U.S. patent application number 10/807556 was filed with the patent office on 2007-01-25 for staphylococcus aureus polynucleotides and sequences.
Invention is credited to Steven C. Barash, Gil H. Choi, Patrick J. Dillon, Michael R. Fannon, Charles A. Kunsch, Craig A. Rosen.
Application Number | 20070020746 10/807556 |
Document ID | / |
Family ID | 21740142 |
Filed Date | 2007-01-25 |
United States Patent
Application |
20070020746 |
Kind Code |
A1 |
Kunsch; Charles A. ; et
al. |
January 25, 2007 |
Staphylococcus aureus polynucleotides and sequences
Abstract
The present invention provides polynucleotide sequences of the
genome of Staphylococcus aureus, polypeptide sequences encoded by
the polynucleotide sequences, corresponding polynucleotides and
polypeptides, vectors and hosts comprising the polynucleotides, and
assays and other uses thereof. The present invention further
provides polynucleotide and polypeptide sequence information stored
on computer readable media, and computer-based systems and methods
which facilitate its use.
Inventors: |
Kunsch; Charles A.;
(Norcross, GA) ; Choi; Gil H.; (Rockville, MD)
; Barash; Steven C.; (Rockville, MD) ; Dillon;
Patrick J.; (Carlsbad, CA) ; Fannon; Michael R.;
(Silver Spring, MD) ; Rosen; Craig A.;
(Laytonsville, MD) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC;INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Family ID: |
21740142 |
Appl. No.: |
10/807556 |
Filed: |
March 24, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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|
08781986 |
Jan 3, 1997 |
6737248 |
|
|
10807556 |
Mar 24, 2004 |
|
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60009861 |
Jan 5, 1996 |
|
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Current U.S.
Class: |
435/252.3 |
Current CPC
Class: |
C12N 15/1093 20130101;
C07K 14/31 20130101; C12N 15/52 20130101; A61K 39/00 20130101 |
Class at
Publication: |
435/252.3 |
International
Class: |
C07H 21/02 20060101
C07H021/02; C07H 21/04 20060101 C07H021/04; C12P 21/04 20060101
C12P021/04; C12N 1/20 20060101 C12N001/20 |
Claims
1. An isolated protein-encoding nucleic acid fragment of the
Staphylococcus aureus genome, wherein said fragment consists of the
nucleotide sequence of any one of the fragments of SEQ ID
NOS:1-5,191 depicted in Tables 2 and 3, or a degenerate variant
thereof.
2. A vector comprising any one of the fragments of claim 1.
3. An isolated fragment of the Staphylococcus aureus genome,
wherein said fragment modulates the expression of an operably
linked open reading frame, wherein said fragment consists of the
nucleotide sequence from about 10 to 200 bases in length which is
5' to any one of the open reading frames depicted in Tables 2 and 3
or a degenerate variant thereof.
4. A vector comprising any one of the fragments of the
Staphylococcus aureus genome of claim 1.
5. An isolated polypeptide encoded by any of the fragments of claim
1.
6. An isolated polynucleotide molecule encoding any one of the
polypeptides of claim 5.
7. An antibody which selectively binds to any one of the
polypeptides of claim 5.
8. An isolated polypeptide comprising an amino acid sequence having
at least 95% identity to a Staphylococcus aureus polypeptide amino
acid sequence selected from the group consisting of SEQ ID
NOS:5,192 to 5,255.
9. The isolated polypeptide of claim 8 wherein the isolated
polypeptide comprises an amino acid sequence identical to that of a
Staphylococcus aureus polypeptide selected from the group
consisting of SEQ ID NOS:5,192 to 5,255.
10. An isolated Staphylococcus aureus polypeptide antigen
comprising at least one epitope derived from a Staphylococcus
aureus polypeptide of claim 9.
11. An isolated polypeptide antigen of claim 10 selected from the
group consisting of the epitopic sequences listed in Table 4.
12. A diagnostic kit for detecting Staphylococcus aureus infection
comprising (a) an isolated polypeptide antigen of claim 10, and (b)
means for detecting the binding of an antibody contained in a
biological fluid to said antigen.
13. A vaccine composition comprising a polypeptide of claim 10
present in a pharmaceutically acceptable carrier.
14. A method of vaccinating an individual against Staphylococcus
aureus infection comprising, administering to an individual the
vaccine composition of claim 13.
15. A method for producing a polypeptide in a host cell comprising
the steps of: (a) incubating a host containing a heterologous
nucleic acid molecule whose nucleotide sequence consists of any one
of the fragments of claim 1, under conditions where said
heterologous nucleic acid molecule is expressed to produce said
protein, and (b) isolating said protein.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 08/781,986, filed Jan. 3, 1997, which claims benefit under 35
U.S.C. section 119(e) of U.S. Provisional Application Ser. No.
60/009,861, filed Jan. 5, 1996.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of molecular
biology. In particular, it relates to, among other things,
nucleotide sequences of Staphylococcus aureus, contigs, ORFs,
fragments, probes, primers and related polynucleotides thereof,
peptides and polypeptides encoded by the sequences, and uses of the
polynucleotides and sequences thereof, such as in fermentation,
polypeptide production, assays and pharmaceutical development,
among others.
BACKGROUND OF THE INVENTION
[0003] The genus Staphylococcus includes at least 20 distinct
species. (For a review see Novick, R. P., The Staphylococcus as a
Molecular Genetic System, Chapter 1, pgs. 1-37 in MOLECULAR BIOLOGY
OF THE STAPHYLOCOCCI, R. Novick, Ed., VCH Publishers, New York
(1990)). Species differ from one another by 80% or more, by
hybridization kinetics, whereas strains within a species are at
least 90% identical by the same measure.
[0004] The species Staphylococcus aureus, a gram-positive,
facultatively aerobic, clump-forming cocci, is among the most
important etiological agents of bacterial infection in humans, as
discussed briefly below.
[0005] Human Health and S. Aureus
[0006] Staphylococcus aureus is a ubiquitous pathogen. (See, for
instance, Mims et al., MEDICAL MICROBIOLOGY, Mosby-Year Book Europe
Limited, London, UK (1993)). It is an etiological agent of a
variety of conditions, ranging in severity from mild to fatal. A
few of the more common conditions caused by S. aureus infection are
burns, cellulitis, eyelid infections, food poisoning, joint
infections, neonatal conjunctivitis, osteomyelitis, skin
infections, surgical wound infection, scalded skin syndrome and
toxic shock syndrome, some of which are described further
below.
[0007] Burns
[0008] Burn wounds generally are sterile initially. However, they
generally compromise physical and immune barriers to infection,
cause loss of fluid and electrolytes and result in local or general
physiological dysfunction. After cooling, contact with viable
bacteria results in mixed colonization at the injury site.
Infection may be restricted to the non-viable debris on the burn
surface ("eschar"), it may progress into full skin infection and
invade viable tissue below the eschar and it may reach below the
skin, enter the lymphatic and blood circulation and develop into
septicaemia. S. aureus is among the most important pathogens
typically found in burn wound infections. It can destroy
granulation tissue and produce severe septicaemia.
[0009] Cellulitis
[0010] Cellulitis, an acute infection of the skin that expands from
a typically superficial origin to spread below the cutaneous layer,
most commonly is caused by S. aureus in conjunction with S.
pyrogenes. Cellulitis can lead to systemic infection. In fact,
cellulitis can be one aspect of synergistic bacterial gangrene.
This condition typically is caused by a mixture of S. aureus and
microaerophilic streptococci. It causes necrosis and treatment is
limited to excision of the necrotic tissue. The condition often is
fatal.
[0011] Eyelid Infections
[0012] S. aureus is the cause of styes and of sticky eye" in
neonates, among other eye infections. Typically such infections are
limited to the surface of the eye, and may occasionally penetrate
the surface with more severe consequences.
[0013] Food Poisoning
[0014] Some strains of S. aureus produce one or more of five
serologically distinct, heat and acid stable enterotoxins that are
not destroyed by digestive process of the stomach and small
intestine (enterotoxins A-E). Ingestion of the toxin, in sufficient
quantities, typically results in severe vomiting, but not
diarrhoea. The effect does not require viable bacteria. Although
the toxins are known, their mechanism of action is not
understood.
[0015] Joint Infections
[0016] S. aureus infects bone joints causing diseases such
osteomyelitis.
[0017] Osteomyelitis
[0018] S. aureus is the most common causative agent of
haematogenous osteomyelitis. The disease tends to occur in children
and adolescents more than adults and it is associated with
non-penetrating injuries to bones. Infection typically occurs in
the long end of growing bone, hence its occurrence in physically
immature populations. Most often, infection is localized in the
vicinity of sprouting capillary loops adjacent to epiphysial growth
plates in the end of long, growing bones.
[0019] Skin Infections
[0020] S. aureus is the most common pathogen of such minor skin
infections as abscesses and boils. Such infections often are
resolved by normal host response mechanisms, but they also can
develop into severe internal infections. Recurrent infections of
the nasal passages plague nasal carriers of S. aureus.
[0021] Surgical Wound Infections
[0022] Surgical wounds often penetrate far into the body. Infection
of such wound thus poses a grave risk to the patient. S. aureus is
the most important causative agent of infections in surgical
wounds. S. aureus is unusually adept at invading surgical wounds;
sutured wounds can be infected by far fewer S. aureus cells then
are necessary to cause infection in normal skin. Invasion of
surgical wound can lead to severe S. aureus septicaemia. Invasion
of the blood stream by S. aureus can lead to seeding and infection
of internal organs, particularly heart valves and bone, causing
systemic diseases, such as endocarditis and osteomyelitis.
[0023] Scalded Skin Syndrome
[0024] S. aureus is responsible for "scalded skin syndrome" (also
called toxic epidermal necrosis, Ritter's disease and Lyell's
disease). This diseases occurs in older children, typically in
outbreaks caused by flowering of S. aureus strains produce
exfoliation (also called scalded skin syndrome toxin). Although the
bacteria initially may infect only a minor lesion, the toxin
destroys intercellular connections, spreads epidermal layers and
allows the infection to penetrate the outer layer of the skin,
producing the desquamation that typifies the diseases. Shedding of
the outer layer of skin generally reveals normal skin below, but
fluid lost in the process can produce severe injury in young
children if it is not treated properly.
[0025] Toxic Shock Syndrome
[0026] Toxic shock syndrome is caused by strains of S. aureus that
produce the so-called toxic shock syndrome toxin. The disease can
be caused by S. aureus infection at any site, but it is too often
erroneously viewed exclusively as a disease solely of women who use
tampons. The disease involves toxaemia and septicaemia, and can be
fatal.
[0027] Nocosomial Infections
[0028] In the 1984 National Nocosomial Infection Surveillance Study
("NNIS") S. aureus was the most prevalent agent of surgical wound
infections in many hospital services, including medicine, surgery,
obstetrics, pediatrics and newborns.
[0029] Resistance to Drugs of S. aureus Strains
[0030] Prior to the introduction of penicillin the prognosis for
patients seriously infected with S. aureus was unfavorable.
Following the introduction of penicillin in the early 1940s even
the worst S. aureus infections generally could be treated
successfully. The emergence of penicillin-resistant strains of S.
aureus did not take long, however. Most strains of S. aureus
encountered in hospital infections today do not respond to
penicillin; although, fortunately, this is not the case for S.
aureus encountered in community infections.
[0031] It is well known now that penicillin-resistant strains of S.
aureus produce a lactamase which converts penicillin to
pencillinoic acid, and thereby destroys antibiotic activity.
Furthermore, the lactamase gene often is propagated episomally,
typically on a plasmid, and often is only one of several genes on
an episomal element that, together, confer multidrug
resistance.
[0032] Methicillins, introduced in the 1960s, largely overcame the
problem of penicillin resistance in S. aureus. These compounds
conserve the portions of penicillin responsible for antibiotic
activity and modify or alter other portions that make penicillin a
good substrate for inactivating lactamases. However, methicillin
resistance has emerged in S. aureus, along with resistance to many
other antibiotics effective against this organism, including
aminoglycosides, tetracycline, chloramphenicol, macrolides and
lincosamides. In fact, methicillin-resistant strains of S. aureus
generally are multiply drug resistant.
[0033] The molecular genetics of most types of drug resistance in
S. aureus has been elucidated (See Lyon et al., Microbiology
Reviews 51: 88-134 (1987)). Generally, resistance is mediated by
plasmids, as noted above regarding penicillin resistance; however,
several stable forms of drug resistance have been observed that
apparently involve integration of a resistance element into the S.
aureus genome itself.
[0034] Thus far each new antibiotic gives rise to resistance
strains, stains emerge that are resistance to multiple drugs and
increasingly persistent forms of resistance begin to emerge. Drug
resistance of S. aureus infections already poses significant
treatment difficulties, which are likely to get much worse unless
new therapeutic agents are developed.
[0035] Molecular Genetics of Staphylococcus Aureus
[0036] Despite its importance in, among other things, human
disease, relatively little is known about the genome of this
organism.
[0037] Most genetic studies of S. aureus have been carried out
using the the strain NCTC8325, which contains prophages psi11,
psi12 and psi13, and the UV-cured derivative of this strain, 8325-4
(also referred to as RN450), which is free of the prophages.
[0038] These studies revealed that the S. aureus genome, like that
of other staphylococci, consists of one circular, covalently
closed, double-stranded DNA and a collection of so-called variable
accessory genetic elements, such as prophages, plasmids,
transposons and the like. Physical characterization of the genome
has not been carried out in any detail. Pattee et al. published a
low resolution and incomplete genetic and physical map of the
chromosome of S. aureus strain NCTC 8325. (Pattee et al. Genetic
and Physical Mapping of Chromosome of Staphylococcus aureus NCTC
8325, Chapter 11, pgs. 163-169 in. MOLECULAR BIOLOGY OF THE
STAPHYLOCOCCI, R. P. Novick, Ed., VCH Publishers, New York, (1990)
The genetic map largely was produced by mapping insertions of Tn551
and Tn4001, which, respectively, confer erythromycin and gentamicin
resistance, and by analysis of SmaI-digested DNA by Pulsed Field
Gel Electrophoresis ("PFGE").
[0039] The map was of low resolution; even estimating the physical
size of the genome was difficult, according to the investigators.
The size of the largest SmaI chromosome fragment, for instance, was
too large for accurate sizing by PFGE. To estimate its size,
additional restriction sites had to be introduced into the
chromosome using a transposon containing a SmaI recognition
sequence.
[0040] In sum, most physical characteristics and almost all of the
genes of Staphylococcus aureus are unknown. Among the few genes
that have been identified, most have not been physically mapped or
characterized in detail. Only a very few genes of this organism
have been sequenced. (See, for instance Thornsberry, J.,
Antimicrobial Chemotherapy 21 Suppl C: 9-16 (1988), current
versions of GENBANK and other nucleic acid databases, and
references that relate to the genome of S. aureus such as those set
out elsewhere herein.)
