EP1114149A1 - Rnase p polypeptides - Google Patents

Abstract

This invention relates to a novel bacterial ribonucleoprotein complex and the component parts thereof. More specifically, this invention relates to RNase P RNA isolated from Staphylococcus aureus and the use of RNase P RNA in screens for the identification of antimicrobial compounds and to the use of such compounds in therapy.

Description

RNASE P POLYPEPTIDES

Field of the Invention:

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

Background of the Invention:

This invention relates to a novel bacterial πbonucleoprotem 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

The 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

The frequency of Staphylococcus aureus infections has nsen dramatically in the past 20 years This has been attnbuted to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems It is no longer uncommon to isolate Staphylococcus aureus strains which are resistant to some or all ofthe standard antibiotics This has created a demand for both new anti-microbial agents and diagnostic tests for this organism

While certain Staphylococcal proteins associated with pathogenicity have been identified, e g . coagulase. hcmolysins, leucocidins and exo- and enterotoxms, additional targets are always useful because it is appreciated that the target of a antimicrobial screen can often bias the outcome Thus, new targets allow for the discovery of new classes of antimicrobials

Brief Description of the Invention: This invention provides a novel πbonucleoprotein complex, particularly such complex from Staphylococcus aureus. and the separately isolated RNA and protein components thereof

In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode the protein and RNA components of such a complex

In particular 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

It is an object of the invention to provide polypeptides that have been identified as novel RNaseP polypeptides by homolog between the amino acid sequence set out in Table 1 [SEQ ID NO 2] and a known amino acid sequence or sequences of other proteins, such as B subtύis RNase P protein

It is a further object of the invention to provide polynucleotides that encode RNase P polypeptides. particularly polynucleotides that encode the polypeptide herein designated RNaseP. as well as polynucleotides that are transcnbed into RNase P RNA. particularly catalytic RNA hi a particularly preferred embodiment of the invention the polynucleotide compnses a region encoding RNaseP polypeptides compns ng the sequence set out in Table 1 [SEQ ID NO 1] which includes a full length gene, or a variant thereof

In another particularly preferred embodiment ofthe invention there is a novel RNaseP protein from Staphylococcus aureus comprising the ammo acid sequence of Table 1 [SEQ ID NO 2]. or a variant thereof In accordance with another aspect of the invention there is provided 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. 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

In accordance with another aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization Among the particularly preferred embodiments of the invention are naturally occurring allelic vanants of RNaseP and polypeptides encoded thereby

Another aspect ofthe invention there are provided novel polypeptides of Staphylococcus aureus referred to herein as RNaseP as well as biologically, diagnostically, prophylactically. clinically or therapeutically useful vanants thereof, and compositions compnsrng the same

Among the particularly preferred embodiments of the invention are vanants of RNaseP polypeptide encoded by naturally occurring alleles ofthe RNaseP gene hi a preferred embodiment ofthe invention there are provided methods for producing the aforementioned RNaseP polypeptides In accordance with yet another aspect of the invention, there are provided inhibitors to such polypeptides. useful as antibacterial agents, including, for example, antibodies

In accordance with certain preferred embodiments of the invention, there are provided products 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. endophthalmitis. preseptal and orbital cellulitis. darcryocystitis). kidney and urinary tract (e g , epiώdymitis, 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. and administering a RNaseP polypeptide or polynucleotide to an organism to raise an lmmunological response against a bactena, especially a Staphylococcus aureus bactena In accordance with certain preferred embodiments of this and other aspects of the invention there are provided polynucleotides that hybndize to RNaseP polynucleotide sequences, particularly under stringent conditions

In certain preferred embodiments of the invention there are provided 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 polynucleotide hi accordance with yet another aspect of the invention, there are provided RNaseP agonists and antagonists, preferably bacteπostatic or bactenocidal agonists and antagonists

In a further aspect ofthe invention there are provided 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

Detailed Description of the Figures: 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

RNA/protein interactions

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:

The following definitions are provided to facilitate understanding of certain tenns used frequently herein Certain other definitions are provided elsewhere herein

"Host cell" is a cell which has been transformed or transfected. or is capable of transformation or transfection by an exogenous polynucleotide sequence

"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences In the art, "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. Griffin, A M , and Griffin. H G . eds , Humana Press. New Jersey. 1994. Sequence Analysis in Molecular Biology, von Heinje, G . Academic Press, 1987, and Sequence Analysis Primer, Gπbskov, M and Devereux, J , eds . M Stockton Press, New York, 1991, and Caπllo, H, and Lφman. D , SIAM J Applied Math , 48 1073 (1988) 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 . et al , NCBI NLM NIH Bethesda. MD 20894, Altschul. S . et al , J Mol Biol 215 403-410 (1990) As an illustration, by a polynucleotide 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 In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% ofthe nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups withm the reference sequence Analogously , by a polypeptide having an ammo acid sequence having at least, for example, 95% identity to a reference amino acid sequence of SEQ ID NO 2 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five ammo acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO 2 In other words, to obtain a polypeptide having an ammo acid sequence at least 95% identical to a reference ammo acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another ammo acid, or a number of amino acids up to 5% of the total ammo acid residues in the reference sequence may be inserted into the reference sequence These alterations of the reference sequence may occur at the amino or carboxy tenninal positions of the reference ammo acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or m one or more contiguous groups withm the reference sequence "Isolated" means altered "by the hand of man" from its natural state, i e , if it occurs in nature, it has been changed or removed from its ong nal environment, or both For example, a polynucleotide or a polypeptide naturally present in a living organism is not '"isolated," but the same polynucleotide or polypeptide separated from the coexisting matenals of its natural state is "isolated", as the term is employed herein

