U.S. patent application number 10/824131 was filed with the patent office on 2004-12-30 for ribg.
This patent application is currently assigned to SmithKline Beecham Corporation. Invention is credited to Black, Michael Terence, Burnham, Martin Karl Russell, Fedon, Jason Craig, Hodgson, John Edward, Knowles, David Justin Charles, Lonetto, Michael Arthur, Nicholas, Richard Oakley, Palmer, Leslie Marie, Pratt, Julie M., Reichard, Raymond Winfield, Rosenberg, Martin, Traini, Christopher Michael, Ward, Judith M., Warren, Richard Lloyd.
Application Number | 20040265872 10/824131 |
Document ID | / |
Family ID | 25526109 |
Filed Date | 2004-12-30 |
United States Patent
Application |
20040265872 |
Kind Code |
A1 |
Black, Michael Terence ; et
al. |
December 30, 2004 |
ribG
Abstract
The invention provides ribG polypeptides and polynucleotides
encoding ribG polypeptides and methods for producing such
polypeptides by recombinant techniques. Also provided are methods
for utilizing ribG polypeptides to screen for antibacterial
compounds.
Inventors: |
Black, Michael Terence;
(Chester Springs, PA) ; Burnham, Martin Karl Russell;
(Barto, PA) ; Fedon, Jason Craig; (Strafford,
PA) ; Hodgson, John Edward; (Malvern, PA) ;
Knowles, David Justin Charles; (Boroughbridge, GB) ;
Lonetto, Michael Arthur; (Collegeville, PA) ;
Nicholas, Richard Oakley; (Collegeville, PA) ;
Palmer, Leslie Marie; (Audubon, PA) ; Pratt, Julie
M.; (Verona, IT) ; Reichard, Raymond Winfield;
(Quakertown, PA) ; Rosenberg, Martin; (Royersford,
PA) ; Traini, Christopher Michael; (Media, PA)
; Ward, Judith M.; (Dorking Surrey, GB) ; Warren,
Richard Lloyd; (Blue Bell, PA) |
Correspondence
Address: |
GLAXOSMITHKLINE
Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Assignee: |
SmithKline Beecham
Corporation
|
Family ID: |
25526109 |
Appl. No.: |
10/824131 |
Filed: |
April 14, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10824131 |
Apr 14, 2004 |
|
|
|
09376633 |
Aug 18, 1999 |
|
|
|
09376633 |
Aug 18, 1999 |
|
|
|
08978456 |
Nov 25, 1997 |
|
|
|
6010881 |
|
|
|
|
Current U.S.
Class: |
435/6.13 ;
435/183; 435/252.3; 435/320.1; 435/69.3; 530/350; 536/23.7 |
Current CPC
Class: |
A61P 31/04 20180101;
C12N 9/0036 20130101; C12Q 1/689 20130101; C07K 2319/00 20130101;
C12N 9/78 20130101; C12Y 105/0103 20130101; A61K 39/00 20130101;
A61P 31/00 20180101; C12Q 2600/158 20130101; C12Y 305/04025
20130101; C12N 15/52 20130101; A61K 38/00 20130101; C07K 16/1275
20130101; C07K 14/31 20130101 |
Class at
Publication: |
435/006 ;
435/069.3; 435/252.3; 435/320.1; 435/183; 530/350; 536/023.7 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/00; C07K 014/31; C12N 015/74 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 19, 1997 |
WO |
PCT/US97/02318 |
Claims
1. An isolated polynucleotide segment comprising: a first
polynucleotide sequence, or the full complement of the entire
length of the first polynucleotide sequence, wherein the first
polynucleotide sequence is selected from the group consisting of:
(a) a polynucleotide consisting of SEQ ID NO: 1; and (b) a nucleic
acid sequence identical to the polynucleotide of (a) except that,
over the entire length corresponding to the polynucleotide of (a),
up to thirty nucleotides are substituted, deleted or inserted for
every 100 nucleotides of the polynucleotide of (a).
2. The isolated polynucleotide segment of claim 1, wherein the
first polynucleotide sequence is selected from the group consisting
of: the polynucleotide of (a); and, a nucleic acid sequence
identical to the polynucleotide of (a) except that, over the entire
length corresponding to the polynucleotide of (a), up to ten
nucleotides are substituted, deleted or inserted for every 100
nucleotides of the polynucleotide of (a).
3. The isolated polynucleotide segment of claim 1, wherein the
first polynucleotide sequence is selected from the group consisting
of: the polynucleotide of (a); and, a nucleic acid sequence
identical to the polynucleotide of (a) except that, over the entire
length corresponding to the polynucleotide of (a), up to five
nucleotides are substituted, deleted or inserted for every 100
nucleotides of the polynucleotide of (a).
4. A vector comprising the isolated polynucleotide segment of claim
1.
5. An isolated host cell comprising the vector of claim 4.
6. An isolated polynucleotide segment, comprising a first
polynucleotide sequence, of the full complement of the entire
length of the first polynucleotide sequence, wherein the first
polynucleotide sequence is selected from the group consisting of:
(a) a polynucleotide which encodes the same mature polypeptide,
expressed by the ribG gene expressed by a polynucleotide comprising
SEQ ID NO: 1 contained in Staphylococcus aureus WCUH 29 contained
in NCIMB Deposit No. 40771; and, (b) a nucleic acid sequence
identical to the polynucleotide of (a) except that, over the entire
length corresponding to the polynucleotide of (a), up to five
nucleotides are substituted, deleted or inserted for every 100
nucleotides of the polynucleotide of (a).
7. The isolated polynucleotide segment of claim 6, wherein the
first polynucleotide sequence is selected from the group consisting
of: the polynucleotide of (a); and, a nucleic acid sequence
identical to the polynucleotide of (a) except that, over the entire
length corresponding to the polynucleotide of (a), up to three
nucleotide are substituted, deleted or inserted for every 100
nucleotides of the polynucleotide of (a).
8. An isolated polynucleotide segment, comprising a first
polynucleotide sequence or the full complement of the entire length
of the first polynucleotide sequence, wherein the first
polynucleotide sequence hybridizes to the full complement of SEQ ID
NO: 1, wherein the hybridization conditions include incubation at
42.degree. C. in a solution comprising: 50% formamide, 5.times.SSC
(150 mM NaCL, 15 mM trisodium citrate), 50 mM sodium phosphate
(pH7.6), 5.times. Denhardt's solution, 10% dextran sulfate, and 20
micrograms/ml denatured, sheared salmon sperm DNA, followed by
washing in 0.1.times.SSC at about 65.degree. C.
9. The isolated polynucleotide segment of claim 8, wherein the
first polynucleotide sequence is identical to SEQ ID NO: 1 except
that, over the entire length corresponding to SEQ ID NO: 1, up to
five nucleotide are substituted, deleted or inserted for every 100
nucleotides of SEQ ID NO: 1.
10. The isolated polynucleotide segment of claim 8, wherein the
first polynucleotide sequence is identical to SEQ ID NO: 1 except
that, over the entire length corresponding to SEQ ID NO: 1, up to
three nucleotide are substituted, deleted or inserted for every 100
nucleotides of SEQ ID NO: 1.
11. An isolated polynucleotide segment, comprising a first
polynucleotide sequence or the full complement of the entire length
of the first polynucleotide sequence, wherein the first
polynucleotide sequence is selected from the group consisting of:
(a) a polynucleotide which encodes a polypeptide comprising the
amino acid sequence set forth in SEQ ID NO:2; and, (b) a nucleic
acid sequence identical to the polynucleotide of (a) except that,
over the entire length corresponding to the polynucleotide of (a),
up to five nucleotides are substituted, deleted or inserted for
every 100 nucleotides of the polynucleotide of (a).
12. A vector comprising the isolated polynucleotide segment of
claim 11.
13. An isolated host cell comprising the vector of claim 12.
14. The isolated polynucleotide segment of claim 11, wherein the
first polynucleotide sequence is selected from the group consisting
of: (a) a polynucleotide which encodes a polypeptide consisting of
the amino acid sequence set forth in SEQ ID NO:2; and, (b) a
nucleoice acid sequence identical to the polynucleotide of (a)
except that, over the entire length corresponding to the
polynucleotide of (a), up to five nucleoitdes are substituted,
deleted or inserted for every 100 nucleotides of the polynucleotide
of (a).
15. A vector comprising the isolated polynucleotide segment of
claim 14.
16. An isolated host cell comprising the vector of claim 15.
17. A composition comprising the isolated polynucleotide segment of
claim 1, which isolated polynucleotide segment is according to the
formula: X--(R.sub.1).sub.m--(R.sub.2)--(R.sub.3).sub.n--Y wherein,
at the 5' end of the molecule, X is hydrogen, and at the 3' end of
the molecule, Y is hydrogen or a metal, R.sub.1 and R.sub.3 are any
nucleic acid residue, m is an integer between I and 3000 or zero, n
is an integer between 1 and 3000 or zero, and R.sub.2 is the first
polynucleotide sequence.
