U.S. patent application number 09/735564 was filed with the patent office on 2002-09-12 for murf2.
Invention is credited to Biswas, Sanjoy, Brown, James Raymond, Burnham, Martin K. R., Chalker, Alison Francis, Holmes, David John, Ingraham, Karen Anne, Mathie, Thomas Berthold, Warren, Richard Lloyd, Zalacain, Magdalena.
Application Number | 20020127596 09/735564 |
Document ID | / |
Family ID | 22282079 |
Filed Date | 2002-09-12 |
United States Patent
Application |
20020127596 |
Kind Code |
A1 |
Biswas, Sanjoy ; et
al. |
September 12, 2002 |
murF2
Abstract
The invention provides murF2 polypeptides and polynucleotides
encoding murF2 polypeptides and methods for producing such
polypeptides by recombinant techniques. Also provided are methods
for utilizing murF2 polypeptides to screen for antibacterial
compounds.
Inventors: |
Biswas, Sanjoy; (Paoli,
PA) ; Brown, James Raymond; (Berwyn, PA) ;
Burnham, Martin K. R.; (Barto, PA) ; Chalker, Alison
Francis; (Trappe, PA) ; Holmes, David John;
(West Chester, PA) ; Ingraham, Karen Anne;
(Auburn, PA) ; Mathie, Thomas Berthold;
(Eagleville, PA) ; Warren, Richard Lloyd; (Blue
Bell, PA) ; Zalacain, Magdalena; (West Chester,
PA) |
Correspondence
Address: |
DECHERT
ATTN: ALLEN BLOOM, ESQ
4000 BELL ATLANTIC TOWER
1717 ARCH STREET
PHILADELPHIA
PA
19103
US
|
Family ID: |
22282079 |
Appl. No.: |
09/735564 |
Filed: |
December 13, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09735564 |
Dec 13, 2000 |
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09305001 |
May 4, 1999 |
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60100894 |
Sep 23, 1998 |
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Current U.S.
Class: |
435/7.1 ;
435/7.21; 530/350; 536/23.1 |
Current CPC
Class: |
C07K 2319/00 20130101;
A61P 35/00 20180101; A61K 48/00 20130101; A61P 31/04 20180101; A61K
38/00 20130101; C07K 14/3156 20130101 |
Class at
Publication: |
435/7.1 ;
435/7.21; 530/350; 536/23.1 |
International
Class: |
G01N 033/53; G01N
033/53; G01N 033/567; C07H 021/02; C07H 021/04; A61K 031/70; A01N
043/04; C07K 001/00; C07K 014/00; C07K 017/00 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
(i) an isolated polypeptide comprising an amino acid having at
least 95% identity to the amino acid sequence of SEQ ID NO:2 over
the entire length of SEQ ID NO:2; (ii) an isolated polypeptide
comprising the amino acid sequence of SEQ ID NO:2, (iii) an
isolated polypeptide that is the amino acid sequence of SEQ ID
NO:2, and (iv) a polypeptide that is encoded by a recombinant
polynucleotide comprising the polynucleotide sequence of SEQ ID
NO:1.
2. An isolated polynucleotide selected from the group consisting
of: (i) an isolated polynucleotide comprising a polynucleotide
sequence encoding a polypeptide that has at least 95% identity to
the amino acid sequence of SEQ ID NO:2, over the entire length of
SEQ ID NO:2; (ii) an isolated polynucleotide comprising a
polynucleotide sequence that has at least 95% identity over its
entire length to a polynucleotide sequence encoding the polypeptide
of SEQ ID NO:2; (iii) an isolated polynucleotide comprising a
nucleotide sequence that has at least 95% identity to that of SEQ
ID NO:1 over the entire length of SEQ ID NO:1; (iv) an isolated
polynucleotide comprising a nucleotide sequence encoding the
polypeptide of SEQ ID NO:2; (v) an isolated polynucleotide that is
the polynucleotide of SEQ ID NO:1; (vi) an isolated polynucleotide
of at least 30 nucleotides in length obtainable by screening an
appropriate library under stringent hybridization conditions with a
probe having the sequence of SEQ ID NO:1 or a fragment thereof of
of at least 30 nucleotides in length; (vii) an isolated
polynucleotide encoding a mature polypeptide expressed by the murF2
gene comprised in the Streptococcus pneumoniae; and (viii) a
polynucleotide sequence complementary to said isolated
polynucleotide of (i), (ii), (iii), (iv), (v), (vi) or (vii).
3. A method for the treatment of an individual: (i) in need of
enhanced activity or expression of or immunological response to the
polypeptide of claim 1 comprising the step of: administering to the
individual a therapeutically effective amount of an antagonist to
said polypeptide; or (ii) having need to inhibit activity or
expression of the polypeptide of claim 1 comprising: (a)
administering to the individual a therapeutically effective amount
of an antagonist to said polypeptide; or (b) administering to the
individual a nucleic acid molecule that inhibits the expression of
a polynucleotide sequence encoding said polypeptide; (c)
administering to the individual a therapeutically effective amount
of a polypeptide that competes with said polypeptide for its
ligand, substrate, or receptor, or (d) administering to the
individual an amount of a polypeptide that induces an immunological
response to said polypeptide in said individual.
4. A process for diagnosing or prognosing a disease or a
susceptibility to a disease in an individual related to expression
or activity of the polypeptide of claim 1 in an individual
comprising the step of: (a) determining the presence or absence of
a mutation in the nucleotide sequence encoding said polypeptide in
an organism in said individual; or (b) analyzing for the presence
or amount of said polypeptide expression in a sample derived from
said individual.
5. A process for producing a polypeptide selected from the group
consisting of: (i) an isolated polypeptide comprising an amino acid
sequence selected from the group having at least 95% identity to
the amino acid sequence of SEQ ID NO:2 over the entire length of
SEQ ID NO:2; (ii) an isolated polypeptide comprising the amino acid
sequence of SEQ ID NO:2; (iii) an isolated polypeptide that it the
amino acid sequence of SEQ ID NO:2, and (iv) a polypeptide that is
encoded by a recombinant polynucleotide comprising the
polynucleotide sequence of SEQ ID NO:1, comprising the step of
culturing a host cell under conditions sufficient for the
production of the polypeptide.
6. A process for producing a host cell comprising an expression
system or a membrane thereof expressing a polypeptide selected from
the group consisting of: (i) an isolated polypeptide comprising an
amino acid sequence selected from the group having at least 95%
identity to the amino acid sequence of SEQ ID NO:2 over the entire
length of SEQ ID NO:2; (ii) an isolated polypeptide comprising the
amino acid sequence of SEQ ID NO:2; (iii) an isolated polypeptide
that is the amino acid sequence of SEQ ID NO:2, and (iv) a
polypeptide that is encoded by a recombinant polynucleotide
comprising the polynucleotide sequence of SEQ ID NO:1, said process
comprising the step of transforming or transfecting a cell with an
expression system comprising a polynucleotide capable of producing
said polypeptide of (i), (ii), (iii) or (iv) when said expression
system is present in a compatible host cell such the host cell,
under appropriate culture conditions, produces said polypeptide of
(i), (ii), (iii) or (iv).
7. A host cell or a membrane expressing a polypeptide selected from
the group consisting of: (i) an isolated polypeptide comprising an
amino acid sequence selected from the group having at least 95%
identity to the amino acid sequence of SEQ ID NO:2 over the entire
length of SEQ ID NO:2; (ii) an isolated polypeptide comprising the
amino acid sequence of SEQ ID NO:2; (iii) an isolated polypeptide
that is the amino acid sequence of SEQ ID NO:2, and (iv) a
polypeptide that is encoded by a recombinant polynucleotide
comprising the polynucleotide sequence of SEQ ID NO:1.
8. An antibody immunospecific for the polypeptide of claim 1.
9. A method for screening to identify compounds that agonize or
that inhibit the function of the polypeptide of claim 1 that
comprises a method selected from the group consisting of: (a)
measuring the binding of a candidate compound to the polypeptide
(or to the cells or membranes bearing the polypeptide) or a fusion
protein thereof by means of a label directly or indirectly
associated with the candidate compound; (b) measuring the binding
of a candidate compound to the polypeptide (or to the cells or
membranes bearing the polypeptide) or a fusion protein thereof in
the presence of a labeled competitor; (c) testing whether the
candidate compound results in a signal generated by activation or
inhibition of the polypeptide, using detection systems appropriate
to the cells or cell membranes bearing the polypeptide; (d) mixing
a candidate compound with a solution comprising a polypeptide of
claim 1, to form a mixture, measuring activity of the polypeptide
in the mixture, and comparing the activity of the mixture to a
standard; or (e) detecting the effect of a candidate compound on
the production of mRNA encoding said polypeptide and said
polypeptide in cells, using for instance, an ELISA assay.
10. An agonist or antagonist to the polypeptide of claim 1.
Description
RELATED APPLICATIONS
[0001] This application claims benefit to U.S. Provisional
Application No. 60/100,894, filed Sep. 23, 1998.
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 polynucleotides and polypeptides of the
mur family, as well as their variants, herein referred to as
"murF2," "murF2 polynucleotide(s)," and "murF2 polypeptide(s)" as
the case may be.
BACKGROUND OF THE INVENTION
[0003] The Streptococci make up a medically important genera of
microbes known to cause several types of disease in humans,
including, for example, otitis media, conjunctivitis, pneumonia,
bacteremia, meningitis, sinusitis, pleural empyema and
endocarditis, and most particularly meningitis, such as for example
infection of cerebrospinal fluid. Since its isolation more than 100
years ago, Streptococcus pneumoniae has been one of the more
intensively studied microbes. For example, much of our early
understanding that DNA is, in fact, the genetic material was
predicated on the work of Griffith and of Avery, Macleod and
McCarty using this microbe. Despite the vast amount of research
with S. pneumoniae, many questions concerning the virulence of this
microbe remain. It is particularly preferred to employ
Streptococcal genes and gene products as targets for the
development of antibiotics.
[0004] The frequency of Streptococcus pneumoniae 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 Streptococcus
pneumoniae strains that are resistant to some or all of the
standard antibiotics. This phenomenon has created an unmet medical
need and demand for new anti-microbial agents, vaccines, drug
screening methods, and diagnostic tests for this organism.