"Polynucleotιde(s)" generally refers to any polynbonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotιde(s)" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of smgle- and double- stranded regions or smgle-. double- and tnple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybnd molecules compnsrng- DNA and RNA that may be single-stranded or, more typically, double-stranded, or tnple-stranded regions, or a mixture of single- and double-stranded regions In addition, "polynucleotide" as used herein 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 "polynucleotιde(s)" also mcludes DNAs or RNAs as descnbed above that contam one or more modified bases Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotιde(s)" as that term is intended herem Moreover. 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 It will be appreciated that a great vanety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art The term "polynucleotιde(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 "Polynucleotιde(s)" also embraces short polynucleotides often referred to as olιgonucleotιde(s) "Polypeptιde(s)" refers to any peptide or protem compnsrng two or more ammo acids joined to each other by peptide bonds or modified peptide bonds "Polypeptιde(s)" refers to both short chams. commonly referred to as peptides, o gopeptides and ohgomers and to longer chains generally referred to as proteins Polypeptides may contam ammo acids other than the 20 gene encoded ammo acids "Polypeptιde(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. as well as m a voluminous research literature, and they are well known to those of skill m the art It will be appreciated that the same type of modification may be present m the same or varying degree at several sites m a given polypeptide Also, 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. gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- nbosylation. selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins, such as arginylation. and ubiquitrnation See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed , T E Creighton. W H Freeman and Company. New York (1993) and Wold, F . Posttranslational Protem Modifications Perspectives and Prospects, pgs 1-12 m POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed . Academic Press, New York (1983), Seifter et al , Meth Enzymol 182 626-646 (1990) and Rattan et al , Protein Synthesis Posttranslational Modifications and Aging. Ann N Y Acad Sci 663 48-62 (1992) Polypeptides may be branched or cyclic, with or without branclimg Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well

'Naπant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the variant may or may not 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, deletions m any combination A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans

Detailed Description of the Invention:

The πbonucleoprotein. 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 endoπbonucleolytic action 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 Bacteπol 177 (8). 1919-1928 (1995)

The mvention relates to novel RNaseP polypeptides and polynucleotides as descnbed m greater detail below In particular, 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

Phylogenetic comparisons readily allow secondary structure modeling and the identification of a minimum consensus structure (Figure 1) Data concerning the RNase P RNA structure are available m the RNase P database on the w w w (http //jwbrown mbio ncsu edu/RNaseP/home html) A polynucleotide of the invention from which the RNA component is transcribed is set forth in Figure 3 [SEQ ID NO 14] In general, few nucleotides are conserved but compensatory base changes m hydrogen bonded regions show that the overall structure is preserved throughout eubacteπa Universal conservation of primary sequences (E cob 61-74, 353-360) together with other conserved or quasi-conserved nucleotides implicate functional importance, the significance of which remain unknown To date all RNase P RNA molecules can be folded to fit a consensus 'cage-like' structure, beyond this domain there is no convincing structural similarity between prokaryotic and eukaryotic RNase P RNAs RNase P Protein Component

The precise functional role of the protein remains unknown While it is appreciated that in vitro the experimental conditions can be established such that the protein component is not ^— necessary for catalytic activity, in vivo, the protein components appears to be required A novel RNase P protein component from S aureus has been identified and is characterized by the amino acid sequence given in Figure 2 [SEQ ID NO 2] in which the S aureus (Sau) sequence is aligned with sequences from other microorganisms

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]

TABLE 1 RNaseP Polynucleotide and Polypeptide Sequences

(A) Sequences from Staphylococcus aureus RNaseP polynucleotide sequence [SEQ ID NO 1]

5 ' -ATG TTA TTG GAA AAA GCT TAC CGA ATT AAA AAG AAT GCA GAT TTT CAG AGA ATA TAT AAA AAA GGT CAT TCT GTA GCC AAC AGA CAA TTT GTT GTA TAC ACT TGT AAT AAT AAA GAA ATA GAC CAT TTT CGC TTA GGT ATT AGT GTT TCT AAA AAA CTA GGT AAT GCA GTG TTA AGA AAC AAG ATT AAA AGA GCA ATA CGT GAA AAT TTC AAA GTA CAT AAG TCG CAT ATA TTG GCC AAA GAT ATT ATT GTA ATA GCA AGA CAG CCA GCT AAA GAT ATG ACG ACT TTA CAA ATA CAG AAT AGT CTT GAG CAC GTA CTT AAA ATT GCC AAA GTT TTT AAT AAA AAG ATT AAG TAA-3 '

(B) RNaseP polypeptide sequence deduced from the polynucleotide sequence in this table [SEQ ID NO 2] NH,- MLLEK VYRIK KNADF GRIYK KGHSV A RQF VVYTC NNKEI DHFRL

GISVS KKLGN AVLRN KIKRA IRENF KVHKS HILAK DIIVI ARQPA KDMTT LQIQN SLEHV LKIAK VFNKK IK- COOH

(C) Polynucleotide sequence embodiments [SEQ ID NO: 1].

X-(Rτ_)_n-ATG TTA TTG GAA AAA GCT TAC CGA ATT AAA AAG AAT GCA

GAT TTT CAG AGA ATA TAT AAA AAA GGT CAT TCT GTA GCC AAC AGA__

CAA TTT GTT GTA TAC ACT TGT AAT AAT AAA GAA ATA GAC CAT TTT

CGC TTA GGT ATT AGT GTT TCT AAA AAA CTA GGT AAT GCA GTG TTA AGA AAC AAG ATT AAA AGA GCA ATA CGT GAA AAT TTC AAA GTA CAT

AAG TCG CAT ATA TTG GCC AAA GAT ATT ATT GTA ATA GCA AGA CAG

CCA GCT AAA GAT ATG ACG ACT TTA CAA ATA CAG AAT AGT CTT GAG CAC GTA CTT AAA ATT GCC AAA GTT TTT AAT AAA AAG ATT AAG TAA-

( R2 ) n- Y

(D) Polypeptide sequence embodiments [SEQ ID NO.2].

X- (R₁)_n-MLLEK VYRIK KNADF GRIYK KGHSV ANRQF VVYTC NNKEI DHFRL GISVS KKLGN AVLRN KIKRA IRENF KVHKS HILAK DIIVI ARQPA KDMTT LQIQN SLEHV LKIAK VFNKK IK-(R₂)_n-Y

(E) Sequences from Staphylococcus aureus RNaseP RNA gene [SEQ ID NO:3].