18. An isolated polynucleotide segment comprising: a first
polynucleotide sequence, or the full complement of the entire
length of the first polynucleotide sequence, wherein the first
polynucleotide sequence is selected form the group consisting of:
(a) a polynucleotide encoding a polypeptide comprising the amino
acid sequence of SEQ ID NO:4; (b) a polynucleotide consisting of
SEQ ID NO:3; and, (c) a nucleic acid sequence identical to the
polynucleotide of (b) except that, over the entire length
corresponding to the polynucleotide of (b), up to thirty
nucleotides are substituted, deleted or inserted for every 100
nucleotides of the polynucleotide of (b).
19. The isolated polynucleotide segment of claim 18, wherein the
first polynucleotide sequence is selected from the group consisting
of: the polynucleotide of (b); and, a nucleic acid sequence
identical to the polynucleotide of (b) except that, over the entire
length corresponding to the polynucleotide of (b), up to ten
nucleotides are substituted, deleted or inserted for every 100
nucleotides of the polynucleotide of (b).
20. The isolated polynucleotide segment of claim 18, wherein the
first polynucleotide sequence is selected from the group consisting
of: the polynucleotide of (b); and, a nucleic acid sequence
identical to the polynucleotide of (b) except that, over the entire
length corresponding to the polynucleotide of (b), up to five
nucleotides are substituted, deleted or inserted for every 100
nucleotides of the polynucleotide of (b).
Description
RELATED APPLICATIONS
[0001] This application claims benefit of PCT Application, Number
PCT/US97/02318, filed Feb. 19, 1997.
FIELD OF THE INVENTION
[0002] This invention relates to newly identified polynucleotides
and polypeptides, and their production and uses, as well as their
variants, agonists and antagonists, and their uses. In particular,
the invention relates to novel polynucleotides and polypeptides of
the riboflavin specific deaminase family, hereinafter referred to
as "ribG".
BACKGROUND OF THE INVENTION
[0003] It is particularly preferred to employ Staphylococcal genes
and gene products as targets for the development of antibiotics.
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 exotoxins as opposed to tissue invasion
and bacteremia. These conditions include: Staphylococcal food
poisoning, scalded skin syndrome and toxic shock syndrome.
[0004] Riboflavin (vitamin B2) is a member of the B complex of
vitamins which function as coenzymes in metabolic reactions.
Riboflavin has two coenzyme forms, flavin mononucleotide (FMN) and
flavin adenine dinucleotide (FAD) which act in oxidation-reduction
reactions such as the cytochrome system of electron transport and
the oxidative degradation of pyruvate, fatty acids and amino acids.
The first committed step in the biosynthesis of riboflavin is the
opening of the imidazole ring of GTP. In the presence of 3 H.sub.2O
and Mg.sup.++, the C-8 of GTP is released as formate accompanied by
the release of pyrophosphate by the action of GTP cyclohyrolase II
(GCH2; EC 3.5.4.25). This enzyme function is encoded by ribA in
bacteria and rib1 in yeast species. Through a series of steps,
involving 3,4-dihydroxy-2-butanone 4 phosphate synthase (ribA),
6,7-dimethyl-8-ribityllumazine synthase (ribH), riboflavin synthase
(ribB), pyrimidine deaminase and pyrimidine reductase (ribG),
enzymes encoded by genes within the riboflavin biosynthesis operon,
riboflavin is formed. Because the genes required for riboflavin
biosynthesis are present in many pathogenic microorganisms, and
since riboflavin biosynthesis has shown to be required for
virulence in the swine pathogen Actinobacillus pleuropneumoniae
(Fuller, T E, et al. (1996) A riboflavin auxotroph of
Actinobacillus pleuropneumoniae is attenuated in swine. Infect.
Immun. 64:4659-4664), these gene products represent broad spectrum
antibacterial as well as antifungal targets.
[0005] The frequency of Staphylococcus aureus infections has risen
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 Staphylococcus aureus strains which
are resistant to some or all of the standard antibiotics. This
phenomenon has created a demand for both new anti-microbial agents,
vaccines, and diagnostic tests for this organism.
[0006] Clearly, there exists a need for factors, such as the nbG
embodiments of the invention, that have a present benefit of being
useful to screen compounds for antibiotic activity. Such factors
are also useful to determine their role in pathogenesis of
infection, dysfunction and disease. There is also a need for
identification and characterization of such factors and their
antagonists and agonists to find ways to prevent, ameliorate or
correct such infection, dysfunction and disease.
[0007] Certain of the polypeptides of the invention possess amino
acid sequence homology to a known Bacillus subtilis ribG
protein.
SUMMARY OF THE INVENTION
[0008] It is an object of the invention to provide polypeptides
that have been identified as novel ribG polypeptides by homology
between the amino acid sequence set out in Table 1 [SEQ ID NO: 2 or
4] and a known amino acid sequence or sequences of other proteins
such as Bacillus subtilis ribG protein.
[0009] It is a further object of the invention to provide
polynucleotides that encode ribG polypeptides, particularly
polynucleotides that encode the polypeptide herein designated
ribG.
[0010] In a particularly preferred embodiment of the invention the
polynucleotide comprises a region encoding ribG polypeptides
comprising a sequence set out in Table 1 [SEQ ID NO: 1 or 3] which
includes a full length gene, or a variant thereof.
[0011] In another particularly preferred embodiment of the
invention there is a novel ribG protein from Staphylococcus aureus
comprising the amino acid sequence of Table 1 [SEQ ID NO:2 or 4],
or a variant thereof.
[0012] 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 WCUH29 on
deposit strain, which nucleic acid is contained in the deposited
strain.
[0013] A further aspect of the invention there are provided
isolated nucleic acid molecules encoding ribG, particularly
Staphylococcus aureus ribG, including mRNAs, cDNAs, genomic DNAs.
Further embodiments of the invention include biologically,
diagnostically, prophylactically, clinically or therapeutically
useful variants thereof, and compositions comprising the same.
[0014] 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 variants of ribG and
polypeptides encoded thereby.
[0015] Another aspect of the invention there are provided novel
polypeptides of Staphylococcus aureus referred to herein as ribG as
well as biologically, diagnostically, prophylactically, clinically
or therapeutically useful variants thereof, and compositions
comprising the same.
[0016] Among the particularly preferred embodiments of the
invention are variants of ribG polypeptide encoded by naturally
occurring alleles of the ribG gene.
[0017] In a preferred embodiment of the invention there are
provided methods for producing the aforementioned ribG
polypeptides.
[0018] In accordance with yet another aspect of the invention,
there are provided inhibitors to such polypeptides, useful as
antibacterial agents, including, for example, antibodies.
[0019] In accordance with certain preferred embodiments of the
invention, there are provided products, compositions and methods
for assessing ribG expression, treating disease, assaying genetic
variation, and administering a ribG polypeptide or polynucleotide
to an organism to raise an immunological response against a
bacteria, especially a Staphylococcus aureus bacteria.
[0020] In accordance with certain preferred embodiments of this and
other aspects of the invention there are provided polynucleotides
that hybridize to ribG polynucleotide sequences, particularly under
stringent conditions.
[0021] In certain preferred embodiments of the invention there are
provided antibodies against ribG polypeptides.
[0022] In other embodiments of the invention there are provided
methods for identifying compounds which bind to or otherwise
interact with and inhibit or activate an activity of a polypeptide
or polynucleotide of the invention comprising: 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 determining 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 of the compound with the polypeptide or
polynucleotide.
[0023] In accordance with yet another aspect of the invention,
there are provided ribg agonists and antagonists, preferably
bacteriostatic or bacteriocidal agonists and antagonists.
[0024] In a further aspect of the invention there are provided
compositions comprising a ribG polynucleotide or a ribG polypeptide
for administration to a cell or to a multicellular organism.
[0025] Various changes and modifications within the spirit and
scope of the disclosed invention will become readily apparent to
those skilled in the art from reading the following descriptions
and from reading the other-parts of the present disclosure
DESCRIPTION OF THE INVENTION
[0026] The invention relates to novel ribG polypeptides and
polynucleotides as described in greater detail below. In
particular, the invention relates to polypeptides and
polynucleotides of a novel ribG of Staphylococcus aureus, which is
related by amino acid sequence homology to Bacillus subtilis ribG
polypeptide. The invention relates especially to ribG having the
nucleotide and amino acid sequences set out in Table 1 as SEQ ID
NO: 1 and SEQ ID NO: 2 respectivelyribG.
Table 1
[0027] ribG Polynucleotide and Polypeptide Sequences
[0028] (A) Sequences from Staphylococcus aureus ribG Polynucleotide
Sequence [SEQ ID NO: 1].