[0005] Moreover, the drug discovery process is currently undergoing
a fundamental revolution as it embraces "functional genomics," that
is, high throughput genome- or gene-based biology. This approach is
rapidly superseding earlier approaches based on "positional
cloning" and other methods. Functional genomics relies heavily on
the various tools of bioinformatics to identify gene sequences of
potential interest from the many molecular biology databases now
available as well as from other sources. There is a continuing and
significant need to identify and characterize further genes and
other polynucleotides sequences and their related polypeptides, as
targets for drug discovery.
[0006] Clearly, there exists a need for polynucleotides and
polypeptides, such as the murF2 embodiments of the invention, that
have a present benefit of, among other things, being useful to
screen compounds for antimicrobial activity. Such factors are also
useful to determine their role in pathogenesis of infection,
dysfunction and disease. 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.
SUMMARY OF THE INVENTION
[0007] The present invention relates to murF2, in particular murF2
polypeptides and murF2 polynucleotides, recombinant materials and
methods for their production. In another aspect, the invention
relates to methods for using such polypeptides and polynucleotides,
including treatment of microbial diseases, amongst others. In a
further aspect, the invention relates to methods for identifying
agonists and antagonists using the materials provided by the
invention, and for treating microbial infections and conditions
associated with such infections with the identified agonist or
antagonist compounds. In a still further aspect, the invention
relates to diagnostic assays for detecting diseases associated with
microbial infections and conditions associated with such
infections, such as assays for detecting murF2 expression or
activity.
[0008] 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.
DISCLOSURE OF THE INVENTION
[0009] The invention relates to murF2 polypeptides and
polynucleotides as described in greater detail below. In
particular, the invention relates to polypeptides and
polynucleotides of a murF2 of Streptococcus pneumoniae, that is
related by amino acid sequence homology to
gi.vertline.1653484.vertline.gnl.vertline.PID.vertline.d1019130
(D90914) hypothetical protein [Synechocystis sp polypeptide. The
invention relates especially to murF2 having a nucleotide and amino
acid sequences set out in Table 1 as SEQ ID NO:1 and SEQ ID NO:2
respectively. Note that sequences recited in the Sequence Listing
below as "DNA" represent an exemplification of the invention, since
those of ordinary skill will recognize that such sequences can be
usefully employed in polynucleotides in general, including
ribopolynucleotides.
1TABLE 1 murF2 Polynucleotide and Polypeptide Sequences (A)
Streptococcus pneumoniae murF2 polynucleotide sequence [SEQ ID NO:
1]. 5'-
ATGAACTTAAAAACTACTTTGGGCCTTCTTGCTGGGCGTTCTTCCCACTTCGTTTTAAGCCGTCTTGGACGT-
GGAAGTAC GCTCCCAGGGAAAGTCGCCCTTCAATTTGATAAAGATATTTTACAAAAC-
CTAGCTAAGAACTACGAGATTGTCGTTGTCA CTGGAACAAATGGAAAAACCCTGACA-
ACTGCCCTCACTGTCGGCATTTTAAAAGAGGTTTATGGTCAAGTTCTAACCAAC
CCAAGCGGTGCCAACATGATTACAGGGATTGCAACAACCTTCCTAACAGCCAAATCTTCTAAAACTGGGAAAA-
ATATTGC CGTCCTCGAAATTGACGAAGCCAGTCTATCTCGTATCTGTGACTATATCC-
AGCCTAGTCTTTTTGTCATTACTAATATCT TCCGTGACCAGATGGACCGTTTCGGTG-
AAATCTATACTACCTATAACATGATATTGGATGCCATTCGGAAAGTTCCAACT
GCTACTGTTCTCCTTAACGGAGACAGTCCACTTTTCTACAAGCCAACTATTCCAAACCCTATAGAGTATTTTG-
GTTTTGA CTTGGAAAAAGGACCAGCCCAACTGGCTCACTACAATACCGAAGGGATTC-
TCTGTCCTGACTGCCAAGGCATCCTCAAAT ATGAGCATAATACCTATGCAAACTTGG-
GTGCCTATATCTGTGAGGGTTGTGGATGTAAACGTCCTGATCTCGACTATCGT
TTGACAAAACTGGTTGAGTTGACCAACAATCGCTCTCGCTTTGTCATAGACGGCCAAGAATACGGTATCCAAA-
TCGGCGG GCTCTATAATATCTATAACGCCCTAGCTGCTGTGGCCATCGCCCGTTTCC-
TAGGTGCCGATTGGCAACTCATCAAACAGG GATTTGACAAGAGCCGTGCTGTCTTTG-
GACGCCAAGAAACCTTTCATATCGGTGACAAGGAATGTACCCTTGTCTTGATT
AAAAATCCAGTCGGTGCAACCCAAGCTATCGAAATGATCAAACTAGCACCTTATCCATTTAGCCTATCTGTCC-
TCCTTAA TGCCAACTATGCAGATGGAATTGACACTAGCTGGATCTGGGATGCAGACT-
TTGAGCAAATCACTGACATGGACATTCCTG AAATCAACGCTGGCGGTGTTCGTCATT-
CTGAAATCGCTCGTCGCCTCCGAGTGACTGGCTATCCAGCTGAGAAAATCACT
GAAACGAGTAATCTGGAGCAAGTTCTCAAGACCATTGAGAATCAAGACTGCAAGCATGCCTATATTCTGGCAA-
CTTATAC TGCCATGCTGGAATTTCGTGAACTGCTGGCTAGTCGTCAGATTGTTAGAA-
AGGAGATGAACTAA-3' (B) Streptococcus pneumoniae murF2 polypeptide
sequence deduced from a polynucleotide sequence in this table [SEQ
ID NO:2]. NH.sub.2-
MNLKTTLGLLAGRSSHFVLSRLGRGSTLPGKVALQFDKDILQNLAKNYEIVVVTGTNGKTLTTALTVGILKEV-
YGQVLTN PSGANMITGTATTFLTAKSSKTGKNIAVLEIDEASLSRICDYIQPSLFVT-
TNIFRDQMDRFGEIYTTYNMILDAIRKVPT ATVLLNGDSPLFYKPTIPNPIEYFGFD-
LEKGPAQLAHYNTEGILCPDCQGILKYEHNTYANLGAYICEGCGCKRPDLDYR
LTKLVELTNNRSRFVTDGQEYGIQIGGLYNIYNALAAVAIARFLGADSQLIKQGFDKSRAVFGRQETFHIGDK-
ECTLVLI KNPVGATQAIEMIKLAPYPFSLSVLLNANYADGIDTSWIWDADFEQITDM-
DIPEINAGGVRHSEIARRLRVTGYPAEKIT ETSNLEQVLKTIENQDCKHAYILATYT-
AMLEFRELLASRQIVRKEMN-COOH
Deposited Materials
[0010] A deposit comprising a Streptococcus pneumoniae 0100993
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 Apr. 11, 1996 and assigned
deposit number 40794. The deposit was described as Streptococcus
pneumoniae 0100993 on deposit.
[0011] On Apr. 17, 1996 a Streptococcus pneumoniae 0100993 DNA
library in E. coli was similarly deposited with the NCIMB and
assigned deposit number 40800. The Streptococcus pneumoniae strain
deposit is referred to herein as "the deposited strain" or as "the
DNA of the deposited strain."
[0012] The deposited strain comprises a full length murF2 gene. The
sequence of the polynucleotides comprised in the deposited strain,
as well as the amino acid sequence of any polypeptide encoded
thereby, are controlling in the event of any conflict with any
description of sequences herein.
[0013] 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 deposited 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. .female.112.
A license may be required to make, use or sell the deposited
strain, and compounds derived therefrom, and no such license is
hereby granted.
[0014] In one aspect of the invention there is provided an isolated
nucleic acid molecule encoding a mature polypeptide expressible by
the Streptococcus pneumoniae 0100993 strain, which polypeptide is
comprised in the deposited strain. Further provided by the
invention are murF2 polynucleotide sequences in the deposited
strain, such as DNA and RNA, and amino acid sequences encoded
thereby. Also provided by the invention are murF2 polypeptide and
polynucleotide sequences isolated from the deposited strain.
Polypeptides
[0015] MurF2 polypeptide of the invention is substantially
phylogenetically related to other proteins of the mur family.
[0016] In one aspect of the invention there are provided
polypeptides of Streptococcus pneumoniae referred to herein as
"murF2" and "murF2 polypeptides" as well as biologically,
diagnostically, prophylactically, clinically or therapeutically
useful variants thereof, and compositions comprising the same.
[0017] Among the particularly preferred embodiments of the
invention are variants of murF2 polypeptide encoded by naturally
occurring alleles of a murF2 gene.
[0018] The present invention further provides for an isolated
polypeptide that: (a) comprises or consists of an amino acid
sequence that has at least 95% identity, most preferably at least
97-99% or exact identity, to that of SEQ ID NO:2 over the entire
length of SEQ ID NO:2; (b) a polypeptide encoded by an isolated
polynucleotide comprising or consisting of a polynucleotide
sequence that has at least 95% identity, even more preferably at
least 97-99% or exact identity to SEQ ID NO:1 over the entire
length of SEQ ID NO:1; (c) a polypeptide encoded by an isolated
polypeptide comprising or consisting of a polynucleotide sequence
encoding a polypeptide that has at least 95% identity, even more
preferably at least 97-99% or exact identity, to the amino acid
sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2.
[0019] The polypeptides of the invention include a polypeptide of
Table 1 [SEQ ID NO:2] (in particular a mature polypeptide) as well
as polypeptides and fragments, particularly those that has a
biological activity of murF2, and also those that have at least 95%
identity to a polypeptide of Table 1 [SEQ ID NO:2] and also include
portions of such polypeptides with such portion of the polypeptide
generally comprising at least 30 amino acids and more preferably at
least 50 amino acids.
[0020] The invention also includes a polypeptide consisting of or
comprising a polypeptide of the formula:
X--(R.sub.1).sub.m--(R.sub.2)--(R.sub.3).sub.n--Y
[0021] wherein, at the amino terminus, X is hydrogen, a metal or
any other moiety described herein for modified polypeptides, and at
the carboxyl terminus, Y is hydrogen, a metal or any other moiety
described herein for modified polypeptides, R.sub.1 and R.sub.3 are
any amino acid residue or modified 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 or modified forms thereof. In the formula above, R.sub.2 is
oriented so that the amino terminal amino acid residue is at the
left, covalently bound to R.sub.1,and its carboxy terminal amino
acid residue is at the right, covalently bound to R.sub.3. Any
stretch of amino acid residues denoted by either R.sub.1 or
R.sub.3, where m and/or n is greater than 1, may be either a
heteropolymer or a homopolymer, preferably a heteropolymer. Other
preferred embodiments of the invention are provided where m is an
integer between 1 and 50, 100 or 500, and n is an integer between 1
and 50, 100, or 500.
