U.S. patent application number 09/923656 was filed with the patent office on 2002-08-29 for gcp.
Invention is credited to Biswas, Sanjoy, Chalker, Alison Frances, Holmes, David, Ingraham, Karen A., Palmer, Leslie Marie, Ray, Jennifer E., Warren, Richard Lloyd, Zalacain, Magdalena.
Application Number | 20020119510 09/923656 |
Document ID | / |
Family ID | 22069974 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119510 |
Kind Code |
A1 |
Biswas, Sanjoy ; et
al. |
August 29, 2002 |
gcp
Abstract
The invention provides gcp polypeptides and polynucleotides
encoding gcp polypeptides and methods for producing such
polypeptides by recombinant techniques. Also provided are methods
for utilizing gcp polypeptides to screen for antibacterial
compounds.
Inventors: |
Biswas, Sanjoy; (Paoli,
PA) ; Chalker, Alison Frances; (Trappe, PA) ;
Holmes, David; (West Chester, PA) ; Ingraham, Karen
A.; (Auburn, PA) ; Palmer, Leslie Marie;
(Audubon, PA) ; Ray, Jennifer E.; (State College,
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: |
22069974 |
Appl. No.: |
09/923656 |
Filed: |
August 7, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09923656 |
Aug 7, 2001 |
|
|
|
09066512 |
Apr 24, 1998 |
|
|
|
Current U.S.
Class: |
435/32 ; 435/183;
435/252.3; 435/320.1; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 31/04 20180101; C07K 14/3156 20130101; Y10S 530/825 20130101;
C12N 9/80 20130101; Y10S 530/82 20130101 |
Class at
Publication: |
435/32 ;
435/69.1; 435/183; 435/252.3; 435/320.1; 536/23.2 |
International
Class: |
C12Q 001/18; C07H
021/04; C12N 009/00; C12P 021/02; C12N 001/21; C12N 015/74 |
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: (a) 70% identity; (b) 80% identity; (c) 90% identity; or (d)
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 which is the amino acid sequence of SEQ ID
NO:2, and (iv) a polypeptide which is encoded by a recombinant
polynucleotide comprising the polyncleotide sequence of SEQ ID
NO:2.
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 (a) 70% identity;
(b) 80% identity; (c) 90% identity; or (d) 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: (a) 70% identity (b) 80%
identity; (c) 90% identity; or (d) 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 which has at least: (a) 70% identity; (b) 80% identity;
(c) 90% identity; or (d) 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; (vi) an isolated polynucleotide which is the polynucleotide
of SEQ ID NO: 1; (vi) an isolated polynucleotide 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; (vii) an isolated polynucleotide encoding a
mature polypeptide expressed by the gcp gene contained in the
Streptococcus pneumoniae; and (viii) a polynucleotide sequence
complementary to said isolated polynucleotide of (i), (ii), (iii),
(iv), (v), (vi) or (vii).
3. An antibody antigenic to or immunospecific for the polypeptide
of claim 1.
4. A method for the treatment of an individual: (i) in need of
enhanced activity or expression of the polypeptide of claim 1
comprising the step of: (a) administering to the individual a
therapeutically effective amount of an agonist to said polypeptide;
or (b) providing to the individual an isolated polynucleotide
comprising a polynucleotide sequence encoding said polypeptide in a
form so as to effect production of said polypeptide activity in
vivo; 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; or (c) administering to the
individual a therapeutically effective amount of a polypeptide that
competes with said polypeptide for its ligand, substrate, or
receptor.
5. 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
the genome of said individual; or (b) analyzing for the presence or
amount of said polypeptide expression in a sample derived from said
individual.
6. A method for screening to identify compounds that activate or
that inhibit the function of the polypeptide of claim 1 which
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 containing 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; (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, or (f) (1) contacting
a composition comprising the polypeptide with the compound to be
screened under conditions to permit interaction between the
compound and the polypeptide to assess the interaction of a
compound, such interaction being associated with a second component
capable of providing a detectable signal in response to the
interaction of the polypeptide with the compound; and (2)
determining whether the compound interacts with and activates or
inhibits an activity of the polypeptide by detecting the presence
or absence of a signal generated from the interaction of the
compound with the polypeptide.
7. An agonist or an antagonist of the activity or expression
polypeptide of claim 1.
8. An expression system comprising a polynucleotide capable of
producing a polypeptide of claim 1 when said expression system is
present in a compatible host cell.
9. A host cell comprising the expression system of claim 8 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: (a) 70% identity;
(b) 80% identity; (c) 90% identity; or (d) 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 which is the
amino acid sequence of SEQ ID NO:2, and (iv) a polypeptide which is
encoded by a recombinant polynucleotide comprising the
polynucleotide sequence of SEQ ID NO:2.
10. 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: (a) 70% identity;
(b) 80% identity; (c) 90% identity; or (d) 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 which is the
amino acid sequence of SEQ ID NO:2, and (iv) a polypeptide which is
encoded by a recombinant polynucleotide comprising the
polynucleotide sequence of SEQ ID NO:2, comprising the step of
culturing a host cell of claim 9 under conditions sufficient for
the production of said polypeptide.
11. A process for producing a host cell comprising the expression
system of claim 8 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: (a) 70% identity; (b) 80% identity; (c) 90% identity; or (d)
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 which is the amino acid sequence of SEQ ID
NO:2, and (iv) a polypeptide which is encoded by a recombinant
polynucleotide comprising the polynucleotide sequence of SEQ ID
NO:2, 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).
12. A host cell produced by the process of claim 11 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: (a) 70% identity; (b) 80%
identity; (c) 90% identity; or (d) 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 which is the amino acid
sequence of SEQ ID NO:2, and (iv) a polypeptide which is encoded by
a recombinant polynucleotide comprising the polynucleotide sequence
of SEQ ID NO:2.
13. A computer readable medium having stored thereon a member
selected from the group consisting of: a polynucleotide comprising
the sequence of SEQ ID NO. 1; a polypeptide comprising the sequence
of SEQ ID NO. 2; a set of polynucleotide sequences wherein at least
one of said sequences comprises the sequence of SEQ ID NO. 1; a set
of polypeptide sequences wherein at least one of said sequences
comprises the sequence of SEQ ID NO. 2; a data set representing a
polynucleotide sequence comprising the sequence of SEQ ID NO. 1; a
data set representing a polynucleotide sequence encoding a
polypeptide sequence comprising the sequence of SEQ ID NO. 2 ; a
polynucleotide comprising the sequence of SEQ ID NO. 1; a
polypeptide comprising the sequence of SEQ ID NO. 2 ; a set of
polynucleotide sequences wherein at least one of said sequences
comprises the sequence of SEQ ID NO. 1; a set of polypeptide
sequences wherein at least one of said sequences comprises the
sequence of SEQ ID NO. 2; a data set representing a polynucleotide
sequence comprising the sequence of SEQ ID NO. 1; a data set
representing a polynucleotide sequence encoding a polypeptide
sequence comprising the sequence of SEQ ID NO. 2.
14. A computer based method for performing homology identification,
said method comprising the steps of providing a polynucleotide
sequence comprising the sequence of SEQ ID NO. 1 in a computer
readable medium; and comparing said polynucleotide sequence to at
least one polynucleotide or polypeptide sequence to identify
homology.
15. A further embodiment of the invention provides a computer based
method for polynucleotide assembly, said method comprising the
steps of: providing a first polynucleotide sequence comprising the
sequence of SEQ ID NO. 1 in a computer readable medium; and
screening for at least one overlapping region between said first
polynucleotide sequence and a second polynucleotide sequence.
Description
FIELD OF THE INVENTION
[0001] 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
glycopeptidase family, as well as their variants, hereinafter
referred to as "gcp," "gcp polynucleotide(s)," and "gcp
polypeptide(s)."
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] Several cell surface associated proteins of the
Staphylococci and Streptococci involved in microbial adhesion to
different host tissues and considered to be important factors in
bacterial pathogenesis have been identified in the last decade (see
Patti, J. M., et al., MSCRAMM-Mediated Adherence of Microorganisms
to Host Tissues (1994) Annu. Rev. Microbiol. 48:85-617).
[0004] Different approaches have been put forward to address such
proteins from Staphylococcus aureus as antibacterial targets, e.g.
fibronectin binding proteins (EP0294349, EP0397633, WO94/18327),
fibrinogen binding protein (WO94106830), collagen binding protein
(WO92/07002) and bone sialoprotein binding protein (WO94/13310).
The binding proteins or binding fragments thereof are used as
antibacterial agents to block binding of the organism to host
tissue, as vaccines to raise antibodies to the organism in the host
animal or as antigens to raise therapeutic antibodies which can be
used to block binding of the organism to host tissue.
[0005] 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 which 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.
[0006] 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.
[0007] Clearly, there exists a need for polynucleotides and
polypeptides, such as the gcp embodiments of the invention, that
have a present benefit of, among other things, being useful to
screen compounds for antibiotic activity. Such factors are also
useful to determine their role in pathogenesis of infection,
dysfunction and disease. There is also a need for identification
and characterization of such factors and their antagonists and
agonists to find ways to prevent, ameliorate or correct such
infection, dysfunction and disease.
[0008] Certain of the polypeptides of the invention possess
significant amino acid sequence homology to a known Pasteurella
haemolytica gcp protein, GenBank D88802, SwissProt P36175, Abdullah
K M, Lo R Y C, Mellors A. J. Bacteriol. 173:5597-5603 (1991),
Rawlings N D, Barrett A J. Meth. Enzymol. 248:183-228 (1995); PCT
Publication WO98/06734, published Feb. 19, 1998; and PCT
Publication WO97/37026, published Oct. 9, 1997.
SUMMARY OF THE INVENTION
[0009] The present invention relates to gcp, in particular gcp
polypeptides and gcp polynucleotides, recombinant materials and
methods for their production. In another aspect, the invention
relates to methods for using such polypeptides and polynucleotides,
including the 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 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 gcp expression or activity.
[0010] Various changes and modifications within the spirit and
scope of the disclosed invention will become readily apparent to
those skilled in the art from reading the following descriptions
and from reading the other parts of the present disclosure.
DESCRIPTION OF THE INVENTION
[0011] The invention relates to gcp polypeptides and
polynucleotides as described in greater detail below. In
particular, the invention relates to polypeptides and
polynucleotides of a gcp of Streptococcus pneumoniae, which is
related by amino acid sequence homology to Pasteurella haemolytica
gcp polypeptide. The invention relates especially to gcp having the
nucleotide and amino acid sequences set out in Table 1 as SEQ ID
NO: 1 and SEQ ID NO: 2 respectively.
1TABLE 1 gcp Polynucleotide and Polypeptide Sequences (A)
Streptococcus pneumoniae gcp polynucleotide sequence [SEQ ID NO:1].