1
ATGGATTATGCGATTCAACTTGCAAATATGGTACAAGGTCAAACAGGTGTTAATCCACCCGTTGG-
CGCTG TTGTAGTTAATGAAGGTAGGATTGTTGGTATTGGTGCACACTTGAGAAAAG-
GTGACAAGCATGCGGAGGT TCAAGCACTTGATATGGCACAACAAAATGCTGAAGGTG-
CGACGATTTATATTACGTTAGAGCCATGTAGT CATTTTGGTTCAACACCACCCTGTG-
TTAACAAAATTATTGATTGTAAGATAGCAAAAGTAGTATACGCAA
CAAAAGACAATTCGTTAGACACACATGGTGATGAGACGTTACGGGCTCACGGTATTGAGGTTGAATGCGT
TGATGATGAACGGGCATCACAATTATACCAAGACTTTTTTAAAGCAAAAGCAAAGCAACT-
GCCACAAATT ACAGTGAAAGTATCTGCAAGTTTAGATGGTAAACAAGCGAATGATAA-
TGGACAAAGTCAATGGATTACTA ACAAAGAGGTTAAACAAGATGTCTATAAGTTAAG-
ACATCGACACGACGCAGTGTTAACTGGAAGACGTAC
AGTTGAATTAGATGATCCACAATATACTACACGTATTCAAGATGGAAAAAACCCTATAAAAGTAATATTG
TCTAAGTCTGGGAATATTCATTTTAATCAGCAAATTTATCAAGATGAATCAACACCAATT-
TGGATATATA CTGAAAATCCAAATTTAACAAGCAATCAAACACATATTGAAATTATT-
TACTTGAAGTCTTGTGATTTAAC AACAATTCTTCACAATTTATATAAAAGAGGAGTT-
GGAACTTTGCTAGTCGAGGCAGGTCCAACCACTACT
TCAGAATTCTCCATCTATTATATAGATGAATTTATTCTCTATTATGCCCCGAAATTAATTGGCGGATCTG
GAAATTATCAATTTTATCAAACAAATGATGTGATTGAGATACCAGATGCGAACCAATTTG-
AAATTGTTCA TTCCGAGTTATTAAATCAAAATGTTAAATTAACTTTACGAAAGAAGT-
GA-3'
[0029] (B) Staphylococcus aureus ribG Polypeptide Sequence Deduced
from the Polynucleotide Sequence in this Table [SEQ ID NO:2].
2 NH.sub.2- MDYAIQLANMVQGQTGVNPPVGAVVVNEGRIVGIGAHL-
RKGDKHAEVQALDMAQQNAEGATIYITLEPCS HFGSTPPCVNKIIDCKIAKVVYATK-
DNSLDTHGDETLPAHGIEVECVDDEPASQLYQDFFKAKAKQLPQI
TVKVSASLDGKQANDNGQSQWITNKEVKQDVYKLRHRHDAVLTGRRTVELDDPQYTTRIQDGKNPIKVIL
SKSGNIHFNQQIYQDESTPIWIYTENPNLTSNQTHIEIIYLKSCDLTTILHNLYKRGVGT-
LLVEAGPTTT SEFSIYYIDEFILYYAPKLIGGSGNYQFYQTNDVIEIPDANQFEIVH-
SELLNQNVKLTLRKK-COOH
[0030] (C) Polynucleotide Sequences Comprising Staphylococcus
aureus ribG ORF Sequence [SEQ ID NO:3].
3 5'- AANCACCAATCCNATTGGGAGGNAATCCAAATCAATNCCCGGAN-
NCCCAATCCAAGTTAATTAAGTCCAA GGTTTTGGAACATTACCAAATATGATTCCGA-
TGAGGTCAAATGNCAANCGGTGTTAATAAACTACGAAAT
GNTGTGNAAATGATAGTAGANCAAGTTGCGCATACAGTNTCTCNATTATATGATGCTTTAGAATCGAATG
AGCAACAACAGCGCAGTTACAATCAATAATTTGTAACTAGAAGATAATAAAGAGAACGCT-
CTATAGAGAC GAATTGAAGGTTTGATTTTAATGTCTGTTAGTAAGAATCATATCAAT-
GAGATGCCTATAGTACTCAGATT ATATTAAATTAAAACCGTCATTAATTGTTTTTTT-
AGAAAACATATAGTATCATTTTAAATGTAGTTGACA
TACTACGTACTCAAATAATCTATAACAATTTCATATATAATTCTTTCGGGGCAGGGTGAAATTCCCAACC
GGCAGTAAATAAAGCCTGCGACCTGCTAATATGTTTCATATTAGTGGCTGATCTAGTGAG-
ATTCTAGAGC CGACAGTTAAAGTCTGGATGGGAGAAAGAATGTTAATTATCGACAAA-
GATAATGTAGCGTATTTGTAAAA ATGTGTACAAATAGGCTTATTTAACGATAA
ATTTTTCTCCTTTGCATCTTAATTCATGATGTGAGGATTTTTTGTTTATAGAGGTGATCAT-
TTGAGTCAA TTTATGGATTATGCGATTCAACTTCCAAATATGGTACAAGGTCANACA-
GGTGTTAATCCACCCGTTGGCG CTGTTGTAGTTAATGAAGGTAGGATTGTTGGTATT-
GGTGCACACTTGAGAAAAGGTGACAAGCATGCGGA
GGTTCAAGCACTTGATATGGCACAACANAATGCTGAAGGTGCGACGATTTATATTACGTTAGAGCCATGT
AGTCATTTTGGTTCAACACCACCCTGTGTTAACAAAATTATTGATTGTAAGATAGCANAA-
GTAGTATTAC NCAACANAAGACAATTCCGTTAGACACACATGGGTGATGAGACGTTA-
CGGGGCTCCACGGTATTTGAGGG TTGAATTGCGTTGGATGATGAACGGGCATCACAA-
TTATACCAAAGACTTTTTTTAAAGCAAAAAGCAAAG
CAACTTGCCACAAAATTACAGTGAAAGTNTCTTGAAAGTTTAGATGGGTAAACAAAGCGAATTGATAATG
GACAAAGTCAATGGATTACTAACAAAGAGGTTAAACAAGATGTCTATAG-3'
[0031] (D) Staphylococcus aureus ribG Polypeptide Sequence Deduced
from the Polynucleotide ORF Sequence in this Table [SEQ ID
NO:4].
4 NH.sub.2- MDYAIQLPNMVQGXTGVNPPVGAVVVNEGRIVGIGAHL-
RKGDKHAEVQALDMAQXNAEGATIYITLEPCS HFGSTPPCVNKIIDCKIAXVVLXNX- RQFR
--COOH
[0032] Deposited Materials
[0033] A deposit containing a Staphylococcus aureus WCUH 29 strain
has been deposited with the National Collections of Industrial and
Marine Bacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive,
Aberdeen AB2 1RY, Scotland on 11 Sep. 1995 and assigned NCIMB
Deposit No. 40771, and referred to as Staphylococcus aureus WCUH29
on deposit. The Staphylococcus aureus strain deposit is referred to
herein as "the deposited strain" or as "the DNA of the deposited
strain."
[0034] The deposited strain contains the full length ribG gene. The
sequence of the polynucleotides contained in the deposited strain,
as well as the amino acid sequence of the polypeptide encoded
thereby, are controlling in the event of any conflict with any
description of sequences herein.
[0035] The deposit of the 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 strain will be irrevocably and without restriction 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. .sctn.112.
[0036] A license may be required to make, use or sell the deposited
strain, and compounds derived therefrom, and no such license is
hereby granted.
[0037] One 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. Further provided by the invention are ribG nucleotide
sequences of the DNA in the deposited strain and amino acid
sequences encoded thereby. Also provided by the invention are ribG
polypeptide sequences isolated from the deposited strain and amino
acid sequences derived therefrom.
[0038] Polypeptides
[0039] The polypeptides of the invention include a polypeptide of
Table 1 [SEQ ID NO:2 or 4] (in particular the mature polypeptide)
as well as polypeptides and fragments, particularly those which
have the biological activity of ribG, and also those which have at
least 70% identity to a polypeptide of Table 1 [SEQ ID NO:1 or 3]or
the relevant portion, preferably at least 80% identity to a
polypeptide of Table 1 [SEQ ID NO:2 or 4and more preferably at
least 90% similarity (more preferably at least 90% identity) to a
polypeptide of Table 1 [SEQ ID NO:2 or 4] and still more preferably
at least 95% similarity (still more preferably at least 95%
identity) to a polypeptide of Table 1 [SEQ ID NO:2 or 4] and also
include portions of such polypeptides with such portion of the
polypeptide generally containing at least 30 amino acids and more
preferably at least 50 amino acids.
[0040] The invention also includes polypeptides of the formula:
--X--(R.sub.1).sub.m--(R.sub.2)--(R.sub.3).sub.n--Y
[0041] wherein, at the amino terminus, X is hydrogen, and at the
carboxyl terminus, Y is hydrogen or a metal, R.sub.1 and R.sub.3
are any amino acid residue, m is an integer between 1 and 1000 or
zero, n is an integer between 1 and 1000 or zero, and R.sub.2 is an
amino acid sequence of the invention, particularly an amino acid
sequence selected from Table 1. In the formula above R.sub.2 is
oriented so that its amino terminal residue is at the left, bound
to R.sub.1, and its carboxy terminal residue is at the right, bound
to R.sub.3. Any stretch of amino acid residues denoted by either R
group, where m and/or n is greater than 1, may be either a
heteropolymer or a homopolymer, preferably a heteropolymer.