5 ' -

GTTCTGATATTTTGGGTAATCGCTATATTATATAGAGGAAAGTCCATGCTCACACAGTCTGAGATGAT

T GTAGTGTTCGTGCTTGATGAAACAATAAATCAAGGCATTAATTTGACGGCAATGAAATATCCTAAGTC

T

TTCGATATGGATAGAGTAATTTGAAAGTGCCACAGTGACGTAGCTTTTATAGAAATATAAAAGGTGGA

A

CGCGGTAAACCCCTCGAGTGAGCAATCCAAATTTGGTAGGAGCACTTGTTTAACGGAATTCAACGTAT AAACGAGACACACTTCGCGAAATGAAGTGGTGTACGACAGATGGTTATCACCTGAGTACCAGTGTGA

CTAGTGCACGTGATGAGTACGATGGAACAGAACATGGCTTATAGAAATATCACTACTA

G-3 '

(F) Polynucleotide sequence embodiments [SEQ ID NO:3] x- ( R₁ ) _n-

GTTCTGATATTTTGGGTAATCGCTATATTATATAGAGGAAAGTCCATGCTCACACAGTCTGAGATGAT

T

GTAGTGTTCGTGCTTGATGAAACAATAAATCAAGGCATTAATTTGACGGCAATGAAATATCCTAAGTC

T TTCGATATGGATAGAGTAATTTGAAAGTGCCACAGTGACGTAGCTTTTATAGAAATATAAAAGGTGGA

A CGCGGTAAACCCCTCGAGTGAGCAATCCAAATTTGGTAGGAGCACTTGTTTAACGGAATTCAACGTAT AAACGAGACACACTTCGCGAAATGAAGTGGTGTACGACAGATGGTTATCACCTGAGTACCAGTGTGA CTAGTGCACGTGATGAGTACGATGGAACAGAACATGGCTTATAGAAATATCACTACTA G-(R₂)_n-Y

Polypeptides ofthe mvention

The 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 As with RNaseP 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 mclude, 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. are also preferred Further preferred are 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

Also preferred are 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 Also included are those fragments that are antigenic or rmmunogenic m an animal, especially m a human Particularly preferred are 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

Usmg the information provided herem, such as a polynucleotide sequence set out in SEQ ID NOS 1, 3. 4 and 14, 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 For example, to obtain a polynucleotide sequence ofthe invention, such as a sequence given m SEQ ID NOS 1, 3. 4 and 14, typically 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 Clones carrying DNA identical to that of the probe can then be distinguished using stringent conditions By sequencing the individual clones thus identified with 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 . Fπtsch, E F and Sambrook et al MOLECULAR CLONING A LABORATORY MANUAL. 2nd Ed . Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York (1989) (see in particular Screening By Hybridization 1 90 and Sequencing Denatured Double-Stranded DNA Templates 13 70) Illustrative of the mvention. the polynucleotides set out in Table 1 [SEQ ID NO 1. 4 and 5] were discovered m a DNA library denved from Staphylococcus aureus WCUH 29

Certain DNA sequences set out m Table 1 [SEQ ID NO 1] contains an open reading frame encoding a protem having about the number of ammo acid residues set forth m Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated usmg ammo acid residue molecular weight values well known m the art The polynucleotide of SEQ ID NO 1, between nucleotide number 1 through number 351 encodes the polypeptide of SEQ ID NO 2 The stop- codon begins at nucleotide number 352 of SEQ ID NO 1

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 For example, a marker sequence that facilitates punfication of the fused polypeptide can be encoded In certain embodiments of the mvention. 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, and 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. 2, 3, 4, 5, 6, 7, 8, 9 or 10 The mvention also provides RNA transcnbed from such polynucleotides, particularly catalytic RNAs The term "polynucleotide encodmg a polypeptide" as used herem 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

In addition to the standard A, G. C. T/U representations for nucleic acid bases, the term "N" is also used "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

Further particularly preferred embodiments are polynucleotides encoding 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

Further preferred embodiments of the mvention are 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 Alternatively, most highly preferred are 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 In this regard, 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 Furthermore, 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, with at least 99% bemg the more 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. 4 or 14, and polynucleotides that are complementary to such polynucleotides Alternatively, most highly preferred are 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 In this regard, 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 Furthermore, 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 In this regard, the mvention especially relates to polynucleotides that hybndize under strmgent conditions to the herem above-descnbed polynucleotides As herem used. the terms "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. 15mM tπsodmm citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10% dextran sulfate. and 20 micrograms/ml denatured, sheared salmon sperm DNA. followed by washing the hybridization support in 0 lx SSC at about 65°C Hybridization and wash conditions are well known and exemplified in Sambrook. et al , Molecular Cloning A Laboratory Manual, Second Edition. Cold Spring Harbor, N Y , (1989), particularly Chapter 11 therein 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

As discussed herem regarding polynucleotide assays of the mvention. for instance, polynucleotides of the mvention as discussed above, 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 Preferably, 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. or a 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 ^~~

A partial homolog to B subtibs RNase P RNA was identified in a proprietary S aureus database comprising sequences from randomly sequenced S aureus DNA library in an E cob host This homolog (A33202 B subtibs RNA nucleotides 31 1-460) is shown m Figure 3 [SEQ ID NO 14]

A PCR primer based on these data was designed to the 3' end ofthe gene (primer 5'- CGC GAA GTG TGT CTC GTT TAT ACG-3¹) [SEQ ID NO 5] and a second based on a universally conserved sequence within the 5' domain (5 '-GAG GAA AGT CCA TGC TC-3') [SEQ ID NO 6] ( available from the RNase P database w w ) permitted recovery of approximately 90%) ofthe gene The complete structural gene was amplified using a degenerate primer (5'-^c/_TGATATTTC^G/_TG^A/_GTAA^T/_cC-3') [SEQ ID NO 13] that would allow the RNA product to form the predicted helices P I and P2 (Figure 1) The structural gene has been cloned behind a T7 promoter, sequenced and shown to be highly related to B subtibs homolog (Figure 2) The precise S aureus genomic sequence encoding (transcribing) helices P I and P2 may be determined by skilled artisans using methods and compounds ofthe invention, such as the degenerate primer described above [SEQ ID NO 13] The exact 5' end has been determined by primer extension analysis using endogenous S aureus RNA isolated from S aureus WCUH29 The sequence ofthe first 20 nucleotides of RNaseP RNA 5' end as determined by this primer extension analysis is

5'-GUUCUGAUAUUUUGGGUAAU-3' [SEQ ID NO 15]

The predicted secondary structure is shown in Figure 4 and based on data available for the putative B subtibs RNAse P RNA secondary structure as well as experimental RNA structure probing analyses Further Description and Definitions

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. Scotland on 11 September 1995 and assigned NCIMB Deposit No 40771 It was referred to as 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

The 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

Thus, the term "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 is a proprotem and is an inactive form of the protem Once the prosequence is cleaved an active mature protem remains

Thus, for example, 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) Further, 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 Alternatively 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

In order to facilitate understanding of the following example certain frequently occurring methods and/or terms will be described "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 In addition, 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 For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme 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 μg of approximately equimolar amounts of the DNA fragments to be hgated