[0022] It is most preferred that a polypeptide of the invention is
derived from Streptococcus pneumoniae, however, it may preferably
be obtained from other organisms of the same taxonomic genus. A
polypeptide of the invention may also be obtained, for example,
from organisms of the same taxonomic family or order.
[0023] A fragment is a variant polypeptide having an amino acid
sequence that is entirely the same as part but not all of any amino
acid sequence of any polypeptide of the invention. As with murF2
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 in a single larger
polypeptide.
[0024] Preferred fragments include, for example, truncation
polypeptides having a portion of an amino acid sequence of Table 1
[SEQ ID NO:2], or of variants thereof, such as a continuous series
of residues that include an amino- and/or carboxyl-terminal amino
acid sequence. Degradation forms of the polypeptides of the
invention produced by or in a host cell, particularly a
Streptococcus pneumoniae, 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.
[0025] Further preferred fragments include an isolated polypeptide
comprising an amino acid sequence having at least 15, 20, 30, 40,
50 or 100 contiguous amino acids from the amino acid sequence of
SEQ ID NO:2, or an isolated polypeptide comprising an amino acid
sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino
acids truncated or deleted from the amino acid sequence of SEQ ID
NO:2.
[0026] 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.
Polynucleotides
[0027] It is an object of the invention to provide polynucleotides
that encode murF2 polypeptides, particularly polynucleotides that
encode a polypeptide herein designated murF2.
[0028] In a particularly preferred embodiment of the invention the
polynucleotide comprises a region encoding murF2 polypeptides
comprising a sequence set out in Table 1 [SEQ ID NO:1] that
includes a full length gene, or a variant thereof. The Applicants
believe that this full length gene is essential to the growth
and/or survival of an organism that possesses it, such as
Streptococcus pneumoniae.
[0029] As a further aspect of the invention there are provided
isolated nucleic acid molecules encoding and/or expressing murF2
polypeptides and polynucleotides, particularly Streptococcus
pneumoniae murF2 polypeptides and polynucleotides, including, for
example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs, genomic
DNAs, B- and Z-DNAs. Further embodiments of the invention include
biologically, diagnostically, prophylactically, clinically or
therapeutically useful polynucleotides and polypeptides, and
variants thereof, and compositions comprising the same.
[0030] Another aspect of the invention relates to isolated
polynucleotides, including at least one full length gene, that
encodes a murF2 polypeptide having a deduced amino acid sequence of
Table 1 [SEQ ID NO:2] and polynucleotides closely related thereto
and variants thereof.
[0031] In another particularly preferred embodiment of the
invention there is a murF2 polypeptide from Streptococcus
pneumoniae comprising or consisting of an amino acid sequence of
Table 1 [SEQ ID NO:2], or a variant thereof.
[0032] Using the information provided herein, such as a
polynucleotide sequence set out in Table 1 [SEQ ID NO:1], a
polynucleotide of the invention encoding murF2 polypeptide may be
obtained using standard cloning and screening methods, such as
those for cloning and sequencing chromosomal DNA fragments from
bacteria using Streptococcus pneumoniae 0100993 cells as starting
material, followed by obtaining a full length clone. For example,
to obtain a polynucleotide sequence of the invention, such as a
polynucleotide sequence given in Table 1 [SEQ ID NO:1], typically a
library of clones of chromosomal DNA of Streptococcus pneumoniae
0100993 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
hybridization conditions. By sequence the individual clones thus
identified by hybridization with sequencing primers designed from
the original polypeptide or polynucleotide sequence it is then
possible to extend the polynucleotide sequence in both directions
to determine a full length gene sequence. Conveniently, such
sequencing is performed, for example, 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). Direct genomic DNA sequencing
may also be performed to obtain a full length gene sequence.
Illustrative of the invention, each polynucleotide set out in Table
1 [SEQ ID NO:1] was discovered in a DNA library derived from
Streptococcus pneumoniae 0100993.
[0033] Moreover, each DNA sequence set out in Table 1 [SEQ ID NO:1]
contains an open reading frame encoding a protein having about the
number of amino acid residues set forth in Table 1 [SEQ ID NO:2]
with a deduced molecular weight that can be calculated using amino
acid residue molecular weight values well known to those skilled in
the art. The polynucleotide of SEQ ID NO:1, between nucleotide
number 1 and the stop codon that begins at nucleotide number 1342
of SEQ ID NO:1, encodes the polypeptide of SEQ ID NO:2.
[0034] In a further aspect, the present invention provides for an
isolated polynucleotide comprising or consisting of: (a) a
polynucleotide sequence that has at least 95% identity, even more
preferably at least 97-99% or exact identity to SEQ ID NO:1 over
the entire length of SEQ ID NO:1, or the entire length of that
portion of SEQ ID NO:1 which encodes SEQ ID NO:2; (b) a
polynucleotide sequence encoding a polypeptide that has at least
95% identity, even more preferably at least 97%, still more
preferably at least 99%, yet still more preferably at least 99.5%
or 100% exact, to the amino acid sequence of SEQ ID NO:2, over the
entire length of SEQ ID NO:2.
[0035] A polynucleotide encoding a polypeptide of the present
invention, including homologs and orthologs from species other than
Streptococcus pneumoniae, may be obtained by a process that
comprises the steps of screening an appropriate library under
stringent hybridization conditions with a labeled or detectable
probe consisting of or comprising the sequence of SEQ ID NO:1 or a
fragment thereof; and isolating a full-length gene and/or genomic
clones comprising said polynucleotide sequence.
[0036] The invention provides a polynucleotide sequence identical
over its entire length to a coding sequence (open reading frame) in
Table 1 [SEQ ID NO:1]. Also provided by the invention is a coding
sequence for a mature polypeptide or a fragment thereof, by itself
as well as a coding sequence for a mature polypeptide or a fragment
in reading frame with another coding sequence, such as a sequence
encoding a leader or secretory sequence, a pre-, or pro- or
prepro-protein sequence. The polynucleotide of the invention may
also comprise at least one non-coding sequence, including for
example, but not limited to at least one non-coding 5' and 3'
sequence, such as the transcribed but non-translated sequences,
termination signals (such as rho-dependent and rho-independent
termination signals), ribosome binding sites, Kozak sequences,
sequences that stabilize mRNA, introns, and polyadenylation
signals. The polynucleotide sequence may also comprise additional
coding sequence encoding additional amino acids. For example, a
marker sequence that facilitates purification of a 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
peptide tag (Wilson et al., Cell 37: 767 (1984), both of that may
be useful in purifying polypeptide sequence fused to them.
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.
[0037] A preferred embodiment of the invention is a polynucleotide
of consisting of or comprising nucleotide 1 to the nucleotide
immediately upstream of or including nucleotide 1342 set forth in
SEQ ID NO:1 of Table 1, both of that encode a murF2
polypeptide.
[0038] The invention also includes a polynucleotide consisting of
or comprising a polynucleotide of the formula:
X--(R.sub.1).sub.m--(R.sub.2)--(R.sub.3).sub.n--Y
[0039] wherein, at the 5' end of the molecule, X is hydrogen, a
metal or a modified nucleotide residue, or together with Y defines
a covalent bond, and at the 3' end of the molecule, Y is hydrogen,
a metal, or a modified nucleotide residue, or together with X
defines the covalent bond, each occurrence of R.sub.1 and R.sub.3
is independently any nucleic acid residue or modified nucleic acid
residue, m is an integer between 1 and 3000 or zero, n is an
integer between 1 and 3000 or zero, and R.sub.2 is a nucleic acid
sequence or modified nucleic acid sequence of the invention,
particularly a nucleic acid sequence selected from Table 1 or a
modified nucleic acid sequence thereof. In the polynucleotide
formula above, R.sub.2 is oriented so that its 5' end nucleic acid
residue is at the left, bound to R.sub.1, and its 3' end nucleic
acid residue is at the right bound to R.sub.3. Any stretch of
nucleic acid residues denoted by either R.sub.1 and/or R.sub.2,
where m and/or n is greater than 1, may be either a heteropolymer
or a homopolymer, preferably a heteropolymer. Where, in a preferred
embodiment, X and Y together define a covalent bond, the
polynucleotide of the above formula is a closed, circular
polynucleotide, that can be a double-stranded polynucleotide
wherein the formula shows a first strand to which the second strand
is complementary. In another preferred embodiment m and/or n is an
integer between 1 and 1000. Other preferred embodiments of the
invention are provided where m is an integer between 1 and 100 or
500, and n is an integer between 1 and 50, 100, or 500.
[0040] It is most preferred that a polynucleotide of the invention
is derived from Streptococcus pneumoniae, however, it may
preferably be obtained from other organisms of the same taxonomic
genus. A polynucleotide of the invention may also be obtained, for
example, from organisms of the same taxonomic family or order.
[0041] 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
Streptococcus pneumoniae murF2 having an amino acid sequence set
out in Table 1 [SEQ ID NO:2]. The term also encompasses
polynucleotides that include a single continuous region or
discontinuous regions encoding the polypeptide (for example,
polynucleotides interrupted by integrated phage, an integrated
insertion sequence, an integrated vector sequence, an integrated
transposon sequence, or due to RNA editing or genomic DNA
reorganization) together with additional regions, that also may
comprise coding and/or non-coding sequences.
[0042] The invention further relates to variants of the
polynucleotides described herein that encode variants of a
polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID
NO:2]. Fragments of polynucleotides of the invention may be used,
for example, to synthesize full-length polynucleotides of the
invention.
[0043] Further particularly preferred embodiments are
polynucleotides encoding murF2 variants, that have the amino acid
sequence of murF2 polypeptide of Table 1 [SEQ ID NO:2] in which
several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid
residues are substituted, modified, deleted and/or added, in any
combination. Especially preferred among these are silent
substitutions, additions and deletions, that do not alter the
properties and activities of murF2 polypeptide.
[0044] Preferred isolated polynucleotide embodiments also include
polynucleotide fragments, such as a polynucleotide comprising a
nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100
contiguous nucleic acids from the polynucleotide sequence of SEQ ID
NO:1, or an polynucleotide comprising a nucleic acid sequence
having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acid
truncated or deleted from the 5' and/or 3' end of the
polynucleotide sequence of SEQ ID NO:1.