[0042] A fragment is a variant polypeptide having an amino acid
sequence that entirely is the same as part but not all of the amino
acid sequence of the aforementioned polypeptides. As with ribG
polypeptides fragments may be "free-standing," or comprised within
a larger polypeptide of which they form a part or region, most
preferably as a single continuous region, a single larger
polypeptide.
[0043] Preferred fragments include, for example, truncation
polypeptides having a portion of an amino acid sequence of Table 1
[SEQ ID NO:2 or 4], or of variants thereof, such as a continuous
series of residues that includes the amino terminus, or a
continuous series of residues that includes the carboxyl terminus.
Degradation forms of the polypeptides of the invention in a host
cell, particularly a Staphylococcus aureus, are also preferred.
Further preferred are fragments characterized by structural or
functional attributes such as fragments that comprise alpha-helix
and alpha-helix forming regions, beta-sheet and beta-sheet-forming
regions, turn and turn-forming regions, coil and coil-forming
regions, hydrophilic regions, hydrophobic regions, alpha
amphipathic regions, beta amphipathic regions, flexible regions,
surface-forming regions, substrate binding region, and high
antigenic index regions.
[0044] Also preferred are biologically active fragments which are
those fragments that mediate activities of ribG, 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 immunogenic in an animal, especially in a
human. Particularly preferred are fragments comprising receptors or
domains of enzymes that confer a function essential for viability
of Staphylococcus aureus or the ability to initiate, or maintain
cause disease in an individual, particularly a human.
[0045] Variants that are fragments of the polypeptides of the
invention may be employed for producing the corresponding
full-length polypeptide by peptide synthesis; therefore, these
variants may be employed as intermediates for producing the
full-length polypeptides of the invention.
[0046] In addition to the standard single and triple letter
representations for amino acids, the term "X" or "Xaa" may also be
used in describing certain polypeptides of the invention. "X" and
"Xaa" mean that any of the twenty naturally occuring amino acids
may appear at such a designated position in the polypeptide
sequence.
[0047] Polynucleotides
[0048] Another aspect of the invention relates to isolated
polynucleotides, including the full length gene, that encode the
ribG polypeptide having a deduced amino acid sequence of Table 1
[SEQ ID NO:2 or 4] and polynucleotides closely related thereto and
variants thereof.
[0049] Using the information provided herein, such as a
polynucleotide sequence set out in Table 1 [SEQ ID NO:1 or 3], a
polynucleotide of the invention encoding ribG polypeptide may be
obtained using standard cloning and screening methods, such as
those for cloning and sequencing chromosomal DNA fragments from
bacteria using Staphylococcus aureus WCUH 29 cells as starting
material, followed by obtaining a full length clone. For example,
to obtain a polynucleotide sequence of the invention, such as a
sequence given in Table 1 (SEQ ID NO: 1 or 3], typically a library
of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in
E.coli or some other suitable host is probed with a radiolabeled
oligonucleotide, 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., Fritsch, E. F.
and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd
Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989). (see in particular Screening By Hybridization 1.90 and
Sequencing Denatured Double-Stranded DNA Templates 13.70).
Illustrative of the invention, the polynucleotide set out in Table
1 [SEQ ID NO:1 or 3] was discovered in a DNA library derived from
Staphylococcus aureus WCUH 29.
[0050] The DNA sequence set out in Table 1 [SEQ ID NO:1 or 3]
contains an open reading frame encoding a protein having about the
number of amino acid residues set forth in Table 1 [SEQ ID NO:2 or
4] with a deduced molecular weight that can be calculated using
amino acid residue molecular weight values well known in the art.
The polynucleotide of SEQ ID NO: 1, between nucleotide number I and
the stop codon which begins at nucleotide number 1027 of SEQ ID NO:
1, encodes the polypeptide of SEQ ID NO:2.
[0051] RibG of the invention is structurally related to other
proteins of the riboflavin specific deaminase family.
[0052] The invention provides a polynucleotide sequence identical
over its entire length to a coding sequence in Table 1 [SEQ ID NO:
1 or 3]. Also provided by the invention is the coding sequence for
the mature polypeptide or a fragment thereof, by itself as well as
the coding sequence for the mature polypeptide or a fragment in
reading frame with other coding sequence, such as those encoding a
leader or secretory sequence, a pre-, or pro- or prepro-protein
sequence. The polynucleotide may also contain non-coding sequences,
including for example, but not limited to non-coding 5' and 3'
sequences, such as the transcribed, non-translated sequences,
termination signals, ribosome binding sites, sequences that
stabilize mRNA, introns, polyadenylation signals, and additional
coding sequence which encode additional amino acids.
[0053] For example, a marker sequence that facilitates purification
of the fused polypeptide can be encoded. In certain embodiments of
the invention, the marker sequence is a hexa-histidine peptide, as
provided in the pQE vector (Qiagen, Inc.) and described 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
invention also include, but are not limited to, polynucleotides
comprising a structural gene and its naturally associated sequences
that control gene expression.
[0054] A preferred embodiment of the invention is a polynucleotide
of comprising nucleotide 1 to the nucleotide immediately upstream
of or including nucleotide 1027 set forth in SEQ ID NO: 1 of Table
1, both of which encode the ribG polypeptide.
[0055] The invention also includes polynucleotides of the
formula:
X--(R.sub.1).sub.m--(R.sub.2)--(R.sub.3).sub.n--Y
[0056] wherein, at the 5' end of the molecule, X is hydrogen, and
at the 3' end of the molecule, Y is hydrogen or a metal, R.sub.1
and R.sub.3 is any 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.sub.2 is a nucleic acid sequence of the invention, particularly a
nucleic acid sequence selected from Table 1. In the polynucleotide
formula above R.sub.2 is oriented so that its 5' end residue is at
the left, bound to R.sub.1, and its 3' end residue is at the right,
bound to R.sub.3. Any stretch of nucleic acid residues denoted by
either R group, where m and/or n is greater than 1, may be either a
heteropolymer or a homopolymer, preferably a heteropolymer. In a
preferred embodiment m and/or n is an integer between 1 and
1000.
[0057] It is most preferred that the polynucleotides of the
inventions are derived from Staphylococcus aureus, however, they
may preferably be obtained from organisms of the same taxonomic
genus. They may also be obtained, for example, from organisms of
the same taxonomic family or order.
[0058] The term "polynucleotide encoding a polypeptide" as used
herein encompasses polynucleotides that include a sequence encoding
a polypeptide of the invention, particularly a bacterial
polypeptide and more particularly a polypeptide of the
Staphylococcus aureus ribG having an amino acid sequence set out in
Table 1 [SEQ ID NO:2 or 4]. The term also encompasses
polynucleotides that include a single continuous region or
discontinuous regions encoding the polypeptide (for example,
interrupted by integrated phage or an insertion sequence or
editing) together with additional regions, that also may contain
coding and/or non-coding sequences.
[0059] The invention further relates to variants of the
polynucleotides described herein that encode for variants of the
polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID
NO:2 or 4]. Variants that are fragments of the polynucleotides of
the invention may be used to synthesize full-length polynucleotides
of the invention.
[0060] Further particularly preferred embodiments are
polynucleotides encoding ribG variants, that have the amino acid
sequence of ribG polypeptide of Table 1 [SEQ ID NO:2 or 4] in which
several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid
residues are substituted, deleted or added, in any combination.
Especially preferred among these are silent substitutions,
additions and deletions, that do not alter the properties and
activities of ribG.
[0061] Further preferred embodiments of the invention are
polynucleotides that are at least 70% identical over their entire
length to a polynucleotide encoding ribG polypeptide having an
amino acid sequence set out in Table 1 [SEQ ID NO:2 or 4], and
polynucleotides that are complementary to such polynucleotides.
Alternatively, most highly preferred are polynucleotides that
comprise a region that is at least 80% identical over its entire
length to a polynucleotide encoding ribG polypeptide 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% being the more preferred.
[0062] Preferred embodiments are polynucleotides that encode
polypeptides that retain substantially the same biological function
or activity as the mature polypeptide encoded by a DNA of Table 1
[SEQ ID NO: 1 or 3].
[0063] The invention further relates to polynucleotides that
hybridize to the herein above-described sequences. In this regard,
the invention especially relates to polynucleotides that hybridize
under stringent conditions to the herein above-described
polynucleotides. As herein used, the terms "stringent conditions"
and "stringent hybridization conditions" mean hybridization 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.degree. C.
in a solution comprising: 50% formamide, 5.times.SSC (150 mM NaCl,
15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5.times.
Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml
denatured, sheared salmon sperm DNA, followed by washing the
hybridization support in 0.1.times.SSC at about 65.degree. 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.
[0064] 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 under stringent
hybridization conditions with a probe having the sequence of said
polynucleotide sequence set forth in SEQ ID NO: 1 or a fragment
thereof; and isolating said DNA sequence. Fragments useful for
obtaining such a polynucleotide include, for example, probes and
primers described elsewhere herein.