The polypeptides and polynucleotides ofthe present invention are preferably provided in an isolated form, and preferably are purified to homogeneity 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

A "double-stranded DNA molecule" refers to the polymeric form of deoxyπbonucleotides (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 Thus, this term includes double-stranded DNA found, inter aba, in linear DNA molecules (e g ,^— restriction fragments), viruses, plasmids, and chromosomes In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscπbed 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 For purposes of defining the present invention, 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 Within the promoter sequence will be found 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

DNA "control sequences" refers collectively to promoter sequences, πbosome 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

A cell has been "transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome ofthe cell In prokaryotes and yeasts, for example, the exogenous DNA may be maintained on an episomal element, such as a plasmid With respect to eukaryotic cells, 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

In accordance with yet a further aspect of the present invention, there is therefore provided a process for producing the polypeptide of the invention by recombinant techniques by expressing a polynucleotide encoding said polypeptide m a host and recovering the expressed product Alternatively, the 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 However, any other vector may be used as long as it is rephcable and viable in the host For recombinant production, host cells can be genetically engmeered to incorporate expression systems or portions thereof or polynucleotides of the mvention Introduction of a polynucleotide mto the host cell can be effected by methods descnbed m many standard laboratory- manuals, such as Davis et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection. microinjection, catiomc lipid- mediated transfection, electroporation, transduction, scrape loading, ballistic mtroduction and infection

Representative examples of appropnate hosts mclude 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

A great vanety of expression systems can be used to produce the polypeptides of the mvention Such 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. and vectors denved from combmations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids The expression system constructs may contam control regions that regulate as well as engender expression Generally, 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 sιte(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 As representative examples of such promoters, 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 πbosome binding site for translation initiation and/or a transcription terminator The vector may also include appropriate sequences for amplifying expression

In addition, 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, πbosome 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 4,431,739, 4,425,437. 4.338,397 Promoter regions can be selected from any desired gene using CAT (chloramphemcol transferase) vectors or other vectors with selectable markers 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. early and late SV40, LTRs from retrovirus, and mouse metallothionem-I Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art In addition to control sequences, it may be desirable to add 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 host cell, e g , the ampicilhn resistance gene of £ cob and S cerevisiae TRPl gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the peπplasmic space or extracellular medium Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e g . stabilization or simplified purification of expressed recombinant product The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein

More particularly, 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 In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available The following vectors are provided by way of example Bacterial pET-3 vectors (Stratagene), pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescnpt, psιX174, pbluescπpt SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia) Eukaryotic pBlueBacIII (Invitrogen), pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) However, any other plasmid or vector may be used as long as they are rephcable and viable m the host ^—

Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bactenophage λ (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 (Saccharomyces), a baculovirus insect cell system, YCpl9 (Saccharomyces) See, generally, "DNA Cloning" Vols I & II, Glover et al ed IRL Press Oxford (1985) (1987) and, T Maniatis et al ("Molecular Cloning" Cold Spring Harbor Laboratory (1982)

In some cases, it 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 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

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, 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

Depending on the expression system and host selected, 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

Depending upon the host employed m a recombinant production procedure, the 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 (RNase P protein) 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 labeled substrate converted mto product, a reporter gene that is responsive to changes m RNaseP polynucleotide or polypeptide activity, and binding assays known m the art

Another example of 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 mclude 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 Examples of small molecules mclude but are not limited to small organic molecules, peptides or peptide-like molecules Other potential antagomsts mclude antisense molecules (see Okano, J Neurochem 56 560 (1991), OL1GODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press. Boca Raton, FL (1988). for a descnption of these molecules) Preferred potential antagomsts mclude compounds related to and vanants of RNaseP Each of the DNA sequences provided herein may be used in the discovery and development of antibacterial compounds The encoded protem, upon expression, can be used as a target for the screening of antibacterial drugs Additionally, 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 In particular 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. for example, initiating phosphorylation of mammalian tyrosme kinases (Rosenshine et al , Infect Immun (50 2211 (1992), to block bacterial adhesion between mammalian extracellular matrix protems and bacterial RNaseP protems that mediate tissue damage and. to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques

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 . empyema, lung abscess), cardiac (e g , infective endocarditis), gastrointestinal (e g , secretory diarrhoea, splenic absces,- retropentoneal abscess), CNS (e g , cerebral abscess), eye (e g , blephantis, conjunctivitis, keratitis, 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 arthntis. osteomyelitis) HTP Screening Strategies

Assays can be developed to detect compounds that inhibit RNase P directed cleavage of RNA substrates Several possible 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 Alternatively, 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 This has the advantage that it is a homogeneous, contmuous assay format and requires no additional manipulations after starting the assay Both formats are useful in the practice of this invention because they use the same RNA substrate RNA Fragment Library Rescue

An effective approach for identifying compounds that interact with RNA is contemplated The concept is based on the over-expression of a drug binding site that is recreated on an RNA fragment, which will sequester the drug and permit the continued functioning of the intact πbozyme (Figure 6) This approach has recently been descπbed in the context of a search for hgands that bind ribosomal RNA (Howard, B-A, et al , Biochem Cell Bio 73(11/12) 1161-1166 (1995)) Following selection the random RNA fragments that apparently present a minimal target structure for drug recognition, are incorporated mto a protocol for rational drug design Accordingly, random fragment libraries based on Ml RNA will be generated and used in HTP screening to identify compounds that disrupt RNA/protem interaction (Figure 7) ^—

Cyclic peptide phage libraries

The incorporation of conformational constraints into flexible lead compounds is a powerful strategy to increase lead potency and is particularly useful m the field of peptidomimetic design ( Al-Obeidi. F et al . J Med Chem 32 2555-2561(1989),Barker, P L , et al . J Med Chem 35 2040-2048 (1992)) Cychzation has been shown to increase the propensity for beta-turn formation m peptides the potential of which has been demonstrated by the identification of high-affinity hgands for streptavidm (Lee, M S . et al , FEBS Lett 359 113-1 18 (1995))

In this case, 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

SELEX Systematic Evolution of Ligands by Exponential Enrichment

This approach may be employed in an attempt to identify RNA recognition motifs for RNase P RNA (Ml RNA) protem binding for structural analysis as an aid to rational drug design and the secondary evaluation of compounds identified via the HTP screens 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 . TIBS 17 89- 93 (1992)) 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 ^—

The identification of a protem/RNA-fragment pair permits the development of a screen for compounds that disrupt their assembly Drug induced disruption of labeled RNA bound to immobilized protein (biotin/streptavidm) would result in the concomitant decrease/loss ofthe signal generated by the presence ofthe RNA (Figure 6 ) RNA/Drug Interactions

