNA to labeled RNaseP polynucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in meltmg temperatures DNA
  • Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and S 1 protection or a chemical cleavage method See, e g , Cotton et al , Proc Natl Acad Sci , USA, 85 4397-4401 (1985) Cells carrying mutations or polymorphisms m the gene of tlie mvention may also be detected at tlie DNA level by a vanety of techniques, to allow for serotyping, for example For example, RT-PCR can be used to detect mutations It is particularly preferred to used RT-PCR m conjunction with automated detection systems, such as, for example.
  • GeneScan RNA or cDNA may also be used for the same purpose, PCR or RT-PCR As an example.
  • PCR primers complementary to a nucleic acid encodmg RNaseP can be used to identify and analyze mutations Examples of representative primers are shown m the Examples The mvention further provides these primers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end These primers may be used for, among other things, amplifying RNaseP DNA isolated from a sample denved from an mdividual
  • the primers may be used to amplify the gene isolated from an infected mdividual such that tlie gene may then be subject to vanous techmques for elucidation ofthe DNA sequence In this way.
  • m tlie DNA sequence may be detected and used to diagnose infection and to serotype and/or classify tlie infectious agent
  • the mvention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections by Staphylococcus aureus, and most preferably disease, such as, infections of the upper respiratory tract (e g , otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e g .
  • endophthalmitis preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e g , epididymitis,- mtrarenal and pennephnc absces, toxic shock syndrome), skm (e g , impetigo, folhcuhtis, cutaneous abscesses, cellulitis, wound infection, bactenal myositis) bone and jomt (e g , septic arth ⁇ tis, osteomyelitis), comprising determining from a sample derived from an individual a increased level of expression of polynucleotide having the sequence of Table 1 [SEQ ID NO 1 ] Increased or decreased expression of RNaseP polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR. RNase protection, Northern blotting and other hybridization methods
  • a diagnostic assay in accordance with the mvention for detectmg over- expression of RNaseP protem compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techniques that can be used to determine levels of a RNaseP protem, m a sample denved from a host are well-known to those of skill m the art Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays Antibodies
  • Antibodies as used herem mcludes monoclonal and polyclonal antibodies, chimenc, smgle cham. simiamzed antibodies and humanized antibodies, as well as Fab fragments, mcluding the products of an Fab immunolglobulin expression library
  • the Fab fragment may also be prepared from its parent monoclonal antibody by enzyme treatment, for example usmg papam to cleave the Fab portion from the Fc portion
  • Antibodies generated agamst the polypeptides of the mvention can be obtained by administenng the polypeptides or epitope-beanng fragments, analogues or cells to an animal, preferably a nonhuman, usmg routine protocols The antibody so obtained will then bind the polypeptides itself In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide
  • any technique known m the art that provides antibodies produced by contmuous cell lme cultures can be used.
  • vanous techmques such as those m Kohler, G and Milstem, C , Nature 256 495-497 (1975), Kozbor et al , Immunology
  • the hyb ⁇ domas are screened to select a cell line with high binding affinity and favorable cross reaction with other staphylococcal species using one or more of the original polypeptide and/or the fusion protein
  • the selected cell line is cultured to obtain the desired
  • phage display technology may be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-RNaseP or from naive libraries (McCafferty. J et al , (1990). Nature 348, 552-554, Marks. J et al , (1992) Biotechnology
  • the antibody should be screened again for high affinity to the polypeptide and/or fusion protem As mentioned above, a fragment ofthe final antibody may be prepared
  • the antibody may be either intact antibody of M r approx 150,000 or a derivative of it, for example a Fab fragment or a Fv fragment as described in Skerra, A and Pluckthun, A .
  • each domain may be directed against a different epitope - termed 'bispecific' antibodies If two antigen binding domains are present each domain may be directed against a different epitope - termed 'bispecific' antibodies
  • polypeptides in which one or more of the amino acid residues are modified may be used.