[0065] As discussed additionally herein regarding polynucleotide
assays of the invention, for instance, polynucleotides of the
invention as discussed above, may be used as a hybridization probe
for RNA, cDNA and genomic DNA to isolate full-length cDNAs and
genomic clones encoding ribG and to isolate cDNA and genomic clones
of other genes that have a high sequence similarity to the ribG
gene. Such probes generally will comprise 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.
[0066] For example, the coding region of the ribG gene may be
isolated by screening using a DNA sequence provided in Table 1 [SEQ
ID NO: 1 or 3] to synthesize an oligonucleotide probe. A labeled
oligonucleotide having a sequence complementary to that of a gene
of the invention is then used to screen a library of cDNA, genomic
DNA or mRNA to determine which members of the library the probe
hybridizes to.
[0067] The polynucleotides and polypeptides of the invention may be
employed, for example, as research reagents and materials for
discovery of treatments of and diagnostics for disease,
particularly human disease, as further discussed herein relating to
polynucleotide assays.
[0068] Polynucleotides of the invention that are oligonucleotides
derived from the sequences of Table 1 [SEQ ID NOS: 1 or 2 or 3 or
4] may be used in the processes herein as described, but preferably
for PCR, to determine 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
in diagnosis of the stage of infection and type of infection the
pathogen has attained.
[0069] The invention also provides polynucleotides that may encode
a polypeptide that is the mature protein plus additional amino or
carboxyl-terminal amino acids, or amino acids interior to the
mature polypeptide (when the mature form has more than one
polypeptide chain, for instance). Such sequences may play a role in
processing of a protein from precursor to a mature form, may allow
protein transport, may lengthen or shorten protein half-life or may
facilitate manipulation of a protein for assay or production, among
other things. As generally is the case in vivo, the additional
amino acids may be processed-away from the mature protein by
cellular enzymes.
[0070] A precursor protein, having the 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 proprotein's.
[0071] In addition to the standard A, G, C, T/U representations for
nucleic acid bases, the term "N" may also be used in describing
certain polynucleotides of the invention. "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 it is preferred that N is not 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.
[0072] In sum, a polynucleotide of the invention may encode a
mature protein, a mature protein plus a leader sequence (which may
be referred to as a preprotein), a precursor of a mature protein
having one or more prosequences that are not the leader sequences
of a preprotein, or a preproprotein, which is a precursor to a
proprotein, having a leader sequence and one or more prosequences,
which generally are removed during processing steps that produce
active and mature forms of the polypeptide.
[0073] Vectors, Host Cells, Expression
[0074] The invention also relates to vectors that comprise a
polynucleotide or polynucleotides of the invention, host cells that
are genetically engineered with vectors of the invention and the
production of polypeptides of the invention by recombinant
techniques. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the invention.
[0075] For recombinant production, host cells can be genetically
engineered to incorporate expression systems or portions thereof or
polynucleotides of the invention. Introduction of a polynucleotide
into the host cell can be effected by methods described in 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,
microinjection, cationic lipid-mediated transfection,
electroporation, transduction, scrape loading, ballistic
introduction and infection.
[0076] Representative examples of appropriate hosts include
bacterial cells, such as streptococci, staphylococci, enterococci
E. coli, streptomyces and Bacillus subtilis 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, C1 27, 3T3, BHK, 293 and Bowes melanoma cells; and plant
cells.
[0077] A great variety of expression systems can be used to produce
the polypeptides of the invention. Such vectors include, among
others, chromosomal, episomal and virus-derived vectors, e.g.,
vectors derived from bacterial plasmids, from bacteriophage, 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
derived from combinations thereof, such as those derived from
plasmid and bacteriophage genetic elements, such as cosmids and
phagemids. The expression system constructs may contain 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 in a host may be
used for expression in this regard. The appropriate DNA sequence
may be inserted into the expression system by any of a variety of
well-known and routine techniques, such as, for example, those set
forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL,
(supra).
[0078] For secretion of the translated protein into the lumen of
the endoplasmic reticulum, into the periplasmic space or into the
extracellular environment, appropriate secretion signals may be
incorporated into the expressed polypeptide. These signals may be
endogenous to the polypeptide or they may be heterologous
signals.
[0079] Polypeptides of the invention can be recovered and purified
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 protein may be
employed to regenerate active conformation when the polypeptide is
denatured during isolation and or purification.
[0080] Diagnostic Assays
[0081] This invention is also related to the use of the ribG
polynucleotides of the invention for use as diagnostic reagents.
Detection of ribG in a eukaryote, particularly a mammal, and
especially a human, will provide a diagnostic method for diagnosis
of a disease. Eukaryotes (herein also "individual(s)"),
particularly mammals, and especially humans, particularly those
infected or suspected to be infected with an organism comprising
the ribG gene may be detected at the nucleic acid level by a
variety of techniques.
[0082] Nucleic acids for diagnosis may be obtained from an infected
individual's cells and tissues, such as bone, blood, muscle,
cartilage, and skin. Genomic DNA may be used directly for detection
or may be amplified enzymatically by using PCR or other
amplification technique prior to analysis. RNA, cDNA and genomic
DNA may also be used in the same ways. Using amplification,
characterization of the species and strain of prokaryote present in
an individual, may be made by an analysis of the genotype of the
prokaryote gene. Deletions and insertions can be detected by a
change in size of the amplified product in comparison to the
genotype of a reference sequence. Point mutations can be identified
by hybridizing amplified DNA to labeled ribG polynucleotide
sequences. Perfectly matched sequences can be distinguished from
mismatched duplexes by RNase digestion or by differences in melting
temperatures. DNA sequence differences may also be detected by
alterations in the electrophoretic mobility of the DNA fragments in
gels, with or without denaturing agents, or by direct DNA
sequencing. 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 S1 protection or a
chemical cleavage method. See, e.g., Cotton et al., Proc. Natl.
Acad. Sci., USA, 85: 43974401 (1985).
[0083] Cells carrying mutations or polymorphisms in the gene of the
invention may also be detected at the DNA level by a variety 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 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 or RT-PCR. As an
example, PCR primers complementary to a nucleic acid encoding ribG
can be used to identify and analyze mutations. Examples of
representative primers are shown below in Table 2.
5TABLE 2 Primers for amplification of ribG polynucleotides SEQ ID
NO PRIMER SEQUENCE 5 5'-ATGAAGGTAGGATTGTTGGTA-3' 6
5'-AGTCTTGGTATAATTGTGATGC-- 3'
[0084] The invention also includes primers of the formula:
X--(R.sub.1).sub.m--(R.sub.2)--(R.sub.3).sub.n--Y
[0085] wherein, at the 5' end of the molecule, X is hydrogen, and
at the 3' end of the molecule, Y is hydrogen or a metal, R.sub.1
and R.sub.3 is any nucleic acid residue, m is an integer between 1
and 20 or zero , n is an integer between 1 and 20 or zero, and
R.sub.2 is a primer sequence of the invention, particularly a
primer sequence selected from Table 2. In the polynucleotide
formula above R.sub.2 is oriented so that its 5' end residue is at
the left, bound to R.sub.1, and its 3' end residue is at the right,
bound to R.sub.3. Any stretch of nucleic acid residues denoted by
either R group, where m and/or n is greater than 1, may be either a
heteropolymer or a homopolymer, preferably a heteropolymer being
complementary to a region of a polynucleotide of Table 1. In a
preferred embodiment m and/or n is an integer between 1 and 10.
[0086] 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 ribG DNA isolated
from a sample derived from an individual. The primers may be used
to amplify the gene isolated from an infected individual such that
the gene may then be subject to various techniques for elucidation
of the DNA sequence. In this way, mutations in the DNA sequence may
be detected and used to diagnose infection and to serotype and/or
classify the infectious agent.
[0087] The invention further provides a process for diagnosing,
disease, preferably bacterial infections, more preferably
infections by Staphylococcus aureus, comprising determining from a
sample derived from an individual a increased level of expression
of polynucleotide having a sequence of Table 1 [SEQ ID NO: 1 or 3].
Increased or decreased expression of ribG polynucleotide can be
measured using any on of the methods well known in the art for the
quantation of polynucleotides, such as, for example, amplification,
PCR, RT-PCR, RNase protection, Northern blotting and other
hybridization methods.
[0088] In addition, a diagnostic assay in accordance with the
invention for detecting over-expression of ribG protein 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 ribG protein, in a sample derived from a host
are well-known to those of skill in the art. Such assay methods
include radioimmunoassays, competitive-binding assays, Western Blot
analysis and ELISA assays.
[0089] Antibodies
[0090] The polypeptides of the invention or variants thereof, or
cells expressing them can be used as an immunogen to produce
antibodies immunospecific for such polypeptides. "Antibodies" as
used herein includes monoclonal and polyclonal antibodies,
chimeric, single chain, simianized antibodies and humanized
antibodies, as well as Fab fragments, including the products of an
Fab immunolglobulin expression library.
[0091] Antibodies generated against the polypeptides of the
invention can be obtained by administering the polypeptides or
epitope-bearing fragments, analogues or cells to an animal,
preferably a nonhuman, using routine protocols. For preparation of
monoclonal antibodies, any technique known in the art that provides
antibodies produced by continuous cell line cultures can be used.