RNase P RNA fragments that confer drug resistance (RNA Fragment Rescue Library supra) 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 πbose 2'-hydroxyl groups of specific nucleotides may be probed via chemical synthesis of the appropriately modified RNA fragment In order to retain the C" -endo configuration characteristic of πbonucleotides, 2'-methoxy and 2'-fluoroπbonucleotιdes analogues can be used, the latter being preferred on steπc 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 "ιndιvιdual(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, characteπzation 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 differences may also be detected by alterations m the electrophoretic mobility ofthe DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencmg See. e g , Myers et al , Science, 230 1242 (1985) 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. mutations 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 . empyema, lung abscess), cardiac (e g , infective endocarditis), gastromtestmal (e g , secretory diarrhoea, splenic absces, retropentoneal abscess), CNS (e g . cerebral abscess), eye (e g , blephaπtis, conjunctivitis, keratitis. 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 arthπtis, 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

In addition, 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

The polypeptides ofthe mvention or vanants thereof, or cells expressing them can be used as an lmmunogen to produce antibodies lmmunospecific for such polypeptides "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

For preparation of monoclonal antibodies, any technique known m the art that provides antibodies produced by contmuous cell lme cultures can be used Examples mclude vanous techmques, such as those m Kohler, G and Milstem, C , Nature 256 495-497 (1975), Kozbor et al , Immunology

Today 4 72 (1983), Cole et al , pg 77-96 m MONOCLONAL ANTIBODIES AND CANCER

THERAPY, Alan R Liss, Inc (1985), )). and the EBV-hybπdoma technique to produce human- monoclonal antibodies (Cole, et al , 1985, m Monoclonal Antibodies and Cancer Therapy,

Alan R Liss, Inc , pp 77-96) The hybπdomas 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

Mab

Techmques for the production of smgle cham antibodies (U S Patent No 4,946,778) can be adapted to produce smgle cham antibodies to polypeptides of this mvention Also, transgemc mice, or other organisms such as other mammals, may be used to express humanized antibodies

Alternatively 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

10, 779-783) The affinity of these antibodies can also be improved by chain shuffling

(Clackson. T et al , (1991) Nature 352, 624-628)

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 .

Science 240 1038-1040 (1988) If two antigen binding domains are present 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

In particular derivatives which are slightly longer or slightly shorter than the native protein or polypeptide fragment of the present invention may be used In addition, 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

Thus, among others, 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 . cerebral abscess), eye (e g , blephantis, conjunctivitis, keratitis, 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 joint (e g , septic arthπtis, osteomyelitis)

Preferably 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 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. Chinese hamster ovary, filamentous or unicellular fungi or insect cell 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. bacteπophages, 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 The term "immunologically equivalent derivative" as used herein 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. such as 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) Alternatively 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

Preferably, the antibody or variant thereof is modified to make it less lmmunogemc m the individual For example, if the individual is human the antibody may most preferably be "humanized", where the comphmentarity deteπnmmg regιon(s) of the hybπdoma-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 The humanized monoclonal antibody, or its fragment havmg binding activity, form a particular aspect of this invention

The modification need not be restricted to one of "humamzation", other primate sequences (for example Newman, R et al . Biotechnology 10 1455-1460 (1992)) may also be used

The use of 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. 1986 83.9551), encapsulation of DNA in various forms of hposomes (Kaneda et al , Science 1989 243,375), particle bombardment (Tang et al , Nature 1992, 356 152, Eisenbraun et al , DNA Cell Biol 1993, 12 791) and in vivo infection usmg cloned retroviral vectors (Seeger et al . PNAS USA 1984 81,5849) Vaccmes

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 accelerating it into the desired cells as a coating on particles or otherwise Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid

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 Thus fused recombinant protein, preferably further comprises an antigenic co-protein, such as hpoprotein D from Hemophilus influenzae. Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-protems which solubihze the protem and facilitate production and purification thereof Moreover, 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

Provided by this invention are 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) Also, provided by this invention are 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 Examples of 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. buffers, bacteπostats and solutes which render the formulation lnsotomc with the bodily fluid, preferably the blood, of the individual, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents 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-dπed 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

Wlπle 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. but are not limited to, salme, buffered salme, dextrose, water, glycerol, ethanol and combmations thereof The formulation should suit tlie mode of admimstration 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

The pharmaceutical 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

In therapy or as a prophylactic, the active agent may be administered to an individual as an mjectable composition, for example as a sterile aqueous dispersion, preferably lsotomc

Alternatively 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 For administration to mammals, and particularly humans, it is expected that 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 exemplary of the average case There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention

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

The 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 duπng the m-body time of the device In addition, the composition could also be used to broaden peπoperative cover for any surgical technique to prevent bacterial wound infections, especially Staphylococcus aureus wound infections

Many orthopaedic surgeons consider that humans with prosthetic joints should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteremia Late deep infection is a serious complication sometimes leading to loss of the prosthetic joint and is accompanied by significant morbidity and mortality It may therefore be possible to extend the use of the active agent as a replacement for prophylactic antibiotics in this situation In addition to the therapy described above, the 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 Alternatively, 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 μg/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 descπbed 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 circumscπbe the scope ofthe disclosed mvention

All examples were earned out usmg standard techmques, which are well known and routme to those of skill m the art, except where otherwise descnbed m detail Routme molecular biology techmques ofthe following examples can be earned out as descπbed m standard laboratory manuals, such as Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd

Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y (1989)

All parts or amounts set out m the following examples are by weight, unless otherwise specified

Unless otherwise stated size separation of fragments m the examples below was earned out usmg standard techmques of agarose and polyacrylamide gel electrophoresis ("PAGE") m Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y (1989) and numerous other references such as, for instance, by Goeddd et ∑ά , Nucleic Acids Res 8 4057 (1980) Example 1. PCR with random primers

This method describes a rapid way to obtain additional sequence data from partial gene fragments independent of probing a genomic library

Random sequencing of a S aureus genomic library followed by sequence homology searching with the B subtibs P-protem sequence resulted in the identification of a 324 base pair (herein "bp") fragment The first 193 nucleotides of this fragment showed significant homology to the C-terminal half of the B subtibs P-protem and other prokaryotic RNase P proteins The N-terminal domain of the putative S aureus RNase P protein was putatively missing Deduced S aureus RNase P protein sequence versus B subtibs shows 58 1% similarity. 34 9% identity (see Figure 2 for an alignment of RNase P protem sequences)

