EP1087984A2 - acpS - Google Patents
acpSInfo
- Publication number
- EP1087984A2 EP1087984A2 EP99953339A EP99953339A EP1087984A2 EP 1087984 A2 EP1087984 A2 EP 1087984A2 EP 99953339 A EP99953339 A EP 99953339A EP 99953339 A EP99953339 A EP 99953339A EP 1087984 A2 EP1087984 A2 EP 1087984A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- polypeptide
- seq
- polynucleotide
- sequence
- isolated
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1288—Transferases for other substituted phosphate groups (2.7.8)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
- C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their vanants, agonists and antagonists, and their uses
- the invention relates to polynucleotides and polypeptides of the acyl earner protern synthase family, as well as their vanants, herein referred to as "acpS,” “acpS polynucleot ⁇ de(s),” and “acpS polypept ⁇ de(s)” as the case may be
- Streptococci make up a medically important genera of microbes known to cause several types of disease in humans including for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid Since its isolation more than 100 years ago, Streptococcus pneumomae has been one of the more intensively studied microbes For example, much of our early understanding that DNA is, in fact, the genetic matenal was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe Despite the vast amount of research with S pneumonwe, many questions concerning the virulence of this microbe remain It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics
- Streptococcus pneumomae infections has nsen dramatically in the past few decades This has been attributed 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 Streptococcus pneumomae strains that are resistant to some or all of the standard antibiotics This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism
- polynucleotides and polypeptides such as the acpS embodiments of the invention that have a present benefit of among other things, being useful to screen compounds for antimicrobial activity
- Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease
- identification and characterization of such factors and their antagonists and agonists to find ways to prevent, amehorate or correct such infection, dysfunction and disease
- the present invention relates to acpS, in particular acpS polypeptides and acpS polynucleotides, recombinant matenals and methods for their production
- the invention relates to methods for using such polypeptides and polynucleotides, including treatment of microbial diseases, amongst others
- the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified agonist or antagonist compounds
- the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting acpS expression or activity
- the invention relates to acpS polypeptides and polynucleotides as descnbed in greater detail below
- the invention relates to polypeptides and polynucleotides of a acpS of Streptococcus pneumomae, which is related b ⁇ ammo acid sequence homology to H pylon acpS polypeptide
- the invention relates especially to acpS having a nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectively
- sequences recited in the Sequence Listing below as "DNA” represent an exemplification of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed m polynucleotides in general, including ⁇ bopolynucleotides
- a deposit compnsing a Streptococcus pneumomae 0100993 strain has been deposited with the National Collections of Industnal and Marine Bactena Ltd (herein "NCIMB"), 23 St Machar Dnve. Aberdeen AB2 IRY, Scotland on 11 Apnl 1996 and assigned deposit number 40794 The deposit was descnbed as Streptococcus pneumomae 0100993 on deposit On 17 Apnl 1996 a Streptococcus pneumomae 0100993 DNA library in E coh v.
- NCIMB National Collections of Industnal and Marine Bactena Ltd
- the Streptococcus pneumomae strain deposit is referred to herein as “the deposited strain” or as “the DNA of the deposited strain "
- the deposited strain compnses a full length acpS gene
- the sequence of the polynucleotides compnsed in the deposited strain, as well as the ammo acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any descnption of sequences herein
- deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure
- the deposited strain will be irrevocabK and without restnction or condition released to the public upon the issuance of a patent
- the deposited strain is provided merely as convenience to those of skill in 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 ma) be required to make, use or sell the deposited strain, and compounds denved therefrom, and no such license is hereby granted
- an isolated nucleic acid molecule encoding a mature polypeptide expressible the Streptococcus pneumomae 0100993 strain, which polypeptide is compnsed in the deposited strain
- acpS polynucleotide sequences in the deposited strain such as DNA and RNA, and amino acid sequences encoded thereby
- amino acid sequences encoded thereby Also provided by the invention are
- acpS Streptococcus pneumomae referred to herem as "acpS” and "acpS polypeptides” as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful vanants thereof, and compositions compnsing the same
- the present invention further provides for an isolated polypeptide which (a) compnses or consists of an ammo acid sequence which has at least 95% identity, most preferably at least 97-99% or exact ldentits , to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (b) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence which has at least 95% identitv.