  • Such peptides may. for example, be prepared by substitution, addition, or rearrangement of ammo acids or by chemical modification thereof All such substitutions and modifications are generally well known to those skilled in the art of peptide chemistry
  • the above-descnbed antibodies may be employed to isolate or to identify clones expressmg the polypeptides to punfy the polypeptides by affinity chromatography
  • antibodies against RNaseP- polypeptide may be employed to treat- infections, particularly bactenal infections and especially disease, such as, infections of the upper respiratory tract (e g , otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis). lower respiratory (e g . empyema, lung abscess), cardiac (e g , infective endocarditis), gastromtestmal (e g , secretory diarrhoea, splemc absces, retropentoneal abscess), CNS (e g .
  • infections of the upper respiratory tract e g , otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis.
  • lower respiratory e g . empyema, lung abscess
  • cardiac e g , infective endocarditis
  • gastromtestmal e g , secretory diarrhoea, s
  • the antibody is prepared by expression of a DNA polymer encoding said antibody in an appropriate expression system such as described above for the expression of polypeptides of the invention
  • an appropriate expression system such as described above for the expression of polypeptides of the invention
  • the choice of vector for the expression system will be determined m part by the host, which may be a prokaryotic cell, such as E cob (preferably stram B) or Streptomyces sp or a eukaryotic cell, such as a mouse C127, mouse myeloma, human HeLa.
  • the host may also be a transgenic animal or a transgenic plant (for example, as described m Hiatt, A et al , Nature 340 76-78(1989)
  • Suitable vectors include plasmids.
  • bacte ⁇ ophages, cosmids and recombinant viruses, derived from, for example, baculoviruses and vaccinia Polypeptide variants include antigemcally.
  • epitopically or immunologically equivalent variants that form a particular aspect of this invention
  • the term "antigemcally equivalent derivative” as used herein encompasses a polypeptide or its equivalent which will be specifically recognized by certain antibodies which, when raised to the protem or polypeptide according to the invention, interfere with the immediate physical interaction between pathogen and mammalian host
  • immunologically equivalent derivative encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host The polypeptide.
  • an antigemcally or immunologically equivalent derivative or a fusion protem thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken
  • the fusion protein may provide stability to the polypeptide
  • the antigen may be associated, for example by conjugation, with an lmmunogemc carrier protein for example bovme serum albumin (BSA) or keyhole limpet haemocyanin (KLH)
  • BSA bovme serum albumin
  • KLH keyhole limpet haemocyanin
  • a multiple antigenic peptide comprising multiple copies of the protem or polypeptide, or an antigemcally or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve lmmunogemcity so as to obviate the use of a carrier
  • the antibody or variant thereof is modified to make it less lmmunogemc m the individual
  • the antibody may most preferably be "humanized", where the comphmentarity dete ⁇ nmmg reg ⁇ on(s) of the hyb ⁇ doma-derived antibody has been transplanted into a human monoclonal antibody , for example as described in Jones. P et al (1986). Nature 321. 522-525 or Tempest et al , (1991) Biotechnology 9, 266-273
  • a polynucleotide of the invention m genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al . Hum Mol Genet 1992. 1 363. Manthorpe et al . Hum Gene Ther 1963 4. 419), delivery of DNA complexed with specific protein carriers (Wu et al , J Biol Chem 1989 264,16985). coprecipitation of DNA with calcium phosphate (Benvemsty & Reshef, PNAS USA.
  • Another aspect of the invention relates to a method for inducing an lmmunological response in an individual, particularly a mammal which comprises inoculating the individual with RNaseP, or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Staphylococcus aureus infection Also provided are methods whereby such immunological response slows bacterial replication Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises delivering to such individual a nucleic acid vector to direct expression of RNaseP, or a fragment or a variant thereof, for expressing RNaseP, or a fragment or a variant thereof in v vo in order to induce an immunological response, such as, to produce antibody and or T cell immune response, including, for example, cytokine-producmg T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established within the- individual or not
  • One way of administering the gene is by
  • a further aspect of the invention relates to an immunological composition which, when introduced mto an individual capable or having induced within it an immunological response, induces an immunological response in such individual to a RNaseP or protem coded therefrom, wherein the composition comprises a recombinant RNaseP or protein coded therefrom comprising DNA which codes for and expresses an antigen of said RNaseP or protein coded therefrom
  • the immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells
  • a RNaseP polypeptide or a fragment thereof may be fused with co-protem which may not by itself produce antibodies, but is capable of stabilizing the first protem and producing a fused protem which will have lmmunogemc and protective properties
  • fused recombinant protein preferably further comprises an antigenic co-protein, such as hpoprotein D from Hemophilus influenzae.