5'-
ATGAAGGATAGATATATTTTAGCATTTGAGACATCCTGTGATGAGACCAGTGTCGCCGTCTTGAAAAACGAC
GATGAGCT CTTGTCCAATGTCATTGCTAGTCAAATTGAGA-
GTCACAAACGTTTTGGTGGCGTAGTGCCCGAAGTAGCCAG TCGTCACC
ATGTCGAGGTCATTACAGCCTGTATCGAGGAGGCATTGGCAGAAGCAGGGATTACCGAAGAGGACG-
TGACAG CTGTTGCG GTTACCTACGGACCAGGCTTGGTCGG-
AGCCTTGCTAGTTGGTTTGTCAGCCGCCAAGGCCTTTGCTTGGGCT CACGGACT
TCCACTGATTCCTGTTAATCACATGGCTGGGCACCTCATGGCAGCTCAGAGTGTGGAGCC-
TTTGGAGTTTCC CTTGCTAG
CCCTTTTAGTCAGTGGTGGGCACACAGAGTTGGTCTATGTTTCTGAGGCTGGCGATTACAAGATTGTTGGAG
AGACACGA GACGATGCAGTTGGGGAGGCTTATGACAAGGT-
CGGTCGTGTCATGGGCTTGACCTATCCTGCAGGTCGTGAG ATTGACGA
GCTGGCTCATCAGGGGCAGGATATTTATGATTTCCCTCGTGCTATGATTAAGGAAGATAATCTGGA-
GTTTTC ATTCTCTG GTTTGAAATCTGCCTTTATCAATCTT-
CACCACAATGCCGAGCAAAAGGGAGAAAGTCTGTCTACAGAGGATT TGTGTGCT
TCCTTCCAAGCAGCTGTACTGGATATTCTCATGGCAAAAACCAAGAAGGCTTTGGAGAAA-
TATCCTGTTAAA ACCCTGGT
TGTGGCAGGTGGTGTGGCAGCCAATAAAGGTCTCAGAGAACGCCTAGCAGCCGAGGTTACAGATGTCAAGGT
CATCATTC CACCTCTGCGCCTCTGCGGAGACAATGCAGGT-
ATGATTGCTTATGCCAGTGTCAGCGAGTGGAACAAAGAAA ACTTTGCA
AACTTGCACCTCAATGCCAAACCAAGCCTCGCTTTTGATACCATGGAATAA-3' (B)
Streptococcus pneumoniae gcp polypeptide sequence deduced from a
polynucleotide sequence in this table [SEQ ID NO:2]. NH.sub.2-
MKDRYILAFETSCDETSVAVLKNDDELLSNVIASQIESHKRFGGV-
VPEVASRHHVEVITACIEEALAEAGIT EEDVTAVA
VTYGPGLVGALLVGLSAAKAFAWAHGLPLIPVNHMAGHLMAAQSVEPLEFPLLALLVSGGHTELVYVSEAGD
YKIVGETR DDAVGEAYDKVGRVMGLTYPAGREIDELAHQG-
QDIYDFPRAMIKEDNLEFSFSGLKSAFINLHHNAEQKGES LSTEDLCA
SFQAAVLDILMAKTKKALEKYPVKTLVVAGGVAANKGLRERLAAEVTDVKVIIPPLRLCGDNAGMI-
AYASVS EWNKENFA NLDLNAKPSLAFDTME-COOH
[0012] Deposited Materials
[0013] A deposit containing 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 AB21RY, Scotland on Apr. 11, 1996 and assigned
deposit number 40794. The deposit was described as Streptococcus
pneumoniae 0100993 on deposit. 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."
[0014] The deposited strain contains the full length gcp gene. The
sequence of the polynucleotides contained 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.
[0015] The deposit of the deposited strain has been made under the
terms of the Budapest Treaty on the International Recognition of
the Deposit of Micro-organisms for Purposes of Patent Procedure.
The strain will be irrevocably and without restriction or condition
released to the public upon the issuance of a patent. The deposited
strain is provided merely as convenience to those of skill in the
art and is not an admission that a deposit is required for
enablement, such as that required under 35 U.S.C. .sctn. 112.
[0016] A license may be required to make, use or sell the deposited
strain, and compounds derived therefrom, and no such license is
hereby granted.
[0017] 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
contained in the deposited strain. Further provided by the
invention are gcp polynucleotide sequences in the deposited strain,
such as DNA and RNA, and amino acid sequences encoded thereby. Also
provided by the invention are gcp polypeptide and polynucleotide
sequences isolated from the deposited strain.
[0018] Polypeptides
[0019] Gcp polypeptide of the invention is substantially
phylogenetically related to other proteins of the glycopeptidase
family.
[0020] In one aspect of the invention there are provided
polypeptides of Streptococcus pneumoniae referred to herein as
"gcp" and "gcp polypeptides" as well as biologically,
diagnostically, prophylactically, clinically or therapeutically
useful variants thereof, and compositions comprising the same.
[0021] Among the particularly preferred embodiments of the
invention are variants of gcp polypeptide encoded by naturally
occurring alleles of the gcp gene.
[0022] The present invention further provides for an isolated
polypeptide which:
[0023] (a) comprises or consists of an amino acid sequence which
has at least 70% identity, preferably at least 80% identity, more
preferably at least 90% identity, yet more preferably 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;
[0024] (b) a polypeptide encoded by an isolated polynucleotide
comprising or consisting of a polynucleotide sequence which has at
least 70% identity, preferably at least 80% identity, more
preferably at least 90% identity, yet more preferably 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;
[0025] (c) a polypeptide encoded by an isolated polynucleotide
comprising or consisting of a polynucleotide sequence encoding a
polypeptide which has at least 70% identity, preferably at least
80% identity, more preferably at least 90% identity, yet more
preferably 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.
[0026] The polypeptides of the invention include a polypeptide of
Table 1 [SEQ ID NO:2] (in particular the mature polypeptide) as
well as polypeptides and fragments, particularly those which have
the biological activity of gcp, and also those which have at least
70% identity to a polypeptide of Table 1 [SEQ ID NO:1 ]or the
relevant portion, preferably at least 80% identity to a polypeptide
of Table 1 [SEQ ID NO:2 and more preferably at least 90% identity
to a polypeptide of Table 1 [SEQ ID NO:2] and still more preferably
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 containing at least 30 amino acids and more
preferably at least 50 amino acids.
[0027] 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
[0028] 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 its 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.
[0029] 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.
[0030] 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 gcp
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.
[0031] 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 includes 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.
[0032] 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.
[0033] Also preferred are biologically active fragments which are
those fragments that mediate activities of gcp, including those
with a similar activity or an improved activity, or with a
decreased undesirable activity. Also included are those fragments
that are antigenic or immunogenic in an animal, especially in a
human. Particularly preferred are fragments comprising receptors or
domains of enzymes that confer a function essential for viability
of Streptococcus pneumoniae or the ability to initiate, or maintain
cause Disease in an individual, particularly a human.
[0034] 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.
[0035] In addition to the standard single and triple letter
representations for amino acids, the term "X" or "Xaa" may also be
used in describing certain polypeptides of the invention. "X" and
"Xaa" mean that any of the twenty naturally occurring amino acids
may appear at such a designated position in the polypeptide
sequence.
[0036] Polynucleotides
[0037] It is an object of the invention to provide polynucleotides
that encode gcp polypeptides, particularly polynucleotides that
encode the polypeptide herein designated gcp.
[0038] In a particularly preferred embodiment of the invention the
polynucleotide comprises a region encoding gcp polypeptides
comprising a sequence set out in Table 1 [SEQ ID NO: 1] which
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 which possesses it, such as
Streptococcus pneumoniae.
[0039] As a further aspect of the invention there are provided
isolated nucleic acid molecules encoding and/or expressing gcp
polypeptides and polynucleotides, particularly Streptococcus
pneumoniae gcp 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.
[0040] Another aspect of the invention relates to isolated
polynucleotides, including at least one full length gene, that
encodes a gcp polypeptide having a deduced amino acid sequence of
Table 1 [SEQ ID NO:2] and polynucleotides closely related thereto
and variants thereof.
[0041] In another particularly preferred embodiment of the
invention there is a gcp polypeptide from Streptococcus pneumoniae
comprising or consisting of an amino acid sequence of Table 1 [SEQ
ID NO:2], or a variant thereof.
[0042] 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 gcp 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 sequencing 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.
[0043] 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 which begins at nucleotide number 1009
of SEQ ID NO:1, encodes the polypeptide of SEQ ID NO:2.
[0044] In a further aspect, the present invention provides for an
isolated polynucleotide comprising or consisting of:
[0045] (a) a polynucleotide sequence which has at least 70%
identity, preferably at least 80% identity, more preferably at
least 90% identity, yet more preferably 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;
[0046] (b) a polynucleotide sequence encoding a polypeptide which
has at least 70% identity, preferably at least 80% identity, more
preferably at least 90% identity, yet more preferably at least 95%
identity, even more preferably at least 97-99% or 100% exact, to
the amino acid sequence of SEQ ID NO:2, over the entire length of
SEQ ID NO:2.
[0047] 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 which
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 containing said polynucleotide sequence.
[0048] 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 contain 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 the fused
polypeptide can be encoded. In certain embodiments of the
invention, the marker sequence is a hexa-histidine peptide, as
provided in the pQE vector (Qiagen, Inc.) and described in Gentz et
al., Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA
peptide tag (Wilson et al., Cell 37: 767 (1984), both of which 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.
[0049] 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 1009 set forth in
SEQ ID NO:1 of Table 1, both of which encode the gcp
polypeptide.
[0050] 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
[0051] 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, which 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 50, 100
or 500, and n is an integer between 1 and 50, 100, or 500.
[0052] 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.
[0053] 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 gcp 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 contain coding and/or non-coding
sequences.
[0054] 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 a polynucleotides of the invention may be used,
for example, to synthesize full-length polynucleotides of the
invention.
[0055] Further particularly preferred embodiments are
polynucleotides encoding gcp variants, that have the amino acid
sequence of gcp 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 gcp polypeptide.
[0056] Further preferred embodiments of the invention are
polynucleotides that are at least 70% identical over their entire
length to a polynucleotide encoding gcp polypeptide having an amino
acid sequence set out in Table 1 [SEQ ID NO:2], and polynucleotides
that are complementary to such polynucleotides. Alternatively, most
highly preferred are polynucleotides that comprise a region that is
at least 80% identical over its entire length to a polynucleotide
encoding gcp polypeptide and polynucleotides complementary thereto.
In this regard, polynucleotides at least 90% identical over their
entire length to the same are particularly preferred, and among
these particularly preferred polynucleotides, those with at least
95% are especially preferred. Furthermore, those with at least 97%
are highly preferred among those with at least 95%, and among these
those with at least 98% and at least 99% are particularly highly
preferred, with at least 99% being the more preferred.
[0057] Preferred embodiments are polynucleotides encoding
polypeptides that retain substantially the same biological function
or activity as the mature polypeptide encoded by a DNA of Table 1
[SEQ ID NO:1].
[0058] In accordance with certain preferred embodiments of this
invention there are provided polynucleotides that hybridize,
particularly under stringent conditions, to gcp polynucleotide
sequences, such as those polynucleotides in Table 1.
[0059] 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 (pH7.6), 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.