- polypeptides of the invention include a polypeptide of Table 1 [SEQ ID NO 2] (m particular a mature polypeptide) as well as polypeptides and fragments, particularly those which have a biological activity of acpS, and also those which have at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2] and also mclude portions of such polypeptides with such portion of the polypeptide generally compnsing at least 30 ammo acids and more preferably at least 50 ammo acids
- the mvention also includes a polypeptide consisting of or compnsing a polypeptide of the formula
- X-(R 1 ) m -(R 2 )-(R 3 ) n -Y wherein, at the ammo terminus, X is hydrogen, a metal or any other moiety descnbed herem for modified polypeptides, and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides, Ri and R3 are any ammo acid residue or modified ammo acid residue, m is an mteger between 1 and 1000 or zero, n is an mteger between 1 and 1000 or zero, and R 2 is an ammo acid sequence of the mvention particularly an ammo acid sequence selected from Table 1 or modified forms thereof In the formula above.
- R 2 is onented so that its ammo terminal ammo acid residue is at the left, covalently bound to Ri and its carboxy terminal ammo acid residue is at the nght. covalently bound to R3
- Any stretch of ammo acid residues denoted by either Ri or R3, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer
- Other preferred embodiments of the mvention are provided where m is an mteger between 1 and 50, 100 or 500, and n is an mteger between 1 and 50, 100, or 500
- a polypeptide of the mvention is denved from Streptococcus pneumomae, however, it may preferably be obtained from other organisms of the same taxonomic genus
- a polypeptide of the mvention may also be obtained, for example, from organisms of the same taxonomic family or order
- a fragment is a vanant polypeptide having an ammo acid sequence that is entirely the same as part but not all of any ammo acid sequence of any polypeptide of the mvention
- fragments may be "free-standing,” or compnsed within a larger polypeptide of which they form a part or region, most preferably as a smgle continuous region in a s gle larger polypeptide
- Preferred fragments include, for example, truncation polypeptides having a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2], or of vanants thereof, such as a continuous senes of residues that mcludes an ammo- and/or carboxyl-termmal ammo acid sequence Degradation forms of the polypeptides of the mvention produced by or in a host cell, particularly a Streptococcus pneumomae, 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- forming regions, coil and coil-foiming regions, hydrophi c regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate b dmg region, and high antigenic mdex regions
- fragments include an isolated polypeptide comprismg an ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids from the ammo acid sequence of SEQ ID NO 2, or an isolated polypeptide compnsmg an ammo acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids truncated or deleted from the ammo acid sequence of SEQ ID NO 2
- Fragments of the polypeptides of the mvention may be employed for producing the corresponding full- length polypeptide by peptide synthesis, therefore, these vanants may be employed as intermediates for producmg the full-length polypeptides of the mvention
- Polynucleotides It is an object of the mvention to provide polynucleotides that encode acpS polypeptides, particularly polynucleotides that encode a polypeptide herem designated acpS
- the polynucleotide compnses a region encoding acpS polypeptides compnsmg a sequence set out m Table 1 [SEQ ID NO 1] which mcludes a full length gene, or a variant thereof The Applicants believe that this full length gene is essential to the growth and/or survival of an organism that possesses it.
- Streptococcus pneumomae Streptococcus pneumomae
- isolated nucleic acid molecules encoding and/or expressmg acpS polypeptides and polynucleotides, particularly Streptococcus pneumomae acpS polypeptides and polynucleotides, including, for example, unprocessed RNAs, nbozyme RNAs. mRNAs, cDNAs. genomic DNAs, B- and Z-DNAs
- acpS polypeptides and polynucleotides particularly Streptococcus pneumomae acpS polypeptides and polynucleotides, including, for example, unprocessed RNAs, nbozyme RNAs. mRNAs, cDNAs. genomic DNAs, B- and Z-DNAs
- Further embodiments of the mvention include biologically, diagnostically, prophylactically, clmically or therapeutically useful polynucleotides and polypeptides, and vanants thereof, and compositions
- Another aspect of the mvention relates to isolated polynucleotides, including at least one full length gene, that encodes a acpS polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and vanants thereof
- acpS polypeptide from Streptococcus pneumomae compnsmg or consistmg of an ammo acid sequence of Table 1 [SEQ ID NO 2], or a variant thereof
- a polynucleotide of the mvention encoding acpS polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bactena usmg Streptococcus pneumomae 0100993 cells as starting matenal, followed by obtaining a full length clone
- a polynucleotide sequence of the invention such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1] typically a library of clones of chromosomal DNA of Streptococcus pneumomae 0100993 m E coh or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, derived