  • Glutathione-S-transferase GST
  • beta-galactosidase relatively large co-protems which solubihze the protem and facilitate production and purification thereof
  • the co-protein may act as an adjuvant in the sense of providing a generalized stimulation ofthe immune system
  • the co-protein may be attached to either the ammo or carboxy terminus ofthe first protein
  • compositions particularly vaccine compositions, and methods comprising the polypeptides or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y et al Science 273 352 (1996)
  • methods using the described polynucleotide or particular fragments thereof which have been shown to encode non-variable regions of bacterial cell surface proteins in DNA constructs used in such genetic immunization experiments m animal models of infection with Staphylococcus aureus will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value from the requisite organ of the animal successfully resisting or clearing infection for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Staphylococcus aureus infection, in mammals, particularly humans
  • the polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue
  • tissue damage include wounds in skm or connective tissue caused, e g , by mechanical, chemical or thermal damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, mammary glands, urethra or vagina
  • the invention also includes a vaccine formulation which comprises an lmmunogenic recombinant protein of the invention together with a suitable carrier Since the protem may be broken down in the stomach, it is preferably administered parenterally. including, for example, administration that is subcutaneous, intramuscular, intravenous, or mtradermal Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored in a freeze-d ⁇ ed condition requiring only the addition of the sterile liquid carrier immediately prior to use
  • the vaccine formulation may also include adjuvant systems for enhancing the lmmunogenicity of the formulation, such as oil-m water systems and other systems known in the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation
  • Wl ⁇ le the invention has been described with reference to certain RNaseP protem, it is to be understood that this covers fragments of the naturally occurring protein and similar protems with additions, deletions or substitutions which do not substantially affect the lmmunogemc properties ofthe recombinant protem Compositions, kits and administration
  • Tlie mvention also relates to compositions compnsrng the polynucleotide or the polypeptides discussed above or their agomsts or antagomsts
  • the polypeptides of the mvention may be employed in combination with a non-stenle or stenle earner or earners for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for admimstration to a subject ⁇
  • Such compositions compnse for instance, a media additive or a therapeutically effective amount of a polypeptide ofthe mvention and a pharmaceutically acceptable earner or excipient
  • Such earners may mclude.
  • the mvention further relates to diagnostic and pharmaceutical packs and kits compnsrng one or more containers filled with one or more of tlie ingredients ofthe aforementioned compositions ofthe mvention
  • Polypeptides and other compounds of the mvention may be employed alone or m conjunction with other compounds, such as therapeutic compounds
  • compositions may be administered in any effective, convenient manner mcludmg, for instance, administration by topical, oral. anal, vagmal. mtravenous, intraperitoneal. intramuscular, subcutaneous, rntranasal or mtradermal routes among others
  • the active agent may be administered to an individual as an mjectable composition, for example as a sterile aqueous dispersion, preferably lsotomc
  • the composition may be formulated for topical application for example m the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash.