A novel two step PCR with two different reverse primers complementary to the known sequence in position 15-39 and 194-215 respectively (shown below, Table 2) as well as random hexamer primers (Gibco) was used to obtain the complete 5' sequence of the S aureus spp gene S aureus genomic DNA partially digested with Hind III or Pst I served as a template Primer #1 (position 194-215) annealed just downstream ofthe stop codon TAA and primer #2 (position 15-39) close to the end ofthe known sequence (see Table 2)

Table 2 Primer Position

5 ' GAATT CCGTG TTAAG AAACA AGATT AAAAG AGCAA TACGT GAAAA TTTCA

3 ' C TTTGT TCTAA TTTTC TCGTT ATGC 5 ' [SEQ ID NO : 7 ] AAGTA—CATAA GTCGN ATATA TTGGN CAAAG ATATT ATTGN TATAG NAAGA CAGGC AGCTA AAGAT ATGAC GACTT TACAA ATACA GNATA GTCTT GAGCA CGTNC TTAAA ATTGG CAAAG TTTTT AATAA AAAGA TTAAG TAAGG ATAGG GTAGG GAATG AAAAC 3 ' CC TATCC CATCC CTTAC TTTTG 5 '

[SEQ ID NO: 8] ATTAA CCCCT CAACG CATCC CGAAG TCTTA CCTCA GACAA ACGTT AGACT GACCC TAGGG TTAAG ACTTG GCCCN AGGGN TATNNTAACT TACTT TAAAA TGTTT TCAC 3'

In the first step, 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

In the second step, 32p-_enci-i_abeled primer #2 and random hexamers were added and the annealing temperature was dropped to 25°C to allow the short random primers to anneal to the DNA

The 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 μl water The sample was concentrated to a final volume of 25 μl under vacuum in a SpeedVac® SCI 10 (Savant) 20μl 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 μg/ml in H2θ) to visualize the DNA and photographed The gel was then dried and exposed to film

There were several defined bands visible both on the ethidium bromide stained gel as well as on the autoradiogram They ranged in size between 50 bp to over 2000 bp. some bands being more intense than others The most prominent bands were approximately 50bp, 150bp. 400bp and 800bp in size

Example 3. Shotgun cloning of the PCR fragments: Cloning and sequencing of the PCR fragments

The PCR fragments were blunt end cloned into Smal cut pUC19 Ligations of 100 ng vector DNA with 1 0 μl and 1 5 μl of PCR products were performed at 16°C overnight E cob XL1 blue cells (α-complementing) were transformed by electroporation and plated onto selective plates containing IPTG and x-gal (for blue/white screening)

Transformation of these cells resulted in approximately 8 5% white colonies in the presence of PCR products, whereas only 4 6% white colonies could be found m the control hgation without insert White colonies had lost the ability to form the β-galactosidase holoenzyme as a result of a reading frame disruption m the lacZ ' gene and were no longer able to cleave x- gal The reading frame was either disrupted by an insert (in the presence of PCR products) or exonuclease activity of the restriction enzyme preparation, destroying the Sma I site of the vector by cleaving terminal nucleotides and resulting in frame shifts after hgation 18 white colonies were picked and grown up as 2 ml cultures to prepare plasmids A sample of each plasmid was linearized with Accl restriction endonuclease (cuts in the multi cloning site) and run on a 1 % TAE agarose gel alongside the original pUC 19 The results showed that 7 plasmids released a short fragment upon restriction digest

As there is only one Accl site m pUC 19. these recombinant plasmids must contain an insert with a restriction site for this enzyme 3 plasmids appeared larger than pUC19 but did not release a fragment The other 8 plasmids were ofthe same size as pUC19 and did not appear to have an insert

The 10 plasmids containing an insert were sequenced both with the forward and reverse pUC/M 13 sequencmg primers ^~~

5 of the 6 plasmids that released a fragment after Accl digest harboured an 213bp insert that started with the primer #2 sequence and the known 14bp followed by a sequence of 174 nucleotides that was common to all of the plasmids (Sequence 1, Table 3) In one ofthe 6 plasmids the insert (Sequence 2, Table 3) was slightly different the first 137 nucleotides were in common with Sequence 1 (dotted line), however the insert sequence extended beyond this and deviated from Sequence 1 (see Table 3)

Table 3

Aplification Product Sequence 1, 2 and 3

Sequence 1 [SEQ ID NO: 9]

5'GTTTT CATTC CCTAC CCTAT CCTTA CTTAA TCTTT TTATT AAAAA GAATG CAGAT TTTCA GAGAA TATAT AAAAA AGGTC ATTCT GTAGC CAACA GACAA TTTGT TGTAT ACACT TGTAA TAATA AAGAA ATAGA CCATT TTCGC TTAGG TATTA GTGTT TCTAA AAAAC TAGGT AATGC AGTGT TAAGA ACAAG ATTAA AAGAG CAATA CGT 3 '

Sequence 2 [SEQ ID NO: 10]

5 ' ACGAT AAATA ACAGT ATGTT ATTGG AAAAA GTCTA CCGAA

TTAAA AAGAA TGCAG ATTTT CAGAG AATAT ATAAA AAAGG TCATT CTGTA

GCCAA CAGAC AATTT GTTGT ATACA CTTGT AATAA TAAAG AAATA GACCA

TTTTC GCTTA GGTAT TAGTG TTTCT AAAAA ACTAG GTAAT GCAGT GTTAA GAACA AGATT AAAAG AGCAA TACGT 3 '

Sequence 3 [SEQ ID NO: 11]

5 ' ... 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)

The predicted translation products of Sequence 1 and 2 showed significant homology— to B subtibs P-protein and indicated that these inserts contain the full sequence information for the 5' domain ofthe spp gene In 2 plasmids the insert started with the sequence of primer #1 followed by 3' half of the spp gene (Sequence 3, Table 3) This sequence was already known but the new sequence showed 9 base changes (shown m bold) m comparison to the original sequence data obtained by random sequencing ofthe S aureus library

2 plasmids contained inserts with different sequences that are a result of unspecific priming during PCR

Example 4 Analysis of the sequence data

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 total length of the gene

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 translated sequence was aligned with known ammo acid sequences of the RNase P protem from 5 different prokaryotes These alignments are shown in Figure 2 The S aureus SP protein sequence matched the B subtibs P-protem sequence well, significant homology to the B subtibs protem Example 5. PCR of the spp gene