[0041] It is clear that the etiology of diseases mediated or
exacerbated by S. aureus infection involves the programmed
expression of S. aureus genes, and that characterizing the genes
and their patterns of expression would add dramatically to our
understanding of the organism and its host interactions. Knowledge
of S. aureus genes and genomic organization would dramatically
improve understanding of disease etiology and lead to improved and
new ways of preventing, ameliorating, arresting and reversing
diseases. Moreover, characterized genes and genomic fragments of S.
aureus would provide reagents for, among other things, detecting,
characterizing and controlling S. aureus infections. There is a
need therefore to characterize the genome of S. aureus and for
polynucleotides and sequences of this organism.
SUMMARY OF THE INVENTION
[0042] The present invention is based on the sequencing of
fragments of the Staphylococcus aureus genome. The primary
nucleotide sequences which were generated are provided in SEQ ID
NOS:1-5,191.
[0043] The present invention provides the nucleotide sequence of
several thousand contigs of the Staphylococcus aureus genome, which
are listed in tables below and set out in the Sequence Listing
submitted herewith, and representative fragments thereof, in a form
which can be readily used, analyzed, and interpreted by a skilled
artisan. In one embodiment, the present invention is provided as
contiguous strings of primary sequence information corresponding to
the nucleotide sequences depicted in SEQ ID NOS:1-5,191.
[0044] The present invention further provides nucleotide sequences
which are at least 95% identical to the nucleotide sequences of SEQ
ID NOS:1-5,191.
[0045] The nucleotide sequence of SEQ ID NOS:1-5,191, a
representative fragment thereof, or a nucleotide sequence which is
at least 95% identical to the nucleotide sequence of SEQ ID
NOS:1-5,191 may be provided in a variety of mediums to facilitate
its use. In one application of this embodiment, the sequences of
the present invention are recorded on computer readable media. Such
media includes, but is not limited to: magnetic storage media, such
as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media.
[0046] The present invention further provides systems, particularly
computer-based systems which contain the sequence information
herein described stored in a data storage means. Such systems are
designed to identify commercially important fragments of the
Staphylococcus aureus genome.
[0047] Another embodiment of the present invention is directed to
fragments of the Staphylococcus aureus genome having particular
structural or functional attributes. Such fragments of the
Staphylococcus aureus genome of the present invention include, but
are not limited to, fragments which encode peptides, hereinafter
referred to as open reading frames or ORFs," fragments which
modulate the expression of an operably linked ORF, hereinafter
referred to as expression modulating fragments or EMFs," and
fragments which can be used to diagnose the presence of
Staphylococcus aureus in a sample, hereinafter referred to as
diagnostic fragments or "DFs."
[0048] Each of the ORFs in fragments of the Staphylococcus aureus
genome disclosed in Tables 1-3, and the EMFs found 5' to the ORFs,
can be used in numerous ways as polynucleotide reagents. For
instance, the sequences can be used as diagnostic probes or
amplification primers for detecting or determining the presence of
a specific microbe in a sample, to selectively control gene
expression in a host and in the production of polypeptides, such as
polypeptides encoded by ORFs of the present invention, particular
those polypeptides that have a pharmacological activity.
[0049] The present invention further includes recombinant
constructs comprising one or more fragments of the Staphylococcus
aureus genome of the present invention. The recombinant constructs
of the present invention comprise vectors, such as a plasmid or
viral vector, into which a fragment of the Staphylococcus aureus
has been inserted.
[0050] The present invention further provides host cells containing
any of the isolated fragments of the Staphylococcus aureus genome
of the present invention. The host cells can be a higher eukaryotic
host cell, such as a mammalian cell, a lower eukaryotic cell, such
as a yeast cell, or a procaryotic cell such as a bacterial
cell.
[0051] The present invention is further directed to isolated
polypeptides and proteins encoded by ORFs of the present invention.
A variety of methods, well known to those of skill in the art,
routinely may be utilized to obtain any of the polypeptides and
proteins of the present invention. For instance, polypeptides and
proteins of the present invention having relatively short, simple
amino acid sequences readily can be synthesized using commercially
available automated peptide synthesizers. Polypeptides and proteins
of the present invention also may be purified from bacterial cells
which naturally produce the protein. Yet another alternative is to
purify polypeptide and proteins of the present invention can from
cells which have been altered to express them.
[0052] The invention further provides polypeptides comprising
Staphylococcus aureus epitopes and vaccine compositions comprising
such polypeptides. Also provided are methods for vacciniating an
individual against Staphylococcus aureus infection.
[0053] The invention further provides methods of obtaining homologs
of the fragments of the Staphylococcus aureus genome of the present
invention and homologs of the proteins encoded by the ORFs of the
present invention. Specifically, by using the nucleotide and amino
acid sequences disclosed herein as a probe or as primers, and
techniques such as PCR cloning and colony/plaque hybridization, one
skilled in the art can obtain homologs.
[0054] The invention further provides antibodies which selectively
bind polypeptides and proteins of the present invention. Such
antibodies include both monoclonal and polyclonal antibodies.
[0055] The invention further provides hybridomas which produce the
above-described antibodies. A hybridoma is an immortalized cell
line which is capable of secreting a specific monoclonal
antibody.
[0056] The present invention further provides methods of
identifying test samples derived from cells which express one of
the ORFs of the present invention, or a homolog thereof. Such
methods comprise incubating a test sample with one or more of the
antibodies of the present invention, or one or more of the Dfs or
antigens of the present invention, under conditions which allow a
skilled artisan to determine if the sample contains the ORF or
product produced therefrom.
[0057] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
above-described assays.
[0058] Specifically, the invention provides a compartmentalized kit
to receive, in close confinement, one or more containers which
comprises: (a) a first container comprising one of the antibodies,
antigens, or one of the DFs of the present invention; and (b) one
or more other containers comprising one or more of the following:
wash reagents, reagents capable of detecting presence of bound
antibodies, antigens or hybridized DFs.
[0059] Using the isolated proteins of the present invention, the
present invention further provides methods of obtaining and
identifying agents capable of binding to a polypeptide or protein
encoded by one of the ORFs of the present invention. Specifically,
such agents include, as further described below, antibodies,
peptides, carbohydrates, pharmaceutical agents and the like. Such
methods comprise steps of: (a) contacting an agent with an isolated
protein encoded by one of the ORFs of the present invention; and
(b) determining whether the agent binds to said protein.
[0060] The present genomic sequences of Staphylococcus aureus will
be of great value to all laboratories working with this organism
and for a variety of commercial purposes. Many fragments of the
Staphylococcus aureus genome will be immediately identified by
similarity searches against GenBank or protein databases and will
be of immediate value to Staphylococcus aureus researchers and for
immediate commercial value for the production of proteins or to
control gene expression.
[0061] The methodology and technology for elucidating extensive
genomic sequences of bacterial and other genomes has and will
greatly enhance the ability to analyze and understand chromosomal
organization. In particular, sequenced contigs and genomes will
provide the models for developing tools for the analysis of
chromosome structure and function, including the ability to
identify genes within large segments of genomic DNA, the structure,
position, and spacing of regulatory elements, the identification of
genes with potential industrial applications, and the ability to do
comparative genomic and molecular phylogeny.
DESCRIPTION OF THE FIGURES
[0062] FIG. 1 is a block diagram of a computer system (102) that
can be used to implement computer-based systems of present
invention.
[0063] FIG. 2 is a schematic diagram depicting the data flow and
computer programs used to collect, assemble, edit and annotate the
contigs of the Staphylococcus aureus genome of the present
invention. Both Macintosh and Unix platforms are used to handle the
AB 373 and 377 sequence data files, largely as described in
Kerlavage et al., Proceedings of the Twenty-Sixth Annual Hawaii
International Conference on System Sciences, 585, IEEE Computer
Society Press, Washington D.C. (1993). Factura (AB) is a Macintosh
program designed for automatic vector sequence removal and
end-trimming of sequence files. The program Loadis runs on a
Macintosh platform and parses the feature data extracted from the
sequence files by Factura to the Unix based Staphylococcus aureus
relational database. Assembly of contigs (and whole genome
sequences) is accomplished by retrieving a specific set of sequence
files and their associated features using extrseq, a Unix utility
for retrieving sequences from an SQL database. The resulting
sequence file is processed by seq_filter to trim portions of the
sequences with more than 2% ambiguous nucleotides. The sequence
files were assembled using TIGR Assembler, an assembly engine
designed at The Institute for Genomic Research (TIGR") for rapid
and accurate assembly of thousands of sequence fragments. The
collection of contigs generated by the assembly step is loaded into
the database with the lassie program. Identification of open
reading frames (ORFs) is accomplished by processing contigs with
zorf. The ORFs are searched against S. aureus sequences from
Genbank and against all protein sequences using the BLASTN and
BLASTP programs, described in Altschul et al., J. Mol. Biol. 215:
403-410 (1990)). Results of the ORF determination and similarity
searching steps were loaded into the database. As described below,
some results of the determination and the searches are set out in
Tables 1-3.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0064] The present invention is based on the sequencing of
fragments of the Staphylococcus aureus genome and analysis of the
sequences. The primary nucleotide sequences generated by sequencing
the fragments are provided in SEQ ID NOS:1-5,191. (As used herein,
the "primary sequence" refers to the nucleotide sequence
represented by the IUPAC nomenclature system.)
[0065] In addition to the aforementioned Staphylococcus aureus
polynucleotide and polynucleotide sequences, the present invention
provides the nucleotide sequences of SEQ ID NOS:1-5,191, or
representative fragments thereof, in a form which can be readily
used, analyzed, and interpreted by a skilled artisan.
[0066] As used herein, a "representative fragment of the nucleotide
sequence depicted in SEQ ID NOS:1-5,191" refers to any portion of
the SEQ ID NOS:1-5,191 which is not presently represented within a
publicly available database. Preferred representative fragments of
the present invention are Staphylococcus aureus open reading frames
(ORFs"), expression modulating fragment (EMFs") and fragments which
can be used to diagnose the presence of Staphylococcus aureus in
sample ("DFs"). A non-limiting identification of preferred
representative fragments is provided in Tables 1-3.
[0067] As discussed in detail below, the information provided in
SEQ ID NOS:1-5,191 and in Tables 1-3 together with routine cloning,
synthesis, sequencing and assay methods will enable those skilled
in the art to clone and sequence all "representative fragments" of
interest, including open reading frames encoding a large variety of
Staphylococcus aureus proteins.
[0068] While the presently disclosed sequences of SEQ ID
NOS:1-5,191 are highly accurate, sequencing techniques are not
perfect and, in relatively rare instances, further investigation of
a fragment or sequence of the invention may reveal a nucleotide
sequence error present in a nucleotide sequence disclosed in SEQ ID
NOS:1-5,191. However, once the present invention is made available
(i.e., once the information in SEQ ID NOS:1-5,191 and Tables 1-3
has been made available), resolving a rare sequencing error in SEQ
ID NOS:1-5,191 will be well within the skill of the art. The
present disclosure makes available sufficient sequence information
to allow any of the described contigs or portions thereof to be
obtained readily by straightforward application of routine
techniques. Further sequencing of such polynucleotide may proceed
in like manner using manual and automated sequencing methods which
are employed ubiquitous in the art. Nucleotide sequence editing
software is publicly available. For example, Applied Biosystem's
(AB) AutoAssembler can be used as an aid during visual inspection
of nucleotide sequences. By employing such routine techniques
potential errors readily may be identified and the correct sequence
then may be ascertained by targeting further sequencing effort,
also of a routine nature, to the region containing the potential
error.
[0069] Even if all of the very rare sequencing errors in SEQ ID
NOS:1-5,191 were corrected, the resulting nucleotide sequences
would still be at least 95% identical, nearly all would be at least
99% identical, and the great majority would be at least 99.9%
identical to the nucleotide sequences of SEQ ID NOS:1-5,191.
[0070] As discussed elsewhere hererin, polynucleotides of the
present invention readily may be obtained by routine application of
well known and standard procedures for cloning and sequencing DNA.
Detailed methods for obtaining libraries and for sequencing are
provided below, for instance. A wide variety of Staphylococcus
aureus strains that can be used to prepare S aureus genomic DNA for
cloning and for obtaining polynucleotides of the present invention
are available to the public from recognized depository
institutions, such as the American Type Culture Collection
(ATCC").
[0071] The nucleotide sequences of the genomes from different
strains of Staphylococcus aureus differ somewhat. However, the
nucleotide sequences of the genomes of all Staphylococcus aureus
strains will be at least 95% identical, in corresponding part, to
the nucleotide sequences provided in SEQ ID NOS:1-5,191. Nearly all
will be at least 99% identical and the great majority will be 99.9%
identical.
[0072] Thus, the present invention further provides nucleotide
sequences which are at least 95%, preferably 99% and most
preferably 99.9% identical to the nucleotide sequences of SEQ ID
NOS:1-5,191, in a form which can be readily used, analyzed and
interpreted by the skilled artisan.
[0073] Methods for determining whether a nucleotide sequence is at
least 95%, at least 99% or at least 99.9% identical to the
nucleotide sequences of SEQ ID NOS:1-5,191 are routine and readily
available to the skilled artisan. For example, the well known fasta
algorithm described in Pearson and Lipman, Proc. Natl. Acad. Sci.
USA 85: 2444 (1988) can be used to generate the percent identity of
nucleotide sequences. The BLASTN program also can be used to
generate an identity score of polynucleotides compared to one
another.
Computer Related Embodiments
[0074] The nucleotide sequences provided in SEQ ID NOS:1-5,191, a
representative fragment thereof, or a nucleotide sequence at least
95%, preferably at least 99% and most preferably at least 99.9%
identical to a polynucleotide sequence of SEQ ID NOS:1-5,191 may be
"provided" in a variety of mediums to facilitate use thereof. As
used herein, provided" refers to a manufacture, other than an
isolated nucleic acid molecule, which contains a nucleotide
sequence of the present invention; i.e., a nucleotide sequence
provided in SEQ ID NOS:1-5,191, a representative fragment thereof,
or a nucleotide sequence at least 95%, preferably at least 99% and
most preferably at least 99.9% identical to a polynucleotide of SEQ
ID NOS:1-5,191. Such a manufacture provides a large portion of the
Staphylococcus aureus genome and parts thereof (e.g., a
Staphylococcus aureus open reading frame (ORF)) in a form which
allows a skilled artisan to examine the manufacture using means not
directly applicable to examining the Staphylococcus aureus genome
or a subset thereof as it exists in nature or in purified form.