- each DNA sequence set out in Table 1 [SEQ ID NO 1] contains an open reading frame encoding a protein having about the number of ammo acid residues set forth in Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated usmg ammo acid residue molecular weight values well known to those skilled m the art
- the present mvention provides for an isolated polynucleotide compnsmg or consisting of (a) a polynucleotide sequence which has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1. or the entire length of that portion of SEQ ID NO 1 which encodes SEQ ID NO 2, (b) a polynucleotide sequence encoding a polypeptide which has at least 95% identity, even more preferably at least 97-99% or 100% exact, to the ammo acid sequence of SEQ ID NO 2. over the entire length of SEQ ID NO 2
- a polynucleotide encoding a polypeptide of the present mvention may be obtained by a process which compnses the steps of screening an appropnate library under stringent hybndrzation conditions with a labeled or detectable probe consisting of or compnsmg the sequence of SEQ ID NO 1 or a fragment thereof, and isolating a full-length gene and/or genomic clones compnsmg said polynucleotide sequence
- the mvention provides a polynucleotide sequence identical over its entire length to a codmg sequence (open readmg frame) m Table 1 [SEQ ID NO 1] Also provided by the mvention is a codmg sequence for a mature polypeptide or a fragment thereof, by itself as well as a codmg sequence for a mature polypeptide or a fragment in readmg frame with another codmg sequence, such as a sequence encoding a leader or secretory sequence, a pre- or pro- or prepro-protein sequence
- the polynucleotide of the mvention may also compnse at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcnbed but non-translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), nbosome bmdmg sites, Kozak sequences, sequences that stabilize mRNA, mtrons
- a preferred embodiment of the mvention is a polynucleotide of consisting of or compnsing nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 361 set forth m SEQ ID NO 1 of Table 1, both of which encode a acpS polypeptide
- the mvention also mcludes a polynucleotide consisting of or compnsmg a polynucleotide of the formula X-(R 1 ) m -(R 2 )-(R 3 ) n -Y wherein at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond and at the 3' end of the molecule.
- Ri and R3 is mdependently any nucleic acid residue or modified nucleic acid residue, m is an integer between 1 and 3000 or zero , n is an integer between 1 and 3000 or zero, and R 2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide formula above, R 2 is oriented so that its 5' end nucleic acid residue is at the left, bound to Rj and its 3' end nucleic acid residue is at the right, bound to R3 Any stretch of nucleic acid residues denoted by either Ri and/or R 2 , where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer Where, m a preferred embodiment, X
- a polynucleotide of the mvention is denved from Streptococcus pneumomae, however, it may preferably be obtained from other organisms of the same taxonomic genus A polynucleotide of the mvention may also be obtained, for example, from organisms of the same taxonomic family or order
- polynucleotide encoding a polypeptide encompasses polynucleotides that mclude a sequence encoding a polypeptide of the mvention, particularly a bactenal polypeptide and more particularly a polypeptide of the Streptococcus pneumomae acpS having an ammo acid sequence set out in Table 1 [SEQ ID NO 2]
- the term also encompasses polynucleotides that mclude a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by mteg
- the mvention further relates to vanants of the polynucleotides descnbed herem that encode vanants of a polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Fragments of polynucleotides of the mvention may be used, for example, to synthesize full-length polynucleotides of the mvention
- polynucleotides encoding acpS vanants that have the ammo acid sequence of acpS 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, modified, deleted and/or added, m anv combmation Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of acpS polypeptide
- polynucleotides that are at least 95 % or 97% identical over their entire length to a polynucleotide encoding acpS polypeptide having an ammo acid sequence set out m Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides
- polynucleotides that compnse a region that is at least 95% are especially preferred
- those with at least 97% are highly preferred among those with at least 95%. and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred
- Preferred embodiments are polvnucleotides encoding polypeptides that retain substantially the same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1]
- the mvention further relates to polynucleotides that hybndize to the polynucleotide sequences provided herem
- the mvention especially relates to polynucleotides that hybndize under strmgent conditions to the polynucleotides descnbed herem
- strmgent conditions and “strmgent hybndization conditions” mean hybndization occurring only if there is at least 95% and preferably at least 97% identity between the sequences
- strmgent hybridization conditions is overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (150mM NaCl, 15mM tnsodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization
- the polynucleotides of the mvention may be used as a hybndization probe for RNA.
- cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding acpS and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a acpS gene
- Such probes generally will compnse at least 15 nucleotide residues or base pairs
- such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs
- Particularly preferred probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base parrs
- a codmg region of a acpS gene may be isolated by screemng usmg a DNA sequence provided in Table 1 [SEQ ID NO 1] to synthesize an ohgonucleotide probe
- a labeled ohgonucleotide havmg a sequence complementary to that of a gene of the mvention is then used to screen a libraiy of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybndizes to
- polynucleotides and polypeptides of the mvention may be employed, for example, as research reagents and matenals for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herem relatmg to polynucleotide assays
- [SEQ ID NOS 1 or 2] may be used m the processes herem as descnbed. but preferably for PCR. to determine whether or not the polynucleotides identified herein in whole or m part are transcnbed in bacteria m infected tissue It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained
- the mvention also provides polynucleotides that encode a polypeptide that is a mature protem plus additional ammo or carboxyl-termmal ammo acids, or ammo acids intenor to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance) Such sequences may play a role m processing 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
- each and even' polynucleotide of the mvention there is provided a polynucleotide complementary to it It is preferred that these complementary' polynucleotides are fully complementary to each polynucleotide with which they are complementary
- a precursor protem, havmg a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proprotems
- a polynucleotide of the 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 preproprotein, which is a precursor to a proprotein, havmg a leader sequence and one or more prosequences, which generally are removed during processmg steps that produce active and mature forms of the polypeptide
- the mvention also relates to vectors that compnse a polynucleotide or polynucleotides of the mvention, host cells that are genetically engmeered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs denved from the DNA constructs of the mvention
- Recombinant polypeptides of the present mvention may be prepared by processes well known m those skilled m the art from genetically engmeered host cells compnsmg expression systems Accordingly, m a further aspect, the present mvention relates to expression systems which compnse a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engmeered with such expression systems, and to the production of polypeptides of the mvention by recombinant techniques
- 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, N Y (1989), such as. calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, cationic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection
- bactenal cells such as cells of streptococci, staphylococci. enterococci E coh, streptomyces, cyanobactena, Bacillus subtihs, and Streptococcus pneumomae
- fungal cells such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Asperg ⁇ lus.
- insect cells such as cells of Drosoph ⁇ a S2 and Spodoptera Sf9
- animal cells such as CHO. COS, HeLa. C127. 3T3, BHK, 293, CV-1 and Bowes melanoma cells
- plant cells such as cells of a gvmnosperm or angiosperm
- vectors include, among others, chromosomal-, episomal- and virus-denved vectors, for example, 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, vaccmia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses.
- viruses such as baculoviruses, papova viruses, such as SV40, vaccmia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses.
- the expression system constructs may compnse control regions that regulate as well as engender expression
- any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression m this regard
- the appropnate DNA sequence may be inserted 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)
- appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
- Polypeptides of the mvention can be recovered and punfied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography. affinity chromatography, hydroxylapatite chromatography, and lectin chromatography Most preferably, high performance liquid chromatography is employed for purification
- Well known techniques for refolding protem may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or punfication Diagnostic, Prognostic, Serotyping and Mutation Assays
- This invention is also related to the use of acpS polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of acpS polynucleotides and/or polypeptides m a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsmg the acpS gene or protem, may be detected at the nucleic acid or ammo acid level by a vanety of well known techniques as well as by methods provided herem
- Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily matenals
- Polynucleotides from any of these sources may be used directly for detection or may be amplified enzymatically by usmg PCR or an ⁇ other amplification technique pnor to analysis RNA, particularly mRNA, cDNA and genomic DNA may also be used m the same ways Usmg amplification, characterization of the species and strain of infectious or resident organism present in an individual, may be made by an analysis of the genotype of a selected polynucleotide of the organism Deletions and insertions can be detected by a change m size of the amplified product m companson to a genotype of a reference sequence selected from a related orgamsm, preferably a different species of the same genus or a different strain of the same species Pomt mutations can be identified by h
- an array of oligonucleotides probes compnsmg acpS nucleotide sequence or fragments thereof can be constructed to conduct efficient screemng of, for example, genetic mutations, serotype, taxonomic classification or identification
- Array technology methods are well known and have general applicability and can be used to address a vanety of questions m molecular genetics including gene expression, genetic linkage, and genetic vanability (see, for example, Chee et al , Science, 274 610 (1996))
- the present invention relates to a diagnostic kit which comprises (a) a polvnucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof , (b) a nucleotide sequence complementary to that of (a), (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or (d) an antibody to a polypeptide of the present mvention, preferably to the polypeptide of SEQ ID NO 2
- a diagnostic kit which comprises (a) a polvnucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof , (b) a nucleotide sequence complementary to that of (a), (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or (d) an antibody to a polypeptide of the present mvention, preferably to
- This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention, preferable, SEQ ID NO 1, which is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, which results from under-expression, over-expression or altered expression of the polynucleotide
- Organisms, particularly infectious organisms, carrymg mutations in such polynucleotide may be detected at the polynucleotide level by a vanety of techniques, such as those descnbed elsewhere herem
- the differences in a polvnucleotide and/or polypeptide sequence between organisms possessmg a first phenotype and organisms possessing a different, second different phenotype can also be determined If a mutation is observed in some or all organisms possessing the first phenotype but not m any organisms possessing the second phenotype, then the mutation is likely to be the causative agent of the first phenotype
- Cells from an organism carrying mutations or polymorphisms (allelic variations) in a polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a variety of techniques, to allow for serotyping, for example.