[0045] Further preferred embodiments of the invention are
polynucleotides that are at least 95% or 97% identical over their
entire length to a polynucleotide encoding murF2 polypeptide having
an amino acid sequence set out in Table 1 [SEQ ID NO:2], and
polynucleotides that are complementary to such polynucleotides.
Most highly preferred are polynucleotides that comprise a region
that is 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.
[0046] Preferred embodiments are polynucleotides encoding
polypeptides that retain substantially the same biological function
or activity as a mature polypeptide encoded by a DNA of Table 1
[SEQ ID NO:1].
[0047] In accordance with certain preferred embodiments of this
invention there are provided polynucleotides that hybridize,
particularly under stringent conditions, to murF2 polynucleotide
sequences, such as those polynucleotides in Table 1.
[0048] The invention further relates to polynucleotides that
hybridize to the polynucleotide sequences provided herein. In this
regard, the invention especially relates to polynucleotides that
hybridize under stringent conditions to the polynucleotides
described herein. As herein used, the terms "stringent conditions"
and "stringent hybridization conditions" mean hybridization
occurring only if there is at least 95% and preferably at least 97%
identity between the sequences. A specific 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 (pH 7.6), 5.times.
Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of
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. Solution hybridization may also be used with the
polynucleotide sequences provided by the invention.
[0049] The invention also provides a polynucleotide consisting of
or comprising a polynucleotide sequence obtained by screening an
appropriate library comprising a 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 polynucleotide sequence. Fragments useful for
obtaining such a polynucleotide include, for example, probes and
primers fully described elsewhere herein.
[0050] As discussed elsewhere herein regarding polynucleotide
assays of the invention, for instance, the polynucleotides of the
invention, may be used as a hybridization probe for RNA, cDNA and
genomic DNA to isolate full-length cDNAs and genomic clones
encoding murF2 and to isolate cDNA and genomic clones of other
genes that have a high identity, particularly high sequence
identity, to a murF2 gene. Such probes generally will comprise at
least 15 nucleotide residues or base pairs. Preferably, such probes
will have at least 30 nucleotide residues or base pairs and may
have at least 50 nucleotide residues or base pairs. Particularly
preferred probes will have at least 20 nucleotide residues or base
pairs and will have lee than 30 nucleotide residues or base
pairs.
[0051] A coding region of a murF2 gene may be isolated by screening
using a DNA sequence provided in Table 1 [SEQ ID NO:1] 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.
[0052] There are several methods available and well known to those
skilled in the art to obtain full-length DNAs, or extend short
DNAs, for example those based on the method of Rapid Amplification
of cDNA ends (RACE) (see, for example, Frohman, et al., PNAS USA
85: 8998-9002, 1988). Recent modifications of the technique,
exemplified by the Marathon.TM. technology (Clontech Laboratories
Inc.) for example, have significantly simplified the search for
longer cDNAs. In the Marathon.TM. technology, cDNAs have been
prepared from mRNA extracted from a chosen tissue and an `adaptor`
sequence ligated onto each end. Nucleic acid amplification (PCR) is
then carried out to amplify the "missing" 5' end of the DNA using a
combination of gene specific and adaptor specific oligonucleotide
primers. The PCR reaction is then repeated using "nested" primers,
that is, primers designed to anneal within the amplified product
(typically an adaptor specific primer that anneals further 3' in
the adaptor sequence and a gene specific primer that anneals
further 5' in the selected gene sequence). The products of this
reaction can then be analyzed by DNA sequencing and a full-length
DNA constructed either by joining the product directly to the
existing DNA to give a complete sequence, or carrying out a
separate full-length PCR using the new sequence information for the
design of the 5' primer.
[0053] 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 diseases,
particularly human diseases, as further discussed herein relating
to polynucleotide assays.
[0054] The polynucleotides of the invention that are
oligonucleotides derived from a sequence of Table 1 [SEQ ID NOS:1
or 2] 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.
[0055] The invention also provides polynucleotides that encode a
polypeptide that is a mature protein plus additional amino or
carboxyl-terminal amino acids, or amino acids interior to a mature
polypeptide (when a 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 a mature protein by cellular
enzymes.
[0056] For each and every polynucleotide of the invention there is
provided a polynucleotide complementary to it. It is preferred that
these complementary polynucleotides are fully complementary to each
polynucleotide with which they are complementary.
[0057] A precursor protein, having a mature form of the polypeptide
fused to one or more prosequences may be an inactive form of the
polypeptide. When prosequences are removed such inactive precursors
generally are activated. Some or all of the prosequences may be
removed before activation. Generally such precursors are called
proproteins.
[0058] As will be recognized, the entire polypeptidde encoded by an
open reading frame is often not required for activity. Accordingly,
it has become routine in molecular biology to map the boundaries of
the primary structure required for activity with N-terminal and
C-terminal deletion experiments. These experiments utilize
exonuclease digestion or convenient restriction sites to cleave
coding nucleic acid sequence. For example, Promega (Madison, Wis.)
sell an Erase-a-base.TM. system that uses Exonuclease III designed
to facilitate analysis of the deletion products (protocol available
at www.promega.com). The digested endpoints can be repaired (e.g.,
by ligation to synthetic linkers) to the extent necessary to
preserve an open reading frame. In this way, the nucleic acid of
SEQ ID NO:1 readily provides contiguous fragments of SEQ ID NO:2
sufficient to provide an activity, such as an enzymatic, binding or
antibody-inducing activity. Nucleic acid sequences encoding such
fragments of SEQ ID NO:2 and variants thereof as described herein
are within the invention, as are polypeptides so encoded.
[0059] 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, that is a precursor to a
proprotein, having a leader sequence and one or more prosequences,
that generally are removed during processing steps that produce
active and mature forms of the polypeptide.
Vectors, Host Cells, Expression Systems
[0060] The invention also relates to vectors that comprise a
polynucleotide or polynucleotides of the invention, host cells that
are generally 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.
[0061] Recombinant polypeptides of the present invention may be
prepared by processes well known in those skilled in the art from
genetically engineered host cells comprising expression systems.
Accordingly, in a further aspect, the present invention relates to
expression systems that comprise a polynucleotide or
polynucleotides of the present invention, to host cells that are
genetically engineered with such expression systems, and to the
production of polypeptides of the invention by recombinant
techniques.
[0062] For recombinant production of the polypeptides of the
invention, 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.
[0063] Representative examples of appropriate hosts include
bacterial cells, such as cells of streptococci, staphylococci,
enterococci E. coli, streptomyces, cyanobacteria, Bacillus
subtilis, and Streptococcus pneumoniae; fungal cells, such as cells
of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida
albicans and Aspergillus; insect cells such as cells of Drosphila
S2 and Spodoptera Sf9; animal cells such as CHO, COS, HeLa, C127,
3T3, BHK, 293, CV-1 and Bowes melanoma cells; and plant cells, such
as cells of a gymnosperm or angiosperm.
[0064] 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, for
example, 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,
picornaviruses 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 comprise control regions that
regulate as well as engender expresssion. 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).
[0065] In recombinant expression systems in eukaryotes, for
secretion of a 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.
[0066] 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.
Diagnostic, Prognostic, Serotyping and Mutation Assays
[0067] This invention is also related to the use of murF2
polynucleotides and polypeptides of the invention for use as
diagnostic reagents. Detection of murF2 polynucleotides and/or
polypeptides in a eukaryote, particularly a mammal, and especially
a human, will provide a diagnostic method for diagnosis of disease,
staging of disease or response of an infectious organism to drugs.
Eukaryotes, particularly mammals, and especially humans,
particularly those infected or suspected to be infected with an
organism comprising the murF2 gene or protein, may be detected at
the nucleic acid or amino acid level by a variety of well known
techniques as well as by methods provided herein.
[0068] Polypeptides and polynucleotides for prognosis, diagnosis or
other analysis may be obtained from a putatively infected and/or
infected individual's bodily materials. Polynucleotides from any of
these sources, particularly DNA or RNA, may be used directly for
detection or may be amplified enzymatically by using PCR or any
other amplification technique prior to analysis. RNA, particularly
mRNA, cDNA and genomic DNA may also be used in the same ways. Using
amplification, characterization of the species and strain of
infectious or resident organism present in an individual, may be
made by an analysis of the genotype of a selected polynucleotide of
the organism. Deletions and insertions can be detected by a change
in size of the amplified product in comparison to a genotype of a
reference sequence selected from a related organism, preferably a
different species of the same genus or a different strain of the
same species. Point mutations can be identified by hybridizing
amplified DNA to labeled murF2 polynucleotide sequences. Perfectly
or significantly matched sequences can be distinguished from
imperfectly or more significantly mismatched duplexes by DNase or
RNase digestion, for DNA or RNA respectively, or by detecting
differences in melting temperatures or renaturation kinetics.
Polynucleotide sequence differences may also be detected by
alterations in the electrophoretic mobility of polynucleotide
fragments in gels as compared to a reference sequence. This may be
carried out with or without denaturing agents. Polynucleotide
differences may also be detected by direct DNA or RNA sequencing.
See, for example, Myers et al., Science, 230: 1242 (1985). Sequence
changes at specific locations also may be revealed by nuclease
protection assays, such as RNase, V1 and S1 protection assay or a
chemical cleavage method. See, for example, Cotton et al., Proc.
Natl. Acad. Sci., USA, 85: 4397-4401 (1985).
[0069] In another embodiment, an array of oligonucleotides probes
comprising murF2 nucleotide sequence or fragments thereof can be
constructed to conduct efficient screening of, for example, genetic
mutations, serotype, taxonomic classification or identification.
Array technology methods are well known and have general
applicability and can be used to address a variety of questions in
molecular genetics including gene expresssion, genetic linkage, and
genetic variability (see, for example, Chee et al., Science, 274:
610 (1996)).
[0070] Thus, in another aspect, the present invention relates to a
diagnostic kit that comprises (a) a polynucleotide of the present
invention, preferably the nucleotide sequence of SEQ ID NO:1, or a
fragment thereof; (b) a nucleotide sequence complementary to that
of (a); (c) a polypeptide of the present invention, preferably the
polynucleotide of SEQ ID NO:2 or a fragment thereof; or (d) an
antibody to a polypeptide of the present invention, preferably to
the polypeptide of SEQ ID NO:2. It will be appreciated than in any
such kit, (a), (b), (c) or (d) may comprise a substantial
component. Such a kit will be of use in diagnosing a disease or
susceptibility to a Disease, among others.