[0060] The invention also provides a polynucleotide consisting of
or comprising a polynucleotide sequence obtained by screening an
appropriate library containing the complete gene for a
polynucleotide sequence set forth in SEQ ID NO:1 under stringent
hybridization conditions with a probe having the sequence of said
polynucleotide sequence set forth in SEQ ID NO:1 or a fragment
thereof; and isolating said polynucleotide sequence. Fragments
useful for obtaining such a polynucleotide include, for example,
probes and primers fully described elsewhere herein.
[0061] 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 gcp and to isolate cDNA and genomic clones of other genes
that have a high identity, particularly high sequence identity, to
the gcp 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.
[0062] A coding region of a gcp 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.
[0063] 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 known 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.
[0064] 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.
[0065] 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.
[0066] The invention also provides polynucleotides that encode a
polypeptide that is the mature protein plus additional amino or
carboxyl-terminal amino acids, or amino acids interior to the
mature polypeptide (when the mature form has more than one
polypeptide chain, for instance). Such sequences may play a role in
processing of a protein from precursor to a mature form, may allow
protein transport, may lengthen or shorten protein half-life or may
facilitate manipulation of a protein for assay or production, among
other things. As generally is the case in vivo, the additional
amino acids may be processed away from the mature protein by
cellular enzymes.
[0067] 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.
[0068] 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.
[0069] In addition to the standard A, G, C, T/U representations for
nucleotides, the term "N" may also be used in describing certain
polynucleotides of the invention. "N" means that any of the four
DNA or RNA nucleotides may appear at such a designated position in
the DNA or RNA sequence, except it is preferred that N is not a
nucleic acid that when taken in combination with adjacent
nucleotide positions, when read in the correct reading frame, would
have the effect of generating a premature termination codon in such
reading frame.
[0070] In sum, a polynucleotide of the invention may encode a
mature protein, a mature protein plus a leader sequence (which may
be referred to as a preprotein), a precursor of a mature protein
having one or more prosequences that are not the leader sequences
of a preprotein, or a preproprotein, which is a precursor to a
proprotein, having a leader sequence and one or more prosequences,
which generally are removed during processing steps that produce
active and mature forms of the polypeptide.
[0071] Vectors, Host Cells, Expression Systems
[0072] The invention also relates to vectors that comprise a
polynucleotide or polynucleotides of the invention, host cells that
are genetically engineered with vectors of the invention and the
production of polypeptides of the invention by recombinant
techniques. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the invention.
[0073] 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 which comprise a polynucleotide or
polynucleotides of the present invention, to host cells which are
genetically engineered with such expression systems, and to the
production of polypeptides of the invention by recombinant
techniques.
[0074] 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 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.
[0075] 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 Drosophila
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.
[0076] 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,
picomaviruses and retroviruses, and vectors derived from
combinations thereof, such as those derived from plasmid and
bacteriophage genetic elements, such as cosmids and phagemids. The
expression system constructs may contain control regions that
regulate as well as engender expression. Generally, any system or
vector suitable to maintain, propagate or express polynucleotides
and/or to express a polypeptide in a host may be used for
expression in this regard. The appropriate DNA sequence may be
inserted into the expression system by any of a variety of
well-known and routine techniques, such as, for example, those set
forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL,
(supra).
[0077] 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.
[0078] 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.
[0079] Diagnostic, Prognostic, Serotyping and Mutation Assays
[0080] This invention is also related to the use of gcp
polynucleotides and polypeptides of the invention for use as
diagnostic reagents. Detection of gcp 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 gcp 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.
[0081] 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 gcp 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, Vi and SI protection assay or a
chemical cleavage method. See, for example, Cotton et al., Proc.
Natl. Acad. Sci., USA, 85: 4397-4401 (1985).
[0082] In another embodiment, an array of oligonucleotides probes
comprising gcp 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 expression, genetic linkage, and
genetic variability (see, for example, Chee et al., Science, 274:
610 (1996)).
[0083] Thus in another aspect, the present invention relates to a
diagnostic kit which comprises:
[0084] (a) a polynucleotide of the present invention, preferably
the nucleotide sequence of SEQ ID NO: 1 , or a fragment
thereof;
[0085] (b) a nucleotide sequence complementary to that of (a);
[0086] (c) a polypeptide of the present invention, preferably the
polypeptide of SEQ ID NO:2 or a fragment thereof; or
[0087] (d) an antibody to a polypeptide of the present invention,
preferably to the polypeptide of SEQ ID NO:2.
[0088] It will be appreciated that 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.
[0089] 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 , which is associated with a disease or pathogenicity will
provide a diagnostic tool that can add to, or define, a diagnosis
of a disease, a prognosis of a course of disease, a determination
of a stage of disease, or a susceptibility to a disease, which
results from under-expression, over-expression or altered
expression of the polynucleotide. Organisms, particularly
infectious organisms, carrying mutations in such polynucleotide may
be detected at the polynucleotide level by a variety of techniques,
such as those described elsewhere herein.
[0090] The nucleotide sequences of the present invention are also
valuable for organism chromosome identification. The sequence is
specifically targeted to, and can hybridize with, a particular
location on an organism's chromosome, particularly to a
Streptococcus pneumoniae chromosome. The mapping of relevant
sequences to chromosomes according to the present invention may be
an important step in correlating those sequences with pathogenic
potential and/or an ecological niche of an organism and/or drug
resistance of an organism, as well as the essentiality of the gene
to the organism. Once a sequence has been mapped to a precise
chromosomal location, the physical position of the sequence on the
chromosome can be correlated with genetic map data. Such data may
be found on-line in a sequence database. The relationship between
genes and diseases that have been mapped to the same chromosomal
region are then identified through known genetic methods, for
example, through linkage analysis (coinheritance of physically
adjacent genes) or mating studies, such as by conjugation.
[0091] 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.
[0092] 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 in the
RNA. It is particularly preferred to use RT-PCR in conjunction with
automated detection systems, such as, for example, GeneScan. RNA,
cDNA or genomic DNA may also be used for the same purpose, PCR. As
an example, PCR primers complementary to a polynucleotide encoding
gcp polypeptide can be used to identify and analyze mutations.
Examples of representative primers are shown below in Table 2.
2TABLE 2 Primers for amplification of gcp polynucleotides SEQ ID NO
PRIMER SEQUENCE 3 5'-GCATTGGCAGAAGCAGGGATTAC-3' 4
5'-CTCGGCATTGTGGTGAAGATTGAT-3'
[0093] The invention also includes primers of the formula:
X-(R.sub.1).sub.m-(R.sub.2)-(R.sub.3).sub.n-Y
[0094] wherein, at the 5' end of the molecule, X is hydrogen, a
metal or a modified nucleotide residue, and at the 3' end of the
molecule, Y is hydrogen, a metal or a modified nucleotide residue,
R.sub.1 and R.sub.3 are any nucleic acid residue or modified
nucleotide residue, m is an integer between 1 and 20 or zero , n is
an integer between 1 and 20 or zero, and R.sub.2 is a primer
sequence of the invention, particularly a primer sequence selected
from Table 2. In the polynucleotide formula above R.sub.2 is
oriented so that its 5' end nucleotide residue is at the left,
bound to R.sub.1, and its 3' end nucleotide residue is at the
right, bound to R.sub.3. Any stretch of nucleic acid residues
denoted by either R group, where m and/or n is greater than 1, may
be either a heteropolymer or a homopolymer, preferably a
heteropolymer being complementary to a region of a polynucleotide
of Table 1. In a preferred embodiment m and/or n is an integer
between 1 and 10.
[0095] The invention further provides these primers with 1, 2, 3
nucleotides removed from the 5' and/or the 3' end. These primers
may be used for, among other things, amplifying gcp 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.
[0096] 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 expression of a gcp polynucleotide can be measured using
any on of the methods well known in the art for the quantitation of
polynucleotides, such as, for example, amplification, PCR, RT-PCR,
RNase protection, Northern blotting, spectrometry and other
hybridization methods.
[0097] In addition, a diagnostic assay in accordance with the
invention for detecting over-expression of gcp 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 gcp 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.
[0098] Differential Expression
[0099] The polynucleotides and polynucleotides of the invention may
be used as reagents for differential screening methods. There are
many differential screening and differential display methods known
in the art in which the polynucleotides and polypeptides of the
invention may be used. For example, the differential display
technique is described by Chuang et al., J. Bacteriol.
175:2026-2036 (1993). This method identifies those genes which are
expressed in an organism by identifying mRNA present using
randomly-primed RT-PCR. By comparing pre-infection and post
infection profiles, genes up and down regulated during infection
can be identified and the RT-PCR product sequenced and matched to
ORF "unknowns."
[0100] In Vivo Expression Technology (IVET) is described by Camilli
et al., Proc. Nat'l. Acad. Sci. USA. 91:2634-2638 (1994). IVET
identifies genes up-regulated during infection when compared to
laboratory cultivation, implying an important role in infection.
ORFs identified by this technique are implied to have a significant
role in infection establishment and/or maintenance. In this
technique random chromosomal fragments of target organism are
cloned upstream of a promoter-less recombinase gene in a plasmid
vector. This construct is introduced into the target organism which
carries an antibiotic resistance gene flanked by resolvase sites.
Growth in the presence of the antibiotic removes from the
population those fragments cloned into the plasmid vector capable
of supporting transcription of the recombinase gene and therefore
have caused loss of antibiotic resistance. The resistant pool is
introduced into a host and at various times after infection
bacteria may be recovered and assessed for the presence of
antibiotic resistance. The chromosomal fragment carried by each
antibiotic sensitive bacterium should carry a promoter or portion
of a gene normally upregulated during infection. Sequencing
upstream of the recombinase gene allows identification of the up
regulated gene.
[0101] RT-PCR may also be used to analyze gene expression patterns.
For RT PCR using the polynucleotides of the invention, messenger
RNA is isolated from bacterial infected tissue, e.g., 48 hour
murine lung infections, and the amount of each MRNA species
assessed by reverse transcription of the RNA sample primed with
random hexanucleotides followed by PCR with gene specific primer
pairs. The determination of the presence and amount of a particular
mRNA species by quantification of the resultant PCR product
provides information on the bacterial genes which are transcribed
in the infected tissue. Analysis of gene transcription can be
carried out at different times of infection to gain a detailed
knowledge of gene regulation in bacterial pathogenesis allowing for
a clearer understanding of which gene products represent targets
for screens for antibacterials. Because of the gene specific nature
of the PCR primers employed it should be understood that the
bacterial mRNA preparation need not be free of mammalian RNA. This
allows the investigator to carry out a simple and quick RNA
preparation from infected tissue to obtain bacterial mRNA species
which are very short lived in the bacterium (in the order of 2
minute halflives). Optimally the bacterial mRNA is prepared from
infected murine lung tissue by mechanical disruption in the
presence of TRIzole (GIBCO-BRL) for very short periods of time,
subsequent processing according to the manufacturers of TRIzole
reagent and DNAase treatment to remove contaminating DNA.