- RT-PCR can be used to detect mutations in the RNA. It is particularly preferred to use RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan.
- RNA, cDNA or genomic DNA may also be used for the same purpose, PCR.
- PCR primers complementary to a polynucleotide encoding acpS polypeptide can be used to identify and analyze mutations.
- the invention 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 acpS DNA and/or RNA isolated from a sample derived from an individual, such as a bodily material.
- the primers may be used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide may then be subject to various techniques for elucidation of the polynucleotide sequence. In this way, mutations in the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent.
- the invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by Streptococcus pneumoniae, comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO: l].
- Increased or decreased expression of a acpS 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, spectrometry and other hybridization methods.
- a diagnostic assay in accordance with the invention for detecting over-expression of acpS polypeptide 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 acpS polypeptide, in a sample derived from a host, such as a bodily material, are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays.
- Polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures.
- substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See, e.g., Coligan et al. Current Protocols in Immunology 1(2): Chapter 5 (1991).
- Polypeptides and polynucleotides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases herein mentioned. It is therefore desirable to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide or polynucleotide.
- the present invention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of a polypeptide or polynucleotide of the mvention, as well as related polypeptides and polynucleotides
- agomsts or antagomsts e g , inhibitors
- Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical branes, and natural product mixtures
- agomsts and antagomsts so-identified may be natural or modified substrates, ligands.
- receptors, enzymes, etc as the case may be, of acpS polypeptides and polynucleotides; or may be structural or functional mimetics thereof (see Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991))
- the screenmg methods may simply measure the bmdmg of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may involve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide or polynucleotide, usmg detection systems appropriate to the cells comprising the polypeptide or polynucleotide Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agomsts, in the absence of an agonist or antagonist, by testing whether the candidate compound results
- the screemng methods may simply compnse the steps of mixing a candidate compound with a solution compnsmg a polypeptide or polynucleotide of the present invention, to form a mixture, measurmg acpS polypeptide and/or polynucleotide activity m the mixture, and comparing the acpS polypeptide and/or polynucleotide activity of the mixture to a standard Fusion proteins, such as those made from Fc portion and acpS polypeptide, as herem descnbed, can also be used for high-throughput screenmg assays to identify antagomsts of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D Bennett et al . J Mol Recognition. 8 52-58 (1995), and K Johanson et al , J Biol Chem, 270(16) 9459-9471 (1995
- the polynucleotides, polypeptides and antibodies that bind to and/or interact with a polypeptide of the present mvention may also be used to configure screenmg methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells
- an ELISA assay may be constructed for measurmg secreted or cell associated levels of polypeptide usmg monoclonal and polyclonal antibodies by standard methods known m the art This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
- the mvention also provides a method of screenmg compounds to identify those which enhance (agonist) or block (antagonist) the action of acpS polypeptides or polynucleotides, particularly those compounds that are bactenstatic and/or bactencidal
- the method of screenmg may mvolve high-throughput techniques For example, to screen for agomsts or antago
- a synthetic reaction mix a cellular compartment, such as a membrane, cell envelope or cell wall or a preparation of any thereof, compnsmg acpS polypeptide and a labeled substrate or ligand of such polypeptide is incubated m the absence or the presence of a candidate molecule that may be a acpS agomst or antagonist
- the ability of the candidate molecule to agonize or antagonize the acpS polypeptide is reflected m decreased bmdmg of the labeled ligand or decreased production of product from such substrate Molecules that bmd gratuitously, i e , without mducmg the effects of acpS polypeptide are most likely to be good antagomsts Molecules that bmd well and, as the case may be, mcrease the rate of product production from substrate, mcrease signal transduction, or mcrease chemical channel activity are agomsts Detection of the rate or
- Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor bindmg techniques known m the art These techniques include, but are not limited to, ligand bmdmg and crosshnkmg assays in which the polypeptide is labeled with a radioactive isotope (for instance, ⁇ 1), chemically modified (for instance, biotmylated), or fused to a peptide sequence suitable for detection or punfication, and mcubated with a source of the putative receptor (e g , cells, cell membranes, cell supernatants, tissue extracts, bodily matenals) Other methods mclude biophysical techniques such as surface plasmon resonance and spectroscopy These screemng methods may also be used to identify agonists and antagomsts of the polypeptide which compete with the bmdmg of the polypeptide to its receptor(s), if any Standard methods for conducting such assays are well
- the fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational correlation time or tumblmg rate Protein complexes, such as formed by acpS polypeptide associating with another acpS polypeptide or other polypeptide.