[0071] This invention also relates to the use of polynucleotides of
the present invention as diagnostic reagents. Detection of a
mutated form of a polynucleotide of the invention, preferable, SEQ
ID NO:1, that is associated with a disease or pathogenicity will
provide a diagnostic tool that can add to, or define, a diagnosis
of a disease, a prognosis of a course of disease, a determination
of a stage of disease, or a susceptibility to a disease, that
results from under-expression, over-expression or altered
expression of the polynucleotide. Organisms, particularly
infectious organisms, carrying mutations in such polynucleotide may
be detected at the polynucleotide level by a variety of techniques,
such as those described elsewhere herein.
[0072] The differences in a polynucleotide and/or polypeptide
sequence between organisms possessing a first phenotype and
organisms possessing a different, second different phenotype can
also be determined. If a mutation is observed in some or all
organisms possessing the first phenotype but not in any organisms
possessing the second phenotype, then the mutation is likely to be
the causative agent of the first phenotype.
[0073] Cells from an organism carrying mutations or polymorphisms
(allelic variations) in a polynucleotide and/or polypeptide of the
invention may also be detected at the polynucleotide or polypeptide
level by a variety of techniques, to allow for serotyping, for
example. For example, RT-PCR can be used to detect mutations the
RNA. It is particularly preferred to use RT-PCR in conjunction with
automated detection systems, such as, for example, GeneScan, RNA,
cDNA or genomic DNA may also be used for the same purpose, PCR. As
an example, PCR primers complementary to a polynucleotide encoding
murF2 polypeptide can be used to identify and analyze mutations.
The invention further provides these primers with 1, 2, 3 or 4
nucleotides removed from the 5' and/or the 3' end. These primers
may be used for, among other things, amplifying murF2 DNA and/or
RNA isolated from a sample derived from an individual, such as a
bodily material. The primers may be used to amplify a
polynucleotide isolated from an infected individual, such that the
polynucleotide may then be subject to various techniques for
elucidation of the polynucleotide sequence. In this way, mutations
in the polynucleotide sequence may be detected and used to diagnose
and/or prognose the infection or its stage or course, or to
serotype and/or classify the infectious agent.
[0074] The invention further provides a process for diagnosing,
disease, preferably bacterial infections, more preferably
infections caused by Streptococcus pneumoniae, comprising
determining from a sample derived from an individual, such as a
bodily material, an increased level of expression of polynucleotide
having a sequence of Table 1 [SEQ ID NO:1]. Increased or decreased
expresssion of a murF2 polynucleotide can be measured using any on
of the methods well known in the art for the quantitation of
polynucleotides, such as, for example, amplifications, PCR, RT-PCR,
RNase protection, Northern blotting, spectrometry and other
hybridization methods.
[0075] In addition, a diagnostic assay in accordance with the
invention for detecting over-expression of murF2 polypeptide
compared to normal control tissue samples may be used to detect the
presence of an infection, for example. Assay techniques that can be
used to determine levels of a murF2 polypeptide, in a sample
derived from a host, such as a bodily material, are well-known to
those of skill in the art. Such assay methods include
radioimmunoassays, competitive-binding assays, Western Blot
analysis, antibody sandwich assays, antibody detection and ELISA
assays.
Antagonists and Agonists--Assays and Molecules
[0076] Polypeptides and polynucleotides of the invention may also
be used to assess the binding of small molecule substrates and
ligands in, for example, cells, cell-free preparations, chemical
libraries, and natural product mixtures. 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).
[0077] Polypeptides and polynucleotides of the present invention
are responsible for many biological functions, including many
disease states, in particular the Diseases herein mentioned. It is
therefore desirable to devise screening methods to identify
compounds that agonize (e.g., stimulate) or that antagonize (e.g.,
inhibit) the function of the polypeptide or polynucleotide.
Accordingly, in a further aspect, the present invention provides
for a method of screening compounds to identify those that agonize
or that antagonize the function of a polypeptide of the invention,
as well as related polypeptides and polynucleotides. In general,
agonists or antagonists (e.g., inhibitors) may be employed for
therapeutic and prophylactic purposes for such Diseases as herein
mentioned. Compounds may be identified form a variety of sources,
for example, cells, cell-free preparations, chemical libraries, and
natural product mixtures. Such agonists and antagonists
so-identified may be natural or modified substrates, ligands,
receptors, enzymes, etc., as the case may be, of murF2 polypeptides
and polynucleotides; or may be structural or functional mimetics
thereof (see Coligan et al., Current Protocols in Immunology
1(2):Chapter 5 (1991)).
[0078] The screening methods may simply measure the binding of a
candidate compound to the polypeptide or polynucleotide, or to
cells or membranes bearing the polypeptide or polynucleotide, or a
fusion protein of the polypeptide by means of a label directly or
indirectly associated with the candidate compound. Alternatively,
the screening method may involve competition with a labeled
competitor. Further, these screening methods may test whether the
candidate compound results in a signal generated by activation or
inhibition of the polypeptide or polynucleotide, using detection
systems appropriate to the cells comprising the polypeptide or
polynucleotide. Inhibitors of activation are generally assayed in
the presence of a known agonist and the effect on activation by the
agonist by the presence of the candidate compound is observed.
Constitutively active polypeptide and/or constitutively expressed
polypeptides and polynucleotides may be employed in screening
methods for inverse agonists, in the absence of an agonist or
antagonist, by testing whether the candidate compound results in
inhibition of activation of the polypeptide or polynucleotide, as
the case may be. Further, the screening methods may simply comprise
the steps of mixing a candidate compound with a solution comprising
a polypeptide or polynucleotide of the present invention, to form a
mixture, measuring murF2 polypeptide and/or polynucleotide activity
in the mixture, and comparing the murF2 polypeptide and/or
polynucleotide activity of the mixture to a standard. Fusion
proteins, such as those made from Fc portion and murF2 polypeptide,
as herein described, can also be used for high-throughput screening
assays to identify antagonists of the polypeptide of the present
invention, as well as of phylogenetically and and/or functionally
related polypeptides (see D. Bennett et al., J Mol Recognition,
8:52-58 (1995); and K. Johanson et al., J Biol Chem,
270(16):9459-9471 (1995)).
[0079] The polynucleotides, polypeptides and antibodies that bind
to and/or interact with a polypeptide of the present invention may
also be used to configure screening methods for detecting the
effect of added compounds on the production of mRNA and/or
polypeptide in cells. For example, an ELISA assay may be
constructed for measuring secreted or cell associated levels of
polypeptide using monoclonal and polyclonal antibodies by standard
methods known in the art. This can be used to discover agents that
may inhibit or enhance the production of polypeptide (also called
antagonist or agonist, respectively) from suitably manipulated
cells or tissues.
[0080] The invention also provides a method of screening compounds
to identify those that enhance (agonist) or block (antagonist) the
action of murF2 polypeptides or polynucleotides, particularly those
compounds that are bacteristatic and/or bactericidal. The method of
screening may involve high-throughput techniques. For example, to
screen for agonists or antagonists, a synthetic reaction mix, a
cellular compartment, such as a membrane, cell envelope or cell
wall, or a preparation of any thereof, comprising murF2 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
murF2 agonist or antagonist. The ability of the candidate molecule
to agonize or antagonize the murF2 polypeptide is reflected in
decreased binding of the labeled ligand or decreased production of
product from such substrate. Molecules that bind gratuitously,
i.e., without inducing the effects of murF2 polypeptide are most
likely to be good antagonists. Molecules that bind well and, as the
case may be, increase the rate of product production from
substrate, increase signal transduction, or increase chemical
channel activity are agonists. Detection of the rate or level of,
as the case may be, production of product from substrate, signal
transduction, or chemical channel activity 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
responsive to changes in murF2 polynucleotide or polypeptide
activity, and binding assays known in the art.
[0081] Polypeptides of the invention may be used to identify
membrane bound or soluble receptors, if any, for such polypeptide,
through standard receptor binding techniques known in the art.
These techniques include, but are not limited to, ligand binding
and crosslinking assays in which the polypeptide is labeled with a
radioactive isotope (for instance, .sup.125I), chemically modified
(for instance, biotinylated), or fused to a peptide sequence
suitable for detection or purification, and incubated with a source
of the putative receptor (e.g., cells, cell membranes, cell
supernatants, tissue extracts, bodily materials). Other methods
include biophysical techniques such as surface plasmon resonance
and spectroscopy. These screening methods may also be used to
identify agonists and antagonists of the polypeptide that compete
with the binding of the polypeptide to its receptor(s), if any.
Standard methods for conducting such assays are well understood in
the art.
[0082] The fluorescence polarization value for a
fluorescently-tagged molecule depends on the rotational correlation
time or tumbling rate. Protein complexes, such as formed by murF2
polypeptide associating with another murF2 polypeptide or other
polypeptide, labeled to comprise a fluorescently-labeled molecule
will have higher polarization values than a fluorescently labeled
monomeric protein. It is preferred that this method be used to
characterize small molecules that disrupt polypeptide
complexes.
[0083] Fluorescence energy transfer may also be used characterize
small molecules that interfere with the formation of murF2
polypeptide dimers, trimers, tetramers or higher order structures,
or structures formed by murF2 polypeptide bound to another product.
MurF2 polypeptide can be labeled with both a donor and acceptor
fluorophore. Upon mixture of the two labeled species and excitation
of the donor fluorophore, fluorescence energy transfer can be
detected by observing fluorescence of the acceptor. Compounds that
block dimerization will inhibit fluorescence energy transfer.
[0084] Surface plasmon resonance can be used to monitor the effect
of small molecules on murF2 polypeptide self-association as well as
an association of murF2 polypeptide and another polypeptide or
small molecule. MurF2 polypeptide can be coupled to a sensor chip
at low site density such that covalently bound molecules will be
monomeric. Solution protein can then passed over the murF2
polypeptide-coated surface and specific binding can be detected in
real-time by monitoring the change in resonance angle caused by a
change in local refractive index. This technique can be used to
characterize the effect of small molecules on kinetic rates and
equilibrium binding constants for murF2 polypeptide
self-association as well as an association of murF2 polypeptide and
another polypeptide or small molecule.