  • impregnated dressings and sutures and aerosols and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams
  • Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions
  • Such carriers may constitute from about l%o to about 98% by weight ofthe formulation, more usually they will constitute up to about 80% by weight ofthe fonnulation
  • the daily dosage level of the active agent will be from 0 01 mg kg to 10 mg/kg, typically around 1 mg/kg
  • the physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual The above dosages are
  • In-dwellmg devices include surgical implants, prosthetic devices and catheters, 1 e , devices that are introduced to the body of an individual and remain in position for an extended time
  • Such devices mclude, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospmal fluid shunts, urinary catheters, continuous ⁇ ambulatory peritoneal dialysis (CAPD) catheters
  • composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an m-dwelhng device Treatment may be continued after surgery du ⁇ ng the m-body time of the device
  • composition could also be used to broaden pe ⁇ operative cover for any surgical technique to prevent bacterial wound infections, especially Staphylococcus aureus wound infections
  • compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use m dental treatment as an alternative to, or m conjunction with, antibiotic prophylaxis
  • the composition of the invention may be used to bathe an indwelling device immediately before insertion
  • the active agent will preferably be present at a concentration of 1 ⁇ g/ml to lOmg/ml for bathing of wounds or indwelling devices
  • a vaccine composition is conveniently in injectable form
  • Conventional adjuvants may be employed to enhance the immune response
  • a suitable unit dose for vaccination is 0 5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals
  • Each reference disclosed herein is incorporated by reference herein in its entirety Any patent application to which this application claims priority is also incorporated by reference herein m its entirety Examples
  • the present mvention is further desc ⁇ bed by the following examples The examples are provided solely to illustrate the mvention by reference to specific embodiments These exemplifications, while illustrating certain specific aspects of the mvention, do not portray the- limitations or circumsc ⁇ be the scope ofthe disclosed mvention
  • This method describes a rapid way to obtain additional sequence data from partial gene fragments independent of probing a genomic library
  • the reverse strand encodmg the C-terminus and upstream the unknown N-termmal sequence of the protein was amplified usmg primer #1, resulting in single stranded products as diagrammed in Figure 9 This amplification of the desired sequence favoured the binding of random primers to that sequence m the next step
  • PCR is expected to generate many different fragments, however the major products should be those primed by primer #2 and a random primer (see below) Only these products were of interest as they should encode the N-terminal half of the protein and they could be monitored since they would be radiolabeled After separation of the PCR products by gel electrophoresis and exposure to film, a limited number of radiolabeled bands should be visible on the autoradiogram This method is diagrammed in Figure 10 These fragments can subsequently be cloned into a suitable vector, e g , pUC19, and sequenced using routine methods Example 2. PCR of the spp 5' domain with random primers
  • the 5' half of the spp gene was amplified in a two step PCR reaction as described herein
  • the PCR products were cleaned using a QIAquick PCR purification column and- recovered in 50 ⁇ l water
  • the sample was concentrated to a final volume of 25 ⁇ l under vacuum in a SpeedVac® SCI 10 (Savant) 20 ⁇ l were loaded onto a 1 6 mm, 8% polyacrylamide gel containing 7M urea and analyzed by gel electrophoresis
  • the gel was stained with ethidium bromide (1 ⁇ g/ml in H2 ⁇ ) to visualize the DNA and photographed The gel was then dried and exposed to film
  • PCR fragments were blunt end cloned into Smal cut pUC19 Ligations of 100 ng vector DNA with 1 0 ⁇ l and 1 5 ⁇ l of PCR products were performed at 16°C overnight E cob XL1 blue cells ( ⁇ -complementing) were transformed by electroporation and plated onto selective plates containing IPTG and x-gal (for blue/white screening)