[0075] In one application of this embodiment, a nucleotide sequence
of the present invention can be recorded on computer readable
media. As used herein, "computer readable media" refers to any
medium which can be read and accessed directly by a computer. Such
media include, but are not limited to: magnetic storage media, such
as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories, such as
magnetic/optical storage media. A skilled artisan can readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide sequence of the present
invention. Likewise, it will be clear to those of skill how
additional computer readable media that may be developed also can
be used to create analogous manufactures having recorded thereon a
nucleotide sequence of the present invention.
[0076] As used herein, "recorded" refers to a process for storing
information on computer readable medium. A skilled artisan can
readily adopt any of the presently know methods for recording
information on computer readable medium to generate manufactures
comprising the nucleotide sequence information of the present
invention.
[0077] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide sequence of the present invention.
The choice of the data storage structure will generally be based on
the means chosen to access the stored information. In addition, a
variety of data processor programs and formats can be used to store
the nucleotide sequence information of the present invention on
computer readable medium. The sequence information can be
represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and MicroSoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. A
skilled artisan can readily adapt any number of data-processor
structuring formats (e.g., text file or database) in order to
obtain computer readable medium having recorded thereon the
nucleotide sequence information of the present invention.
[0078] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium. Thus, by providing in computer readable
form the nucleotide sequences of SEQ ID NOS:1-5,191, a
representative fragment thereof, or a nucleotide sequence at least
95%, preferably at least 99% and most preferably at least 99.9%
identical to a sequence of SEQ ID NOS:1-5,191 the present invention
enables the skilled artisan routinely to access the provided
sequence information for a wide variety of purposes.
[0079] The examples which follow demonstrate how software which
implements the BLAST (Altschul et al., J. Mol. Biol. 215:403-410
(1990)) and BLAZE (Brutlag et al., Comp. Chem. 17:203-207 (1993))
search algorithms on a Sybase system was used to identify open
reading frames (ORFs) within the Staphylococcus aureus genome which
contain homology to ORFs or proteins from both Staphylococcus
aureus and from other organisms. Among the ORFs discussed herein
are protein encoding fragments of the Staphylococcus aureus genome
useful in producing commercially important proteins, such as
enzymes used in fermentation reactions and in the production of
commercially useful metabolites.
[0080] The present invention further provides systems, particularly
computer-based systems, which contain the sequence information
described herein. Such systems are designed to identify, among
other things, commercially important fragments of the
Staphylococcus aureus genome.
[0081] As used herein, "a computer-based system" refers to the
hardware means, software means, and data storage means used to
analyze the nucleotide sequence information of the present
invention. The minimum hardware means of the computer-based systems
of the present invention comprises a central processing unit (CPU),
input means, output means, and data storage means. A skilled
artisan can readily appreciate that any one of the currently
available computer-based system are suitable for use in the present
invention.
[0082] As stated above, the computer-based systems of the present
invention comprise a data storage means having stored therein a
nucleotide sequence of the present invention and the necessary
hardware means and software means for supporting and implementing a
search means.
[0083] As used herein, "data storage means" refers to memory which
can store nucleotide sequence information of the present invention,
or a memory access means which can access manufactures having
recorded thereon the nucleotide sequence information of the present
invention.
[0084] As used herein, "search means" refers to one or more
programs which are implemented on the computer-based system to
compare a target sequence or target structural motif with the
sequence information stored within the data storage means. Search
means are used to identify fragments or regions of the present
genomic sequences which match a particular target sequence or
target motif. A variety of known algorithms are disclosed publicly
and a variety of commercially available software for conducting
search means are and can be used in the computer-based systems of
the present invention. Examples of such software includes, but is
not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA). A
skilled artisan can readily recognize that any one of the available
algorithms or implementing software packages for conducting
homology searches can be adapted for use in the present
computer-based systems.
[0085] As used herein, a "target sequence" can be any DNA or amino
acid sequence of six or more nucleotides or two or more amino
acids. A skilled artisan can readily recognize that the longer a
target sequence is, the less likely a target sequence will be
present as a random occurrence in the database. The most preferred
sequence length of a target sequence is from about 10 to 100 amino
acids or from about 30 to 300 nucleotide residues. However, it is
well recognized that searches for commercially important fragments,
such as sequence fragments involved in gene expression and protein
processing, may be of shorter length.
[0086] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
three-dimensional configuration which is formed upon the folding of
the target motif. There are a variety of target motifs known in the
art. Protein target motifs include, but are not limited to, enzymic
active sites and signal sequences. Nucleic acid target motifs
include, but are not limited to, promoter sequences, hairpin
structures and inducible expression elements (protein binding
sequences).
[0087] A variety of structural formats for the input and output
means can be used to input and output the information in the
computer-based systems of the present invention. A preferred format
for an output means ranks fragments of the Staphylococcus aureus
genomic sequences possessing varying degrees of homology to the
target sequence or target motif. Such presentation provides a
skilled artisan with a ranking of sequences which contain various
amounts of the target sequence or target motif and identifies the
degree of homology contained in the identified fragment.
[0088] A variety of comparing means can be used to compare a target
sequence or target motif with the data storage means to identify
sequence fragments of the Staphylococcus aureus genome. In the
present examples, implementing software which implement the BLAST
and BLAZE algorithms, described in Altschul et al., J. Mol. Biol.
215: 403-410 (1990), was used to identify open reading frames
within the Staphylococcus aureus genome. A skilled artisan can
readily recognize that any one of the publicly available homology
search programs can be used as the search means for the
computer-based systems of the present invention. Of course,
suitable proprietary systems that may be known to those of skill
also may be employed in this regard.
[0089] FIG. 1 provides a block diagram of a computer system
illustrative of embodiments of this aspect of present invention.
The computer system 102 includes a processor 106 connected to a bus
104. Also connected to the bus 104 are a main memory 108
(preferably implemented as random access memory, RAM) and a variety
of secondary storage devices 110, such as a hard drive 112 and a
removable medium storage device 114. The removable medium storage
device 114 may represent, for example, a floppy disk drive, a
CD-ROM drive, a magnetic tape drive, etc. A removable storage
medium 116 (such as a floppy disk, a compact disk, a magnetic tape,
etc.) containing control logic and/or data recorded therein may be
inserted into the removable medium storage device 114. The computer
system 102 includes appropriate software for reading the control
logic and/or the data from the removable medium storage device 114,
once it is inserted into the removable medium storage device
114.
[0090] A nucleotide sequence of the present invention may be stored
in a well known manner in the main memory 108, any of the secondary
storage devices 110, and/or a removable storage medium 116. During
execution, software for accessing and processing the genomic
sequence (such as search tools, comparing tools, etc.) reside in
main memory 108, in accordance with the requirements and operating
parameters of the operating system, the hardware system and the
software program or programs.
Biochemical Embodiments
[0091] Other embodiments of the present invention are directed to
isolated fragments of the Staphylococcus aureus genome. The
fragments of the Staphylococcus aureus genome of the present
invention include, but are not limited to fragments which encode
peptides; hereinafter open reading frames (ORFs), fragments which
modulate the expression of an operably linked ORF, hereinafter
expression modulating fragments (EMFs) and fragments which can be
used to diagnose the presence of Staphylococcus aureus in a sample,
hereinafter diagnostic fragments (DFs).
[0092] As used herein, an "isolated nucleic acid molecule" or an
"isolated fragment of the Staphylococcus aureus genome" refers to a
nucleic acid molecule possessing a specific nucleotide sequence
which has been subjected to purification means to reduce, from the
composition, the number of compounds which are normally associated
with the composition. Particularly, the term refers to the nucleic
acid molecules having the sequences set out in SEQ ID NOS:1-5,191,
to representative fragments thereof as described above, to
polynucleotides at least 95%, preferably at least 99% and
especially preferably at least 99.9% identical in sequence thereto,
also as set out above.
[0093] A variety of purification means can be used to generated the
isolated fragments of the present invention. These include, but are
not limited to methods which separate constituents of a solution
based on charge, solubility, or size.
[0094] In one embodiment, Staphylococcus aureus DNA can be
mechanically sheared to produce fragments of 15-20 kb in length.
These fragments can then be used to generate an Staphylococcus
aureus library by inserting them into lambda clones as described in
the Examples below. Primers flanking, for example, an ORF, such as
those enumerated in Tables 1-3 can then be generated using
nucleotide sequence information provided in SEQ ID NOS:1-5,191.
Well known and routine techniques of PCR cloning then can be used
to isolate the ORF from the lambda DNA library of Staphylococcus
aureus genomic DNA. Thus, given the availability of SEQ ID
NOS:1-5,191, the information in Tables 1, 2 and 3, and the
information that may be obtained readily by analysis of the
sequences of SEQ ID NOS:1-5,191 using methods set out above, those
of skill will be enabled by the present disclosure to isolate any
ORF-containing or other nucleic acid fragment of the present
invention.
[0095] The isolated nucleic acid molecules of the present invention
include, but are not limited to single stranded and double stranded
DNA, and single stranded RNA.
[0096] As used herein, an "open reading frame," ORF, means a series
of triplets coding for amino acids without any termination codons
and is a sequence translatable into protein.
[0097] Tables 1, 2 and 3 list ORFs in the Staphylococcus aureus
genomic contigs of the present invention that were identified as
putative coding regions by the GeneMark software using
organism-specific second-order Markov probability transition
matrices. It will be appreciated that other criteria can be used,
in accordance with well known analytical methods, such as those
discussed herein, to generate more inclusive, more restrictive or
more selective lists.
[0098] Table 1 sets out ORFs in the Staphylococcus aureus contigs
of the present invention that are at least 80 amino acids long and
over a continuous region of at least 50 bases which are 95% or more
identical (by BLAST analysis) to an S. aureus nucleotide sequence
available through Genbank in November 1996.
[0099] Table 2 sets out ORFs in the Staphylococcus aureus contigs
of the present invention that are not in Table 1 and match, with a
BLASTP probability score of 0.01 or less, a polypeptide sequence
available through Genbank by September 1996.
[0100] Table 3 sets out ORFs in the Staphylococcus aureus contigs
of the present invention that do not match significantly, by BLASTP
analysis, a polypeptide sequence available through Genbank by
September 1996.
[0101] In each table, the first and second columns identify the ORF
by, respectively, contig number and ORF number within the contig;
the third column indicates the reading frame, taking the first 5'
nucleotide of the contig as the start of the +1 frame; the fourth
column indicates the first nucleotide of the ORF, counting from the
5' end of the contig strand; and the fifth column indicates the
length of each ORF in nucleotides.
[0102] In Tables 1 and 2, column six, lists the Reference" for the
closest matching sequence available through Genbank. These
reference numbers are the databases entry numbers commonly used by
those of skill in the art, who will be familiar with their
denominators. Descriptions of the nomenclature are available from
the National Center for Biotechnology Information. Column seven in
Tables 1 and 2 provides the gene name" of the matching sequence;
column eight provides the BLAST identity" score from the comparison
of the ORF and the homologous gene; and column nine indicates the
length in nucleotides of the highest scoring segment pair"
identified by the BLAST identity analysis.
[0103] In Table 3, the last column, column six, indicates the
length of each ORF in amino acid residues.
[0104] The concepts of percent identity and percent similarity of
two polypeptide sequences is well understood in the art. For
example, two polypeptides 10 amino acids in length which differ at
three amino acid positions (e.g., at positions 1, 3 and 5) are said
to have a percent identity of 70%. However, the same two
polypeptides would be deemed to have a percent similarity of 80%
if, for example at position 5, the amino acids moieties, although
not identical, were "similar" (i.e., possessed similar biochemical
characteristics). Many programs for analysis of nucleotide or amino
acid sequence similarity, such as fasta and BLAST specifically list
percent identity of a matching region as an output parameter. Thus,
for instance, Tables 1 and 2 herein enumerate the percent identity"
of the highest scoring segment pair" in each ORF and its listed
relative. Further details concerning the algorithms and criteria
used for homology searches are provided below and are described in
the pertinent literature highlighted by the citations provided
below.
[0105] It will be appreciated that other criteria can be used to
generate more inclusive and more exclusive listings of the types
set out in the tables. As those of skill will appreciate, narrow
and broad searches both are useful. Thus, a skilled artisan can
readily identify ORFs in contigs of the Staphylococcus aureus
genome other than those listed in Tables 1-3, such as ORFs which
are overlapping or encoded by the opposite strand of an identified
ORF in addition to those ascertainable using the computer-based
systems of the present invention.
[0106] As used herein, an "expression modulating fragment," EMF,
means a series of nucleotide molecules which modulates the
expression of an operably linked ORF or EMF.
[0107] As used herein, a sequence is said to "modulate the
expression of an operably linked sequence" when the expression of
the sequence is altered by the presence of the EMF. EMFs include,
but are not limited to, promoters, and promoter modulating
sequences (inducible elements). One class of EMFs are fragments
which induce the expression or an operably linked ORF in response
to a specific regulatory factor or physiological event.
[0108] EMF sequences can be identified within the contigs of the
Staphylococcus aureus genome by their proximity to the ORFs
provided in Tables 1-3. An intergenic segment, or a fragment of the
intergenic segment, from about 10 to 200 nucleotides in length,
taken from any one of the ORFs of Tables 1-3 will modulate the
expression of an operably linked ORF in a fashion similar to that
found with the naturally linked ORF sequence. As used herein, an
"intergenic segment" refers to fragments of the Staphylococcus
aureus genome which are between two ORF(s) herein described. EMFs
also can be identified using known EMFs as a target sequence or
target motif in the computer-based systems of the present
invention. Further, the two methods can be combined and used
together.
[0109] The presence and activity of an EMF can be confirmed using
an EMF trap vector. An EMF trap vector contains a cloning site
linked to a marker sequence. A marker sequence encodes an
identifiable phenotype, such as antibiotic resistance or a
complementing nutrition auxotrophic factor, which can be identified
or assayed when the EMF trap vector is placed within an appropriate
host under appropriate conditions. As described above, a EMF will
modulate the expression of an operably linked marker sequence. A
more detailed discussion of various marker sequences is provided
below.
[0110] A sequence which is suspected as being an EMF is cloned in
all three reading frames in one or more restriction sites upstream
from the marker sequence in the EMF trap vector. The vector is then
transformed into an appropriate host using known procedures and the
phenotype of the transformed host in examined under appropriate
conditions. As described above, an EMF will modulate the expression
of an operably linked marker sequence.
[0111] As used herein, a "diagnostic fragment," DF, means a series
of nucleotide molecules which selectively hybridize to
Staphylococcus aureus sequences. DFs can be readily identified by
identifying unique sequences within contigs of the Staphylococcus
aureus genome, such as by using well-known computer analysis
software, and by generating and testing probes or amplification
primers consisting of the DF sequence in an appropriate diagnostic
format which determines amplification or hybridization
selectivity.