Preferably the process is optimized by finding those conditions
which give a maximum amount of Streptococcus pneumoniae 16S
ribosomal RNA as detected by probing Northerns with a suitably
labeled sequence specific oligonucleotide probe. Typically a 5' dye
labeled primer is used in each PCR primer pair in a PCR reaction
which is terminated optimally between 8 and 25 cycles. The PCR
products are separated on 6% polyacrylamide gels with detection and
quantification using GeneScanner (manufactured by ABI).
[0102] Gridding and Polynucleotide Subtraction
[0103] Methods have been described for obtaining information about
gene expression and identity using so called "high density DNA
arrays" or grids. See, e.g., M. Chee et al., Science, 274:610-614
(1996) and other references cited therein. Such gridding assays
have been employed to identify certain novel gene sequences,
referred to as Expressed Sequence Tags (EST) (Adams et a., Science,
252:1651-1656 (1991)). A variety of techniques have also been
described for identifying particular gene sequences on the basis of
their gene products. For example, see International Patent
Application No. WO91/07087, published May 30, 1991. In addition,
methods have been described for the amplification of desired
sequences. For example, see International Patent Application No.
WO91/17271, published Nov. 14, 1991.
[0104] The polynucleotides of the invention may be used as
components of polynucleotide arrays, preferably high density arrays
or grids. These high density arrays are particularly useful for
diagnostic and prognostic purposes. For example, a set of spots
each comprising a different gene, and further comprising a
polynucleotide or polynucleotides of the invention, may be used for
probing, such as using hybridization or nucleic acid amplification,
using a probes obtained or derived from a bodily sample, to
determine the presence of a particular polynucleotide sequence or
related sequence in an individual. Such a presence may indicate the
presence of a pathogen, particularly Streptococcus pneumoniae, and
may be useful in diagnosing and/or prognosing disease or a course
of disease. A grid comprising a number of variants of the
polynucleotide sequence of SEQ ID NO:1 are preferred. Also
preferred is a comprising a number of variants of a polynucleotide
sequence encoding the polypeptide sequence of SEQ ID NO:2.
[0105] Antibodies
[0106] The polypeptides and polynucleotides of the invention or
variants thereof, or cells expressing the same can be used as
immunogens to produce antibodies immunospecific for such
polypeptides or polynucleotides respectively.
[0107] In certain preferred embodiments of the invention there are
provided antibodies against gcp polypeptides or
polynucleotides.
[0108] Antibodies generated against the polypeptides or
polynucleotides of the invention can be obtained by administering
the polypeptides and/or polynucleotides of the invention, or
epitope-bearing fragments of either or both, analogues of either or
both, or cells expressing either or both, to an animal, preferably
a nonhuman, using routine protocols. For preparation of monoclonal
antibodies, any technique known in the art that provides antibodies
produced by continuous cell line cultures can be used. Examples
include various techniques, such as those in Kohler, G. and
Milstein, C., Nature 256: 495-497 (1975); Kozbor et al., Immunology
Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES
AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
[0109] Techniques for the production of single chain antibodies
(U.S. Pat. No. 4,946,778) can be adapted to produce single chain
antibodies to polypeptides or polynucleotides of this invention.
Also, transgenic mice, or other organisms such as other mammals,
may be used to express humanized antibodies immunospecific to the
polypeptides or polynucleotides of the invention.
[0110] Alternatively, phage display technology may be utilized to
select antibody genes with binding activities towards a polypeptide
of the invention either from repertoires of PCR amplified v-genes
of lymphocytes from humans screened for possessing anti-gcp or from
naive libraries (McCafferty, et al., (1990), Nature 348, 552-554;
Marks, et al., (1992) Biotechnology 10, 779-783). The affinity of
these antibodies can also be improved by, for example, chain
shuffling (Clackson et al., (1991) Nature 352: 628).
[0111] The above-described antibodies may be employed to isolate or
to identify clones expressing the polypeptides or polynucleotides
of the invention to purify the polypeptides or polynucleotides by,
for example, affinity chromatography.
[0112] Thus, among others, antibodies against gcp-polypeptide or
gep-polynucleotide may be employed to treat infections,
particularly bacterial infections.
[0113] Polypeptide variants include antigenically, epitopically or
immunologically equivalent variants form a particular aspect of
this invention.
[0114] A polypeptide or polynucleotide of the invention, such as an
antigenically or immunologically equivalent derivative or a fusion
protein of the polypeptide is used as an antigen to immunize a
mouse or other animal such as a rat or chicken. The fusion protein
may provide stability to the polypeptide. The antigen may be
associated, for example by conjugation, with an immunogenic carrier
protein for example bovine serum albumin, keyhole limpet
haemocyanin or tetanus toxoid. Alternatively, a multiple antigenic
polypeptide comprising multiple copies of the polypeptide, or an
antigenically or immunologically equivalent polypeptide thereof may
be sufficiently antigenic to improve immunogenicity so as to
obviate the use of a carrier.
[0115] Preferably, the antibody or variant thereof is modified to
make it less immunogenic in the individual. For example, if the
individual is human the antibody may most preferably be
"humanized," where the complimentarily determining region or
regions of the hybridoma-derived antibody has been transplanted
into a human monoclonal antibody, for example as described in Jones
et al. (1986), Nature 321, 522-525 or Tempest et al., (1991)
Biotechnology 9, 266-273.
[0116] In accordance with an aspect of the invention, there is
provided the use of a polynucleotide of the invention for
therapeutic or prophylactic purposes, in particular genetic
immunization. Among the particularly preferred embodiments of the
invention are naturally occurring allelic variants of gcp
polynucleotides and polypeptides encoded thereby.
[0117] The use of a polynucleotide of the invention in genetic
immunization will preferably employ a suitable delivery method such
as direct injection of plasmid DNA into muscles (Wolff et al., Hum
Mol Genet (1992) 1: 363, Manthorpe et al., Hum. Gene Ther. (1983)
4: 419), delivery of DNA complexed with specific protein carriers
(Wu et al., J Biol Chem. (1989) 264: 16985), coprecipitation of DNA
with calcium phosphate (Benvenisty & Reshef, PNAS USA, (1986)
83: 9551), encapsulation of DNA in various forms of liposomes
(Kaneda et al., Science (1989) 243: 375), particle bombardment
(Tang et al., Nature (1992) 356:152, Eisenbraun et al., DNA Cell
Biol (1993) 12: 791) and in vivo infection using cloned retroviral
vectors (Seeger et al., PNAS USA (1984) 81: 5849).
[0118] Antagonists and Agonists--Assays and Molecules
[0119] 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).
[0120] Polypeptides and polynucleotides of the present invention
are responsible for many biological functions, including many
disease states, in particular the Diseases hereinbefore mentioned.
It is therefore desirable to devise screening methods to identify
compounds which stimulate or which 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 which stimulate or which inhibit the function of
a polypeptide or polynucleotide of the invention, as well as
related polypeptides and polynucleotides. In general, agonists or
antagonists may be employed for therapeutic and prophylactic
purposes for such Diseases as hereinbefore mentioned. Compounds may
be identified from a variety of sources, for example, cells,
cell-free preparations, chemical libraries, and natural product
mixtures. Such agonists, antagonists or inhibitors so-identified
may be natural or modified substrates, ligands, receptors, enzymes,
etc., as the case may be, of gcp polypeptides and polynucleotides;
or may be structural or functional mimetics thereof (see Coligan et
al., Current Protocols in Immunology 1(2):Chapter 5 (1991)).
[0121] 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 or inhibitors, in the absence of an
agonist or inhibitor, 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 containing a polypeptide or polynucleotide of the present
invention, to form a mixture, measuring gcp polypeptide and/or
polynucleotide activity in the mixture, and comparing the gcp
polypeptide and/or polynucleotide activity of the mixture to a
standard. Fusion proteins, such as those made from Fe portion and
gcp polypeptide, as hereinbefore 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)).
[0122] 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 which
may inhibit or enhance the production of polypeptide (also called
antagonist or agonist, respectively) from suitably manipulated
cells or tissues.
[0123] The invention also provides a method of screening compounds
to identify those which enhance (agonist) or block (antagonist) the
action of gcp polypeptides or polynucleotides, particularly those
compounds that are bacteriostatic 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 gcp polypeptide
and a labeled substrate or ligand of such polypeptide is incubated
in the absence or the presence of a candidate molecule that may be
a gcp agonist or antagonist. The ability of the candidate molecule
to agonize or antagonize the gcp polypeptide is reflected in
decreased binding of the labeled ligand or decreased production of
product from such substrate. Molecules that bind gratuitously, ie.,
without inducing the effects of gcp 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 calorimetric, labeled substrate converted
into product, a reporter gene that is responsive to changes in gcp
polynucleotide or polypeptide activity, and binding assays known in
the art.
[0124] 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 which 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.
[0125] In other embodiments of the invention there are provided
methods for identifying compounds which bind to or otherwise
interact with and inhibit or activate an activity or expression 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.
[0126] Another example of an assay for gcp agonists is a
competitive assay that combines gcp and a potential agonist with
gcp-binding molecules, recombinant gcp binding molecules, natural
substrates or ligands, or substrate or ligand mimetics, under
appropriate conditions for a competitive inhibition assay. Gcp can
be labeled, such as by radioactivity or a colorimetric compound,
such that the number of gcp molecules bound to a binding molecule
or converted to product can be determined accurately to assess the
effectiveness of the potential antagonist.
[0127] Potential antagonists include, among others, small organic
molecules, peptides, polypeptides and antibodies that bind to a
polynucleotide and/or polypeptide of the invention and thereby
inhibit or extinguish its activity or expression. Potential
antagonists also may be small organic molecules, a peptide, a
polypeptide such as a closely related protein or antibody that
binds the same sites on a binding molecule, such as a binding
molecule, without inducing gcp-induced activities, thereby
preventing the action or expression of gcp polypeptides and/or
polynucleotides by excluding gcp polypeptides and/or
polynucleotides from binding.
[0128] Potential antagonists include a small molecule that binds to
and occupies the binding site of the polypeptide thereby preventing
binding to cellular binding molecules, such that normal biological
activity is prevented. Examples of small molecules include but are
not limited to small organic molecules, peptides or peptide-like
molecules. Other potential antagonists include antisense molecules
(see Okano, J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS
ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton,
Fla. (1988), for a description of these molecules). Preferred
potential antagonists include compounds related to and variants of
gcp.
[0129] Other examples of potential polypeptide antagonists include
antibodies or, in some cases, oligonucleotides or proteins which
are closely related to the ligands, substrates, receptors, enzymes,
etc., as the case may be, of the polypeptide, e.g., a fragment of
the ligands, substrates, receptors, enzymes, etc.; or small
molecules which bind to the polypeptide of the present invention
but do not elicit a response, so that the activity of the
polypeptide is prevented.
[0130] Certain of the polypeptides of the invention are
biomimetics, functional mimetics of the natural gcp polypeptide.