Examples include various techniques, such as those in Kohler, G.
and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al.,
Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONAL
ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
[0092] Techniques for the production of single chain antibodies
(U.S. Pat. No. 4,946,778) can be adapted to produce single chain
antibodies to polypeptides of this invention. Also, transgenic
mice, or other organisms such as other mammals, may be used to
express humanized antibodies.
[0093] 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-ribG 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).
[0094] If two antigen binding domains are present each domain may
be directed against a different epitope--termed `bispecific`
antibodies.
[0095] The above-described antibodies may be employed to isolate or
to identify clones expressing the polypeptides to purify the
polypeptides by affinity chromatography.
[0096] Thus, among others, antibodies against ribG-polypeptide may
be employed to treat infections, particularly bacterial
infections.
[0097] Polypeptide variants include antigenically, epitopically or
immunologically equivalent variants that form a particular aspect
of this invention. The term "antigenically equivalent derivative"
as used herein encompasses a polypeptide or its equivalent which
will be specifically recognized by certain antibodies which, when
raised to the protein 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.
[0098] The polypeptide, such as an antigenically or immunologically
equivalent derivative or a fusion protein 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 immunogenic carrier protein for example bovine
serum albumin (BSA) or keyhole limpet haemocyanin (KLH).
Alternatively a multiple antigenic peptide comprising multiple
copies of the protein or polypeptide, or an antigenically or
immunologically equivalent polypeptide thereof may be sufficiently
antigenic to improve immunogenicity so as to obviate the use of a
carrier.
[0099] Preferably, the antibody or variant thereof is modified to
make it less immunogenic in the individual. For example, if the
individual is human the antibody may most preferably be
"humanized"; where the complimentarity determining region(s) of the
hybridoma-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.
[0100] The use of a polynucleotide of the invention in 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 (Benvenisty & Reshef, PNAS USA,
1986:83,9551), encapsulation of DNA in various forms of liposomes
(Kaneda et al., Science 1989:243,375), particle bombardment (Tang
et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol
1993, 12:79 1) and in vivo infection using cloned retroviral
vectors (Seeger et al., PNAS USA 1984:81,5849).
[0101] Antagonists and Agonists--Assays and Molecules
[0102] Polypeptides 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. These 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).
[0103] The invention also provides a method of screening compounds
to identify those which enhance (agonist) or block (antagonist) the
action of ribG polypeptides or polynucleotides, particularly those
compounds that are bacteriostatic and/or bacteriocidal. The method
of screening may involve high-throughput techniques. For example,
to screen for agonists or antagoists, a synthetic reaction mix, a
cellular compartment, such as a membrane, cell envelope or cell
wall, or a preparation of any thereof, comprising ribG polypeptide
and a labeled substrate or ligand of such polypeptide is incubated
in the absence or the presence of a candidate molecule that may be
a ribg agonist or antagonist. The ability of the candidate molecule
to agonize or antagonize the ribG polypeptide is reflected in
decreased binding of the labeled ligand or decreased production of
product from such substrate. Molecules that bind gratuitously, ie.,
without inducing the effects of ribG polypeptide are most likely to
be good antagonists. Molecules that bind well and increase the rate
of product production from substrate are agonists. Detection of the
rate or level of production of product from substrate may be
enhanced by using a reporter system. Reporter systems that may be
useful in this regard include but are not limited to colorimetric
labeled substrate converted into product, a reporter gene that is
resporisive to changes in ribG polynucleotide or polypeptide
activity, and binding assays known in the art.
[0104] Another example of an assay for ribG antagonists is a
competitive assay that combines ribG and a potential antagonist
with ribG-binding molecules, recombinant ribG binding molecules,
natural substrates or ligands, or, substrate or ligand mimetics,
under appropriate conditions for a competitive inhibition assay.
RibG can be labeled, such as by radioactivity or a calorimetric
compound, such that the number of ribG molecules bound to a binding
molecule or converted to product can be determined accurately to
assess the effectiveness of the potential antagonist.
[0105] Potential antagonists include small organic molecules,
peptides, polypeptides and antibodies that bind to a polynucleotide
or polypeptide of the invention and thereby inhibit or extinguish
its activity. Potential antagonists also may be small organic
molecules, a peptide, a polypeptide such as a closely related
protein or antibody that binds the same sites on a binding
molecule, such as a binding molecule, without inducing ribG-induced
activities, thereby preventing the action of ribG by excluding ribg
from binding.
[0106] Potential antagonists include a small molecule that binds to
and occupies the binding site of the polypeptide thereby preventing
binding to cellular binding molecules, such that normal biological
activity is prevented. Examples of small molecules include but are
not limited to small organic molecules, peptides or peptide-like
molecules. Other potential antagonists include antisense molecules
(see Okano, J Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS
ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton,
Fla. (1988), for a description of these molecules). Preferred
potential antagonists include compounds related to and variants of
ribG.
[0107] Each of the DNA sequences provided herein may be used in the
discovery and development of antibacterial compounds. The encoded
protein, upon expression, can be used as a target for the screening
of antibacterial drugs. Additionally, the DNA sequences encoding
the amino terminal regions of the encoded protein or Shine-Delgarno
or other translation facilitating sequences of the respective mRNA
can be used to construct antisense sequences to control the
expression of the coding sequence of interest.
[0108] The invention also provides the use of the polypeptide,
polynucleotide or inhibitor of the invention to interfere with the
initial physical interaction between 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 in-dwelling devices or to
extracellular matrix proteins in wounds; to block ribG
protein-mediated mammalian cell invasion by, for example,
initiating phosphorylation of mammalian tyrosine kinases
(Rosenshine et al., Infect. Immun. 60:2211 (1992); to block
bacterial adhesion between mammalian extracellular matrix proteins
and bacterial ribG proteins 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.
[0109] The antagonists and agonists: of the invention may be
employed, for instance, to inhibit and treat diseases.
[0110] Helicobacter pylori (herein H. pylori) bacteria infect the
stomachs of over one-third of the world's population causing
stomach cancer, ulcers, and gastritis (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. pylon and gastric
adenocarcinoma, classifying the bacterium as a Group I (definite)
carcinogen. Preferred antimicrobial compounds of the invention
(agonists and antagonists of ribG) found using screens provided by
the invention, particularly broad-spectrum antibiotics, should be
useful in the treatment of H. pylori infection. Such treatment
should decrease the advent of H. pylori-induced cancers, such as
gastrointestinal carcinoma. Such treatment should also cure gastric
ulcers and gastritis.
[0111] Vaccines
[0112] Another aspect of the invention relates to a method for
inducing an immunological response in an individual, particularly a
mammal which comprises inoculating the individual with ribG, 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
ribG, or a fragment or a variant thereof, for expressing ribG, or a
fragment or a variant thereof in vivo in order to induce an
immunological response, such as, to produce antibody and/ or T cell
immune response, including, for example, cytokine-producing 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.
[0113] A further aspect of the invention relates to an
immunological composition which, when introduced into an individual
capable or having induced within it an immunological response,
induces an immunological response in such individual to a ribG or
protein coded therefrom, wherein the composition comprises a
recombinant ribG or protein coded therefrom comprising DNA which
codes for and expresses an antigen of said ribG 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.
[0114] A ribG polypeptide or a fragment thereof may be fused with
co-protein which may not by itself produce antibodies, but is
capable of stabilizing the first protein and producing a fused
protein which will have immunogenic and protective properties. Thus
fused recombinant protein, preferably further comprises an
antigenic co-protein, such as lipoprotein D from Hemophilus
influenzae, Glutathione-S-transferase (GST) or beta-galactosidase,
relatively large co-proteins which solubilize the protein and
facilitate production and purification thereof. Moreover, the
co-protein may act as an adjuvant in the sense of providing a
generalized stimulation of the immune system. The co-protein may be
attached to either the amino or carboxy terminus of the first
protein.
[0115] 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).
[0116] 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 in 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.
[0117] 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 skin
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.
[0118] The invention also includes a vaccine formulation which
comprises an immunogenic recombinant protein of the invention
together with a suitable carrier. Since the protein may be broken
down in the stomach, it is preferably administered parenterally,
including, for example, administration that is subcutaneous,
intramuscular, intravenous, or intradermal. Formulations suitable
for parenteral administration include aqueous and non-aqueous
sterile injection solutions which may contain anti-oxidants,
buffers, bacteriostats and solutes which render the formulation
insotonic 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-dried 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
immunogenicity of the formulation, such as oil-in 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.
[0119] While the invention has been described with reference to
certain ribg protein, it is to be understood that this covers
fragments of the naturally occurring protein and similar proteins
with additions, deletions or substitutions which do not
substantially affect the immunogenic properties of the recombinant
protein.
[0120] Compositions, Kits and Administration
[0121] The invention also relates to compositions comprising the
polynucleotide or the polypeptides discussed above or their
agonists or antagonists. The polypeptides of the invention may be
employed in combination with a non-sterile or sterile carrier or
carriers for use with cells, tissues or organisms, such as a
pharmaceutical carrier suitable for administration to a subject.