  • the 10 plasmids containing an insert were sequenced both with the forward and reverse pUC/M 13 sequencmg primers ⁇
  • GAAAA TTTCA AAGTA CATAA GTCGC ATATA TTGGC CAAAG ATTGT AATAG CAAGA CAGCC AGCTA AAGAT ATGAC GACTT TACAA ATACA GAATA GTCTT GAGCA CGTAC TTAAA ATTGC CAAAG TTTTT AATAA AAAGA TTAAG TAAGG ATAGG GTAGG GAATG AAAAC 3 ' stop codon (underlined)
  • Primer #2 sequence is shown in bold and also the known 14 bases underlined (3 changes to known sequence m italic)
  • 2 plasmids contained inserts with different sequences that are a result of unspecific priming during PCR
  • the newly obtained sequence data was drawn together and predicted to represent the complete sequence of the spp structural gene
  • the reading frame was determined from a partial sequence ofthe 3' domain According to this reading frame the start codon ATG could be identified 174bp upstream of primer #2 sequence (see above)
  • the nucleotide sequence of the spp gene is shown in Table 1 [SEQ ID NO 2], the start codon (ATG) and stop codon (TAA) are both shown in bold
  • the deduced translation product of the spp gene (SP protein) is 117 amino acids is also shown m Table 1 [SEQ ID NO 2]
  • the newly obtained sequence data was used to design PCR primers for the amplification ofthe full length spp structural gene
  • the PCR resulted in a single band of approximately 360bp with a total DNA yield of 2 ⁇ g as determined by spectrophotomet ⁇ cal measurement
  • the DNA fragment was cleaned using a QIAquick PCR purification column and recovered in 80 ⁇ l water
  • the pMalc2 vector provides a method for expressing and purifying a protem produced from a cloned gene (New England BioLabs) (see also, Guan, C , Li, P , Riggs, P D _ and lnouye, H (1987) Gene 67, 21-30, Mama, C V et al (1988) Gene 74, 365-373 Riggs, P in Ausubel, F M et al (eds) Current prot in Molecular Biol (1992) Kellerman and Ferenci (1982) Methods in Enzymol 90, 459-463, Yamsch-Perron, C et al (1985) Gene 33, 103-119, Zagursky, R J and Berman, M L (1984) Gene 27, 183-191, regarding the vector and its uses)
  • the cloned gene is inserted downstream from the malE gene of E cob, which encodes the maltose-bindmg-protein (MBP). resulting in the expression of an MBP fusion protem
  • the method uses the strong "tac" promotor under the control of the lac repressor and a one step purification of the fusion protein using MBP's affinity for maltose Unique restriction sites are available between malE and lacZa for inserting the gene of interest, allowing blue/white screening of transformants
  • the vector also contains a recognition site for the specific Factor Xa protease located just 5' to the polyhnker This allows MBP to be cleaved after purification without leaving any vector derived residues attached to the protem of interest when the gene is cloned mto the Xmnl site Example 7. Ligation of the spp PCR product into pMalc2
  • the spp PCR product was digested with BamHI and cleaned using a QIAquick PCR purification column Vector and insert were hgated overnight m a 20 ⁇ l reaction at 16°C The molar ratio of vector to insert was 1 5
  • the hgation was desalted usmg QIAquick Nucleotide Removal columns
  • a 1 liter culture of £ cob XL-1 Blue pMalc2 spp was grown at 37°C, 220 rpm and expression of the MBP-SPP fusion induced by adding ImM IPTG at an A 600 of 0 6 Induction was continued for 2 hours The A" ⁇ was monitored over that time period to ensure that cells were still growing during induction lml samples were removed from the culture after 0 5. 1 0 1 5 and 2 hours, the cells pelleted and the pellet resuspended in 100 ⁇ l SDS gel loading buffer 3 ⁇ l of the samples were analysed by SDS-PAGE with Coomassie staining
  • the cells were not effected by the overexpression of the fusion protem, they kept growing and after 2 hours of IPTG induction reached an A"" ⁇ of 1 43
  • Protem extracts from samples were separated by SDS-PAGE and transferred onto a mtro-cellulose membrane by Western transfer
  • the MBP-SPP fusion protem could be lmmuno-detected by incubation of the membrane with anti-MBP rabbit serum and HRP- coupled anti-rabbit antibodies followed by ECL detection
  • the Western Blot revealed that 2 protems were induced upon IPTG addition
  • the major protem was of the expected size of approximately 56kD.