These functional mimetics may be used for, among other things,
antagonizing the activity of gcp polypeptide or as a antigen or
immunogen in a manner described elsewhere herein. Functional
mimetics of the polypeptides of the invention include but are not
limited to truncated polypeptides. For example, preferred
functional mimetics include, a polypeptide comprising the
polypeptide sequence set forth in SEQ ID NO:2 lacking 20, 30, 40,
50, 60, 70 or 80 amino- or carboxy-terminal amino acid residues,
including fusion proteins comprising one or more of these truncated
sequences. Polynucleotides encoding each of these functional
mimetics may be used as expression cassettes to express each
mimetic polypeptide. It is preferred that these cassettes comprise
5' and 3' restriction sites to allow for a convenient means to
ligate the cassettes together when desired. It is further preferred
that these cassettes comprise gene expression signals known in the
art or described elsewhere herein.
[0131] Thus, in another aspect, the present invention relates to a
screening kit for identifying agonists, antagonists, ligands,
receptors, substrates, enzymes, etc. for a polypeptide and/or
polynucleotide of the present invention; or compounds which
decrease or enhance the production of such polypeptides and/or
polynucleotides , which comprises:
[0132] (a) a polypeptide and/or a polynucleotide of the present
invention;
[0133] (b) a recombinant cell expressing a polypeptide and/or
polynucleotide of the present invention;
[0134] (c) a cell membrane expressing a polypeptide and/or
polynucleotide of the present invention; or
[0135] (d) antibody to a polypeptide and/or polynucleotide of the
present invention; which polypeptide is preferably that of SEQ ID
NO:2, and which polynucleotide is preferably that of SEQ ID
NO:1.
[0136] It will be appreciated that in any such kit, (a), (b), (c)
or (d) may comprise a substantial component.
[0137] It will be readily appreciated by the skilled 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,
antagonist or inhibitor of the polypeptide and/or polynucleotide,
by:
[0138] (a) determining in the first instance the three-dimensional
structure of the polypeptide and/or polynucleotide, or complexes
thereof;
[0139] (b) deducing the three-dimensional structure for the likely
reactive site(s), binding site(s) or motif(s) of an agonist,
antagonist or inhibitor;
[0140] (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
[0141] (d) testing whether the candidate compounds are indeed
agonists, antagonists or inhibitors. 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.
[0142] 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 gcp polypeptide and/or
polynucleotide.
[0143] 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 gcp polypeptide and/or
polypeptide.
[0144] In a further aspect, the present invention relates to
genetically engineered soluble fusion proteins comprising a
polypeptide of the present invention, or a fragment thereof, and
various portions of the constant regions of heavy or light chains
of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
Preferred as an immunoglobulin is the constant part of the heavy
chain of human IgG, particularly IgG1, where fusion takes place at
the hinge region. In a particular embodiment, the Fc part can be
removed simply by incorporation of a cleavage sequence which can be
cleaved with blood clotting factor Xa. Furthermore, this invention
relates to processes for the preparation of these fusion proteins
by genetic engineering, and to the use thereof for drug screening,
diagnosis and therapy. A further aspect of the invention also
relates to polynucleotides encoding such fusion proteins. Examples
of fusion protein technology can be found in International Patent
Application Nos. WO94/29458 and WO94/22914.
[0145] In still another approach, expression of the gene encoding
endogenous gcp 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 which 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.
[0146] 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-Delgamo 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.
[0147] 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 on
in-dwelling devices or to extracellular matrix proteins in wounds;
to block gcp protein-mediated mammalian cell invasion by, for
example, initiating phosphorylation of mammalian tyrosine kinases
(Rosenshine et al., Infect. Immun. 60:2211 (1992); to block
bacterial adhesion between eukaryotic, preferably mammalian,
extracellular matrix proteins and bacterial gcp 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.
[0148] In accordance with yet another aspect of the invention,
there are provided gcp agonists and antagonists, preferably
bacteriostatic or bacteriocidal agonists and antagonists.
[0149] The antagonists and agonists of the invention may be
employed, for instance, to prevent, inhibit and/or treat
diseases.
[0150] Helicobacter pylori (herein "H. pylori") bacteria infect the
stomachs of over one-third of the world's population causing
stomach cancer, ulcers, and gastritis (International Agency for
Research on Cancer (1994) Schistosomes, Liver Flukes and
Helicobacter Pylori (International Agency for Research on Cancer,
Lyon, France, http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the
International Agency for Research on Cancer recently recognized a
cause-and-effect relationship between H. pylori and gastric
adenocarcinoma, classifying the bacterium as a Group I (definite)
carcinogen. Preferred antimicrobial compounds of the invention
(agonists and antagonists of gcp 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.
[0151] Vaccines
[0152] There are provided by the invention, products, compositions
and methods for assessing gcp expression, treating disease,
assaying genetic variation, and administering a gcp polypeptide
and/or polynucleotide to an organism to raise an immunological
response against a bacteria, especially a Streptococcus pneumoniae
bacteria.
[0153] Another aspect of the invention relates to a method for
inducing an immunological response in an individual, particularly a
mammal which comprises inoculating the individual with gcp
polynucleotide and/or polypeptide, or a fragment or variant
thereof, adequate to produce antibody and/or T cell immune response
to protect said individual from infection, particularly bacterial
infection and most particularly Streptococcus pneumoniae infection.
Also provided are methods whereby such immunological response slows
bacterial replication. Yet another aspect of the invention relates
to a method of inducing immunological response in an individual
which comprises delivering to such individual a nucleic acid
vector, sequence or ribozyme to direct expression of gcp
polynucleotide and/or polypeptide, or a fragment or a variant
thereof, for expressing gcp polynucleotide and/or polypeptide, or a
fragment or a variant thereof in vivo in order to induce an
immunological response, such as, to produce antibody and/or T cell
immune response, including, for example, cytokine-producing T cells
or cytotoxic T cells, to protect said individual, preferably a
human, from disease, whether that disease is already established
within the individual or not. One example of administering the gene
is by accelerating it into the desired cells as a coating on
particles or otherwise. Such nucleic acid vector may comprise DNA,
RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, a
DNA-protein complex or an RNA-protein complex.
[0154] A further aspect of the invention relates to an
immunological composition that when introduced into an individual,
preferably a human, capable of having induced within it an
immunological response, induces an immunological response in such
individual to a gcp polynucleotide and/or polypeptide encoded
therefrom, wherein the composition comprises a recombinant gcp
polynucleotide and/or polypeptide encoded therefrom and/or
comprises DNA and/or RNA which encodes and expresses an antigen of
said gop polynucleotide, polypeptide encoded therefrom, or other
polypeptide of the invention. The immunological response may be
used therapeutically or prophylactically and may take the form of
antibody immunity and/or cellular immunity, such as cellular
immunity arising from CTL or CD4+T cells.
[0155] A gcp polypeptide or a fragment thereof may be fused with
co-protein or chemical moiety which may or may not by itself
produce antibodies, but which is capable of stabilizing the first
protein and producing a fused or modified protein which will have
antigenic and/or immunogenic properties, and preferably protective
properties. Thus fused recombinant protein, preferably further
comprises an antigenic co-protein, such as lipoprotein D from
Hemophilus influenzae, ; Glutathione-S-transferase (GST) or
beta-galactosidase, or any other relatively large co-protein which
solubilizes the protein and facilitates production and purification
thereof. Moreover, the co-protein may act as an adjuvant in the
sense of providing a generalized stimulation of the immune system
of the organism receiving the protein. The co-protein may be
attached to either the amino- or carboxy-terminus of the first
protein.
[0156] Provided by this invention are compositions, particularly
vaccine compositions, and methods comprising the polypeptides
and/or polynucleotides of the invention and immunostimulatory DNA
sequences, such as those described in Sato, Y. et al. Science 273:
352 (1996).
[0157] Also, provided by this invention are methods using the
described polynucleotide or particular fragments thereof, which
have been shown to encode non-variable regions of bacterial cell
surface proteins, in polynucleotide constructs used in such genetic
immunization experiments in animal models of infection with
Streptococcus pneumoniae. Such experiments will be particularly
useful for identifying protein epitopes able to provoke a
prophylactic or therapeutic immune response. It is believed that
this approach will allow for the subsequent preparation of
monoclonal antibodies of particular value, derived from the
requisite organ of the animal successfully resisting or clearing
infection, for the development of prophylactic agents or
therapeutic treatments of bacterial infection, particularly
Streptococcus pneumoniae infection, in mammals, particularly
humans.
[0158] A polypeptide of the invention may be used as an antigen for
vaccination of a host to produce specific antibodies which protect
against invasion of bacteria, for example by blocking adherence of
bacteria to damaged tissue. Examples of tissue damage include
wounds in skin or connective tissue caused, for example, by
mechanical, chemical, thermal or radiation damage or by
implantation of indwelling devices, or wounds in the mucous
membranes, such as the mouth, throat, mammary glands, urethra or
vagina.
[0159] The invention also includes a vaccine formulation which
comprises an immunogenic recombinant polypeptide and/or
polynucleotide of the invention together with a suitable carrier,
such as a pharmaceutically acceptable carrier. Since the
polypeptides and polynucleotides may be broken down in the stomach,
each is preferably administered parenterally, including, for
example, administration that is subcutaneous, intramuscular,
intravenous, or intradermal. Formulations suitable for parenteral
administration include aqueous and non-aqueous sterile injection
solutions which may contain anti-oxidants, buffers, bacteriostatic
compounds and solutes which render the formulation isotonic with
the bodily fluid, preferably the blood, of the individual; and
aqueous and non-aqueous sterile suspensions which may include
suspending agents or thickening agents. The formulations may be
presented in unit-dose or multi-dose containers, for example,
sealed ampoules and vials and may be stored in a freeze-dried
condition requiring only the addition of the sterile liquid carrier
immediately prior to use. The vaccine formulation may also include
adjuvant systems for enhancing the immunogenicity of the
formulation, such as oil-in water systems and other systems known
in the art. The dosage will depend on the specific activity of the
vaccine and can be readily determined by routine
experimentation.
[0160] While the invention has been described with reference to
certain gcp polypeptides and polynucleotides, it is to be
understood that this covers fragments of the naturally occurring
polypeptides and polynucleotides, and similar polypeptides and
polynucleotides with additions, deletions or substitutions which do
not substantially affect the immunogenic properties of the
recombinant polypeptides or polynucleotides.
[0161] Compositions, Kits and Administration
[0162] In a further aspect of the invention there are provided
compositions comprising a gcp polynucleotide and/or a gcp
polypeptide for administration to a cell or to a multicellular
organism.
[0163] The invention also relates to compositions comprising a
polynucleotide and/or a polypeptides discussed herein or their
agonists or antagonists. The polypeptides and polynucleotides of
the invention may be employed in combination with a non-sterile or
sterile carrier or carriers for use with cells, tissues or
organisms, such as a pharmaceutical carrier suitable for
administration to an individual. Such compositions comprise, for
instance, a media additive or a therapeutically effective amount of
a polypeptide and/or polynucleotide of the invention and a
pharmaceutically acceptable carrier or excipient. Such carriers may
include, but are not limited to, saline, buffered saline, dextrose,
water, glycerol, ethanol and combinations thereof. The formulation
should suit the mode of administration. The invention further
relates to diagnostic and pharmaceutical packs and kits comprising
one or more containers filled with one or more of the ingredients
of the aforementioned compositions of the invention.