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 μg as determined by spectrophotometπcal measurement The DNA fragment was cleaned using a QIAquick PCR purification column and recovered in 80μl water

Example 6. Cloning spp into pMalc2

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

1 μg of the protein fusion vector pMalc2 was cut with Xmnl and BamHI restriction endonucleases to enable directional cloning of the spp structural gene To remove the small 15 bp insert the digest was cleaned using QIAquick PCR purification column and the DNA recovered in 90 μl water A sample was analysed by agarose gel electrophoresis to ensure complete digestion of the vector After 30 mm of incubation ^{at 37}°^{c lth both} enzymes the vector was completely cut

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 μl reaction at 16°C The molar ratio of vector to insert was 1 5 To prepare the DNA for electroporation, the hgation was desalted usmg QIAquick Nucleotide Removal columns Example 8. Transformation of E. coli XL-1 Blue cells 40 μl electrocompetent E cob XL-1 Blue cells were transformed with 2 μl desalted hgation reaction Cells were recovered in lml SOC medium after electroporation 50 μl of a 1 100 dilution were plated onto an LB plate containing ampicilhn. IPTG and x-gal and incubated at 37°C overnight 1 1 white and 1 blue colony were recovered The 11 white colonies were grown up and the plasmids were isolated from the clones Sequencing of the plasmids showed that 6 of the 11 plasmids contained the correct spp insert The resulting plasmid was denoted pMalc2 spp, and the fusion protein product MBP-SPP Example 9 Growth of cells and fusion protein induction in E. coli XL-1 Blue pMa\c2::spp

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 A⁶⁰⁰ 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 μl SDS gel loading buffer 3 μl 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

Another E cob strain was available that constituively expresses the ArgU tRNA (tRNA^ucu) and IleX tRNA (tRNA^UAU) from the plasmid pRJ952 Example 10. Transformation of E. coli W3110 pRI952

50 μl electrocompetent E cob W3110 pRI952 cells were transformed with 2 μl desalted hgation reaction Cells were recovered in lml SOC medium after electroporation - 100 μl of the culture were plated onto selective LB plates (ampilhn, cam) containing IPTG and x-gal and incubated at 37°C overnight Too many colonies grew on the plate and could not be counted 5 white colonies were selected and their plasmids isolated Sequencing of the plasmids showed that all contained the correct spp insert

Example 11. Growth of cells and fusion protein induction in E. coli W3110 pRI952 pMalc2::sp/7

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

Example 12. Purification of the MBP-SPP fusion by affinity chromatography

Following optimal induction, the cells (E cob W3110 pRI952 pMalc2 spp) were harvested and disrupted by sonication after a freeze-thaw cycle to weaken the cell wall using known methods 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

Example 13. Factor Xa cleavage ofthe fusion protein and separation of MBP and SPP

500 μg Factor Xa protease were added to the solution and the fusion protem was ^~~ cleaved overnight at 4°C A sample was analysed by SDS-PAGE before and after Factor Xa incubation to monitor complete cleavage After 15 hours almost all the fusion protem was cleaved resulting in the 42 kD MBP and SPP of approximately 14kD

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. In vitro transcription of the SP RNA

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 MEGAscπpt™ 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 μl 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

In vitro transcription of SP RNA from pSPR resulted m a smgle RNA product ofthe expected size of 401 nucleotides The total RNA yield was 140 μg (determined spectrophotometπcally) After cleaning, the RNA was recovered in 40 μl water resulting m a

29 μM solution, that was stored at -20°C

Example 15. In vitro transcription of E. coli ptRNA^met incorporating [α-^^P] UTP

E cob ptRNA^met, a substrate for RNase P. was transcribed in vitro by T7 RNA polymerase (Epicenter) in the presence of [α-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 μl reaction at 37°C for 60mιn To remove unincorporated nucleotides 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]

GGGCG AAUUC GCCUC GGCUA CGUAG CUCAG UUGGU UAGAG CACAU CACUC AUAAU GAUGG GGUCA CAGGU UCGAA UCCCG UCGUA GCCAC CAG

Transcription of ptRNA^1*' resulted in a 2μM (6 ng/μl) solution 64 3%o of the nucleotides were incorporated in the transcript and the specific activity was 2 171 10⁸ cpm/μg Example 16. 5' end-labeling of p6AT-l The chemically synthesized 42nt RNA molecule p6AT-l was 5' end-labeled with high specific activity [γ-32pj ATP (6000μCι/μl) using bactenophage T4 polynucleotide kmase The labeling was performed in a 10 μl reaction at 37°C for 30 mm The enzyme was heat- mactivated at 95 °C for 2 mm To remove salts, unincorporated nucleotides and the enzyme, 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 μl water 0 5 μl were spotted onto a filter paper disk and counted m a scintillation counter (Cherenkov analysis)

The end-labeling resulted m a p6AT-l preparation with an activity of 640.000 cpm/μl

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 Tπs Cl pH 7 5. lOOmM MgCl₂ 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 πbozyme

Cleavage reactions (20 μl) were all performed at 37°C for 30 mm with lOOnM SP RNA and an additional 200nM SP protem in the holoenzyme reactions Substrates (ptRNA^met or p6AT-l) were added m trace amounts (smgle-turnover reactions) Cleavage was monitored by size resolution of the mtact precursor and the cleaved leader sequence by denaturing gel elctrophoresis and autoradiography or visualization on the phosphoπmager (Molecular Dynamics Storm 860) Example 18 Effect of KC1 versus NH4CI on p6AT-l cleavage

All experiments were performed in lOOmM Tπs Cl pH7 5, lOOmM MgC12 and 1M or 2M monovalent salt (KC1 or NH4C1)

There was hardly any cleavage product detectable at 1 M KC1 or NH4C1 Cleavage occurred when 2M monovalent salt were used Potassium chloride gave better results than ammonium chloride

Further optimization of the monovalent salt concentration was investigated following optimization of additional parameters described in the following sections Example 19. Effect of the pH on p6AT-l cleavage

Buffers containing lOOmM MgC12. 2M KC1 and lOOmM Tπs 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

Example 20. Influence of PEG 8,000 on p6AT-l cleavage

The addition of polyethylene glycol has been shown to improve substrate cleavage by E cob Ml RNA Here the effect of different PEG 8.000 concentrations on p6AT-l cleavage by SP RNA was demonstrated 1%, 2 5% and 5% PEG were added to reactions containing 1M KC1, lOOmM Tπs CI pH8, lOOmM MgC12