[0112] The sequences falling within the scope of the present
invention are not limited to the specific sequences herein
described, but also include allelic and species variations thereof.
Allelic and species variations can be routinely determined by
comparing the sequences provided in SEQ ID NOS:1-5,191, a
representative fragment thereof, or a nucleotide sequence at least
99% and preferably 99.9% identical to SEQ ID NOS:1-5,191, with a
sequence from another isolate of the same species. Furthermore, to
accommodate codon variability, the invention includes nucleic acid
molecules coding for the same amino acid sequences as do the
specific ORFs disclosed herein. In other words, in the coding
region of an ORF, substitution of one codon for another which
encodes the same amino acid is expressly contemplated.
[0113] Any specific sequence disclosed herein can be readily
screened for errors by resequencing a particular fragment, such as
an ORF, in both directions (i.e., sequence both strands).
Alternatively, error screening can be performed by sequencing
corresponding polynucleotides of Staphylococcus aureus origin
isolated by using part or all of the fragments in question as a
probe or primer.
[0114] Each of the ORFs of the Staphylococcus aureus genome
disclosed in Tables 1, 2 and 3, and the EMFs found 5' to the ORFs,
can be used as polynucleotide reagents in numerous ways. For
example, the sequences can be used as diagnostic probes or
diagnostic amplification primers to detect the presence of a
specific microbe in a sample, particular Staphylococcus aureus.
Especially preferred in this regard are ORF such as those of Table
3, which do not match previously characterized sequences from other
organisms and thus are most likely to be highly selective for
Staphylococcus aureus. Also particularly preferred are ORFs that
can be used to distinguish between strains of Staphylococcus
aureus, particularly those that distinguish medically important
strain, such as drug-resistant strains.
[0115] In addition, the fragments of the present invention, as
broadly described, can be used to control gene expression through
triple helix formation or antisense DNA or RNA, both of which
methods are based on the binding of a polynucleotide sequence to
DNA or RNA. Triple helix-formation optimally results in a shut-off
of RNA transcription from DNA, while antisense RNA hybridization
blocks translation of an mRNA molecule into polypeptide.
Information from the sequences of the present invention can be used
to design antisense and triple helix-forming oligonucleotides.
Polynucleotides suitable for use in these methods are usually 20 to
40 bases in length and are designed to be complementary to a region
of the gene involved in transcription, for triple-helix formation,
or to the mRNA itself, for antisense inhibition. Both techniques
have been demonstrated to be effective in model systems, and the
requisite techniques are well known and involve routine procedures.
Triple helix techniques are discussed in, for example, Lee et al.,
Nucl. Acids Res. 6: 3073 (1979); Cooney et al., Science 241: 456
(1988); and Dervan et al., Science 251: 1360 (1991). Antisense
techniques in general are discussed in, for instance, Okano, J.
Neurochem. 56: 560 (1991) and OLIGODEOXYNUCLEOTIDES AS ANTISENSE
INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, Fla.
(1988)).
[0116] The present invention further provides recombinant
constructs comprising one or more fragments of the Staphylococcus
aureus genomic fragments and contigs of the present invention.
Certain preferred recombinant constructs of the present invention
comprise a vector, such as a plasmid or viral vector, into which a
fragment of the Staphylococcus aureus genome has been inserted, in
a forward or reverse orientation. In the case of a vector
comprising one of the ORFs of the present invention, the vector may
further comprise regulatory sequences, including for example, a
promoter, operably linked to the ORF. For vectors comprising the
EMFs of the present invention, the vector may further comprise a
marker sequence or heterologous ORF operably linked to the EMF.
[0117] Large numbers of suitable vectors and promoters are known to
those of skill in the art and are commercially available for
generating the recombinant constructs of the present invention. The
following vectors are provided by way of example. Useful bacterial
vectors include phagescript, PsiX 174, pBluescript SK and KS (+ and
-), pNH8a, pNH 16a, pNH 18a, pNH46a (available from Stratagene);
pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (available from
Pharmacia). Useful eukaryotic vectors include pWLneo, pSV2cat,
pOG44, pXT1, pSG (available from Stratagene) pSVK3, pBPV, pMSG,
pSVL (available from Pharmacia).
[0118] Promoter regions can be selected from any desired gene using
CAT (chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are pKK232-8 and pCM7.
Particular named bacterial promoters include lacI, lacZ, T3, T7,
gpt, lambda PR, and trc. Eukaryotic promoters include CMV immediate
early, HSV thymidine kinase, early and late SV40, LTRs from
retrovirus, and mouse metallothionein-I. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art.
[0119] The present invention further provides host cells containing
any one of the isolated fragments of the Staphylococcus aureus
genomic fragments and contigs of the present invention, wherein the
fragment has been introduced into the host cell using known
methods. The host cell can be a higher eukaryotic host cell, such
as a mammalian cell, a lower eukaryotic host cell, such as a yeast
cell, or a procaryotic cell, such as a bacterial cell.
[0120] A polynucleotide of the present invention, such as a
recombinant construct comprising an ORF of the present invention,
may be introduced into the host by a variety of well established
techniques that are standard in the art, such as calcium phosphate
transfection, DEAE, dextran mediated transfection and
electroporation, which are described in, for instance, Davis, L. et
al., BASIC METHODS IN MOLECULAR BIOLOGY (1986).
[0121] A host cell containing one of the fragments of the
Staphylococcus aureus genomic fragments and contigs of the present
invention, can be used in conventional manners to produce the gene
product encoded by the isolated fragment (in the case of an ORF) or
can be used to produce a heterologous protein under the control of
the EMF.
[0122] The present invention further provides isolated polypeptides
encoded by the nucleic acid fragments of the present invention or
by degenerate variants of the nucleic acid fragments of the present
invention. By "degenerate variant" is intended nucleotide fragments
which differ from a nucleic acid fragment of the present invention
(e.g., an ORF) by nucleotide sequence but, due to the degeneracy of
the Genetic Code, encode an identical polypeptide sequence.
[0123] Preferred nucleic acid fragments of the present invention
are the ORFs depicted in Tables 2 and 3 which encode proteins.
[0124] A variety of methodologies known in the art can be utilized
to obtain any one of the isolated polypeptides or proteins of the
present invention. At the simplest level, the amino acid sequence
can be synthesized using commercially available peptide
synthesizers. This is particularly useful in producing small
peptides and fragments of larger polypeptides. Such short fragments
as may be obtained most readily by synthesis are useful, for
example, in generating antibodies against the native polypeptide,
as discussed further below.
[0125] In an alternative method, the polypeptide or protein is
purified from bacterial cells which naturally produce the
polypeptide or protein. One skilled in the art can readily employ
well-known methods for isolating polpeptides and proteins to
isolate and purify polypeptides or proteins of the present
invention produced naturally by a bacterial strain, or by other
methods. Methods for isolation and purification that can be
employed in this regard include, but are not limited to,
immunochromatography, HPLC, size-exclusion chromatography,
ion-exchange chromatography, and immuno-affinity
chromatography.
[0126] The polypeptides and proteins of the present invention also
can be purified from cells which have been altered to express the
desired polypeptide or protein. As used herein, a cell is said to
be altered to express a desired polypeptide or protein when the
cell, through genetic manipulation, is made to produce a
polypeptide or protein which it normally does not produce or which
the cell normally produces at a lower level. Those skilled in the
art can readily adapt procedures for introducing and expressing
either recombinant or synthetic sequences into eukaryotic or
prokaryotic cells in order to generate a cell which produces one of
the polypeptides or proteins of the present invention.
[0127] Any host/vector system can be used to express one or more of
the ORFs of the present invention. These include, but are not
limited to, eukaryotic hosts such as HeLa cells, CV-1 cell, COS
cells, and Sf9 cells, as well as prokaryotic host such as E. coli
and B. subtilis. The most preferred cells are those which do not
normally express the particular polypeptide or protein or which
expresses the polypeptide or protein at low natural level.
[0128] "Recombinant," as used herein, means that a polypeptide or
protein is derived from recombinant (e.g., microbial or mammalian)
expression systems. "Microbial" refers to recombinant polypeptides
or proteins made in bacterial or fungal (e.g., yeast) expression
systems. As a product, "recombinant microbial"defines a polypeptide
or protein essentially free of native endogenous substances and
unaccompanied by associated native glycosylation. Polypeptides or
proteins expressed in most bacterial cultures, e.g., E. coli, will
be free of glycosylation modifications; polypeptides or proteins
expressed in yeast will have a glycosylation pattern different from
that expressed in mammalian cells.
[0129] "Nucleotide sequence" refers to a heteropolymer of
deoxyribonucleotides. Generally, DNA segments encoding the
polypeptides and proteins provided by this invention are assembled
from fragments of the Staphylococcus aureus genome and short
oligonucleotide linkers, or from a series of oligonucleotides, to
provide a synthetic gene which is capable of being expressed in a
recombinant transcriptional unit comprising regulatory elements
derived from a microbial or viral operon.
[0130] Recombinant expression vehicle or vector" refers to a
plasmid or phage or virus or vector, for expressing a polypeptide
from a DNA (RNA) sequence. The expression vehicle can comprise a
transcriptional unit comprising an assembly of (1) a genetic
regulatory elements necessary for gene expression in the host,
including elements required to initiate and maintain transcription
at a level sufficient for suitable expression of the desired
polypeptide, including, for example, promoters and, where
necessary, an enhancers and a polyadenylation signal; (2) a
structural or coding sequence which is transcribed into mRNA and
translated into protein, and (3) appropriate signals to initiate
translation at the beginning of the desired coding region and
terminate translation at its end. Structural units intended for use
in yeast or eukaryotic expression systems preferably include a
leader sequence enabling extracellular secretion of translated
protein by a host cell. Alternatively, where recombinant protein is
expressed without a leader or transport sequence, it may include an
N-terminal methionine residue. This residue may or may not be
subsequently cleaved from the expressed recombinant protein to
provide a final product.
[0131] "Recombinant expression system" means host cells which have
stably integrated a recombinant transcriptional unit into
chromosomal DNA or carry the recombinant transcriptional unit extra
chromosomally. The cells can be prokaryotic or eukaryotic.
Recombinant expression systems as defined herein will express
heterologous polypeptides or proteins upon induction of the
regulatory elements linked to the DNA segment or synthetic gene to
be expressed.
[0132] Mature proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the present invention. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described in
Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2.sup.nd
Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1989), the disclosure of which is hereby incorporated by
reference in its entirety.
[0133] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), alpha-factor, acid phosphatase, or heat shock
proteins, among others. The heterologous structural sequence is
assembled in appropriate phase with translation initiation and
termination sequences, and preferably, a leader sequence capable of
directing secretion of translated protein into the periplasmic
space or extracellular medium. Optionally, the heterologous
sequence can encode a fusion protein including an N-terminal
identification peptide imparting desired characteristics, e.g.,
stabilization or simplified purification of expressed recombinant
product.
[0134] Useful expression vectors for bacterial use are constructed
by inserting a structural DNA sequence encoding a desired protein
together with suitable translation initiation and termination
signals in operable reading phase with a functional promoter. The
vector will comprise one or more phenotypic selectable markers and
an origin of replication to ensure maintenance of the vector and,
when desirable, provide amplification within the host.
[0135] Suitable prokaryotic hosts for transformation include
strains of Staphylococcus aureus, E. coli, B. subtilis, Salmonella
typhimurium and various species within the genera Pseudomonas,
Streptomyces, and Staphylococcus. Others may, also be employed as a
matter of choice.
[0136] As a representative but non-limiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 (available form Pharmacia
Fine Chemicals, Uppsala, Sweden) and GEM 1 (available from Promega
Biotec, Madison, Wis., USA). These pBR322 "backbone" sections are
combined with an appropriate promoter and the structural sequence
to be expressed.
[0137] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter, where it is inducible, is derepressed or induced
by appropriate means (e.g., temperature shift or chemical
induction) and cells are cultured for an additional period to
provide for expression of the induced gene product. Thereafter
cells are typically harvested, generally by centrifugation,
disrupted to release expressed protein, generally by physical or
chemical means, and the resulting crude extract is retained for
further purification.
[0138] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described in Gluzman, Cell 23: 175 (1981), and other cell lines
capable of expressing a compatible vector, for example, the C127,
3T3, CHO, HeLa and BHK cell lines.
[0139] Mammalian expression vectors will comprise an origin of
replication, a suitable promoter and enhancer, and also any
necessary ribosome binding sites, polyadenylation site, splice
donor and acceptor sites, transcriptional termination sequences,
and 5' flanking nontranscribed sequences. DNA sequences derived
from the SV40 viral genome, for example, SV40 origin, early
promoter, enhancer, splice, and polyadenylation sites may be used
to provide the required nontranscribed genetic elements.
[0140] Recombinant polypeptides and proteins produced in bacterial
culture is usually isolated by initial extraction from cell
pellets, followed by one or more salting-out, aqueous ion exchange
or size exclusion chromatography steps. Microbial cells employed in
expression of proteins can be disrupted by any convenient method,
including freeze-thaw cycling, sonication, mechanical disruption,
or use of cell lysing agents. Protein refolding steps can be used,
as necessary, in completing configuration of the mature protein.
Finally, high performance liquid chromatography (HPLC) can be
employed for final purification steps.
[0141] An additional aspect of the invention includes
Staphylococcus aureus polypeptides which are useful as
immunodiagnostic antigens and/or immunoprotective vaccines,
collectively "immunologically useful polypeptides". Such
immunologically useful polypeptides may be selected from the ORFs
disclosed herein based on techniques well known in the art and
described elsewhere herein. The inventors have used the following
criteria to select several immunologically useful polypeptides:
[0142] As is known in the art, an amino terminal type I signal
sequence directs a nascent protein across the plasma and outer
membranes to the exterior of the bacterial cell. Such outermembrane
polypeptides are expected to be immunologically useful. According
to Izard, J. W. et al., Mol. Microbiol. 13, 765-773; (1994),
polypeptides containing type I signal sequences contain the
following physical attributes: The length of the type I signal
sequence is approximately 15 to 25 primarily hydrophobic amino acid
residues with a net positive charge in the extreme amino terminus;
the central region of the signal sequence must adopt an
alpha-helical conformation in a hydrophobic environment; and the
region surrounding the actual site of cleavage is ideally six
residues long, with small side-chain amino acids in the -1 and -3
positions.
[0143] Also known in the art is the type IV signal sequence which
is an example of the several types of functional signal sequences
which exist in addition to the type I signal sequence detailed
above. Although functionally related, the type IV signal sequence
possesses a unique set of biochemical and physical attributes
(Strom, M. S. and Lory, S., J. Bacteriol. 174, 7345-7351; 1992)).