  • the second protem, produced in lower amounts was 10-15 kD larger
  • Analysis of the spp sequence revealed an accumulation of AUA (ile) and AGA (arg) codons that are rare in E cob
  • the normal frequency of AGA in E cob is 0 27%o. of AUA 0 51%)
  • the spp sequence consists to 4 3%o of AGA and 5 1% of AUA These codons may cause frame shifts and other altered translation events when overexpressing a protein with a high abundance of rare codons
  • the cells were grown and the expression of the fusion protein was induced as described above
  • the cells kept growing during the induction period from an A°00 of 0 6 to 1 45
  • Samples were taken from the culture at 0 0. 0 5, 1 5 and 2 5 hours after IPTG addition and protein extracts analysed by SDS-PAGE with Coomassie staining, as well as Western transfer with lmmunodection
  • Overexpression of the rare tRNAs IleX and ArgU resulted in only one MBP-SPP fusion protem being overexpressed during IPTG induction as detected by the anti-MBP serum
  • the cells E cob W3110 pRI952 pMalc2 spp
  • the cell debris was removed by centrifugation and the protein concentration of the crude extract determined using the BIO-RAD Protem Assay The total amount of protem m the crude extract was 86 mg
  • a sample of the crude extract was analysed by SDS- PAGE with Coomassie staining which showed that the fusion protem was released from the cells during sonication
  • the crude extract was then pumped through the amylose resin column, to which the MBP-SPP fusion bound, and unbound protein was removed from the column by extensive washing with column buffer
  • the fusion protein was then eluted with column buffer supplemented with lOmM maltose using methods described herein
  • the eluate was collected as 3 ml fractions Samples were removed during the purification procedure and analyzed by SDS-PAGE with Coomassie staining Almost all of the fusion protem bound to the amylose resin, only trace amounts were found m the flow-through and no fusion protein was washed off the column Fractions 4- 11 contained reasonable amounts of protein and were pooled. yielding in 25 ml protem solution with a concentration of 1 mg/ml
  • the SP protem readily precipitated upon Factor Xa cleavage and could be separated from MBP by centrifugation
  • the SPP pellet was washed 3 times and then resuspended in 5ml column buffer
  • the MBP remained m solution and was found m the supernatant whereas the pellet consisted mainly ofthe SP protem and one minor contaminating protein, resulting in a reasonably pure preparation of SPP
  • the total SPP yield was 2 18 mg/hter culture
  • the SPP preparation was diluted with column buffer to a final concentration of 0 32 mg/ml ImM DTT was added to prevent mtermolecular disulfide bond formation between cystein residues
  • the addition of 7M urea denatured and apparently completely resolubihzed the protem
  • the protein was ahquoted, snap-frozen and stored at -80°C Example 14.
  • SP RNA was transcribed in vitro from the plasmid pSPR using T7 RNA polymerase pSPR is a pUC19 derivative that harbours the S aureus spr gene behind a T7 promotor
  • the plasmid was linearized with BamHI restriction endonuclease to enable run off transcription
  • the Ambion MEGAsc ⁇ ptTM T7 kit was used for large scale transcription of the SP RNA
  • the transcription reaction was performed at 30 C to allow slow folding ofthe RNA mto the correct conformation during synthesis
  • the RNA was cleaned under non-denaturmg conditions using CLONTECH Chroma-Spm 30 columns and the RNA recoverd in 40 ⁇ l water
  • the quality of the transcript was monitored by gel electrophoresis on a 5% polyacrylamide TBE gel with 7M urea
  • the RNA was visualized by UV-shadowing over a TLC plate