- labeled to comprise a fluorescently- labeled molecule will have higher polarization values than a fluorescently labeled monome ⁇ c protein It is preferred that this method be used to characterize small molecules that disrupt polypeptide complexes
- Fluorescence energv transfer may also be used characterize small molecules that interfere with the formation of acpS polypeptide dimers, t ⁇ mers. tetramers or higher order structures, or structures formed by acpS polypeptide bound to another polypeptide acpS polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixing of the two labeled species and excitation of the donor fluorophore, fluorescence energy transfer can be detected by observing fluorescence of the acceptor Compounds that block dime ⁇ zation will inhibit fluorescence energy transfer
- acpS polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monome ⁇ c Solution protein can then passed over the acpS polypeptide -coated surface and specific bmdmg can be detected m real-time by monitoring the change in resonance angle caused by a change in local refractive index
- This technique can be used to characterize the effect of small molecules on kinetic rates and equihbnum bmdmg constants for acpS polypeptide self-association as well as an association of acpS polypeptide and another polypeptide or small molecule
- a scintillation proximity assay may be used to characterize the interaction between an association of acpS polypeptide with another acpS polypeptide or a different polypeptide acpS polypeptide can be coupled to a scintillation-filled bead Addition of radio-labeled acpS polypeptide results in bind g where the radioactive source molecule is m close proximity to the scintillation fluid Thus, signal is emitted upon acpS polypeptide bmdmg and compounds that prevent acpS polypeptide self-association or an association of acpS polypeptide and another polypeptide or small molecule will diminish signal
- an assay for acpS agomsts is a competitive assay that combmes acpS and a potential agomst with acpS-bmdmg molecules recombinant acpS bmdmg molecules, natural substrates or ligands. or substrate or ligand mimetics, under appropnate conditions for a competitive inhibition assay acpS can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of acpS molecules bound to a bmdmg molecule or converted to product can be determmed accurately to assess the effectiveness of the potential antagonist
- a polypeptide and/or polynucleotide of the present invention may also be used in a method for the structure-based design of an agomst or antagonist of the polypeptide and/or polynucleotide, by (a) determimng m the first instance the three-dimensional structure of the polypeptide and/or polynucleotide, or complexes thereof, (b) deducmg the three- dimensional structure for the likely reactive s ⁇ te(s), bmdmg s ⁇ te(s) or mot ⁇ f(s) of an agonist or antagonist, (c) synthesizing candidate compounds that are predicted to bmd to or react with the deduced bmdmg s ⁇ te(s). reactive s ⁇ te(s), and/or mot ⁇ f(s), and (d) testing whether the candidate compounds are indeed agonists or antagomsts
- the present mvention provides methods of treatmg abnormal conditions such as. for instance, a Disease, related to either an excess of, an under-expression of, an elevated activity of, or a decreased activity of acpS polypeptide and/or polynucleotide
- Each of the polynucleotide sequences provided herein may be used m the discovery and development of antibacterial compounds
- the encoded protem upon expression, can be used as a target for the screening of antibacterial drugs
- the polynucleotide sequences encoding the ammo terminal regions of the encoded protein or Shme-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the codmg sequence of interest
- the invention also provides the use of the polypeptide, polynucleotide, agomst or antagonist of the invention to interfere with the initial physical mteraction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of mfection
- the molecules of the mvention may be used m the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matnx protems on m-d ellmg devices or to extracellular matrix proteins m wounds, to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial acpS protems that mediate tissue damage and/or, to block the normal progression of pathogenesis in mfections initiated other than by the implantation of m-dwellmg devices or by other surgical techniques
- acpS agomsts and antagomsts preferably bactenstatic or bactencidal agomsts and antagomsts
- the antagomsts and agomsts of the mvention may be employed, for instance, to prevent, inhibit and/or treat diseases Helicobacter pylori (herein "H pylori”) bacteria mfect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastntis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori (International Agency for Research on Cancer, Lyon, France, http //www uicc ch/ecp/ecp2904 htm) Moreover, the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylori and gastnc adenocarcmoma, classifymg the bactenum as a Group I (defimte) carcinogen Preferred antimicrobial compounds of the invention (agomsts and antagomsts of acpS polypeptides and/or polyn
- Bodily mate ⁇ al(s) means any mate ⁇ al denved from an individual or from an orgamsm infecting, infesting or inhabiting an individual, including but not limited to, cells, tissues and waste, such as, bone, blood, serum, cerebrospinal fluid, semen, saliva muscle, cartilage, organ tissue, skin, urine, stool or autopsy matenals
- D ⁇ sease(s) means any disease caused by or related to infection by a bacte ⁇ a, including , for example, otitis media, conjunctivitis, pneumoma. bacteremia, meningitis, smusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid
- “Host cell(s)” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence
- Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determmed 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 determmed by the match between strings of such sequences "Identity” can be readily calculated by known methods, including but not limited to those described m (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Bwcomputtng 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 Hemje, G , Academic Press, 1987.
- Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified m publicly available computer programs Computer program methods to determine identity 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 (Altschul, S F et al , J Molec Bwl 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
- Gap Length Penalty 4 A program useful with these parameters is publicly available as the "gap” program from Genetics
- Parameters for polynucleotide companson include the following Algorithm Needleman and
- Polynucleotide embodiments further include an isolated polynucleotide compnsmg a polynucleotide sequence having at least a 50, 60. 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1 , wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, mcludmg transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides m the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleot
- n n is the number of nucleotide alterations
- x n is the total number of nucleotides m SEQ ID NO 1.
- y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest mteger prior to subtracting it from x n
- Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
- a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 1, that is it may be 100% identical, or it may include up to a certain integer number of nucleic acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity
- Such alterations are selected from the group consistmg of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions interspersed either individually among the nucleic acids m the reference sequence or in one or more contiguous groups withm the reference sequence
- the number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of nucleic acids in SEQ ID NO 1 by the mteger defining the percent identity divided by 100 and then subtracting that product from said total number of nucleic acids in SEQ ID NO 1, or
- n n is the number of nucleic acid alterations
- x n is the total number of nucleic acids in SEQ ID NO 1
- y is, for instance 0 95 for 95%. 0 97 for 97% or 1 00 for 100%, etc
- • is the symbol for the multiplication operator, and wherem any non-mteger product of x n and y is rounded down to the nearest integer prior to subtracting it from x n
- Polypeptide embodiments further mclude an isolated polypeptide compnsmg a polypeptide having at least a 95 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may include up to a certain integer number of ammo acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consistmg of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or m one or more contiguous groups withm the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of ammo acids in SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amm
- n a is the number of ammo acid alterations
- x a is the total number of amino acids m SEQ ID NO 2
- y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
- • is the symbol for the multiplication operator, and wherem any non-mteger product of x a and y is rounded down to the nearest integer pnor to subtracting it from x a
- a polypeptide sequence of the present mvention may be identical to the reference sequence of SEQ ID NO 2. that is it may be 100% identical, or it may mclude up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity
- Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-termrnal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or m one or more contiguous groups withm the reference sequence
- the number of amino acid alterations for a given % identity is determmed by multiplying the total number of amino acids m SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids in SEQ ID NO 2, or
- n a is the number of ammo acid alterations
- x a is the total number of ammo acids in SEQ ID NO 2
- y is. for instance 0 95 for 95%. 0 97 for 97% or 1 00 for 100%, etc
- • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a
- “Indrv ⁇ dual(s)" means a multicellular eukaryote, including, but not limited to a metazoan, a mammal, an ovid. a bovid. a simian, a primate, and a human
- Isolated means altered “by the hand of man” from its natural state, i e , if it occurs m nature, it has been changed or removed from its onginal environment, or both
- a polynucleotide or a polypeptide naturally present m a living orgamsm is not “isolated,” but the same polynucleotide or polypeptide separated from the coexistmg matenals of its natural state is “isolated", as the term is employed herem
- a polynucleotide or polypeptide that is mtroduced mto an orgamsm by transformation, genetic manipulation or b ⁇ any other recombinant method is "isolated” even if it is still present m said orgamsm, which organism may be Irving or non-Irving "Organ ⁇ sm(s)” means a (1) prokaryote, mcludmg but not limited to, a member of the genus Streptococcus Sta
- mcludmg but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces cenviseae, Kluveromyces lactis or Candida albicans
- Polynucleot ⁇ de(s) generally refers to any polynbonucleotide or polydeoxynbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
- Polynucleot ⁇ de(s)” mclude, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double-stranded regions or single-, double- and tnple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybnd molecules compnsmg DNA and RNA that may be single-stranded or.