These are typically six to eight amino acids with a net basic
charge followed by an additional sixteen to thirty primarily
hydrophobic residues. The cleavage site of a type IV signal
sequence is typically after the initial six to eight amino acids at
the extreme amino terminus. In addition, all type IV signal
sequences contain a phenylalanine residue at the +1 site relative
to the cleavage site.
[0144] Studies of the cleavage sites of twenty-six bacterial
lipoprotein precursors has allowed the definition of a consensus
amino acid sequence for lipoprotein cleavage. Nearly three-fourths
of the bacterial lipoprotein precursors examined contained the
sequence L-(A,S)-(G,A)-C at positions -3 to +1, relative to the
point of cleavage (Hayashi, S. and Wu, H. C. Lipoproteins in
bacteria. J Bioenerg. Biomembr. 22, 451-471; 1990).
[0145] It well known that most anchored proteins found on the
surface of gram-positive bacteria possess a highly conserved
carboxy terminal sequence. More than fifty such proteins from
organisms such as S. pyogenes, S. mutans, E. faecalis, S.
pneumoniae, and others, have been identified based on their
extracellular location and carboxy terminal amino acid sequence
(Fischetti, V. A. Gram-positive commensal bacteria deliver antigens
to elicit mucosal and systemic immunity. ASM News 62, 405-410;
1996). The conserved region is comprised of six charged amino acids
at the extreme carboxy terminus coupled to 15-20 hydrophobic amino
acids presumed to function as a transmembrane domain. Immediately
adjacent to the transmembrane domain is a six amino acid sequence
conserved in nearly all proteins examined. The amino acid sequence
of this region is L-P-X-T-G-X, where X is any amino acid.
[0146] Amino acid sequence similarities to proteins of known
function by BLAST enables the assignment of putative functions to
novel amino acid sequences and allows for the selection of proteins
thought to function outside the cell wall. Such proteins are well
known in the art and include "lipoprotein", "periplasmic", or
"antigen".
[0147] An algorithm for selecting antigenic and immunogenic
Staphylococcus aureus polypeptides including the foregoing criteria
was developed by the present inventors. Use of the algorithm by the
inventors to select immunologically useful Staphylococcus aureus
polypeptides resulted in the selection of several ORFs which are
predicted to be outermembrane-associated proteins. These proteins
are identified in Table 4, below, and shown in the Sequence Listing
as SEQ ID NOS:5,192 to 5,255. Thus the amino acid sequence of each
of several antigenic Staphylococcus aureus polypeptides listed in
Table 4 can be determined, for example, by locating the amino acid
sequence of the ORF in the Sequence Listing. Likewise the
polynucleotide sequence encoding each ORF can be found by locating
the corresponding polynucleotide SEQ ID in Tables 1, 2, or 3, and
finding the corresponding nucleotide sequence in the sequence
listing.
[0148] As will be appreciated by those of ordinary skill in the
art, although a polypeptide representing an entire ORF may be the
closest approximation to a protein found in vivo, it is not always
technically practical to express a complete ORF in vitro. It may be
very challenging to express and purify a highly hydrophobic protein
by common laboratory methods. As a result, the immunologically
useful polypeptides described herein as SEQ ID NOS:5,192-5,255 may
have been modified slightly to simplify the production of
recombinant protein, and are the preferred embodiments. In general,
nucleotide sequences which encode highly hydrophobic domains, such
as those found at the amino terminal signal sequence, are excluded
for enhanced in vitro expression of the polypeptides. Furthermore,
any highly hydrophobic amino acid sequences occurring at the
carboxy terminus are also excluded. Such truncated polypeptides
include for example the mature forms of the polypeptides expected
to exist in nature.
[0149] Those of ordinary skill in the art can identify soluble
portions the polypeptide identified in Table 4, and in the case of
truncated polypeptides sequences shown as SEQ ID NOS:5,255, may
obtain the complete predicted amino acid sequence of each
polypeptide by translating the corresponding polynucleotides
sequences of the corresponding ORF listed in Tables 1, 2 and 3 and
found in the sequence listing.
[0150] Accordingly, polypeptides comprising the complete amino acid
sequence of an immunologically useful polypeptide selected from the
group of polypeptides encoded by the ORFs identified in Table 4, or
an amino acid sequence at least 95% identical thereto, preferably
at least 97% identical thereto, and most preferably at least 99%
identical thereto form an embodiement of the invention; in
addition, polypeptides comprising an amino acid sequence selected
from the group of amino acid sequences shown in the sequence
listing as SEQ ID NOS:5,255, or an amino acid sequence at least 95%
identical thereto, preferably at least 97% identical thereto and
most preferably 99% identical thereto, form an embodiment of the
invention. Polynucleotides encoding the foregoing polypeptides also
form part of the invention.
[0151] In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide
of the invention, particularly those epitope-bearing portions
(antigenic regions) identified in Table 4. The epitope-bearing
portion is an immunogenic or antigenic epitope of a polypeptide of
the invention. An "immunogenic epitope" is defined as a part of a
protein that elicits an antibody response when the whole protein is
the immunogen. On the other hand, a region of a protein molecule to
which an antibody can bind is defined as an "antigenic epitope."
The number of immunogenic epitopes of a protein generally is less
than the number of antigenic epitopes. See, for instance, Geysen et
al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).
[0152] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in that
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See, for instance,
Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A.
(1983) "Antibodies that react with predetermined sites on
proteins", Science, 219:660-666. Peptides capable of eliciting
protein-reactive sera are frequently represented in the primary
sequence of a protein, can be characterized by a set of simple
chemical rules, and are confined neither to immunodominant regions
of intact proteins (i.e., immunogenic epitopes) nor to the amino or
carboxyl terminals. Antigenic epitope-bearing peptides and
polypeptides of the invention are therefore useful to raise
antibodies, including monoclonal antibodies, that bind specifically
to a polypeptide of the invention. See, for instance, Wilson et
al., Cell 37:767-778 (1984) at 777.
[0153] Antigenic epitope-bearing peptides and polypeptides of the
invention preferably contain a sequence of at least seven, more
preferably at least nine and most preferably between about 15 to
about 30 amino acids contained within the amino acid sequence of a
polypeptide of the invention. Non-limiting examples of antigenic
polypeptides or peptides that can be used to generate S. aureus
specific antibodies include: a polypeptide comprising peptides
shown in Table 4 below. These polypeptide fragments have been
determined to bear antigenic epitopes of indicated S. aureus
proteins by the analysis of the Jameson-Wolf antigenic index, a
representative sample of which is shown in FIG. 3.
[0154] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means. See, e.g.,
Houghten, R. A. (1985) General method for the rapid solid-phase
synthesis of large numbers of peptides: specificity of
antigen-antibody interaction at the level of individual amino
acids. Proc. Natl. Acad. Sci. USA 82:5131-5135; this "Simultaneous
Multiple Peptide Synthesis (SMPS)" process is further described in
U.S. Pat. No. 4,631,211 to Houghten et al. (1986). Epitope-bearing
peptides and polypeptides of the invention are used to induce
antibodies according to methods well known in the art. See, for
instance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M.
et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et
al., J. Gen. Virol. 66:2347-2354 (1985). Immunogenic
epitope-bearing peptides of the invention, i.e., those parts of a
protein that elicit an antibody response when the whole protein is
the immunogen, are identified according to methods known in the
art. See, for instance, Geysen et al., supra. Further still, U.S.
Pat. No. 5,194,392 to Geysen (1990) describes a general method of
detecting or determining the sequence of monomers (amino acids or
other compounds) which is a topological equivalent of the epitope
(i.e., a "mimotope") which is complementary to a particular
paratope (antigen binding site) of an antibody of interest. More
generally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a
method of detecting or determining a sequence of monomers which is
a topographical equivalent of a ligand which is complementary to
the ligand binding site of a particular receptor of interest.
Similarly, U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996)
on Peralkylated Oligopeptide Mixtures discloses linear C1-C7-alkyl
peralkylated oligopeptides and sets and libraries of such peptides,
as well as methods for using such oligopeptide sets and libraries
for determining the sequence of a peralkylated oligopeptide that
preferentially binds to an acceptor molecule of interest. Thus,
non-peptide analogs of the epitope-bearing peptides of the
invention also can be made routinely by these methods.
[0155] Table 4 lists immunologically useful polypeptides identified
by an algorithm which locates novel Staphylococcus aureus
outermembrane proteins, as is described above. Also listed are
epitopes or "antigenic regions" of each of the identified
polypeptides. The antigenic regions, or epitopes, are delineated by
two numbers x-y, where x is the number of the first amino acid in
the open reading frame included within the epitope and y is the
number of the last amino acid in the open reading frame included
within the epitope. For example, the first epitope in ORF 168-6 is
comprised of amino acids 36 to 45 of SEQ ID NO:5,192, as is
described in Table 4. The inventors have identified several
epitopes for each of the antigenic polypeptides identified in Table
4. Accordingly, forming part of the present invention are
polypeptides comprising an amino acid sequence of one or more
antigenic regions identified in Table 4. The invention further
provides polynucleotides encoding such polypeptides.
[0156] The present invention further includes isolated
polypeptides, proteins and nucleic acid molecules which are
substantially equivalent to those herein described. As used herein,
substantially equivalent can refer both to nucleic acid and amino
acid sequences, for example a mutant sequence, that varies from a
reference sequence by one or more substitutions, deletions, or
additions, the net effect of which does not result in an adverse
functional dissimilarity between reference and subject sequences.
For purposes of the present invention, sequences having equivalent
biological activity, and equivalent expression characteristics are
considered substantially equivalent. For purposes of determining
equivalence, truncation of the mature sequence should be
disregarded.
[0157] The invention further provides methods of obtaining homologs
from other strains of Staphylococcus aureus, of the fragments of
the Staphylococcus aureus genome of the present invention and
homologs of the proteins encoded by the ORFs of the present
invention. As used herein, a sequence or protein of Staphylococcus
aureus is defined as a homolog of a fragment of the Staphylococcus
aureus fragments or contigs or a protein encoded by one of the ORFs
of the present invention, if it shares significant homology to one
of the fragments of the Staphylococcus aureus genome of the present
invention or a protein encoded by one of the ORFs of the present
invention. Specifically, by using the sequence disclosed herein as
a probe or as primers, and techniques such as PCR cloning and
colony/plaque hybridization, one skilled in the art can obtain
homologs.
[0158] As used herein, two nucleic acid molecules or proteins are
said to "share significant homology" if the two contain regions
which prossess greater than 85% sequence (amino acid or nucleic
acid) homology. Preferred homologs in this regard are those with
more than 90% homology. Especially preferred are those with 93% or
more homology. Among especially preferred homologs those with 95%
or more homology are particularly preferred. Very particularly
preferred among these are those with 97% and even more particularly
preferred among those are homologs with 99% or more homology. The
most preferred homologs among these are those with 99.9% homology
or more. It will be understood that, among measures of homology,
identity is particularly preferred in this regard.
[0159] Region specific primers or probes derived from the
nucleotide sequence provided in SEQ ID NOS:1-5,191 or from a
nucleotide sequence at least 95%, particularly at least 99%,
especially at least 99.5% identical to a sequence of SEQ ID
NOS:1-5,191 can be used to prime DNA synthesis and PCR
amplification, as well as to identify colonies containing cloned
DNA encoding a homolog. Methods suitable to this aspect of the
present invention are well known and have been described in great
detail in many publications such as, for example, Innis et al., PCR
PROTOCOLS, Academic Press, San Diego, Calif. (1990)).
[0160] When using primers derived from SEQ ID NOS:1-5,191 or from a
nucleotide sequence having an aforementioned identity to a sequence
of SEQ ID NOS:1-5,191, one skilled in the art will recognize that
by employing high stringency conditions (e.g., annealing at
50-60.degree. C. in 6.times.SSPC and 50% formamide, and washing at
50-65.degree. C. in 0.5.times.SSPC) only sequences which are
greater than 75% homologous to the primer will be amplified. By
employing lower stringency conditions (e.g., hybridizing at
35-37.degree. C. in 5.times.SSPC and 40-45% formamide, and washing
at 42.degree. C. in 0.5.times.SSPC), sequences which are greater
than 40-50% homologous to the primer will also be amplified.
[0161] When using DNA probes derived from SEQ ID NOS:1-5,191, or
from a nucleotide sequence having an aforementioned identity to a
sequence of SEQ ID NOS:1-5,191, for colony/plaque hybridization,
one skilled in the art will recognize that by employing high
stringency conditions (e.g., hybridizing at 50-65.degree. C. in
5.times.SSPC and 50% formamide, and washing at 50-65.degree. C. in
0.5.times.SSPC), sequences having regions which are greater than
90% homologous to the probe can be obtained, and that by employing
lower stringency conditions (e.g., hybridizing at 35-37.degree. C.
in 5.times.SSPC and 40-45% formamide, and washing at 42.degree. C.
in 0.5.times.SSPC), sequences having regions which are greater than
35-45% homologous to the probe will be obtained.
[0162] Any organism can be used as the source for homologs of the
present invention so long as the organism naturally expresses such
a protein or contains genes encoding the same. The most preferred
organism for isolating homologs are bacterias which are closely
related to Staphylococcus aureus.
[0163] Illustrative Uses of Compositions of the Invention
[0164] Each ORF provided in Tables 1 and 2 is identified with a
function by homology to a known gene or polypeptide. As a result,
one skilled in the art can use the polypeptides of the present
invention for commercial, therapeutic and industrial purposes
consistent with the type of putative identification of the
polypeptide. Such identifications permit one skilled in the art to
use the Staphylococcus aureus ORFs in a manner similar to the known
type of sequences for which the identification is made; for
example, to ferment a particular sugar source or to produce a
particular metabolite. A variety of reviews illustrative of this
aspect of the invention are available, including the following
reviews on the industrial use of enzymes, for example, BIOCHEMICAL
ENGINEERING AND BIOTECHNOLOGY HANDBOOK, 2nd Ed., Macmillan
Publications, Ltd. NY (1991) and BIOCATALYSTS IN ORGANIC SYNTHESES,
Tramper et al., Eds., Elsevier Science Publishers, Amsterdam, The
Netherlands (1985). A variety of exemplary uses that illustrate
this and similar aspects of the present invention are discussed
below.
[0165] 1. Biosynthetic Enzymes
[0166] Open reading frames encoding proteins involved in mediating
the catalytic reactions involved in intermediary and macromolecular
metabolism, the biosynthesis of small molecules, cellular processes
and other functions includes enzymes involved in the degradation of
the intermediary products of metabolism, enzymes involved in
central intermediary metabolism, enzymes involved in respiration,
both aerobic and anaerobic, enzymes involved in fermentation,
enzymes involved in ATP proton motor force conversion, enzymes
involved in broad regulatory function, enzymes involved in amino
acid synthesis, enzymes involved in nucleotide synthesis, enzymes
involved in cofactor and vitamin synthesis, can be used for
industrial biosynthesis.