Such compositions comprise, for instance, a media additive or a
therapeutically effective amount of a polypeptide of the invention
and a pharmaceutically acceptable carrier or excipient. Such
carriers may include, but are not limited to, saline, buffered
saline, dextrose, water, glycerol, ethanol and combinations
thereof. The formulation should suit the mode of administration.
The invention further relates to diagnostic and pharmaceutical
packs and kits comprising one or more containers filled with one or
more of the ingredients of the aforementioned compositions of the
invention.
[0122] Polypeptides and other compounds of the invention may be
employed alone or in conjunction with other compounds, such as
therapeutic compounds.
[0123] The pharmaceutical compositions may be administered in any
effective, convenient manner including, for instance,
administration by topical, oral, anal, vaginal, intravenous,
intraperitoneal, intramuscular, subcutaneous, intranasal or
intradermal routes among others.
[0124] In therapy or as a prophylactic, the active agent may be
administered to an individual as an injectable composition, for
example as a sterile aqueous dispersion, preferably isotonic.
[0125] Alternatively the composition may be formulated for topical
application for example in 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 1% to about 98% by weight of the formulation; more
usually they will constitute up to about 80% by weight of the
formulation.
[0126] 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.
[0127] In-dwelling devices include surgical implants, prosthetic
devices and catheters, i.e., devices that are introduced to the
body of an individual and remain in position for an extended time.
Such devices include, for example, artificial joints, heart valves,
pacemakers, vascular grafts, vascular catheters, cerebrospinal
fluid shunts, urinary catheters, continuous ambulatory peritoneal
dialysis (CAPD) catheters.
[0128] The composition of the invention may be administered by
injection to achieve a systemic effect against relevant bacteria
shortly before insertion of an in-dwelling device. Treatment may be
continued after surgery during the in-body time of the device. In
addition, the composition could also be used to broaden
perioperative cover for any surgical technique to prevent bacterial
wound infections, especially Staphylococcus aureus wound
infections.
[0129] 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.
[0130] 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 in dental treatment as an
alternative to, or in conjunction with, antibiotic prophylaxis.
[0131] 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
.mu.g/ml to 10 mg/ml for bathing of wounds or indwelling
devices.
[0132] 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 prteferably 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.
[0133] Each reference disclosed herein is incorporated by reference
herein in its entirety. Any patent application to which this
application claims priority is also incorporated by reference
herein in its entirety.
[0134] Glossary
[0135] The following definitions are provided to facilitate
understanding of certain terms used frequently herein.
[0136] "Disease(s)" means and disease caused by or related to
infection by a bacteria, including disease, such as, infections of
the upper respiratory tract (e.g., otitis media, bacterial
tracheitis, acute epiglottitis, thyroiditis), lower respiratory
(e.g., empyema, lung abscess), cardiac (e.g., infective
endocarditis), gastrointestinal (e.g., secretory diarrhoea, splenic
absces, retroperitoneal abscess), CNS (e.g., cerebral abscess), eye
(e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis,
preseptal and orbital cellulitis, darcryocystitis), kidney and
urinary tract (e.g., epididymitis, intrarenal and perinephric
absces, toxic shock syndrome), skin (e.g., impetigo, folliculitis,
cutaneous abscesses, cellulitis, wound infection, bacterial
myositis) bone and joint (e.g., septic arthritis,
osteomyelitis).
[0137] "Host cell" is a cell which has been transformed or
transfected, or is capable of transformation or transfection by an
exogenous polynucleotide sequence.
[0138] "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, Gribskov, M. and Devereux, J.,
eds., M Stockton Press, New York, 1991; and Carillo, H., and
Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred
methods to determine identity are designed to give the largest
match between the sequences tested. Methods to determine identity
and similarity are codified in 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% of the 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 within the reference sequence. Analogously, by a
polypeptide having an amino 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 amino 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 amino acid sequence at
least 95% identical to a reference amino acid sequence, up to 5% of
the amino acid residues in the reference sequence may be deleted or
substituted with another amino acid, or a number of amino acids up
to 5% of the total amino 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 terminal
positions of the reference amino acid sequence or anywhere between
those terminal positions, interspersed either individually among
residues in the reference sequence or in one or more contiguous
groups within the reference sequence.
[0139] "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 original environment, or both. For example, a
polynucleotide or a polypeptide naturally present in a living
organism is not "isolated," but the same polynucleotide or
polypeptide separated from the coexisting materials of its natural
state is "isolated", as the term is employed herein. Moreover, a
polynucleotide or polypeptide that is introduced into an organism
by transformation, genetic manipulation or by any other recombinant
method is "isolated" even if it is still present in said organism,
which organism may be living or non-living.
[0140] "Polynucleotide(s)" generally refers to any
polyribonucleotide or polydeoxribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide(s)"
include, without limitation, single- and double-stranded DNA, DNA
that is a mixture of single- and double-stranded regions or
single-, double- and triple-stranded regions, single- and
double-stranded RNA, and RNA that is mixture of single- and
double-stranded regions, hybrid molecules comprising DNA and RNA
that may be single-stranded or, more typically, double-stranded, or
triple-stranded regions, or a mixture of single- and
double-stranded regions. In addition, "polynucleotide" as used
herein refers to triple-stranded regions comprising 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 of the molecules, but more typically involve only a
region of some of the molecules. One of the molecules of a
triple-helical region often is an oligonucleotide. As used herein,
the term "polynucleotide(s)" also includes DNAs or RNAs as
described above that contain one or more modified bases. Thus, DNAs
or RNAs with backbones modified for stability or for other reasons
are "polynucleotide(s)" as that term is intended herein. Moreover,
DNAs or RNAs comprising unusual bases, such as inosine, or modified
bases, such as tritylated bases, to name just two examples, are
polynucleotides as the term is used herein. It will be appreciated
that a great variety of modifications have been made to DNA and RNA
that serve many useful purposes known to those of skill in the art.
The term "polynucleotide(s)" as it is employed herein embraces such
chemically, enzymatically or metabolically modified forms of
polynucleotides, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells, including,. for example,
simple and complex-cells. "Polynucleotide(s)" also embraces short
polynucleotides often referred to as oligonucleotide(s).
[0141] "Polypeptide(s)" refers to any peptide or protein comprising
two or more amino acids joined to each other by peptide bonds or
modified peptide bonds. "Polypeptide(s)" refers to both short
chains, commonly referred to as peptides, oligopeptides and
oligomers and to longer chains generally referred to as proteins.
Polypeptides may contain amino acids other than the 20 gene encoded
amino acids. "Polypeptide(s)" include those modified either by
natural processes, such as processing and other post-translational
modifications, but also by chemical modification techniques. Such
modifications are well described in basic texts and in more
detailed monographs, as well as in a voluminous
research-literature, and they are well known to those of skill in
the art. It will be appreciated that the same type of modification
may be present in the same or varying degree at several sites in a
given polypeptide. Also, a given polypeptide may contain many types
of modifications. Modifications can occur anywhere in a
polypeptide, including the peptide backbone, the amino acid
side-chains, and the amino or carboxyl termini. Modifications
include, for example, acetylation, acylation, ADP-ribosylation,
amidation, covalent attachment of flavin, covalent attachment of a
heme moiety, covalent attachment of a nucleotide or nucleotide
derivative, covalent attachment of a lipid or lipid derivative,
covalent attachment of phosphotidylinositol, cross-linking,
cyclization, disulfide bond formation, demethylation, formation of
covalent cross-links, formation of cysteine, formation of
pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI
anchor formation, hydroxylation, iodination, methylation,
myristoylation, oxidation, proteolytic processing, phosphorylation,
prenylation, racemization, glycosylation, lipid attachment,
sulfation, gamma-carboxylation of glutamic acid residues,
hydroxylation and ADP-ribosylation, selenoylation, sulfation,
transfer-RNA mediated addition of amino acids to proteins, such as
arginylation, and ubiquitination. See, for instance,
PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,
Posttranslational Protein Modifications: Perspectives and
Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF
PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983);
Seifter et al., Meth. 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 branching. Cyclic, branched and
branched circular polypeptides may result from post-translational
natural processes and may be made by entirely synthetic methods, as
well.
[0142] "Variant(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 amino acid
sequence of a polypeptide encoded by the reference polynucleotide.
Nucleotide changes may result in amino 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 amino 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, in many regions, identical. A
variant and reference polypeptide may differ in amino acid sequence
by one or more substitutions, additions, deletions in any
combination. A substituted or inserted amino acid residue may or
may not be one encoded by the genetic code. A variant of a
polynucleotide or polypeptide may be a naturally occurring such as
an allelic 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.
EXAMPLES
[0143] The examples below are carried out using standard
techniques, which are well known and routine to those of skill in
the art, except where otherwise described in detail. The examples
are illustrative, but do not limit the invention.
Example 1
Strain selection, Library Production and Sequencing
[0144] The polynucleotide having a DNA sequence given in Table 1
[SEQ ID NO: 1 or 3] was obtained from a library of clones of
chromosomal DNA of Staphylococcus aureus in E. coli. The sequencing
data from two or more clones containing overlapping Staphylococcus
aureus DNAs was used to construct the contiguous DNA sequence in
SEQ ID NO: 1. Libraries may be prepared by routine methods, for
example:
[0145] Methods 1 and 2 Below.