  • E cob ptRNA met a substrate for RNase P. was transcribed in vitro by T7 RNA polymerase (Epicenter) in the presence of [ ⁇ -32p] T pp o internally label the RNA
  • the DNA template pGem3Z-ptRNA was linearized with BstNI restriction endonuclease to allow run-off transcription
  • the transcription was performed m a 20 ⁇ l reaction at 37°C for 60m ⁇ n
  • the 93nt RNA (sequence shown below) was cleaned under non-denaturing conditions using a CLONTECH Chroma-Spin 10 column
  • To calculate the specific activity of the RNA probe lul samples were removed before and after cleaning, mixed with 5 ml scintillation cocktail and counted in a scintillation counter ptRNA met in pGem-3Z, sequence of the transcript (93mer) 5'-» 3' [SEQ ID NO 12]
  • RNA molecule p6AT-l was 5' end-labeled with high specific activity [ ⁇ -32pj ATP (6000 ⁇ C ⁇ / ⁇ l) using bactenophage T4 polynucleotide kmase
  • the labeling was performed in a 10 ⁇ l reaction at 37°C for 30 mm
  • the enzyme was heat- mactivated at 95 °C for 2 mm
  • p6AT-l was gel purified from a 20% polyacrylamide TBE gel with 7M urea
  • the RNA was extracted from the gel slice and precipitated with 300mM NaOAc and 2 5 volumes 100% ethanol After a spin at 14,000g and 4°C the RNA pellet was washed twice with chilled 70%o ethanol The pellet was air dried and subsequently resuspended in 50 ⁇ l water 0 5 ⁇ l were spotted onto a filter paper disk and counted
  • Example 17 Determination of the optimal buffer conditions for SP RNA reactions E cob Ml RNA is able to cleave ptRNA met or the minimal substrate p6AT-l m lOOmM NH4C1. lOOmM T ⁇ s Cl pH 7 5. lOOmM MgCl 2
  • the conditions for cleavage of either substrate by S aureus SP RNA were unknown and clearly deviated from those optimized for E cob
  • a large number of different buffer conditions were investigated in order to determine optimal buffer conditions for either substrate cleavage by the S aureus ⁇ bozyme
  • Buffers containing lOOmM MgC12. 2M KC1 and lOOmM T ⁇ s Cl at a pH of 7 0, 7 5 and 8 0 were tested for the ability to promote p6AT-l cleavage by SP RNA
  • the substrate was cleaved in all three buffers but cleavage was very poor at pH 7 0 and optimal at pH 8 0
  • the MgC12 concentration was successively reduced from lOOmM to lOmM to determine the lowest magnesium ion concentration at which the SP RNA could still process the substrate lOOmM. 50mM. 25mM and lOmM MgC12 were tested in a buffer containing 2M KCl. 1 OOmM T ⁇ s CI pH8 and 5% PEG
  • ptRNAmet could be processed at KCl concentrations below 400mM Some cleavage could already be detected at 20mM KCl, whereas no cleavage of p6AT-l was observed under these conditions
  • the cleavage rate increased with the KCl concentration At 150mM KCl more than 50% of the substrate was processed, reaching 82% at 800mM KCl At concentrations of 1M or above 85% of the ptRNA were cleaved, and no further increase could be detected, indicating that 15% of the substrate was uncleavable E cob Ml RNA as a positive control cleaved 80% of the substrate in lOOmM T ⁇ s CI pH7 5, lOOmM NH4C1, lOOm
  • the S aureus RNase P holoenzyme cleavage of p6AT-l was investigated over a range of different KCl concentrations at low magnesium ion concentrations All reactions were performed in lOOmM T ⁇ s Cl pH8, lOmM MgC12, 5% PEG and 40mM to 1 5M KCl Cleavage could already be observed at 40mM KCl
  • the cleavage rate increased with the concentration of monovalent salt, peaking at 150mM, where 50% of the substrate was processed
  • the cleavage rate decreased again and p6AT-l processing did not occur at KCl concentrations of 600mM and above
  • the E cob holoenzyme control cleaved 57% of the p6AT-l withm 20m ⁇ n in lOOmM T ⁇ s CI pH7 5, lOOmM NH4C1.