[0167] The various metabolic pathways present in Staphylococcus
aureus can be identified based on absolute nutritional requirements
as well as by examining the various enzymes identified in Table 1-3
and SEQ ID NOS:1-5,191.
[0168] Of particular interest are polypeptides involved in the
degradation of intermediary metabolites as well as
non-macromolecular metabolism. Such enzymes include amylases,
glucose oxidases, and catalase.
[0169] Proteolytic enzymes are another class of commercially
important enzymes. Proteolytic enzymes find use in a number of
industrial processes including the processing of flax and other
vegetable fibers, in the extraction, clarification and
depectinization of fruit juices, in the extraction of vegetables'
oil and in the maceration of fruits and vegetables to give
unicellular fruits. A detailed review of the proteolytic enzymes
used in the food industry is provided in Rombouts et al., Symbiosis
21: 79 (1986) and Voragen et al. in BIOCATALYSTS IN AGRICULTURAL
BIOTECHNOLOGY, Whitaker et al., Eds., American Chemical Society
Symposium Series 389: 93 (1989).
[0170] The metabolism of sugars is an important aspect of the
primary metabolism of Staphylococcus aureus. Enzymes involved in
the degradation of sugars, such as, particularly, glucose,
galactose, fructose and xylose, can be used in industrial
fermentation. Some of the important sugar transforming enzymes,
from a commercial viewpoint, include sugar isomerases such as
glucose isomerase. Other metabolic enzymes have found commercial
use such as glucose oxidases which produces ketogulonic acid (KGA).
KGA is an intermediate in the commercial production of ascorbic
acid using the Reichstein's procedure, as described in Krueger et
al., Biotechnology 6(A), Rhine et al., Eds., Verlag Press,
Weinheim, Germany (1984).
[0171] Glucose oxidase (GOD) is commercially available and has been
used in purified form as well as in an immobilized form for the
deoxygenation of beer. See, for instance, Hartmeir et al.,
Biotechnology Letters 1: 21 (1979). The most important application
of GOD is the industrial scale fermentation of gluconic acid.
Market for gluconic acids which are used in the detergent, textile,
leather, photographic, pharmaceutical, food, feed and concrete
industry, as described, for example, in Bigelis et al., beginning
on page 357 in GENE MANIPULATIONS AND FUNGI; Benett et al., Eds.,
Academic Press, New York (1985). In addition to industrial
applications, GOD has found applications in medicine for
quantitative determination of glucose in body fluids recently in
biotechnology for analyzing syrups from starch and cellulose
hydrosylates. This application is described in Owusu et al.,
Biochem. et Biophysica. Acta. 872: 83 (1986), for instance.
[0172] The main sweetener used in the world today is sugar which
comes from sugar beets and sugar cane. In the field of industrial
enzymes, the glucose isomerase process shows the largest expansion
in the market today. Initially, soluble enzymes were used and later
immobilized enzymes were developed (Krueger et al., Biotechnology,
The Textbook of Industrial Microbiology, Sinauer Associated
Incorporated, Sunderland, Mass. (1990)). Today, the use of
glucose-produced high fructose syrups is by far the largest
industrial business using immobilized enzymes. A review of the
industrial use of these enzymes is provided by Jorgensen, Starch
40:307 (1988).
[0173] Proteinases, such as alkaline serine proteinases, are used
as detergent additives and thus represent one of the largest
volumes of microbial enzymes used in the industrial sector. Because
of their industrial importance, there is a large body of published
and unpublished information regarding the use of these enzymes in
industrial processes. (See Faultman et al., Acid Proteases
Structure Function and Biology, Tang, J., ed., Plenum Press, New
York (1977) and Godfrey et al., Industrial Enzymes, MacMillan
Publishers, Surrey, UK (1983) and Hepner et al, Report Industrial
Enzymes by 1990, Hel Hepner & Associates, London (1986)).
[0174] Another class of commercially usable proteins of the present
invention are the microbial lipases, described by, for instance,
Macrae et al., Philosophical Transactions of the Chiral Society of
London 310:227 (1985) and Poserke, Journal of the American Oil
Chemist Society 61:1758 (1984). A major use of lipases is in the
fat and oil industry for the production of neutral glycerides using
lipase catalyzed inter-esterification of readily available
triglycerides. Application of lipases include the use as a
detergent additive to facilitate the removal of fats from fabrics
in the course of the washing procedures.
[0175] The use of enzymes, and in particular microbial enzymes, as
catalyst for key steps in the synthesis of complex organic
molecules is gaining popularity at a great rate. One area of great
interest is the preparation of chiral intermediates. Preparation of
chiral intermediates is of interest to a wide range of synthetic
chemists particularly those scientists involved with the
preparation of new pharmaceuticals, agrochemicals, fragrances and
flavors. (See Davies et al., Recent Advances in the Generation of
Chiral Intermediates Using Enzymes, CRC Press, Boca Raton, Fla.
(1990)). The following reactions catalyzed by enzymes are of
interest to organic chemists: hydrolysis of carboxylic acid esters,
phosphate esters, amides and nitrites, esterification reactions,
trans-esterification reactions, synthesis of amides, reduction of
alkanones and oxoalkanates, oxidation of alcohols to carbonyl
compounds, oxidation of sulfides to sulfoxides, and carbon bond
forming reactions such as the aldol reaction.
[0176] When considering the use of an enzyme encoded by one of the
ORFs of the present invention for biotransformation and organic
synthesis it is sometimes necessary to consider the respective
advantages and disadvantages of using a microorganism as opposed to
an isolated enzyme. Pros and cons of using a whole cell system on
the one hand or an isolated partially purified enzyme on the other
hand, has been described in detail by Bud et al., Chemistry in
Britain (1987), p. 127.
[0177] Amino transferases, enzymes involved in the biosynthesis and
metabolism of amino acids, are useful in the catalytic production
of amino acids. The advantages of using microbial based enzyme
systems is that the amino transferase enzymes catalyze the
stereo-selective synthesis of only L-amino acids and generally
possess uniformly high catalytic rates. A description of the use of
amino transferases for amino acid production is provided by
Roselle-David, Methods of Enzymology 136:479 (1987).
[0178] Another category of useful proteins encoded by the ORFs of
the present invention include enzymes involved in nucleic acid
synthesis, repair, and recombination. A variety of commercially
important enzymes have previously been isolated from members of
Staphylococcus aureus. These include Sau3A and Sau96I.
[0179] 2. Generation of Antibodies
[0180] As described here, the proteins of the present invention, as
well as homologs thereof, can be used in a variety procedures and
methods known in the art which are currently applied to other
proteins. The proteins of the present invention can further be used
to generate an antibody which selectively binds the protein. Such
antibodies can be either monoclonal or polyclonal antibodies, as
well fragments of these antibodies, and humanized forms.
[0181] The invention further provides antibodies which selectively
bind to one of the proteins of the present invention and hybridomas
which produce these antibodies. A hybridoma is an immortalized cell
line which is capable of secreting a specific monoclonal
antibody.
[0182] In general, techniques for preparing polyclonal and
monoclonal antibodies as well as hybridomas capable of producing
the desired antibody are well known in the art (Campbell, A. M.,
MONOCLONAL ANTIBODY TECHNOLOGY: LABORATORY TECHNIQUES IN
BIOCHEMISTRY AND MOLECULAR BIOLOGY, Elsevier Science Publishers,
Amsterdam, The Netherlands (1984); St. Groth et al., J. Immunol.
Methods 35: 1-21 (1980), Kohler and Milstein, Nature 256: 495-497
(1975)), the trioma technique, the human B-cell hybridoma technique
(Kozbor et al., Immunology Today 4: 72 (1983), pgs. 77-96 of Cole
et al., in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,
Inc. (1985)).
[0183] Any animal (mouse, rabbit, etc.) which is known to produce
antibodies can be immunized with the pseudogene polypeptide.
Methods for immunization are well known in the art. Such methods
include subcutaneous or interperitoneal injection of the
polypeptide. One skilled in the art will recognize that the amount
of the protein encoded by the ORF of the present invention used for
immunization will vary based on the animal which is immunized, the
antigenicity of the peptide and the site of injection.
[0184] The protein which is used as an immunogen may be modified or
administered in an adjuvant in order to increase the protein's
antigenicity. Methods of increasing the antigenicity of a protein
are well known in the art and include, but are not limited to
coupling the antigen with a heterologous protein (such as globulin
or galactosidase) or through the inclusion of an adjuvant during
immunization.
[0185] For monoclonal antibodies, spleen cells from the immunized
animals are removed, fused with myeloma cells, such as SP2/0-Ag14
myeloma cells, and allowed to become monoclonal antibody producing
hybridoma cells.
[0186] Any one of a number of methods well known in the art can be
used to identify the hybridoma cell which produces an antibody with
the desired characteristics. These include screening the hybridomas
with an ELISA assay, western blot analysis, or radioimmunoassay
(Lutz et al., Exp. Cell Res. 175: 109-124 (1988)).
[0187] Hybridomas secreting the desired antibodies are cloned and
the class and subclass is determined using procedures known in the
art (Campbell, A. M., Monoclonal Antibody Technology: Laboratory
Techniques in Biochemistry and Molecular Biology, Elsevier Science
Publishers, Amsterdam, The Netherlands (1984)).
[0188] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce
single chain antibodies to proteins of the present invention.
[0189] For polyclonal antibodies, antibody containing antisera is
isolated from the immunized animal and is screened for the presence
of antibodies with the desired specificity using one of the
above-described procedures.
[0190] The present invention further provides the above-described
antibodies in detectably labelled form. Antibodies can be
detectably labelled through the use of radioisotopes, affinity
labels (such as biotin, avidin, etc.), enzymatic labels (such as
horseradish peroxidase, alkaline phosphatase, etc.) fluorescent
labels (such as FITC or rhodamine, etc.), paramagnetic atoms, etc.
Procedures for accomplishing such labelling are well-known in the
art, for example see Sternberger et al., J. Histochem. Cytochem.
18:315 (1970); Bayer, E. A. et al., Meth. Enzym. 62:308 (1979);
Engval, E. et al., Immunol. 109:129 (1972); Goding, J. W. J.
Immunol. Meth. 13:215 (1976)).
[0191] The labeled antibodies of the present invention can be used
for in vitro, in vivo, and in situ assays to identify cells or
tissues in which a fragment of the Staphylococcus aureus genome is
expressed.
[0192] The present invention further provides the above-described
antibodies immobilized on a solid support. Examples of such solid
supports include plastics such as polycarbonate, complex
carbohydrates such as agarose and sepharose, acrylic resins and
such as polyacrylamide and latex beads. Techniques for coupling
antibodies to such solid supports are well known in the art (Weir,
D. M. et al., "Handbook of Experimental Immunology" 4th Ed.,
Blackwell Scientific Publications, Oxford, England, Chapter 10
(1986); Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press, N.Y.
(1974)). The immobilized antibodies of the present invention can be
used for in vitro, in vivo, and in situ assays as well as for
immunoaffinity purification of the proteins of the present
invention.
[0193] 3. Diagnostic Assays and Kits
[0194] The present invention further provides methods to identify
the expression of one of the ORFs of the present invention, or
homolog thereof, in a test sample, using one of the DFs, antigens
or antibodies of the present invention.
[0195] In detail, such methods comprise incubating a test sample
with one or more of the antibodies, or one or more of the DFs, or
one or more antigens of the present invention and assaying for
binding of the DFs, antigens or antibodies to components within the
test sample.
[0196] Conditions for incubating a DF, antigen or antibody with a
test sample vary. Incubation conditions depend on the format
employed in the assay, the detection methods employed, and the type
and nature of the DF or antibody used in the assay. One skilled in
the art will recognize that any one of the commonly available
hybridization, amplification or immunological assay formats can
readily be adapted to employ the Dfs, antigens or antibodies of the
present invention. Examples of such assays can be found in Chard,
T., An Introduction to Radioimmunoassay and Related Techniques,
Elsevier Science Publishers, Amsterdam, The Netherlands (1986);
Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic
Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985);
Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory
Techniques in Biochemistry; PCT publication WO95/32291, and
Molecular Biology, Elsevier Science Publishers, Amsterdam, The
Netherlands (1985), all of which are hereby incorporated herein by
reference.
[0197] The test samples of the present invention include cells,
protein or membrane extracts of cells, or biological fluids such as
sputum, blood, serum, plasma, or urine. The test sample used in the
above-described method will vary based on the assay format, nature
of the detection method and the tissues, cells or extracts used as
the sample to be assayed. Methods for preparing protein extracts or
membrane extracts of cells are well known in the art and can be
readily be adapted in order to obtain a sample which is compatible
with the system utilized.
[0198] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention.
[0199] Specifically, the invention provides a compartmentalized kit
to receive, in close confinement, one or more containers which
comprises: (a) a first container comprising one of the Dfs,
antigens or antibodies of the present invention; and (b) one or
more other containers comprising one or more of the following: wash
reagents, reagents capable of detecting presence of a bound DF,
antigen or antibody.
[0200] In detail, a compartmentalized kit includes any kit in which
reagents are contained in separate containers. Such containers
include small glass containers, plastic containers or strips of
plastic or paper. Such containers allows one to efficiently
transfer reagents from one compartment to another compartment such
that the samples and reagents are not cross-contaminated, and the
agents or solutions of each container can be added in a
quantitative fashion from one compartment to another. Such
containers will include a container which will accept the test
sample, a container which contains the antibodies used in the
assay, containers which contain wash reagents (such as phosphate
buffered saline, Tris-buffers, etc.), and containers which contain
the reagents used to detect the bound antibody, antigen or DF.
[0201] Types of detection reagents include labelled nucleic acid
probes, labelled secondary antibodies, or in the alternative, if
the primary antibody is labelled, the enzymatic, or antibody
binding reagents which are capable of reacting with the labelled
antibody. One skilled in the art will readily recognize that the
disclosed Dfs, antigens and antibodies of the present invention can
be readily incorporated into one of the established kit formats
which are well known in the art.
[0202] 4. Screening Assay for Binding Agents
[0203] Using the isolated proteins of the present invention, the
present invention further provides methods of obtaining and
identifying agents which bind to a protein encoded by one of the
ORFs of the present invention or to one of the fragments and the
Staphylococcus aureus fragment and contigs herein described.
[0204] In general, such methods comprise steps of:
[0205] (a) contacting an agent with an isolated protein encoded by
one of the ORFs of the present invention, or an isolated fragment
of the Staphylococcus aureus genome; and
[0206] (b) determining whether the agent binds to said protein or
said fragment.