[0164] Polypeptides, polynucleotides and other compounds of the
invention may be employed alone or in conjunction with other
compounds, such as therapeutic compounds.
[0165] The pharmaceutical compositions may be administered in any
effective, convenient manner including, for instance,
administration by topical, oral, anal, vaginal, intravenous,
intraperitoneal, intramuscular, subcutaneous, intranasal or
intradermal routes among others.
[0166] In therapy or as a prophylactic, the active agent may be
administered to an individual as an injectable composition, for
example as a sterile aqueous dispersion, preferably isotonic.
[0167] Alternatively the composition may be formulated for topical
application for example in the form of ointments, creams, lotions,
eye ointments, eye drops, ear drops, mouthwash, impregnated
dressings and sutures and aerosols, and may contain appropriate
conventional additives, including, for example, preservatives,
solvents to assist drug penetration, and emollients in ointments
and creams. Such topical formulations may also contain compatible
conventional carriers, for example cream or ointment bases, and
ethanol or oleyl alcohol for lotions. Such carriers may constitute
from about 1% to about 98% by weight of the formulation; more
usually they will constitute up to about 80% by weight of the
formulation.
[0168] In a further aspect, the present invention provides for
pharmaceutical compositions comprising a therapeutically effective
amount of a polypeptide and/or polynucleotide, such as the soluble
form of a polypeptide and/or polynucleotide of the present
invention, agonist or antagonist peptide or small molecule
compound, in combination with a pharmaceutically acceptable carrier
or excipient. Such carriers include, but are not limited to,
saline, buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof. The invention further relates to
pharmaceutical packs and kits comprising one or more containers
filled with one or more of the ingredients of the aforementioned
compositions of the invention. Polypeptides, polynucleotides and
other compounds of the present invention may be employed alone or
in conjunction with other compounds, such as therapeutic
compounds.
[0169] The composition will be adapted to the route of
administration, for instance by a systemic or an oral route.
Preferred forms of systemic administration include injection,
typically by intravenous injection. Other injection routes, such as
subcutaneous, intramuscular, or intraperitoneal, can be used.
Alternative means for systemic administration include transmucosal
and transdermal administration using penetrants such as bile salts
or fusidic acids or other detergents. In addition, if a polypeptide
or other compounds of the present invention can be formulated in an
enteric or an encapsulated formulation, oral administration may
also be possible. Administration of these compounds may also be
topical and/or localized, in the form of salves, pastes, gels, and
the like.
[0170] For administration to mammals, and particularly humans, it
is expected that the daily dosage level of the active agent will be
from 0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The
physician in any event will determine the actual dosage which will
be most suitable for an individual and will vary with the age,
weight and response of the particular individual. The above dosages
are exemplary of the average case. There can, of course, be
individual instances where higher or lower dosage ranges are
merited, and such are within the scope of this invention.
[0171] In-dwelling devices include surgical implants, prosthetic
devices and catheters, i.e., devices that are introduced to the
body of an individual and remain in position for an extended time.
Such devices include, for example, artificial joints, heart valves,
pacemakers, vascular grafts, vascular catheters, cerebrospinal
fluid shunts, urinary catheters, continuous ambulatory peritoneal
dialysis (CAPD) catheters.
[0172] The composition of the invention may be administered by
injection to achieve a systemic effect against relevant bacteria
shortly before insertion of an in-dwelling device. Treatment may be
continued after surgery during the in-body time of the device. In
addition, the composition could also be used to broaden
perioperative cover for any surgical technique to prevent bacterial
wound infections, especially Streptococcus pneumoniae wound
infections.
[0173] Many orthopedic surgeons consider that humans with
prosthetic joints should be considered for antibiotic prophylaxis
before dental treatment that could produce a bacteremia. Late deep
infection is a serious complication sometimes leading to loss of
the prosthetic joint and is accompanied by significant morbidity
and mortality. It may therefore be possible to extend the use of
the active agent as a replacement for prophylactic antibiotics in
this situation.
[0174] In addition to the therapy described above, the compositions
of this invention may be used generally as a wound treatment agent
to prevent adhesion of bacteria to matrix proteins exposed in wound
tissue and for prophylactic use in dental treatment as an
alternative to, or in conjunction with, antibiotic prophylaxis.
[0175] Alternatively, the composition of the invention may be used
to bathe an indwelling device immediately before insertion. The
active agent will preferably be present at a concentration of 1
.mu.g/ml to 10 mg/ml for bathing of wounds or indwelling
devices.
[0176] A vaccine composition is conveniently in injectable form.
Conventional adjuvants may be employed to enhance the immune
response. A suitable unit dose for vaccination is 0.5-5
microgram/kg of antigen, and such dose is preferably administered
1-3 times and with an interval of 1-3 weeks. With the indicated
dose range, no adverse toxicological effects will be observed with
the compounds of the invention which would preclude their
administration to suitable individuals.
[0177] Sequence Databases, Sequences in a Tangible Medium, and
Algorithms
[0178] Polynucleotide and polypeptide sequences form a valuable
information resource with which to determine their 2- and
3-dimensional structures as well as to identify further sequences
of similar homology. These approaches are most easily facilitated
by storing the sequence in a computer readable medium and then
using the stored data in a known macromolecular structure program
or to search a sequence database using well known searching tools,
such as GCC.
[0179] The polynucleotide and polypeptide sequences of the
invention are particularly useful as components in databases useful
for search analyses as well as in sequence analysis algorithms. As
used in this section entitled "Sequence Databases, Sequences in a
Tangible Medium, and Algorithms," and in claims related to this
section, the terms "polynucleotide of the invention" and
"polynucleotide sequence of the invention" mean any detectable
chemical or physical characteristic of a polynucleotide of the
invention that is or may be reduced to or stored in a tangible
medium, preferably a computer readable form. For example,
chromatographic scan data or peak data, photographic data or scan
data therefrom, called bases, and mass spectrographic data. As used
in this section entitled Databases and Algorithms and in claims
related thereto, the terms "polypeptide of the invention" and
"polypeptide sequence of the invention" mean any detectable
chemical or physical characteristic of a polypeptide of the
invention that is or may be reduced to or stored in a tangible
medium, preferably a computer readable form. For example,
chromatographic scan data or peak data, photographic data or scan
data therefrom, and mass spectrographic data.
[0180] The invention provides a computer readable medium having
stored thereon polypeptide sequences of the invention and/or
polynucleotide sequences of the invention. For example, a computer
readable medium is provided comprising and having stored thereon a
member selected from the group consisting of: a polynucleotide
comprising the sequence of a polynucleotide of the invention; a
polypeptide comprising the sequence of a polypeptide sequence of
the invention; a set of polynucleotide sequences wherein at least
one of the sequences comprises the sequence of a polynucleotide
sequence of the invention; a set of polypeptide sequences wherein
at least one of the sequences comprises the sequence of a
polypeptide sequence of the invention; a data set representing a
polynucleotide sequence comprising the sequence of polynucleotide
sequence of the invention; a data set representing a polynucleotide
sequence encoding a polypeptide sequence comprising the sequence of
a polypeptide sequence of the invention; a polynucleotide
comprising the sequence of a polynucleotide sequence of the
invention; a polypeptide comprising the sequence of a polypeptide
sequence of the invention; a set of polynucleotide sequences
wherein at least one of the sequences comprises the sequence of a
polynucleotide sequence of the invention; a set of polypeptide
sequences wherein at least one of said sequences comprises the
sequence of a polypeptide sequence of the invention; a data set
representing a polynucleotide sequence comprising the sequence of a
polynucleotide sequence of the invention; a data set representing a
polynucleotide sequence encoding a polypeptide sequence comprising
the sequence of a polypeptide sequence of the invention. The
computer readable medium can be any composition of matter used to
store information or data, including, for example, commercially
available floppy disks, tapes, chips, hard drives, compact disks,
and video disks.
[0181] Also provided by the invention are methods for the analysis
of character sequences or strings, particularly genetic sequences
or encoded genetic sequences. Preferred methods of sequence
analysis include, for example, methods of sequence homology
analysis, such as identity and similarity analysis, RNA structure
analysis, sequence assembly, cladistic analysis, sequence motif
analysis, open reading frame determination, nucleic acid base
calling, nucleic acid base trimming, and sequencing chromatogram
peak analysis.
[0182] A computer based method is provided for performing homology
identification. This method comprises the steps of providing a
polynucleotide sequence comprising the sequence a polynucleotide of
the invention in a computer readable medium; and comparing said
polynucleotide sequence to at least one polynucleotide or
polypeptide sequence to identify homology.
[0183] A computer based method is also provided for performing
homology identification, said method comprising the steps of:
providing a polypeptide sequence comprising the sequence of a
polypeptide of the invention in a computer readable medium; and
comparing said polypeptide sequence to at least one polynucleotide
or polypeptide sequence to identify homology.
[0184] A computer based method is still further provided for
polynucleotide assembly, said method comprising the steps of:
providing a first polynucleotide sequence comprising the sequence
of a polynucleotide of the invention in a computer readable medium;
and screening for at least one overlapping region between said
first polynucleotide sequence and a second polynucleotide
sequence.
[0185] A further embodiment of the invention provides a computer
based method for performing homology identification, said method
comprising the steps of: providing a polynucleotide sequence
comprising the sequence of a polynucleotide of the invention in a
computer readable medium; and comparing said polynucleotide
sequence to at least one polynucleotide or polypeptide sequence to
identify homology.
[0186] A further embodiment of the invention provides a computer
based method for performing homology identification, said method
comprising the steps of: providing a polypeptide sequence
comprising the sequence of a polypeptide of the invention in a
computer readable medium; and comparing said polypeptide sequence
to at least one polynucleotide or polypeptide sequence to identify
homology.
[0187] A further embodiment of the invention provides a computer
based method for polynucleotide assembly, said method comprising
the steps of: providing a first polynucleotide sequence comprising
the sequence of a polynucleotide of the invention in a computer
readable medium; and screening for at least one overlapping region
between said first polynucleotide sequence and a second
polynucleotide sequence.
[0188] In another preferred embodiment of the invention there is
provided a computer readable medium having stored thereon a member
selected from the group consisting of: a polynucleotide comprising
the sequence of SEQ ID NO. 1; a polypeptide comprising the sequence
of SEQ ID NO. 2 ; a set of polynucleotide sequences wherein at
least one of said sequences comprises the sequence of SEQ ID NO. 1
; a set of polypeptide sequences wherein at least one of said
sequences comprises the sequence of SEQ ID NO. 2 ; a data set
representing a polynucleotide sequence comprising the sequence of
SEQ ID NO. 1; a data set representing a polynucleotide sequence
encoding a polypeptide sequence comprising the sequence of SEQ ID
NO. 2 ; a polynucleotide comprising the sequence of SEQ ID NO. 1 ;
a polypeptide comprising the sequence of SEQ ID NO. 2 ; a set of
polynucleotide sequences wherein at least one of said sequences
comprises the sequence of SEQ ID NO. 1; a set of polypeptide
sequences wherein at least one of said sequences comprises the
sequence of SEQ ID NO. 2; a data set representing a polynucleotide
sequence comprising the sequence of SEQ ID NO. 1; a data set
representing a polynucleotide sequence encoding a polypeptide
sequence comprising the sequence of SEQ ID NO. 2. A further
preferred embodiment of the invention provides a computer based
method for performing homology identification, said method
comprising the steps of providing a polynucleotide sequence
comprising the sequence of SEQ ID NO. 1 in a computer readable
medium; and comparing said polynucleotide sequence to at least one
polynucleotide or polypeptide sequence to identify homology.