  • lOmM MgC12 The optimal buffer for p6AT-l cleavage by the holoenzyme was 150mM KCl, lOOmM T ⁇ s CI pH 8 0. lOmM MgCl 2 , 5% PEG 8000 p6AT-l was a " better substrate for the holoenzyme than for SP RNA, as under optimal conditions only 27% p6AT-l was cleaved as opposed to 50% by the S aureus RNase P holoenzyme Example 25. MgCl2 requirements for ptRNA cleavage by the holoenzyme
  • the S aureus RNase P holoenzyme failed to cleave ptRNA met under the same conditions employed for p6AT-l processing
  • the holoenzyme was not able to cleave the substrate at lOmM MgCl 2 under any given KCl concentration
  • the concentration of magnesium ions was increased to 20mM and ptRNA cleavage by the holoenzyme as well as the SP RNA ribozyme were tested at different KCl concentrations
  • the buffers contained lOOmM T ⁇ s CI pH8, 20mM MgCl 2 , 5% PEG and lOmM, 50mM, 150mM, 600mM or 1 5M KCl
  • the S aureus RNase P holoenzyme was tested for its ability to cleave ptRNA" 16 ' under different KCl concentrations ranging from 20mM to 1 5M
  • the buffers contained lOOmM T ⁇ s CI pH8, 20mM MgCl 2 , 5% PEG 8000 and xM KCl
  • the holoenzyme was able to cleave ptRNA me t at KCl concentrations as low as 20mM
  • the cleavage rate increased with the salt concentration with a peak at 150mM where 55% of the substrate was processed
  • the cleavage rate decreased at KCl concentrations above 200mM but improved again at 1M KCl reaching a maximum at 1 5M
  • the cleavage at high salt concentrations was not due to holoenzyme activity but result of SP RNA ribozyme activity at high KCl concentrations
  • ptRNA me t was a better substrate for SP RNA than for the S aureus RNase P holoenzyme
  • the optimal buffer conditions for ptRNAmet cleavage by the holoenzyme were 150mM KCUOOmM T ⁇ s CI pH8, 20mM MgCl 2 . 5% PEG
  • the polynucleotide havmg the DNA sequence given in SEQ ID NO 1 was obtained ⁇ from a library of clones of chromosomal DNA of Staphylococcus aureus m E cob
  • the sequencing data from two or more clones containing overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence m SEQ ID NO 1 Libraries may be prepared by routine methods, for example Methods 1 and 2 below
  • Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 according to standard procedures and size-fractionated by either of two methods
  • Total cellular DNA is mechanically sheared by passage through a needle in order to size-fractionate according to standard procedures
  • DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are ligated mto the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E cob infected with the packaged library
  • the library is amplified by standard procedures Method 2
  • Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning mto library vectors (e g . Rsal, Pall, Alul. Bshl235I), and such fragments are size-fractionated according to standard procedures EcoRI linkers are ligated to the DNA and the fragments then ligated mto the vector Lambda ZapII that have been cut with EcoRI. the library packaged by standard procedures, and E cob infected with the packaged library The library

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Abstract

L'invention concerne un nouveau complexe de ribonucléoprotéines bactérien et les éléments du composé correspondants. Plus spécifiquement, l'invention concerne la RNase P ARN isolée à partir de Staphylococcus aureus et l'utilisation de la RNase P sur des écrans servant à l'identification de composés antimicrobiens, ainsi que l'utilisation de ces composés en thérapie.
PCT/US1999/021644 1998-09-17 1999-09-17 Polypeptides rnase p Ceased WO2000015775A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2000570302A JP2002525048A (ja) 1998-09-17 1999-09-17 Ｒｎａｓｅｐポリペプチド
EP99948325A EP1114149A1 (fr) 1998-09-17 1999-09-17 Polypeptides rnase p

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US15614598A 1998-09-17 1998-09-17
US09/156,145 1998-09-17

Publications (1)

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WO2000015775A1 true WO2000015775A1 (fr) 2000-03-23

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EP (1) EP1114149A1 (fr)
JP (1) JP2002525048A (fr)
WO (1) WO2000015775A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1183333A4 (fr) * 1999-05-19 2002-09-11 Smithkline Beecham Corp Procedes d'utilisation des mecanismes reactifs d'action de la rnase p

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112021005788A2 (pt) * 2018-09-26 2021-10-26 Case Western Reserve University Métodos e composições para aumentar a expressão de proteínas e/ou tratar um distúrbio de haploinsuficiência

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 8 January 1997 (1997-01-08), HAAS E. ET AL.: "Staphylococcus Aureus Ribonuclease P RNA (rnpB) gene, partical sequence" *
HAAS E. ET AL.: "Structure and evolution of ribonuclease P RNA in Gram-positive bacteria", NUCLEIC ACIDS RESEARCH, vol. 24, no. 23, December 1996 (1996-12-01), pages 4775 - 4782, XP002924721 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1183333A4 (fr) * 1999-05-19 2002-09-11 Smithkline Beecham Corp Procedes d'utilisation des mecanismes reactifs d'action de la rnase p

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EP1114149A1 (fr) 2001-07-11

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