- polynucleotide refers to tnple-stranded regions compnsmg RNA or DNA or both RNA and DNA
- the strands m such regions may be from the same molecule or from different molecules
- the regions may mclude all of one or more of the molecules but more typically mvolve only a region of some of the molecules
- polynucleot ⁇ de(s) also mcludes DNAs or RNAs as descnbed above that compnse one or more modified bases Thus.
- DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleot ⁇ de(s)" as that term is intended herem Moreover. DNAs or RNAs compnsmg unusual bases, such as mos e, 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 m the art
- the term "polynucleot ⁇ de(s)" as it is employed herem 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, mcludmg, for example, simple and complex cells
- Polynucleot ⁇ de(s) also embraces short polynucleotides often refened to as oh
- Polypept ⁇ de(s) refers to any peptide or protem compnsmg two or more ammo acids joined to each other by peptide bonds or modified peptide bonds
- Polypept ⁇ de(s) refers to both short chains, commonly refened to as peptides.
- Polypeptides may compnse ammo acids other than the 20 gene encoded ammo acids "Polypept ⁇ de(s)" mclude those modified either by natural processes, such as processmg and other post-translational modifications, but also by chemical modification techniques Such modifications are well descnbed m basic texts and in 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 var ing degree at several sites m a given polypeptide Also, a given polypeptide may compnse many types of modifications Modifications can occur anywhere m a polypeptide, mcludmg the peptide backbone, the amino acid side-chains, and the ammo or carboxyl termini Modifications mclude, for example, acetylauon, acylation, ADP
- covalent attachment of a lipid or lipid denvative covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma- carboxylation, GPI anchor formation, hydroxylation. lodination, methylation, mynstoylation, oxidation, proteohtic processmg. phosphorylation, prenylation.
- Polypeptides may be branched or cyclic with or without branching Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well "Recombinant expression system(s)" refers to expression systems or portions thereof or polynucleotides of the mvention mtroduced or transformed mto a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the mvention
- Vanant(s) is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
- a typical variant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the vanant 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, fusion protems and truncations in the polypeptide encoded by the reference sequence, as discussed below
- a typical va ⁇ ant 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 m ammo acid sequence by one
- vanants m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added m any combination
- a vanant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic va ⁇ ant, or it may be a variant that is not known to occur naturally
- Non-naturally occurring vanants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans EXAMPLES
- Total cellular DNA is mechanically sheared by passage through a needle in order to size- fractionate accordmg to standard procedures
- DNA fragments of up to l lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are hgated mto the vector Lambda ZapII that has been cut with EcoRI. the library packaged by standard procedures and
- Method 2 Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a senes 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 hgated to the DNA and the fragments then hgated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coh infected with the packaged library The library is amplified by standard procedures
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Abstract
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WO1998006734A1 (en) * | 1996-08-16 | 1998-02-19 | Smithkline Beecham Corporation | Novel prokaryotic polynucleotides, polypeptides and their uses |
WO1998018931A2 (en) * | 1996-10-31 | 1998-05-07 | Human Genome Sciences, Inc. | Streptococcus pneumoniae polynucleotides and sequences |
WO1998026072A1 (en) * | 1996-12-13 | 1998-06-18 | Eli Lilly And Company | Streptococcus pneumoniae dna sequences |
WO2000018952A1 (en) * | 1998-09-30 | 2000-04-06 | Millennium Pharmaceuticals, Inc. | Use of s-ydcb and b-ydcb, essential bacterial genes |
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WO1998006734A1 (en) * | 1996-08-16 | 1998-02-19 | Smithkline Beecham Corporation | Novel prokaryotic polynucleotides, polypeptides and their uses |
WO1998018931A2 (en) * | 1996-10-31 | 1998-05-07 | Human Genome Sciences, Inc. | Streptococcus pneumoniae polynucleotides and sequences |
WO1998026072A1 (en) * | 1996-12-13 | 1998-06-18 | Eli Lilly And Company | Streptococcus pneumoniae dna sequences |
WO2000018952A1 (en) * | 1998-09-30 | 2000-04-06 | Millennium Pharmaceuticals, Inc. | Use of s-ydcb and b-ydcb, essential bacterial genes |
Non-Patent Citations (2)
Title |
---|
MCALLISTER, K.A. ET AL.: "Biochemical and Molecular Analyses of the Streptococcus pneumoniae Acyl Carrier Protein Synthase, an Enzyme Essential for Fatty Acid Biosynthesis" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 275, no. 40, 6 October 2000 (2000-10-06), pages 30864-30872, XP002308240 * |
See also references of WO9961452A2 * |
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