[0189] A still further preferred embodiment of the invention
provides a computer based method for performing homology
identification, said method comprising the steps of: providing a
polypeptide sequence comprising the sequence of SEQ ID NO. 2 in a
computer readable medium; and comparing said polypeptide sequence
to at least one polynucleotide or polypeptide sequence to identify
homology.
[0190] A further embodiment of the invention provides a computer
based method for polynucleotide assembly, said method comprising
the steps of: providing a first polynucleotide sequence comprising
the sequence of SEQ ID NO. 1 in a computer readable medium; and
screening for at least one overlapping region between said first
polynucleotide sequence and a second polynucleotide sequence.
[0191] A further embodiment of the invention provides a computer
based method for performing homology identification, said method
comprising the steps of: providing a polynucleotide sequence
comprising the sequence of SEQ ID NO. 1 in a computer readable
medium; and comparing said polynucleotide sequence to at least one
polynucleotide or polypeptide sequence to identify homology.
[0192] A further embodiment of the invention provides a computer
based method for performing homology identification, said method
comprising the steps of: providing a polypeptide sequence
comprising the sequence of SEQ ID NO. 2 in a computer readable
medium; and comparing said polypeptide sequence to at least one
polynucleotide or polypeptide sequence to identify homology.
[0193] A further embodiment of the invention provides a computer
based method for polynucleotide assembly, said method comprising
the steps of: providing a first polynucleotide sequence comprising
the sequence of SEQ ID NO. 1 in a computer readable medium; and
[0194] screening for at least one overlapping region between said
first polynucleotide sequence and a second polynucleotide
sequence.
[0195] 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 described above for
publications and references.
[0196] GLOSSARY
[0197] The following definitions are provided to facilitate
understanding of certain terms used frequently herein.
[0198] "Antibody(ies)" as used herein includes polyclonal and
monoclonal antibodies, chimeric, single chain, and humanized
antibodies, as well as Fab fragments, including the products of an
Fab or other immunoglobulin expression library.
[0199] "Antigenically equivalent derivative(s)" as used herein
encompasses a polypeptide, polynucleotide, or the equivalent of
either which will be specifically recognized by certain antibodies
which, when raised to the protein, polypeptide or polynucleotide
according to the invention, interferes with the immediate physical
interaction between pathogen and mammalian host.
[0200] "Bispecific antibody(ies)" means an antibody comprising at
least two antigen binding domains, each domain directed against a
different epitope.
[0201] "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 materials.
[0202] "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.
[0203] "Fusion protein(s)" refers to a protein encoded by two,
often unrelated, fused genes or fragments thereof. In one example,
EP-A-0464 discloses fusion proteins comprising various portions of
constant region of immunoglobulin molecules together with another
human protein or part thereof. In many cases, employing an
immunoglobulin Fc region as a part of a fusion protein is
advantageous for use in therapy and diagnosis resulting in, for
example, improved pharmacokinetic properties [see, e.g., EP-A
0232262]. On the other hand, for some uses it would be desirable to
be able to delete the Fc part after the fusion protein has been
expressed, detected and purified.
[0204] "Host cell(s)" is a cell which has been transformed or
transfected, or is capable of transformation or transfection by an
exogenous polynucleotide sequence.
[0205] "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.
[0206] Parameters for polypeptide sequence comparison include the
following:
[0207] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453
(1970)
[0208] Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff,
Proc. Natl. Acad. Sci. USA.
[0209] 89:10915-10919 (1992)
[0210] Gap Penalty: 12
[0211] Gap Length Penalty: 4
[0212] 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).
[0213] Parameters for polynucleotide comparison include the
following:
[0214] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453
(1970)
[0215] Comparison matrix: matches=+10, mismatch=0
[0216] Gap Penalty: 50
[0217] Gap Length Penalty: 3
[0218] Available as: The "gap" program from Genetics Computer
Group, Madison Wis. These are the default parameters for nucleic
acid comparisons.
[0219] A preferred meaning for "identity" for polynucleotides and
polypeptides, as the case may be, are provided in (1) and (2)
below.
[0220] (1) Polynucleotide embodiments further include an isolated
polynucleotide comprising a polynucleotide sequence having at least
a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference
sequence of SEQ ID NO:1, wherein said polynucleotide sequence may
be identical to the reference sequence of SEQ ID NO: 1 or may
include up to a certain integer number of nucleotide alterations as
compared to the reference sequence, wherein said alterations are
selected from the group consisting of at least one nucleotide
deletion, substitution, including transition and transversion, or
insertion, and wherein said alterations may occur at the 5'
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 that
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)
[0221] 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
0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for
85%, 0.90 for 90%, 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.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.
[0222] By way of example, a polynucleotide sequence of the present
invention may be identical to the reference sequence of SEQ ID
NO:2, that is it may be 100% identical, or it may include up to a
certain integer number of amino acid alterations as compared to the
reference sequence such that the percent identity is less than 100%
identity. Such alterations are selected from the group consisting
of at least one nucleic acid deletion, substitution, including
transition and transversion, or insertion, and wherein said
alterations may occur at the 5' terminal positions of the reference
polynucleotide sequence or anywhere between those terminal
positions, interspersed either individually among the nucleic acids
in the reference sequence or in one or more contiguous groups
within the reference sequence. The number of nucleic acid
alterations for a given percent identity 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.n.ltoreq.x.sub.n-(x.sub.n.multidot.y),
[0223] wherein n.sub.n is the number of amino acid alterations,
x.sub.n is the total number of amino acids in SEQ ID NO:2, y is,
for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc.,
.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.
[0224] (2) Polypeptide embodiments further include an isolated
polypeptide comprising a polypeptide having at least a 50,60, 70,
80, 85, 90, 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
substitution, 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),
[0225] 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
0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for
85%, 0.90 for 90%, 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.
[0226] By way of example, a polypeptide sequence of the present
invention may be identical to the reference sequence of SEQ ID
NO:2, that is it may be 100% identical, or it may include up to a
certain integer number of amino acid alterations as compared to the
reference sequence such that the percent identity is less than 100%
identity. Such alterations are selected from the group consisting
of at least one amino acid deletion, substitution, including
conservative and non-conservative substitution, or insertion, and
wherein said alterations may occur at the amino- or
carboxy-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. The
number of amino acid alterations for a given % identity 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)
[0227] 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,
for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., 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.
[0228] "Immunologically equivalent derivative(s)" as used herein
encompasses a polypeptide, polynucleotide, or the equivalent of
either which when used in a suitable formulation to raise
antibodies in a vertebrate, the antibodies act to interfere with
the immediate physical interaction between pathogen and mammalian
host.
[0229] "Immunospecific" means that characteristic of an antibody
whereby it possesses substantially greater affinity for the
polypeptides of the invention or the polynucleotides of the
invention than its affinity for other related polypeptides or
polynucleotides respectively, particularly those polypeptides and
polynucleotides in the prior art.
[0230] "Individual(s)" means a multicellular eukaryote, including,
but not limited to a metazoan, a marmal, an ovid, a bovid, a
simian, a primate, and a human.
[0231] "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.
[0232] "Organism(s)" means a (i) prokaryote, including but not
limited to, a member of the genus 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 not 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 aegvptius, Haemophilus
parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi,
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.
[0233] "Polynucleotide(s)" generally refers to any
polyribonucleotide or polydeoxyribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide(s)"
include, without limitation, single- and double-stranded DNA, DNA
that is a mixture of single- and double-stranded regions or
single-, double- and triple-stranded regions, single- and
double-stranded RNA, and RNA that is mixture of single- and
double-stranded regions, hybrid molecules comprising DNA and RNA
that may be single-stranded or, more typically, double-stranded, or
triple-stranded regions, or a mixture of single- and
double-stranded regions. In addition, "polynucleotide" as used
herein refers to triple-stranded regions comprising RNA or DNA or
both RNA and DNA. The strands in such regions may be from the same
molecule or from different molecules. The regions may include all
of one or more of the molecules, but more typically involve only a
region of some of the molecules. One of the molecules of a
triple-helical region often is an oligonucleotide. As used herein,
the term "polynucleotide(s)" also includes DNAs or RNAs as
described above that contain one or more modified bases. Thus, DNAs
or RNAs with backbones modified for stability or for other reasons
are "polynucleotide(s)" as that term is intended herein. Moreover,
DNAs or RNAs comprising unusual bases, such as inosine, or modified
bases, such as tritylated bases, to name just two examples, are
polynucleotides as the term is used herein. It will be appreciated
that a great variety of modifications have been made to DNA and RNA
that serve many useful purposes known to those of skill in the art.
The term "polynucleotide(s)" as it is employed herein embraces such
chemically, enzymatically or metabolically modified forms of
polynucleotides, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells, including, for example, simple
and complex cells. "Polynucleotide(s)" also embraces short
polynucleotides often referred to as oligonucleotide(s).
[0234] "Polypeptide(s)" refers to any peptide or protein comprising
two or more amino acids joined to each other by peptide bonds or
modified peptide bonds. "Polypeptide(s)" refers to both short
chains, commonly referred to as peptides, oligopeptides and
oligomers and to longer chains generally referred to as proteins.
Polypeptides may contain amino acids other than the 20 gene encoded
amino acids. "Polypeptide(s)" include those modified either by
natural processes, such as processing and other post-translational
modifications, but also by chemical modification techniques. Such
modifications are well described in basic texts and in more
detailed monographs, as well as in a voluminous research
literature, and they are well known to those of skill in the art.
It will be appreciated that the same type of modification may be
present in the same or varying degree at several sites in a given
polypeptide. Also, a given polypeptide may contain many types of
modifications. Modifications can occur anywhere in a polypeptide,
including the peptide backbone, the amino acid side-chains, and the
amino or carboxyl termini. Modifications include, for example,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
cross-links, formation of cysteine, formation of pyroglutamate,
formylation, gamma-carboxylation, 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.
[0235] "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.
[0236] "Subtraction set" is one or more, but preferably less than
100, polynucleotides comprising at least one polynucleotide of the
invention
[0237] "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 like 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 techniques, by direct
synthesis, and by other recombinant methods known to skilled
artisans.
EXAMPLES
[0238] The examples below are carried out using standard
techniques, which are well known and routine to those of skill in
the art, except where otherwise described in detail. The examples
are illustrative, but do not limit the invention.
Example 1
Strain Selection, Library Production and Sequencing
[0239] 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 containing 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:
[0240] Methods 1 and 2 below.