[0085] A scintillation proximity assay may be used to characterize
the interaction between an association of murF2 polypeptide with
another murF2 polypeptide or a different polypeptide. MurF2
polypeptide can be coupled to a scintillation-filled bead. Addition
of radio-labeled murF2 polypeptide results in binding where the
radioactive source molecule is in close proximity to the
scintillation fluid. Thus, signal is emitted upon murF2 polypeptide
binding and compounds that prevent murF2 polypeptide
self-association or an association of murF2 polypeptide and another
polypeptide or small molecule will diminish signal.
[0086] In other embodiments of the invention there are provided
methods for identifying compounds that bind to or otherwise
interact with and inhibit or activate an activity or expresssion of
a polypeptide and/or polynucleotide of the invention comprising:
contacting a polypeptide and/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
and/or polynucleotide to assess the binding to or other interaction
with the compound, such binding or interaction preferably being
associated with a second component capable of providing a
detectable signal in response to the binding or interaction of the
polypeptide and/or polynucleotide with the compound; and
determining whether the compound binds to or otherwise interacts
with and activates or inhibits an activity or expression of the
polypeptide and/or polynucleotide by detecting the presence or
absence of a signal generated from the binding or interaction of
the compound with the polypeptide and/or polynucleotide.
[0087] Another example of an assay for murF2 agonists is a
competitive assay that combines murF2 and a potential agonist with
murF2-binding molecules, recombinant murF2 binding molecules,
natural substrates or ligands, or substrate or ligand mimetics,
under appropriate conditions for a competitive inhibition assay.
MurF2 can be labeled, such as by radioactivity or a colorimetric
compound, such that the number of murF2 molecules bound to a
binding molecule or converted to product can be determined
accurately to assess the effectiveness of the potential
antagonist.
[0088] It will be readily appreciated by the skilled in artisan
that a polypeptide and/or polynucleotide of the present invention
may also be used in a method for the structure-based design of an
agonist or antagonist of the polypeptide and/or polynucleotide, by:
(a) determining in the first instance the three-dimensional
structure of the polypeptide and/or polynucleotide, or complexes
thereof; (b) deducing the three-dimensional structure for the
likely reactive site(s), binding site(s) or motif(s) of an agonist
or antagonist; (c) synthesizing candidate compounds that are
predicted to bind to or react with the deduced binding site(s),
reactive site(s), and/or motif(s); and (d) testing whether the
candidate compounds are indeed agonists or antagonists.
[0089] It will be further appreciated that this will normally be an
iterative process, and this iterative process may be performed
using automated and computer-controlled steps.
[0090] In a further aspect, the present invention provides methods
of treating abnormal conditions such as, for instance, a Disease,
related to either an excess of, an under-expression of, an elevated
activity of, or a decreased activity of murF2 polypeptide and/or
polynucleotide.
[0091] If the expression and/or activity of the polypeptide and/or
polynucleotide is in excess, several approaches are available. One
approach comprises administering to an individual in need thereof
an inhibitor compound (antagonist) as herein described, optionally
in combination with a pharmaceutically acceptable carrier, in an
amount effective to inhibit the function and/or expression of the
polypeptide and/or polynucleotide, such as, for example, by
blocking the binding of ligands, substrates, receptors, enzymes,
etc., or by inhibiting a second signal, and thereby alleviating the
abnormal condition. In another approach, soluble forms of the
polypeptides still capable of binding the ligand, substrate,
enzymes, receptors, etc. in competition with endogenous polypeptide
and/or polynucleotide may be administered. Typical examples of such
competitors include fragments of the murF2 polypeptide and/or
polypeptide.
[0092] In still another approach, expression of the gene encoding
endogenous murF2 polypeptide can be inhibited using expression
blocking techniques. This blocking may be targeted against any step
in gene expression, but is preferably targeted against
transcription and/or translation. An examples of a known technique
of this sort involve the use of antisense sequences, either
internally generated or separately administered (see, for example,
O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as
Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton,
Fla. (1988)). Alternatively, oligonucleotides that form triple
helices with the gene can be supplied (see, for example, Lee et
al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)
241:456; Dervan et al., Science (1991) 251: 1360). These oligomers
can be administered per se or the relevant oligomers can be
expressed in vivo.
[0093] Each of the polynucleotide 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
polynucleotide 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.
[0094] The invention also provides the use of the polypeptide,
polynucleotide, agonist or antagonist of the invention to interfere
with the initial physical interaction between a pathogen or
pathogens and a eukaryotic, preferably 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 and/or gram negative bacteria, to
eukaryotic, preferably mammalian, extracellular matrix proteins or
in-dwelling devices or to extracellular matrix proteins in wounds;
to block bacterial adhesion between eukaryotic, preferably
mammalian, extracellular matrix proteins and bacterial murF2
proteins that mediate tissue damage and/or; to block the normal
progression of pathogenesis in infections initiated other than by
the implantation of in-dwelling devices or by other surgical
techniques.
[0095] In accordance with yet another aspect of the invention,
there are provided murF2 agonists and antagonists, preferably
bacteristatic or bactericidal agonists and antagonists.
[0096] The antagonists and agonists of the invention may be
employed, for instance, to prevent, inhibit and/or treat
diseases.
[0097] 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, Franc, http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the
International Agency for Research on Cancer recently recognized a
cause-and-effect relationship between H. pylori and gastric
adenocarcinoma, classifying the bacterium as a Group I (definite)
carcinogen. Preferred antimocrobial compounds of the invention
(agonists and antagonists of murF2 polypeptides and/or
polynucleotides) found using screens provided by the invention, or
known in the art, particularly narrow-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 prevent,
inhibit and/or cure gastric ulcers and gastritis.
[0098] All publications and references, including but not limited
to patents and patent applications, cited in this specification are
herein incorporated by reference in their entirety as if each
individual publication or reference were specifically and
individually indicated to be incorporated by reference herein as
being fully set forth. Any patent application to which this
application claims priority is also incorporated by reference
herein in its entirety in the manner describe above for
publications and references.
GLOSSARY
[0099] The following definitions are provided to facilitate
understanding of certain terms used frequently herein.
[0100] "Bodily material(s) means any material derived from an
individual or from an organism infecting, infesting or inhabiting
an individual, including but not limited to, cells, tissues and
waste, such as, bone, blood, serum, cerebrospinal fluid, semen,
saliva, muscle, cartilage, organ tissue, skin, urine, stool or
autopsy material.
[0101] "Disease(s)" means any disease caused by or related to
infection by a bacteria, including, for example, otitis media,
conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis,
pleural empyema and endocarditis, and most particularly meningitis,
such as for example infection of cerebrospinal fluid.
[0102] "Host cell(s)" is a cell that has been introduced (e.g.,
transformed or transfected) or is capable of introduction (e.g.,
transformation or transfection) by an exogenous polynucleotide
sequence.
[0103] "Identity," as known in the art, is a relationship between
two or more polypeptide sequences or two or more polynucleotide
sequences, as the case may be, 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" 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). Methods to determine identity are
designed to give the largest match between the sequences tested.
Moreover, methods to determine identity are codified in publicly
available computer programs. Computer program methods to determine
identity between two sequences include, but are not limited to, the
GCG program package (Devereux, J., et al., Nucleic Acids Research
12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et
al., J. Molec. 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). The well known Smith
Waterman algorithm may also be used to determine identity.
[0104] Parameters for polypeptide sequence comparison include the
following:
[0105] Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453
(1970)
[0106] Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff,
Proc. Natl. Acad. Sci. USA, 89:10915-10919 (1992)
[0107] Gap Penalty: 12
[0108] Gap Length Penalty: 4
[0109] A program useful with these parameters is publicly available
as the "gap" program from Genetics Computer Group, Madison Wis. The
aforementioned parameters are the default parameters for peptide
comparisons (along with no penalty for end gaps).
[0110] Parameters for polynucleotide comparison include the
following:
[0111] Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453
(1970)
[0112] Comparison matrix: matches=+10, mismatch=0
[0113] Gap Penalty: 50
[0114] Gap Length Penalty: 3
[0115] Available as: The "gap" program From Genetics Computer
Group, Madison, Wis. These are the default parameters for nucleic
acid comparisons.
[0116] A preferred meaning for "identity" for polynucleotides and
polypeptides, as the case may be, are provided in (1) and (2)
below.
[0117] (1) Polynucleotide embodiments further include an isolated
polynucleotide comprising a polynucleotide sequence having at least
95, 97, 99, 99.5 or 100% identity to the reference sequence of SEQ
ID NO:1, wherein said polynucleotide sequence may be identical to
the reference sequence of SEQ ID NO:1 or may include up to a
certain integer number of nucleotide alterations as compared to the
reference sequence, wherein said alterations are selected from the
group consisting of at least one nucleotide deletion, substitution,
including transition and transversion, or insertion, and wherein
said alterations may occur at the 5' or 3' terminal positions of
the reference nucleotide sequence or anywhere between those
terminal positions, interspersed either individually among the
nucleotides in the reference sequence or in one or more contiguous
groups within the reference sequence, and wherein said number of
nucleotide alterations is determined by multiplying the total
number of nucleotides in SEQ ID NO:1 by the integer defining the
percent identity divided by 100 and then subtracting the product
from said total number of nucleotides in SEQ ID NO:1, or:
n.sub.n.ltoreq.x.sub.n-(x.sub.n.multidot.y),
[0118] wherein n.sub.n is the number of nucleotide alterations,
x.sub.n is the total number of nucleotides in SEQ ID NO:1, y is
0.95 for 95%, 0.97 for 97%, 0.99 for 99%, 0.995 for 99.5% or 1.00
for 100%, and .multidot. is the symbol for the multiplication
operator, and wherein any non-integer product of x.sub.n and y is
rounded down to the nearest integer prior to subtracting it from
x.sub.n. Alterations of a polynucleotide sequence encoding the
polypeptide of SEQ ID NO:2 may create nonsense, missense or
frameshift mutations in this coding sequence and thereby alter the
polypeptide encoded by the polynucleotide following such
alterations.