[0207] The agents screened in the above assay can be, but are not
limited to, peptides, carbohydrates, vitamin derivatives, or other
pharmaceutical agents. The agents can be selected and screened at
random or rationally selected or designed using protein modeling
techniques.
[0208] For random screening, agents such as peptides,
carbohydrates, pharmaceutical agents and the like are selected at
random and are assayed for their ability to bind to the protein
encoded by the ORF of the present invention.
[0209] Alternatively, agents may be rationally selected or
designed. As used herein, an agent is said to be "rationally
selected or designed" when the agent is chosen based on the
configuration of the particular protein. For example, one skilled
in the art can readily adapt currently available procedures to
generate peptides, pharmaceutical agents and the like capable of
binding to a specific peptide sequence in order to generate
rationally designed antipeptide peptides, for example see Hurby et
al., Application of Synthetic Peptides: Antisense Peptides," In
Synthetic Peptides, A User's Guide, W. H. Freeman, NY (1992), pp.
289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or
pharmaceutical agents, or the like.
[0210] In addition to the foregoing, one class of agents of the
present invention, as broadly described, can be used to control
gene expression through binding to one of the ORFs or EMFs of the
present invention. As described above, such agents can be randomly
screened or rationally designed/selected. Targeting the ORF or EMF
allows a skilled artisan to design sequence specific or element
specific agents, modulating the expression of either a single ORF
or multiple ORFs which rely on the same EMF for expression
control.
[0211] One class of DNA binding agents are agents which contain
base residues which hybridize or form a triple helix by binding to
DNA or RNA. Such agents can be based on the classic phosphodiester,
ribonucleic acid backbone, or can be a variety of sulfhydryl or
polymeric derivatives which have base attachment capacity.
[0212] Agents suitable for use in these methods usually contain 20
to 40 bases and are designed to be complementary to a region of the
gene involved in transcription (triple helix--see Lee et al., Nucl.
Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988);
and Dervan et al., Science 251: 1360 (1991)) or to the mRNA itself
(antisense--Okano, J. Neurochem. 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988)). Triple helix-formation
optimally results in a shut-off of RNA transcription from DNA,
while antisense RNA hybridization blocks translation of an mRNA
molecule into polypeptide. Both techniques have been demonstrated
to be effective in model systems. Information contained in the
sequences of the present invention can be used to design antisense
and triple helix-forming oligonucleotides, and other DNA binding
agents.
[0213] 5. Pharmaceutical Compositions and Vaccines
[0214] The present invention further provides pharmaceutical agents
which can be used to modulate the growth or pathogenicity of
Staphylococcus aureus, or another related organism, in vivo or in
vitro. As used herein, a "pharmaceutical agent" is defined as a
composition of matter which can be formulated using known
techniques to provide a pharmaceutical compositions. As used
herein, the "pharmaceutical agents of the present invention" refers
the pharmaceutical agents which are derived from the proteins
encoded by the ORFs of the present invention or are agents which
are identified using the herein described assays.
[0215] As used herein, a pharmaceutical agent is said to "modulate
the growth or pathogenicity of Staphylococcus aureus or a related
organism, in vivo or in vitro," when the agent reduces the rate of
growth, rate of division, or viability of the organism in question.
The pharmaceutical agents of the present invention can modulate the
growth or pathogenicity of an organism in many fashions, although
an understanding of the underlying mechanism of action is not
needed to practice the use of the pharmaceutical agents of the
present invention. Some agents will modulate the growth or
pathogenicity by binding to an important protein thus blocking the
biological activity of the protein, while other agents may bind to
a component of the outer surface of the organism blocking
attachment or rendering the organism more prone to act the bodies
nature immune system. Alternatively, the agent may comprise a
protein encoded by one of the ORFs of the present invention and
serve as a vaccine. The development and use of vaccines derived
from membrane associated polypeptides are well known in the art.
The inventors have identified particularly preferred immunogenic
Staphylococcus aureus polypeptides for use as vaccines. Such
immunogenic polypeptides are described above and summarized in
Table 4, below.
[0216] As used herein, a "related organism" is a broad term which
refers to any organism whose growth or pathogenicity can be
modulated by one of the pharmaceutical agents of the present
invention. In general, such an organism will contain a homolog of
the protein which is the target of the pharmaceutical agent or the
protein used as a vaccine. As such, related organisms do not need
to be bacterial but may be fungal or viral pathogens.
[0217] The pharmaceutical agents and compositions of the present
invention may be administered in a convenient manner, such as by
the oral, topical, intravenous, intraperitoneal, intramuscular,
subcutaneous, intranasal or intradermal routes. The pharmaceutical
compositions are administered in an amount which is effective for
treating and/or prophylaxis of the specific indication. In general,
they are administered in an amount of at least about 1 mg/kg body
weight and in most cases they will be administered in an amount not
in excess of about 1 g/kg body weight per day. In most cases, the
dosage is from about 0.1 mg/kg to about 10 g/kg body weight daily,
taking into account the routes of administration, symptoms,
etc.
[0218] The agents of the present invention can be used in native
form or can be modified to form a chemical derivative. As used
herein, a molecule is said to be a "chemical derivative" of another
molecule when it contains additional chemical moieties not normally
a part of the molecule. Such moieties may improve the molecule's
solubility, absorption, biological half life, etc. The moieties may
alternatively decrease the toxicity of the molecule, eliminate or
attenuate any undesirable side effect of the molecule, etc.
Moieties capable of mediating such effects are disclosed in, among
other sources, REMINGTON'S PHARMACEUTICAL SCIENCES (1980) cited
elsewhere herein.
[0219] For example, such moieties may change an immunological
character of the functional derivative, such as affinity for a
given antibody. Such changes in immunomodulation activity are
measured by the appropriate assay, such as a competitive type
immunoassay. Modifications of such protein properties as redox or
thermal stability, biological half-life, hydrophobicity,
susceptibility to proteolytic degradation or the tendency to
aggregate with carriers or into multimers also may be effected in
this way and can be assayed by methods well known to the skilled
artisan.
[0220] The therapeutic effects of the agents of the present
invention may be obtained by providing the agent to a patient by
any suitable means (e.g., inhalation, intravenously,
intramuscularly, subcutaneously, enterally, or parenterally). It is
preferred to administer the agent of the present invention so as to
achieve an effective concentration within the blood or tissue in
which the growth of the organism is to be controlled. To achieve an
effective blood concentration, the preferred method is to
administer the agent by injection. The administration may be by
continuous infusion, or by single or multiple injections.
[0221] In providing a patient with one of the agents of the present
invention, the dosage of the administered agent will vary depending
upon such factors as the patient's age, weight, height, sex,
general medical condition, previous medical history, etc. In
general, it is desirable to provide the recipient with a dosage of
agent which is in the range of from about 1 pg/kg to 10 mg/kg (body
weight of patient), although a lower or higher dosage may be
administered. The therapeutically effective dose can be lowered by
using combinations of the agents of the present invention or
another agent.
[0222] As used herein, two or more compounds or agents are said to
be administered "in combination" with each other when either (1)
the physiological effects of each compound, or (2) the serum
concentrations of each compound can be measured at the same time.
The composition of the present invention can be administered
concurrently with, prior to, or following the administration of the
other agent.
[0223] The agents of the present invention are intended to be
provided to recipient subjects in an amount sufficient to decrease
the rate of growth (as defined above) of the target organism.
[0224] The administration of the agent(s) of the invention may be
for either a "prophylactic" or "therapeutic" purpose. When provided
prophylactically, the agent(s) are provided in advance of any
symptoms indicative of the organisms growth. The prophylactic
administration of the agent(s) serves to prevent, attenuate, or
decrease the rate of onset of any subsequent infection. When
provided therapeutically, the agent(s) are provided at (or shortly
after) the onset of an indication of infection. The therapeutic
administration of the compound(s) serves to attenuate the
pathological symptoms of the infection and to increase the rate of
recovery.
[0225] The agents of the present invention are administered to a
subject, such as a mammal, or a patient, in a pharmaceutically
acceptable form and in a therapeutically effective concentration. A
composition is said to be "pharmacologically acceptable" if its
administration can be tolerated by a recipient patient. Such an
agent is said to be administered in a "therapeutically effective
amount" if the amount administered is physiologically significant.
An agent is physiologically significant if its presence results in
a detectable change in the physiology of a recipient patient.
[0226] The agents of the present invention can be formulated
according to known methods to prepare pharmaceutically useful
compositions, whereby these materials, or their functional
derivatives, are combined in admixture with a pharmaceutically
acceptable carrier vehicle. Suitable vehicles and their
formulation, inclusive of other human proteins, e.g., human serum
albumin, are described, for example, in REMINGTON'S PHARMACEUTICAL
SCIENCES, 16.sup.th Ed., Osol, A., Ed., Mack Publishing, Easton Pa.
(1980). In order to form a pharmaceutically acceptable composition
suitable for effective administration, such compositions will
contain an effective amount of one or more of the agents of the
present invention, together with a suitable amount of carrier
vehicle.
[0227] Additional pharmaceutical methods may be employed to control
the duration of action. Control release preparations may be
achieved through the use of polymers to complex or absorb one or
more of the agents of the present invention. The controlled
delivery may be effectuated by a variety of well known techniques,
including formulation with macromolecules such as, for example,
polyesters, polyamino acids, polyvinyl, pyrrolidone,
ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or
protamine, sulfate, adjusting the concentration of the
macromolecules and the agent in the formulation, and by appropriate
use of methods of incorporation, which can be manipulated to
effectuate a desired time course of release. Another possible
method to control the duration of action by controlled release
preparations is to incorporate agents of the present invention into
particles of a polymeric material such as polyesters, polyamino
acids, hydrogels, poly(lactic acid) or ethylene vinylacetate
copolymers. Alternatively, instead of incorporating these agents
into polymeric particles, it is possible to entrap these materials
in microcapsules prepared, for example, by coacervation techniques
or by interfacial polymerization with, for example,
hydroxymethylcellulose or gelatine-microcapsules and
poly(methylmethacylate) microcapsules, respectively, or in
colloidal drug delivery systems, for example, liposomes, albumin
microspheres, microemulsions, nanoparticles, and nanocapsules or in
macroemulsions. Such techniques are disclosed in REMINGTON'S
PHARMACEUTICAL SCIENCES (1980).
[0228] The invention further provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Associated with such container(s) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceuticals or biological products, which notice
reflects approval by the agency of manufacture, use or sale for
human administration.
[0229] In addition, the agents of the present invention may be
employed in conjunction with other therapeutic compounds.
[0230] 6. Shot-Gun Approach to Megabase DNA Sequencing
[0231] The present invention further demonstrates that a large
sequence can be sequenced using a random shotgun approach. This
procedure, described in detail in the examples that follow, has
eliminated the up front cost of isolating and ordering overlapping
or contiguous subclones prior to the start of the sequencing
protocols.
[0232] Certain aspects of the present invention are described in
greater detail in the examples that follow. The examples are
provided by way of illustration. Other aspects and embodiments of
the present invention are contemplated by the inventors, as will be
clear to those of skill in the art from reading the present
disclosure.
ILLUSTRATIVE EXAMPLES
[0233] Libraries and Sequencing
[0234] 1. Shotgun Sequencing Probability Analysis
[0235] The overall strategy for a shotgun approach to whole genome
sequencing follows from the Lander and Waterman (Landerman and
Waterman, Genomics 2: 231 (1988)) application of the equation for
the Poisson distribution. According to this treatment, the
probability, P.sub.0, that any given base in a sequence of size L,
in nucleotides, is not sequenced after a certain amount, n, in
nucleotides, of random sequence has been determined can be
calculated by the equation P.sub.0=e.sup.-m, where m is L/n, the
fold coverage." For instance, for a genome of 2.8 Mb, m=1 when 2.8
Mb of sequence has been randomly generated (1.times. coverage). At
that point, P.sub.0=e.sup.-1=0.37. The probability that any given
base has not been sequenced is the same as the probability that any
region of the whole sequence L has not been determined and,
therefore, is equivilent to the fraction of the whole sequence that
has yet to be determined. Thus, at one-fold coverage, approximately
37% of a polynucleotide of size L, in nucleotides has not been
sequenced. When 14 Mb of sequence has been generated, coverage is
5.times.for a .2.8 Mb and the unsequenced fraction drops to 0.0067
or 0.67%. 5.times.coverage of a 2.8 Mb sequence can be attained by
sequencing approximately 17,000 random clones from both insert ends
with an average sequence read length of 410 bp.
[0236] Similarly, the total gap length, G, is determined by the
equation G=Le.sup.-m, and the average gap size, g, follows the
equation, g=L/n. Thus, 5.times.coverage leaves about 240 gaps
averaging about 82 bp in size in a sequence of a polynucleotide 2.8
Mb long.
[0237] The treatment above is essentially that of Lander and
Waterman, Genomics 2: 231 (1988).
[0238] Random Library Construction
[0239] In order to approximate the random model described above
during actual sequencing, a nearly ideal library of cloned genomic
fragments is required. The following library construction procedure
was developed to achieve this end.
[0240] Staphylococcus aureus DNA was prepared by phenol extraction.
A mixture containing 600 ug DNA in 3.3 ml of 300 mM sodium acetate,
10 mM Tris-HCl, 1 mM Na-EDTA, 30% glycerol was sonicated for 1 min.
at 0.degree. C. in a Branson Model 450 Sonicator at the lowest
energy setting using a 3 mm probe. The sonicated DNA was ethanol
precipitated and redissolved in 500 ul TE buffer.
[0241] To create blunt-ends, a 100 ul aliquot of the resuspended
DNA was digested with 5 units of BAL31 nuclease (New England
BioLabs) for 10 min at 30.degree. C. in 200 ul BAL31 buffer. The
digested DNA was phenol-extracted, ethanol-precipitated,
redissolved in 100 ul TE buffer, and then size-fractionated by
electrophoresis through a 1.0% low melting temperature agarose gel.
The section containing DNA fragments 1.6-2.0 kb in size was excised
from the gel, and the LGT agarose was melted and the resulting
solution was extracted with phenol to separate the agarose from the
DNA. DNA was ethanol precipitated and redissolved in 20 ul of TE
buffer for ligation to vector.
[0242] A two-step ligation procedure was used to produce a plasmid
library with 97% inserts, of which >99% were single inserts. The
first ligation mixture (50 ul) contained 2 ug of DNA fragments, 2
ug pUC 18 DNA (Pharmacia) cut with SmaI and dephosphorylated with
bacterial alkaline phosphatase, and 10 units of T4 ligase
(GIBCO/BRL) and was incubated at 14.degree. C. for 4 hr. The
ligation mixture then was phenol extracted and ethanol
precipitated, and the precipitated DNA was dissolved in 20 ul TE
buffer and electrophoresed on a 1.0% low melting agarose gel.