The ability to cleave the substrate improved with increasing PEG concentrations, and therefore 5% PEG was selected to be included in subsequent reactions Example 21. M C-2 requirements for p6AT-l processing

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 Tπs CI pH8 and 5% PEG

Cleavage results were obtained at lOOmM MgC12 However SP RNA was still able to process p6AT-l at 50mM and to some extend at 25mM MgC12 No cleavage could be- observed at lOmM MgC12 Example 22. Determination of KCl requirements for p6AT-l cleavage The KCl concentration was successively increased from 40mM to 1 5M in lOOmM

Tπs CI pH8. lOOmM MgC12 and 5% PEG

No cleavage occurred at KCl concentrations below 400mM At 400mM KCl p6AT-l processing was still very poor but increased with the salt concentration to reach a maximal level of 27% at 1 5M KCl That was comparable to p6AT-l cleavage by E cob Ml RNA, where 35% of the substrate was cleaved after 20mιn The best buffer conditions for p6AT-l cleage by SP RNA were 1 5M KCl, lOOmM Tπs CI pH8, lOOmM MgC12, 5% PEG Example 23. KCl requirements for ptRNA^met cleavage by SP RNA ptRNA"^** was cleaved under the same buffer conditions as described above ptRNA"^*' was a better substrate than p6AT-l By contrast to p6AT-l, 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 Tπs CI pH7 5, lOOmM NH4C1, lOOmM MgC12 withm 20mιn The best buffer conditions for ptRNAmet cleavage by SP RNA were 1M KCl. lOOmM Tπs Cl pH8, lOOmM MgC12. 5% PEG Example 24. Determination of the optimal buffer conditions for S. aureus RNase P holoenzyme reactions: p6AT-l cleavage by the holoenzyme

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 Tπs 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 At higher KCl concentrations 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 20mιn in lOOmM Tπs CI pH7 5, lOOmM NH4C1. lOmM MgC12 The optimal buffer for p6AT-l cleavage by the holoenzyme was 150mM KCl, lOOmM Tπs CI pH 8 0. lOmM MgCl₂, 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 MgCl₂ 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 Tπs CI pH8, 20mM MgCl₂, 5% PEG and lOmM, 50mM, 150mM, 600mM or 1 5M KCl

Cleavage of ptRNA by the holoenzyme at 1 OmM MgCl₂ and low KCl concentrations was very poor At 20mM MgC12 and 150mM KCl the holoenzyme processed ptRNA very well, 54%> of the substrate was cleaved withm a 20mm incubation Only a low percentage of substrate processmg could be detected at 600mM KCl, but good cleavage occurred at 1 5M KCl The SP RNA alone was also able to cleave ptRNA under these conditions but was unable to cleave ptRNA at lower salt concentrations Therefore the optimal MgCl₂ concentration for ptRNA^me^ cleavage by the holoenzyme was 2 OmM Example 26 Determination of the optimal KCl concentration

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 Tπs CI pH8, 20mM MgCl₂, 5% PEG 8000 and xM KCl

The holoenzyme was able to cleave ptRNA^met 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^met 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 Tπs CI pH8, 20mM MgCl₂. 5% PEG

Example 26 Strain selection, Library Production and Sequencing

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

Method 1

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

Claims

WHAT IS CLAIMED IS:

1 An isolated polynucleotide compnsrng a polynucleotide sequence selected from the group consistmg of

(a) a polynucleotide havmg at least a 90%o identity to a polynucleotide compnsrng the nucleic acid sequence of SEQ ID NO 3,

(b) a polynucleotide havmg at least a 90%o identity to a polynucleotide compnsrng the ^~~ same catalytic RNA expressed by the RNaseP RNA gene contamed m the Staphylococcus aureus ofthe deposited stram,

(c) a polynucleotide which is complementary to the polynucleotide of (a) or (b), and

(d) a polynucleotide compnsrng at least 30 sequential bases ofthe polynucleotide of (a). (b) or (c)

2 The polynucleotide of Claim 1 wherein the polynucleotide is DNA

3 The polynucleotide of Claim 1 wherein the polynucleotide is RNA

4 The polynucleotide of Claim 2 compnsrng the nucleic acid sequence set forth m SEQ ID NO 3

5 A vector compnsrng the polynucleotide of Claim 1

6 A host cell compnsrng the vector of Claim 5

7 A process for producmg a polypeptide compnsrng expressing from the host cell of Claim 6 a polypeptide encoded by said DNA

8 A process for producing a RNaseP RNA or catalytic fragment comprising culturmg a host of claim 6 under conditions sufficient for the production of said RNA or catalytic fragment

9 A polynucleotide compnsrng a polynucleotide sequence which is at least 90% identical to the polynucleotide sequence of SEQ ID NO 3

10 An antagonist which mhibits the activity or expression of the RNA transcnbed from the polynucleotide of claim 9

11 A method for the treatment of an mdividual havmg need to inhibit RNaseP polynucleotide compnsrng administering to the mdividual a therapeutically effective amount of the antagonist of Claim 10

12 A process for diagnosing a disease related to expression or activity of the polynucleotide of claim 9 m an mdividual compnsrng (a) determining an RNA sequence encoded said polynucleotide. and/or

(b) analyzing for the catalytic activity, presence or amount of said RNA in a sample denved from an mdividual

13 A method for identifying compounds which mteract with and inhibit or activate an activity ofthe polynucleotide of claim 1 compnsrng contacting a composition compnsrng the polynucleotide with the compound to be screened under conditions to permit mteraction between the compound and the polynucleotide to assess the- mteraction of a compound, such mteraction bemg associated with a second component capable of providmg a detectable signal m response to the mteraction of the polynucleotide with the compound, and determining whether the compound interacts with and activates or mhibits an activity ofthe polynucleotide by detectmg the presence or absence of a signal generated from the mteraction ofthe compound with the polynucleotide

14 An isolated RNase P RNA from Staphylococcus aureus

15 An RNase P homoenzyme comprising an RNA component of the sequence of SEQ ID NO 3

16 An RNase P holoenzyme comprising a protein component of the sequence of SEQ ID NO 2 and an RNA component encoded by the DNA of SEQ ID NO 3

17 An isolated DNA encoding the components of Claim 16

18 A method for individual or co-expression ofthe DNA of Claim 17

19 A screen for the identification of inhibitors of RNase P comprising the DNA or SEQ ID NO 3 or an RNA encoded therefrom