[0119] (2) Polypeptide embodiments further include an isolated
polypeptide comprising a polypeptide having at least a 95, 97 or
100% identity to a polypeptide reference sequence of SEQ ID NO:2,
wherein said polypeptide sequence may be identical to the reference
sequence of SEQ ID NO:2 or may include up to a certain integer
number of amino acid alterations as compared to the reference
sequence, wherein said alterations are selected from the group
consisting of at least one amino acid deletion, substitution,
including conservative and non-conservative substitutions, or
insertion, and wherein said alterations may occur at the amino- or
carboxy-terminal positions of the reference polypeptide sequence or
anywhere between those terminal positions, interspersed either
individually among the amino acids in the reference sequence or in
one or more contiguous groups within the reference sequence, and
wherein said number of amino acid alterations is determined by
multiplying the total number of amino acids in SEQ ID NO:2 by the
integer defining the percent identity divided by 100 and then
subtracting that product from said total number of amino acids in
SEQ ID NO:2, or:
n.sub.a.ltoreq.x.sub.a-(x.sub.a.multidot.y),
[0120] wherein n.sub.a is the number of amino acid alterations,
x.sub.a is the total number of amino acids in SEQ ID NO:2, y is
0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and .multidot. is the
symbol for the multiplication operator, and wherein any non-integer
product of x.sub.a and y is rounded down to the nearest integer
prior to subtracting it from x.sub.a.
[0121] "Individual(s)" means a multicellular eukaryote, including,
but not limited to a metazoan, a mammal, an ovid, a bovid, a
simian, a primate, and a human.
[0122] "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.
[0123] "Organism(s)" means a (i) prokaryote, including but not
limited to, a member of the genes Streptococcus, Staphylococcus,
Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus,
Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia,
Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix,
Branhamella, Actinobacillus, Streptobacillus, Listeria,
Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema,
Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia,
Borrelia, Leptospira, Spirillum, Campylobacter, Shigella,
Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia
and Mycoplasma, and further including, but limited to, a member of
the species or group, Group A Streptococcus, Group B Streptococcus,
Group C Streptococcus, Group D Streptococcus, Group G
Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,
Streptococcus agalactiae, Streptococcus faecalis, Streptococcus
faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria
meningitidis, Staphylococcus aureus, Staphylococcus epidermidis,
Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium
tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans,
Mycobacterium leprae, Actinomyctes israelii, Listeria
monocytogenes, Bordetella pertusis, Bordatella parapertusis,
Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae,
Haemophilus influenzae, Haemophilus aegyptius, Haemophilus
parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typi,
Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia
pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia
liquefaciens, Vibrio cholera, Shigella dysenterii, Shigella
flexneri, Pseudomonas aeruginosa, Franscisella tularensis, Brucella
abortis, Bacillus anthracis, Bacillus cereus, Clostridium
perfringens, Clostridium tetani, Clostridium botulinum, Treponema
pallidum, Rickettsia rickettsii and Chlamydia trachomitis, (ii) an
archaeon, including but not limited to Archaebacter, and (iii) a
unicellular or filamentous eukaryote, including but not limited to,
a protozoan, a fungus, a member of the genus Saccharomyces,
Kluveromyces, or Candida, and a member of the species Saccharomyces
ceriviseae, Kluveromyces lactis, or Candida albicans.
[0124] "Polynucleotide(s)" generally refers to any
polyribonucleotide or polydeoxyribonucleotide, that 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-standard 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 comprise 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 a 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).
[0125] "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 comprise amino acids other than then 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 comprise 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, 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.
[0126] "Recombinant expression system(s)" refers to expression
systems or portions thereof or polynucleotides of the invention
introduced or transformed into a host cell or host cell lysate for
the production of the polynucleotides and polypeptides of the
invention.
[0127] "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, fusion proteins 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. The present
invention also includes include variants of each of the
polypeptides of the invention, that is polypeptides that vary from
the referents by conservative amino acid substitutions, whereby a
residue is substituted by another with the characteristics. Typical
such substitutions are among Ala, Val, Leu and Ile; among Ser and
Thr; among the acidic residues Asp and Glu; among Asn and Gln; and
among the basic residues Lys and Arg; or aromatic residues Phe and
Tyr. Particularly preferred are variants in which several, 5-10,
1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added
in any combination. 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 technique, by direct synthesis, and by other
recombinant methods known to skilled artisans.
EXAMPLES
[0128] The examples below are carried out using standard
techniques, that 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
[0129] The polynucleotide having a DNA sequence given in Table 1
[SEQ ID NO:1] was obtained from a library of clones of chromosomal
DNA of Streptococcus pneumoniae in E. coli. The sequencing data
from two or more clones comprising overlapping Streptococcus
pneumoniae DNAs was used to construct the contiguous DNA sequence
in SEQ ID NO:1. Libraries may be prepared by routine methods, for
example:
[0130] Methods 1 and 2 below.
[0131] Total cellular DNA is isolated from Streptococcus pneumoniae
0100993 according to standard procedures and size-fractionated by
either of two methods.
[0132] Method 1
[0133] 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.
[0134] Method 2
[0135] Total cellular DNA is partially hydrolyzed with one or a
combination of restriction enzymes appropriate to generate a series
of fragments for cloning into library vectors (e.g., RsaI, PalI,
AluI, Bshl235I), 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
murF2 Characterization
[0136] The S. pneumoniae murF2 Gene is Expressed During Infection
in a Respiratory Test Infection Model
[0137] The determination of expression during infection of a gene
from Streptococcus pneumoniae
[0138] Excised lungs from a 48 hour respiratory tract infection of
Streptococcus pneumoniae 0100993 in the mouse is efficiently
disrupted and processed in the presence of chaotropic agents and
RNAse inhibitor to provide a mixture of animal and bacterial RNA.
The optimal conditions for disruption and processing to give stable
preparations and high yields of bacterial RNA are followed by the
use of hybridisation to a radiolabelled oligonucleotide specific to
Streptococcus pneumoniae 16S RNA on Northern blots. The RNAse free,
DNAse free, DNA and protein free preparations of RNA obtained are
suitable for Reverse Transcription PCR (RT-PCR) using unique primer
pairs designed from the sequence of each gene of Streptococcus
pneumoniae 0100993.
[0139] a) Isolation of Tissue Infected with Streptococcus
pneumoniae 0100993 from a Mouse animal Model of Infection
(Lungs)
[0140] Streptococcus pneumoniae 0100993 is seeded onto TSA (Tryptic
Soy Agar, BBL) plates containing 5% horse blood and allowed to grow
overnight at 37.degree. C. in a CO2 incubator. Bacterial growth is
scraped into 5 ml of phosphate-buffered saline (PBS) and adjusted
to an A600.about.0.6 (4.times.106/ml). Mice (male CBA/J-1 mice,
approximately 20 g) were anaesthetized with isoflurane and 50
microliters of the prepared bacterial inoculum is delivered by
intranasal instillation. Animals are allowed to recover and
observed twice daily for signs of moribundancy. Forty-eight hours
after infection the animals are euthanized by carbon dioxide
overdose and their torsos swabbed with ethanol and then RNAZap. The
torso is then opened, and the lungs are aseptically removed. Half
of each pair of lungs is placed in a cryovial and immediately
frozen in liquid nitrogen; the other half is used for bacterial
enumeration after homogenization of the tissue in 1 ml of PBS.
[0141] b) Isolation of Streptococcus pneumoniae 0100993 RNA From
Infected Tissue Samples
[0142] Infected tissue samples, in 2-ml cryo-strorage tubes, are
removed from -80.degree. C. storage into a dry ice ethanol bath. In
a microbiological safety cabinet the samples are disrupted up to
eight at a time while the remaining samples are kept frozen in the
dry ice ethanol bath. To disrupt the bacteria within the tissue
sample, 50-100 mg of the tissue is transfered to a FastRNA tube
containing a silica/ceramic matrix (BIO101). Immediately, 1 ml of
extraction reagents (FastRNA reagents, BIO101) are added to give a
sample to reagent volume ratio of approximately 1 to 20. The tubes
are shaken in a reciprocating shaker (FastPrep FP120, BIO101) at
6000 rpm for 20-120 sec. The crude RNA preparation is extracted
with chloroform/isoamyl alcohol, and precipitated with
DEPC-treated/Isopropanol Precipitation Solution (BIO101). RNA
preparations are stored in this isopropanol solution at -80.degree.
C. if necessary. The RNA is pelleted (12,000 g for 10 min.), washed
with 75% ethanol (v/v in DEPC-treated water), air-dried for 5-10
min, and resuspended in 0.1 ml of DEPC-treated water, followed by
5-10 minutes at 55.degree. C. Finally, after at least 1 minute on
ice, 200 units of Rnasin (Promega) is added.
[0143] RNA preparations are stored at -80.degree. C. for up to one
month. For longer term storage the RNA precipitate can be stored at
the wash stage of the protocol in 75% ethanol for at least one year
at -20.degree. C.
[0144] Quality in the RNA isolated is assessed by running samples
on 1% agarose gels. 1.times.TBE gels stained with ethidium bromide
are used to visualise total RNA yields. To demonstrate the
isolation of bacterial RNA from the infected tissue 1.times.MOPS,
2.2M formaldehyde gels are run and vacuum blotted to Hybond-N
(Amersham). The blot is then hybridised with a 32P-labelled
oligonucleotide probe, of sequence 5' AACTGAGACTGGCTTAAGAGATTA 3'
[SEQ ID NO:3], specific to 16S rRNA of Streptococcus pneumoniae.
The size of the hybridising band is compared to that of control RNA
isolated from in vitro growth Streptococcus pneumoniae 0100993 in
the Northern blot. Correct sized bacterial 16S rRNA bands can be
detected in total RNA samples which show degradation of the
mammalian RNA when visualised on TBE gels.
[0145] c) The Removal of DNA from Streptococcus pneumoniae-Derived
RNA
[0146] DNA was removed from 50 microgram samples of RNA by a 30
minute treatment at 37.degree. C. with 20 units of RNAase-free
DNAaseI (GenHunter) in the buffer supplied in a final volume of 57
microliters.
[0147] The DNAase was inactivated and removed by treatment with
TRIzol LS Reagent (Gibco BRL, Life Technologies) according to the
manufacturers protocol.
[0148] DNAase treated RNA was resuspended in 100 microliters of
DEPC treated water with the addition of Rnasin as described
before.