[0241] Total cellular DNA is isolated from Streptococcus pneumoniae
0100993 according to standard procedures and size-fractionated by
either of two methods.
[0242] Method 1
[0243] 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.
[0244] Method 2
[0245] Total cellular DNA is partially hydrolyzed with a one or a
combination of restriction enzymes appropriate to generate a series
of fragments for cloning into library vectors (e.g., RsaI, PalI,
AluI, 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
gcp Characterization
[0246] The Determination of Expression During Infection of a Gene
from Streptococcus pneumoniae
[0247] Necrotic fatty tissue from a four day groin infection of
Streptococcus pneumoniae 0100993 in the mouse is efficiently
disrupted and processed in the presence of chaotropic agents and
RNAase 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 RNAase
free, DNAase 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.
[0248] a) Isolation of Tissue Infected with Streptococcus
pneumoniae 0100993 from a Mouse Animal Model of Infection
[0249] 10 ml. volumes of sterile nutrient broth (No.2 Oxoid) are
seeded with isolated, individual colonies of Streptococcus
pneumoniae 0100993 from an agar culture plate. The cultures are
incubated aerobically (static culture) at 37.degree. C. for 16-20
hours. 4 week old mice (female,18 g-22 g, strain MF1) are each
infected by subcutaneous injection of 0.5 ml. of this broth culture
of Streptococcus pneumoniae 0100993 (diluted in broth to
approximately 108 cfu/ml.) into the anterior, right lower quadrant
(groin area). Mice should be monitored regularly during the first
24 hours after infection, then daily until termination of study.
Animals with signs of systemic infection, i.e. lethargy, ruffled
appearance, isolation from group, should be monitored closely and
if signs progress to moribundancy, the animal should be culled
immediately.
[0250] Visible external signs of lesion development will be seen
24-48 h after infection. Examination of the abdomen of the animal
will show the raised outline of the abscess beneath the skin. The
localised lesion should remain in the right lower quadrant, but may
occasionally spread to the left lower quadrant, and superiorly to
the thorax. On occasions, the abscess may rupture through the
overlying skin layers. In such cases the affected animal should be
culled immediately and the tissues sampled if possible. Failure to
cull the animal may result in the necrotic skin tissue overlying
the abscess being sloughed off, exposing the abdominal muscle
wall.
[0251] Approximately 96 hours after infection, animals are killed
using carbon dioxide asphyxiation. To minimise delay between death
and tissue processing/storage, mice should be killed individually
rather than in groups.The dead animal is placed onto its back and
the fur swabbed liberally with 70% alcohol. An initial incision
using scissors is made through the skin of the abdominal left lower
quadrant, travelling superiorly up to, then across the thorax. The
incision is completed by cutting inferiorly to the abdominal lower
right quadrant. Care should be taken not to penetrate the abdominal
wall. Holding the skin flap with forceps, the skin is gently pulled
way from the abdomen. The exposed abscess, which covers the
peritoneal wall but generally does not penetrate the muscle sheet
completely, is excised, taking care not to puncture the viscera
[0252] The abscess/muscle sheet and other infected tissue may
require cutting in sections, prior to flash-freezing in liquid
nitrogen, thereby allowing easier storage in plastic collecting
vials.
[0253] b) Isolation of Streptococcus pneumoniae 0100993 RNA from
Infected Tissue Samples
[0254] 4-6 infected tissue samples(each approx 0.5-0.7 g) in 2 ml
screw-cap tubes are removed from -80.degree. C. storage into a dry
ice ethanol bath In a microbiological safety cabinet the samples
are disrupted individually whilst the remaining samples are kept
cold in the dry ice ethanol bath. To disrupt the bacteria within
the tissue sample 1 ml of TRIzol Reagent (Gibco BRL, Life
Technologies) is added followed by enough 0.1 mm zirconia/silica
beads to almost fill the tube, the lid is replaced taking care not
to get any beads into the screw thread so as to ensure a good seal
and eliminate aerosol generation. The sample is then homogenised in
a Mini-BeadBeater Type BX-4 (Biospec Products). Necrotic fatty
tissue isstrain treated for 100 seconds at 5000 rpm in order to
achieve bacterial lysis. In vivo grown bacteria require longer
treatment than in vitro grown Streptococcus pneumoniae
Streptococcus which are disrupted by a 30 second bead-beat.
[0255] After bead-beating the tubes are chilled on ice before
opening in a fume-hood as heat generated during disruption may
degrade the TRIzol and release cyanide.
[0256] 200 microlitres of chloroform is then added and the tubes
shaken by hand for 15 seconds to ensure complete mixing. After 2-3
minutes at room temperature the tubes are spun down at
12,000.times. g, 4.degree. C. for 15 minutes and RNA extraction is
then continued according to the method given by the manufacturers
of TRIzol Reagent i.e.:--The aqueous phase, approx 0.6 ml, is
transferred to a sterile eppendorf tube and 0.5 ml of isopropanol
is added. After 10 minutes at room temperature the samples are spun
at 12,000.times. g, 4.degree. C. for 10 minutes. The supernatant is
removed and discarded then the RNA pellet is washed with 1 ml 75%
ethanol. A brief vortex is used to mix the sample before
centrifuging at 7,500.times. g, 4.degree. C. for 5 minutes. The
ethanol is removed and the RNA pellet dried under vacuum for no
more than 5 minutes. Samples are then resuspended by repeated
pipetting in 100 microlitres 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.
[0257] 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.
[0258] Quality of 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 32 P labelled
oligonucletide probe specific to 16s rRNA of Streptococcus
pneumoniae (K.Greisen, M. Loeffelholz, A. Purohit and D. Leong.
J.Clin. (1994) Microbiol. 32 335-351). The oligonucleotide of the
sequence is used as a probe. The size of the hybridising band is
compared to that of control RNA isolated from in vitro grown
Streptococcus pneumoniae 0100993 in the Northern blot. Correct
sized bacterial 16s rRNA bands can be detected in total RNA samples
which show extensive degradation of the mammalian RNA when
visualised on TBE gels.
[0259] c) The Removal of DNA from Streptococcus pneumoniae
0100993-derived RNA
[0260] DNA was removed from 73 microlitre samples of RNA by a 15
minute treatment on ice with 3 units of DNAaseI, amplification
grade (Gibco BRL, Life Technologies) in the buffer supplied with
the addition of 200 units of Rnasin (Promega) in a final volume of
90 microlitres.
[0261] The DNAase was inactivated and removed by treatment with
TRIzol LS Reagent (Gibco BRL, Life Technologies) according to the
manufacturers protocol. DNAase treated RNA was resuspended in 73
microlitres of DEPC treated water with the addition of Rnasin as
described in Method 1.
[0262] d) The preparation of cDNA from RNA Samples Derived from
Infected Tissue
[0263] 10 microlitre 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. 1 nanogram of random hexamers is
used to prime each reaction. Controls without the addition of
SuperScriptII reverse transcriptase are also run. Both +/-RT
samples are treated with RNaseH before proceeding to the PCR
reaction
[0264] e) The use of PCR to Determine the Presence of a Bacterial
cDNA Species
[0265] PCR reactions are set up on ice in 0.2 ml tubes by adding
the following components: 45 microlitres PCR SUPERMIX (Gibco BRL,
Life Technologies); 1 microlitre 50 mM MgC12 , to adjust final
concentration to 2.5 mM; 1 microlitre PCR primers(optimally 18-25
basepairs in length and designed to possess similar annealing
temperatures), each primer at 10 mM initial concentration; and 2
microlitres cDNA.
[0266] PCR reactions are run on a Perkin Elmer GeneAmp PCR System
9600 as follows: 5 minutes at 95.degree. C., then 50 cycles of 30
seconds each at 94.degree. C., 42.degree. C. and 72.degree. C.
followed by 3 minutes at 72.degree. C. and then a hold temperature
of 4.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 microlitre 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 PrismTM 377 Sequencer using GeneScanTM
software as supplied by Perkin Elmer).
[0267] 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.
[0268] 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 and 35 cycles of PCR.
[0269] 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.
[0270] Two polynucleotide sequences of the invention, SEQ ID NOS: 1
and 3, were identified in the above test as transcribed in vivo.
SEQ ID NO:2 was deduced from the polynucleotide sequence given as
SEQ ID NO: 1. SEQ ID NO:4 was deduced from the polynucleotide
sequence given as SEQ ID NO:3. The pair of PCR primers used to
identify the gene are given as SEQ ID NOS:5 and 6.
[0271] By this method it was shown that the S. pneumoniae gcp gene
is transcribed in vivo.
[0272] The Determination of Gene Product Essentiality in S.
pneumoniae
[0273] An allelic replacement cassette is generated using PCR
technology. The cassette typically consists of a pair of 500 bp
chromosomal DNA fragments flanking an erythromycin resistance gene.
The chromosomal DNA sequences are usually the 500 bp preceding and
following the gene of interest.
[0274] Attempts are made to introduce the allelic replacement
cassette into S. pneumoniae R6 or S. pneumoniae 100993 by
transformation. Competent cells are prepared according to published
protocols. DNA is introduced into the cells by incubation of 500 ng
of allelic replacement cassette with 10.sup.6 cells at 30.degree.
C. for 20 minutes. The cells are transferred to 37.degree. C. for
90 minutes to allow expression of the erythromycin resistance gene.
Cells are plated in agar containing 1 .mu.g erythromycin per ml.
Following incubation at 37.degree. C. for 36 hours, any observed
colonies are picked and grown overnight in Todd-Hewitt broth
supplemented with 0.5% yeast extract. Typically, in positive
control experiments carried out in parallel which target a
non-essential gene, 102-103 transformants containing the
appropriate allelic replacement are obtained. If erythromycin
resistant colonies are only observed in transformation experiments
using S. pneumoniae R6, DNA from these cells are used to transform
S. pneumoniae 100993. The transformation procedure is identical to
that for S. pneumoniae R6 except that a competence stimulating
heptadecapeptide (Havarstein et al., (1995) P.N.A.S. 92,
11140-11144) is added at a concentration of 1 .mu.g/ml in the
initial transformation mix. Mutants are selected by their ability
to grow in agar containing 1 .mu.g erythromycin per ml.
[0275] If no transformants are obtained in three separate
transformation experiments, then the target gene is considered as
being essential in vitro.
[0276] However, if colonies are obtained chromosomal DNA is
prepared from these cells and examined using diagnostic PCR.
Oligonucleotides designed to hybridize to sequences within the
allelic replacement cassette are used in conjunction with DNA
primers hybridizing to chromosomal sequences outside the cassette
to generate DNA products amplified by PCR of characteristic size.
This chromosomal DNA is also subject to Southern analysis in order
to verify that the appropriate chromosomal DNA rearrangement has
occurred.
[0277] In order to demonstrate that the mutation is stably
maintained, the defective strain is grown for many generations in
the absence of selective pressure and then assayed for its ability
to grow in the absence and presence of erythromycin.
[0278] By this method it is considered that the gcp gene product is
essential in vitro.
Sequence CWU 1
1
* * * * *
References