[0146] Total cellular DNA is isolated from Staphylococcus aureus
WCUH 29 according to standard procedures and size-fractionated by
either of two methods.
[0147] Method 1
[0148] 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 11 kbp in size are rendered
blunt by treatment with exonuclease and DNA polymerase, and EcoRI
linkers added. Fragments are ligated into the vector Lambda ZapII
that has been cut with EcoRI, the library packaged by standard
procedures and E.coli infected with the packaged library. The
library is amplified by standard procedures.
[0149] Method 2
[0150] Total cellular DNA is partially hydrolyzed with a one or a
combination of restriction enzymes appropriate to generate a series
of fragments for cloning into library vectors (e.g., RsaI, PalI,
AluI, BshI2351), and such. fragments are size-fractionated
according to standard procedures. EcoRI linkers are ligated to the
DNA and the fragments then ligated into the vector Lambda ZapII
that have been cut with EcoRI, the library packaged by standard
procedures, and E.coli infected with the packaged library. The
library is amplified by standard procedures.
Example 2
ribG Characterization
[0151] Riboflavin Biosynthesis Operon
[0152] This ORF is part of an operon which encodes genes ribg,
ribB, ribA and ribH. Gene ribG starts at nucleotide 1 and ends at
nucleotide 1029. Gene ribB starts at nucleotide 1036 and ends at
nucleotide 1668. Gene ribA starts at nucleotide 1679 and ends at
nucleotide 2860. Gene ribH starts at nucleotide 2873 and ends at
nucleotide 3337. The operon sequence below is SEQ ID NO:7.
6 ATGGATTATGCGATTCAACTTGCAAATATGGTACAAGGTCAAACAGGTGTTAATCCA-
CCCGTTGGCGCTG TTGTAGTTAATGAAGGTAGGATTGTTGGTATTGGTGCACACTT-
GAGAAAAGGTGACAAGCATGCGGAGGT TCAAGCACTTGATATGGCACAACAAAATGC-
TGAAGGTGCGACGATTTATATTACGTTAGAGCCATGTAGT
CATTTTGGTTCAACACCACCCTGTGTTAACAAAATTATTGATTGTAAGATAGCAAAAGTAGTATACGCAA
CAAAAGACAATTCGTTAGACACACATGGTGATGAGACGTTACGGGCTCACGGTATTGAGG-
TTGAATGCGT TGATGATGAACGGGCATCACAATTATACCAAGACTTTTTTAAAGCAA-
AAGCAAAGCAACTGCCACAAATT ACAGTGAAAGTATCTGCAAGTTTAGATGGTAAAC-
AAGCGAATGATAATGGACAAAGTCAATGGATTACTA
ACAAAGAGGTTAAACAAGATGTCTATAAGTTAAGACATCGACACGACGCAGTGTTAACTGGAAGACGTAC
AGTTGAATTAGATGATCCACAATATACTACACGTATTCAAGATGGAAAAAACCCTATAAA-
AGTAATATTG TCTAAGTCTGGGAATATTCATTTTAATCAGCAAATTTATCAAGATGA-
ATCAACACCAATTTGGATATATA CTGAAAATCCAAATTTAACAAGCAATCAAACACA-
TATTGAAATTATTTACTTGAAGTCTTGTGATTTAAC
AACAATTCTTCACAATTTATATAAAAGAGGAGTTGGAACTTTGCTAGTCGAGGCAGGTCCAACCACTACT
TCAGAATTCTCCATCTATTATATAGATGAATTTATTCTCTATTATGCCCCGAAATTAATT-
GGCGGATCTG GAAATTATCAATTTTATCAAACAAATGATGTGATTGAGATACCAGAT-
GCGAACCAATTTGAAATTGTTCA TTCCGAGTTATTAAATCAAAATGTTAAATTAACT-
TTACGAAAGAAGTGATGATGCATGTTTACTGGCATC
GTTGAAGAAATAGGTGTCGTTAAAAGTGTTCAAATTCGTCAATCAGTCAGGACGATTGAAATCGAAGCAC
ATAAGATTACGGCAGATATGCATATTGGTGATTCCATCAGTGTTAATGGTGCATGTTTAA-
CAGTGATTGA TTTTGATCAGACATCTTTTACTGTTCAAGTAATTAAAAGCACTGAAA-
ATAAAACCTATTTAGCAGATGTT AAGCGACAATCAGAAGTAAATTTAGAGCGTGCCA-
TGAGTGGTAACGGTAGGTTTGGTGGACATTTTGTCC TCGGTCATGTAGATGAACTAGGAACAGTTT
CAAAAATAAATGAAACAGCCAATGCC-
AAAATTATTACGATTCAATGTAGCCAACATATTAATAATCAGTT
AGTTAAGCAAGGGTCTATTACTGTGGATGGTGTAAGTCTAACGGTATTTGATAAGCATGATAACAGTTTT
GACATTCATCTTATTCCAGAAACGAGGCGTTCAACGATTTTATCATCCAAAAAATTAGGA-
GATAAAGTAC ATTTAGAAACAGACGTTTTGTTTAAATATGTTGAAAATATTTTAAAT-
AAAGATAAAGACCAATTATCTGT AGATAAATTAAGAGCATTTGGTTTTTAGGAGGGG-
TAGCATGCAATTCGATAATATTGACAGTGCTTTAAT
GGCTTTAAAAAATGGAGAAACAATTATTGTAGTAGATGATGAGAATCGTGAAAATGAAGGTGATTTAGTA
GCGGTTACTGAATGGATGAACGATAATACCATTAATTTTATGGCGAAAGAAGCAAGGGGA-
TTAATATGCG CACCAGTGTCTAAAGATATTGCACAACGTTTGGATTTGGTACAAATG-
GTTGATGATAACTCCGACATCTT TGGTACGCAATTTACAGTGAGTATTGATCATGTA-
GATACAACAACAGGAATTAGTGCTTATGAACGTACA
TTGACTGCCAAAAAGCTCATTGATCCTAGTAGTGAAGCTAAAGATTTTAATCGTCCTGGTCATTTATTTC
CATTAGTAGCACAAGATAAAGGCGTATTAGCTAGAAATGGACACACAGAAGCGGCTGTTG-
ATTTAGCTAA ACTTACTGGTGCCAAGCCCGCTGGTGTCATTTGTGAGATTATGAATG-
ATGACGGCACGATGGCGAAAGGA CAAGATTTACAAAATTTTAAAGAAAAACATCAAT-
TAAAGATGATTACGATTGATGATTTAATTGAATATC
GTAAAAAATTAGAACCAGAAATTGAATTTAAGGCAAAAGTGAAAATGCCTACAGATTTCGGAACATTTGA
TATGTATGGTTTTAAAGCGACATACACAGATGAAGAGATAGTTGTACTGACAAAAGGTGC-
AATTCGACAA CATGAAAATGTACGCTTACATTCTGCGTGCCTTACAGGCGATATTTT-
CCATAGTCAACGTTGTGATTGTG GTGCTCAACTTGAATCGTCTATGAAGTATATCAA-
TGAACATGGTGGCATGATTATTTATCTACCTCAAGA
AGGTCGTGGCATAGGATTGTTAAACAAATTACGCGCATATGAATTAATTGAGCAAGGATATGATACAGTA
ACTGCAAATTTAGCATTAGGTTTTGATGAAGATTTACGAGATTATCATATTGCTGCACAG-
ATTTTAAAAT ATTTTAACATCGAACATATCAATTTATTAAGTAATAATCCAAGTAAA-
TTTGAGGGATTAAAACAATATGG CATTGATATTGCAGAAAGAATTGAAGTTATCGTA-
CCAGAAACGGTACATAATCATGATTATATGGTAACG
AAAAAAATAAAAATGGGTCATTTAATATAGGAGGACTTTAACATGAATTTTGAAGGTAAATTAATTGGAA
AAGATTTGAAAGTTGCAATCGTAGTTAGTCGATTTAATGATTTTATCACTGGAAGATTAC-
TTGAAGGTGC AAAAGATACTTTGATTCGACATGATGTTAATGAAGACAATATTGATG-
TAGCATTTGTTCCTGGTGCGTTT GAAATTCCTTTAGTAGCTAAAAAATTAGCCTCAT-
CAGGAAATTATGATGCAATAATTACATTAGGATGCG
TAATTCGCGGTGCTACGTCTCATTATGATTATGTTTGTAATGAAGTGCGAAAGGTGTTTCTAAAGTAAAT
GATCAAACTAATGTACCAGTCATATTTGGTATTTTAACGACTGAAAGTATTGAACAAGCT-
GTGGAAAGAG CAGGTACGAAAGCTGGTAATAAAGGTGCCGAAGCAGCAGTAAGTGCA-
ATTGAAATGGCTAATTTATTAAA ATCTATAAAAGCATAG
[0153]
Sequence CWU 1
1
* * * * *
References