[0149] d) The Preparation of cDNA from RNA Samples Derived from
Infected Tissue
[0150] 3 microgram samples of DNAase treated RNA are reverse
transcribed using a SuperScript Preamplification System for First
Strand cDNA Synthesis kit (Gibco BRL, Life Technologies) according
to the manufacturers instructions. 150 nanogram of random hexamers
is used to prime each reaction. Controls without the addition of
SuperSciptII reverse transcriptase are also run. Both +/-RT samples
are treated with RNaseH before proceeding to the PCR reaction
[0151] e) The Use of PCR to Determine the Presence of a Bacterial
cDNA Species
[0152] PCR reaction are set up on ice in 0.2 ml tubes by adding the
following components: 43 microliters PCR Master Mix (Advanced
Biotechnologies Ltd.); 1 microliter PCR primers (optimally 18-25
basepairs in length and designed to possess similar annealing
temperatures), each primer at 10 mM initial concentration; and 5
microliters cDNA.
[0153] PCR reactions are run on a Perkin Elmer GeneAmp PCR System
9600 as follows: 2 minutes at 94.degree. C., then 50 cycles of 30
seconds each at 94.degree. C., 50.degree. C. and 72.degree. C.
followed by 7 minutes at 72.degree. C. and then a hold temperature
of 20.degree. C. (the number of cycles is optimally 30-50 to
determine the appearance or lack of a PCR product and optimally
8-30 cycles if an estimation of the starting quantity of cDNA from
the RT reaction is to be made); 10 microliters aliquots are then
run out on 1% 1.times.TBE gels stained with ethidium bromide, with
PCR product, if present, sizes estimated by comparison to a 100 bp
DNA Ladder (Gibco BRL, Life Technologies). Alternatively if the PCR
products are conveniently labelled by the use of a labelled PCR
primer (e.g. labelled at the 5'end with a dye) a suitable aliquot
of the PCR product is run out on a polyacrylamide sequencing gel
and its presence and quantity detected using a suitable gel
scanning system (e.g. ABI Prism TM377 Sequencer using GeneScan.TM.
software as supplied by Perkin Elmer).
[0154] RT/PCR controls may include +/- reverse transcriptase
reactions, 16S rRNA primers or DNA specific primer pairs designed
to produce PCR products from non-transcribed Streptococcus
pneumoniae 0100993 genomic sequences.
[0155] To test the efficiency of the primer pairs they are used in
DNA PCR with Streptococcus pneumoniae 0100993 total DNA. PCR
reactions are set up and run as described above using approx. 1
microgram of DNA in place of the cDNA.
[0156] Primer pairs which fail to give the predicted sized product
in either DNA PCR or RT/PCR are PCR failures and as such are
uninformative. Of those which give the correct size product with
DNA PCR two classes are distinguished in RT/PCR: 1. Genes which are
not transcribed in vivo reproducibly fail to give a product in
RT/PCR; and 2. Genes which are transcribed in vivo reproducibly
give the correct size product in RT/PCR and show a stronger signal
in the +RT samples than the signal (if at all present) in -RT
controls.
Example 3
The murF2 Gene is Essential for S. pneumoniae in Vitro Growth
[0157] Demonstration of gene essentiality to bacterial
viability
[0158] An allelic replacement cassette was generated using PCR
technology. The cassette consisted of a pair of 500 bp chromosomal
DNA fragments flanking an erythromycin resistance gene. The
chromosomal DNA sequences are the 500 bp preceding and following
the DNA sequence encoding the murF2 gene contained in Seq. ID
NO:1
[0159] The allelic replacement cassette was introduced into S.
pneumoniae R6 by transformation. Competent cells were prepared
according to published protocols. DNA was introduced into the cells
by incubation of ng quantities of allelic replacement cassette with
10.sup.6 cells at 30.degree. C. for 30 minutes. The cells were
transferred to 37.degree. C. for 90 minutes to allow expression of
the erythromycin resistance gene. Cells were plated in agar
containing 1 ug erythromycin per ml. Following incubation at
37.degree. C. for 36 hours, colonies are picked and grown overnight
in Todd-Hewitt broth supplemented with 0.5% yeast extract.
Typically 1000 transformants containing the appropriate allelic
replacement are obtained. If no transformants are obtained in three
separate transformation experiments as was the case for this gene
murF2, then the gene is considered as being essential in vitro.
Sequence CWU 1
1
3 1 1344 DNA Streptococcus pneumoniae 1 atgaacttaa aaactacttt
gggccttctt gctgggcgtt cttcccactt cgttttaagc 60 cgtcttggac
gtggaagtac gctcccaggg aaagtcgccc ttcaatttga taaagatatt 120
ttacaaaacc tagctaagaa ctacgagatt gtcgttgtca ctggaacaaa tggaaaaacc
180 ctgacaactg ccctcactgt cggcatttta aaagaggttt atggtcaagt
tctaaccaac 240 ccaagcggtg ccaacatgat tacagggatt gcaacaacct
tcctaacagc caaatcttct 300 aaaactggga aaaatattgc cgtcctcgaa
attgacgaag ccagtctatc tcgtatctgt 360 gactatatcc agcctagtct
ttttgtcatt actaatatct tccgtgacca gatggaccgt 420 ttcggtgaaa
tctatactac ctataacatg atattggatg ccattcggaa agttccaact 480
gctactgttc tccttaacgg agacagtcca cttttctaca agccaactat tccaaaccct
540 atagagtatt ttggttttga cttggaaaaa ggaccagccc aactggctca
ctacaatacc 600 gaagggattc tctgtcctga ctgccaaggc atcctcaaat
atgagcataa tacctatgca 660 aacttgggtg cctatatctg tgagggttgt
ggatgtaaac gtcctgatct cgactatcgt 720 ttgacaaaac tggttgagtt
gaccaacaat cgctctcgct ttgtcataga cggccaagaa 780 tacggtatcc
aaatcggcgg gctctataat atctataacg ccctagctgc tgtggccatc 840
gcccgtttcc taggtgccga ttcgcaactc atcaaacagg gatttgacaa gagccgtgct
900 gtctttggac gccaagaaac ctttcatatc ggtgacaagg aatgtaccct
tgtcttgatt 960 aaaaatccag tcggtgcaac ccaagctatc gaaatgatca
aactagcacc ttatccattt 1020 agcctatctg tcctccttaa tgccaactat
gcagatggaa ttgacactag ctggatctgg 1080 gatgcagact ttgagcaaat
cactgacatg gacattcctg aaatcaacgc tggcggtgtt 1140 cgtcattctg
aaatcgctcg tcgcctccga gtgactggct atccagctga gaaaatcact 1200
gaaacgagta atctggagca agttctcaag accattgaga atcaagactg caagcatgcc
1260 tatattctgg caacttatac tgccatgctg gaatttcgtg aactgctggc
tagtcgtcag 1320 attgttagaa aggagatgaa ctaa 1344 2 447 PRT
Streptococcus pneumoniae 2 Met Asn Leu Lys Thr Thr Leu Gly Leu Leu
Ala Gly Arg Ser Ser His 1 5 10 15 Phe Val Leu Ser Arg Leu Gly Arg
Gly Ser Thr Leu Pro Gly Lys Val 20 25 30 Ala Leu Gln Phe Asp Lys
Asp Ile Leu Gln Asn Leu Ala Lys Asn Tyr 35 40 45 Glu Ile Val Val
Val Thr Gly Thr Asn Gly Lys Thr Leu Thr Thr Ala 50 55 60 Leu Thr
Val Gly Ile Leu Lys Glu Val Tyr Gly Gln Val Leu Thr Asn 65 70 75 80
Pro Ser Gly Ala Asn Met Ile Thr Gly Ile Ala Thr Thr Phe Leu Thr 85
90 95 Ala Lys Ser Ser Lys Thr Gly Lys Asn Ile Ala Val Leu Glu Ile
Asp 100 105 110 Glu Ala Ser Leu Ser Arg Ile Cys Asp Tyr Ile Gln Pro
Ser Leu Phe 115 120 125 Val Ile Thr Asn Ile Phe Arg Asp Gln Met Asp
Arg Phe Gly Glu Ile 130 135 140 Tyr Thr Thr Tyr Asn Met Ile Leu Asp
Ala Ile Arg Lys Val Pro Thr 145 150 155 160 Ala Thr Val Leu Leu Asn
Gly Asp Ser Pro Leu Phe Tyr Lys Pro Thr 165 170 175 Ile Pro Asn Pro
Ile Glu Tyr Phe Gly Phe Asp Leu Glu Lys Gly Pro 180 185 190 Ala Gln
Leu Ala His Tyr Asn Thr Glu Gly Ile Leu Cys Pro Asp Cys 195 200 205
Gln Gly Ile Leu Lys Tyr Glu His Asn Thr Tyr Ala Asn Leu Gly Ala 210
215 220 Tyr Ile Cys Glu Gly Cys Gly Cys Lys Arg Pro Asp Leu Asp Tyr
Arg 225 230 235 240 Leu Thr Lys Leu Val Glu Leu Thr Asn Asn Arg Ser
Arg Phe Val Ile 245 250 255 Asp Gly Gln Glu Tyr Gly Ile Gln Ile Gly
Gly Leu Tyr Asn Ile Tyr 260 265 270 Asn Ala Leu Ala Ala Val Ala Ile
Ala Arg Phe Leu Gly Ala Asp Ser 275 280 285 Gln Leu Ile Lys Gln Gly
Phe Asp Lys Ser Arg Ala Val Phe Gly Arg 290 295 300 Gln Glu Thr Phe
His Ile Gly Asp Lys Glu Cys Thr Leu Val Leu Ile 305 310 315 320 Lys
Asn Pro Val Gly Ala Thr Gln Ala Ile Glu Met Ile Lys Leu Ala 325 330
335 Pro Tyr Pro Phe Ser Leu Ser Val Leu Leu Asn Ala Asn Tyr Ala Asp
340 345 350 Gly Ile Asp Thr Ser Trp Ile Trp Asp Ala Asp Phe Glu Gln
Ile Thr 355 360 365 Asp Met Asp Ile Pro Glu Ile Asn Ala Gly Gly Val
Arg His Ser Glu 370 375 380 Ile Ala Arg Arg Leu Arg Val Thr Gly Tyr
Pro Ala Glu Lys Ile Thr 385 390 395 400 Glu Thr Ser Asn Leu Glu Gln
Val Leu Lys Thr Ile Glu Asn Gln Asp 405 410 415 Cys Lys His Ala Tyr
Ile Leu Ala Thr Tyr Thr Ala Met Leu Glu Phe 420 425 430 Arg Glu Leu
Leu Ala Ser Arg Gln Ile Val Arg Lys Glu Met Asn 435 440 445 3 25
DNA Streptococcus pneumoniae 3 aactgagact ggctttaaga gatta 25
* * * * *
References