Discrete bands in a ladder were visualized by ethidium
bromide-staining and UV illumination and identified by size as
insert (i), vector (v), v+i, v+2i, v+3i, etc. The portion of the
gel containing v+i DNA was excised and the v+i DNA was recovered
and resuspended into 20 ul TE. The v+i DNA then was blunt-ended by
T4 polymerase treatment for 5 min. at 37.degree. C. in a reaction
mixture (50 ul) containing the v+i linears, 500 uM each of the 4
dNTPs, and 9 units of T4 polymerase (New England BioLabs), under
recommended buffer conditions. After phenol extraction and ethanol
precipitation the repaired v+i linears were dissolved in 20 ul TE.
The final ligation to produce circles was carried out in a 50 ul
reaction containing 5 ul of v+i linears and 5 units of T4 ligase at
14.degree. C. overnight. After 10 min. at 70.degree. C. the
following day, the reaction mixture was stored at -20.degree.
C.
[0243] This two-stage procedure resulted in a molecularly random
collection of single-insert plasmid recombinants with minimal
contamination from double-insert chimeras (<1%) or free vector
(<3%).
[0244] Since deviation from randomness can arise from propagation
the DNA in the host, E. coli host cells deficient in all
recombination and restriction functions (A. Greener, Strategies 3
(1):5 (1990)) were used to prevent rearrangements, deletions, and
loss of clones by restriction. Furthermore, transformed cells were
plated directly on antibiotic diffusion plates to avoid the usual
broth recovery phase which allows multiplication and selection of
the most rapidly growing cells.
[0245] Plating was carried out as follows. A 100 ul aliquot of
Epicurian Coli SURE II Supercompetent Cells (Stratagene 200152) was
thawed on ice and transferred to a chilled Falcon 2059 tube on ice.
A 1.7 ul aliquot of 1.42 M beta-mercaptoethanol was added to the
aliquot of cells to a final concentration of 25 mM. Cells were
incubated on ice for 10 min. A 1 ul aliquot of the final ligation
was added to the cells and incubated on ice for 30 min. The cells
were heat pulsed for 30 sec. at 42.degree. C. and placed back on
ice for 2 min. The outgrowth period in liquid culture was
eliminated from this protocol in order to minimize the preferential
growth of any given transformed cell. Instead the transformation
mixture was plated directly on a nutrient rich SOB plate containing
a 5 ml bottom layer of SOB agar (5% SOB agar: 20 g tryptone, 5 g
yeast extract, 0.5 g NaCl, 1.5% Difco Agar per liter of media). The
5 ml bottom layer is supplemented with 0.4 ml of 50 mg/ml
ampicillin per 100 ml SOB agar. The 15 ml top layer of SOB agar is
supplemented with 1 ml X-Gal (2%), 1 ml MgCl.sub.2 (1 M), and 1 ml
MgSO.sub.4/100 ml SOB agar. The 15 ml top layer was poured just
prior to plating. Our titer was approximately 100 colonies/10 ul
aliquot of transformation.
[0246] All colonies were picked for template preparation regardless
of size. Thus, only clones lost due to "poison" DNA or deleterious
gene products would be deleted from the library, resulting in a
slight increase in gap number over that expected.
[0247] Random DNA Sequencing
[0248] High quality double stranded DNA plasmid templates were
prepared using an alkaline lysis method developed in collaboration
with 5Prime---->3Prime Inc. (Boulder, Colo.). Plasmid
preparation was performed in a 96-well format for all stages of DNA
preparation from bacterial growth through final DNA purification.
Average template concentration was determined by running 25% of the
samples on an agarose gel. DNA concentrations were not
adjusted.
[0249] Templates were also prepared from a Staphylococcus aureus
lambda genomic library. An unamplified library was constructed in
Lambda DASH II vector (Stratagene). Staphylococcus aureus DNA
(>100 kb) was partially digested in a reaction mixture (200 ul)
containing 50 ug DNA, 1.times.Sau3AI buffer, 20 units Sau3AI for 6
min. at 23 C. The digested DNA was phenol-extracted and centrifuges
over a 10-40% sucroce gradient. Fractions containing genomic DNA of
15-25 kb were recovered by precipitation. One ul of fragments was
used with 1 ul of DASHII vector (Stratagene) in the recommended
ligation reaction. One ul of the ligation mixture was used per
packaging reaction following the recommended protocol with the
Gigapack II XL Packaging Extract Phage were plated directly without
amplification from the packaging mixture (after dilution with 500
ul of recommended SM buffer and chloroform treatment). Yield was
about 2.5.times.10.sup.9 pfu/ul.
[0250] An amplified library was prepared from the primary packaging
mixture according to the manufactureer's protocol. The amplified
library is stored frozen in 7% dimethylsulfoxide. The phage titer
is approximately 1.times.10.sup.9 pfu/ml.
[0251] Mini-liquid lysates (0.1 ul) are prepared from randomly
selected plaques and template is prepared by long range PCR.
Samples are PCR amplified using modified T3 and T7 primers, and
Elongase Supermix (LTI).
[0252] Sequencing reactions are carried out on plasmid templates
using a combination of two workstations (BIOMEK 1000 and Hamilton
Microlab 2200) and the Perkin-Elmer 9600 thermocycler with Applied
Biosystems PRISM Ready Reaction Dye Primer Cycle Sequencing Kits
for the M13 forward (M13-21) and the M13 reverse (M13RP1) primers.
Dye terminator sequencing reactions are carried out on the lambda
templates on a Perkin-Elmer 9600 Thermocycler using the Applied
Biosystems Ready Reaction Dye Terminator Cycle Sequencing kits.
Modified T7 and T3 primers are used to sequence the ends of the
inserts from the Lambda DASH II library. Sequencing reactions are
on a combination of AB 373 DNA Sequencers and ABI 377 DNA
sequencers. All of the dye terminator sequencing reactions are
analyzed using the 2.times. 9 hour module on the AB 377. Dye primer
reactions are analyzed on a combination of ABI 373 and ABI 377 DNA
sequencers. The overall sequencing success rate very approximately
is about 85% for M13-21 and M13RP1 sequences and 65% for
dye-terminator reactions. The average usable read length is 485 bp
for M13-21 sequences, 445 bp for M13RP 1 sequences, and 375 bp for
dye-terminator reactions.
[0253] Protocol for Automated Cycle Sequencing
[0254] The sequencing was carried out using Hamilton Microstation
2200, Perkin Elmer 9600 thermocyclers, ABI 373 and ABI 377
Automated DNA Sequencers. The Hamilton combines pre-aliquoted
templates and reaction mixes consisting of deoxy- and
dideoxynucleotides, the thermostable Taq DNA polymerase,
fluorescently-labelled sequencing primers, and reaction buffer.
Reaction mixes and templates were combined in the wells of a
96-well thermocycling plate and transferred to the Perkin Elmer
9600 thermocycler. Thirty consecutive cycles of linear
amplification (i.e., one primer synthesis) steps were performed
including denaturation, annealing of primer and template, and
extension; i.e., DNA synthesis. A heated lid with rubber gaskets on
the thermocycling plate prevents evaporation without the need for
an oil overlay.
[0255] Two sequencing protocols were used: one for dye-labelled
primers and a second for dye-labelled dideoxy chain terminators.
The shotgun sequencing involves use of four dye-labelled sequencing
primers, one for each of the four terminator nucleotide. Each
dye-primer was labelled with a different fluorescent dye,
permitting the four individual reactions to be combined into one
lane of the 373 or 377 DNA Sequencer for electrophoresis,
detection, and base-calling. ABI currently supplies pre-mixed
reaction mixes in bulk packages containing all the necessary
non-template reagents for sequencing. Sequencing can be done with
both plasmid and PCR-generated templates with both dye-primers and
dye-terminators with approximately equal fidelity, although plasmid
templates generally give longer usable sequences.
[0256] Thirty-two reactions were loaded per ABI 373 Sequencer each
day and 96 samples can be loaded on an ABI 377 per day.
Electrophoresis was run overnight (ABI 373) or for 21/2 hours (ABI
377) following the manufacturer's protocols. Following
electrophoresis and fluorescence detection, the ABI 373 or ABI 377
performs automatic lane tracking and base-calling. The
lane-tracking was confirmed visually. Each sequence
electropherogram (or fluorescence lane trace) was inspected
visually and assessed for quality. Trailing sequences of low
quality were removed and the sequence itself was loaded via
software to a Sybase database (archived daily to 8 mm tape).
Leading vector polylinker sequence was removed automatically by a
software program. Average edited lengths of sequences from the
standard ABI 373 or ABI 377 were around 400 bp and depend mostly on
the quality of the template used for the sequencing reaction.
[0257] Informatics
[0258] 1. Data Management
[0259] A number of information management systems for a large-scale
sequencing lab have been developed. (For review see, for instance,
Kerlavage et al., Proceedings of the Twenty-Sixth Annual Hawaii
International Conference on System Sciences, IEEE Computer Society
Press, Washington D.C., 585 (1993)) The system used to collect and
assemble the sequence data was developed using the Sybase
relational database management system and was designed to automate
data flow whereever possible and to reduce user error. The database
stores and correlates all information collected during the entire
operation from template preparation to final analysis of the
genome. Because the raw output of the ABI 373 Sequencers was based
on a Macintosh platform and the data management system chosen was
based on a Unix platform, it was necessary to design and implement
a variety of multi-user, client-server applications which allow the
raw data as well as analysis results to flow seamlessly into the
database with a minimum of user effort.
[0260] Assembly
[0261] An assembly engine (TIGR Assembler) developed for the rapid
and accurate assembly of thousands of sequence fragments was
enployed to generate contigs. The TIGR assembler simultaneously
clusters and assembles fragments of the genome. In order to obtain
the speed necessary to assemble more than 10.sup.4 fragments, the
algorithm builds a hash table of 12 bp oligonucleotide subsequences
to generate a list of potential sequence fragment overlaps. The
number of potential overlaps for each fragment determines which
fragments are likely to fall into repetitive elements. Beginning
with a single seed sequence fragment, TIGR Assembler extends the
current contig by attempting to add the best matching fragment
based on oligonucleotide content. The contig and candidate fragment
are aligned using a modified version of the Smith-Waterman
algorithm which provides for optimal gapped alignments (Waterman,
M. S., Methods in Enzymology 164: 765 (1988)). The contig is
extended by the fragment only if strict criteria for the quality of
the match are met. The match criteria include the minimum length of
overlap, the maximum length of an unmatched end, and the minimum
percentage match. These criteria are automatically lowered by the
algorithm in regions of minimal coverage and raised in regions with
a possible repetitive element. The number of potential overlaps for
each fragment determines which fragments are likely to fall into
repetitive elements. Fragments representing the boundaries of
repetitive elements and potentially chimeric fragments are often
rejected based on partial mismatches at the ends of alignments and
excluded from the current contig. TIGR Assembler is designed to
take advantage of clone size information coupled with sequencing
from both ends of each template. It enforces the constraint that
sequence fragments from two ends of the same template point toward
one another in the contig and are located within a certain ranged
of base pairs (definable for each clone based on the known clone
size range for a given library).
[0262] Identifying Genes
[0263] The predicted coding regions of the Staphylococcus aureus
genome were initially defined with the program zorf, which finds
ORFs of a minimum length. The predicted coding region sequences
were used in searches against a database of all Staphylococcus
aureus nucleotide sequences from GenBank (release 92.0), using the
BLASTN search method to identify overlaps of 50 or more nucleotides
with at least a 95% identity. Those ORFs with nucleotide sequence
matches are shown in Table 1. The ORFs without such matches were
translated to protein sequences and and compared to a non-redundant
database of known proteins generated by combining the Swiss-prot,
PIR and GenPept databases. ORFs of at least 80 amino acids that
matched a database protein with BLASTP probability less than or
equal to 0.01 are shown in Table 2. The table also lists assigned
functions based on the closest match in the databases. ORFs of at
least 120 amino acids that did not match protein or nucleotide
sequences in the databases at these levels are shown in Table
3.
[0264] Illustrative Applications
[0265] 1. Production of an Antibody to a Staphylococcus aureus
Protein
[0266] Substantially pure protein or polypeptide is isolated from
the transfected or transformed cells using any one of the methods
known in the art. The protein can also be produced in a recombinant
prokaryotic expression system, such as E. coli, or can by
chemically synthesized. Concentration of protein in the final
preparation is adjusted, for example, by concentration on an Amicon
filter device, to the level of a few micrograms/ml. Monoclonal or
polyclonal antibody to the protein can then be prepared as
follows.
[0267] Monoclonal Antibody Production by Hybridoma Fusion
[0268] Monoclonal antibody to epitopes of any of the peptides
identified and isolated as described can be prepared from murine
hybridomas according to the classical method of Kohler, G. and
Milstein, C., Nature 256:495 (1975) or modifications of the methods
thereof. Briefly, a mouse is repetitively inoculated with a few
micrograms of the selected protein over a period of a few weeks.
The mouse is then sacrificed, and the antibody producing cells of
the spleen isolated. The spleen cells are fused by means of
polyethylene glycol with mouse myeloma cells, and the excess
unfused cells destroyed by growth of the system on selective media
comprising aminopterin (HAT media). The successfully fused cells
are diluted and aliquots of the dilution placed in wells of a
microtiter plate where growth of the culture is continued.
Antibody-producing clones are identified by detection of antibody
in the supernatant fluid of the wells by immunoassay procedures,
such as ELISA, as originally described by Engvall, E., Meth.
Enzymol. 70:419 (1980), and modified methods thereof. Selected
positive clones can be expanded and their monoclonal antibody
product harvested for use. Detailed procedures for monoclonal
antibody production are described in Davis, L. et al. Basic Methods
in Molecular Biology Elsevier, N.Y. Section 21-2 (1989).
[0269] Polyclonal Antibody Production by Immunization
[0270] Polyclonal antiserum containing antibodies to heterogenous
epitopes of a single protein can be prepared by immunizing suitable
animals with the expressed protein described above, which can be
unmodified or modified to enhance immunogenicity. Effective
polyclonal antibody production is affected by many factors related
both to the antigen and the host species. For example, small
molecules tend to be less immunogenic than other and may require
the use of carriers and adjuvant. Also, host animals vary in
response to site of inoculations and dose, with both inadequate or
excessive doses of antigen resulting in low titer antisera. Small
doses (ng level) of antigen administered at multiple intradermal
sites appears to be most reliable. An effective immunization
protocol for rabbits can be found in Vaitukaitis, J. et al., J.
Clin. Endocrinol. Metab. 33:988-991 (1971).
[0271] Booster injections can be given at regular intervals, and
antiserum harvested when antibody titer thereof, as determined
semi-quantitatively, for example, by double immunodiffusion in agar
against known concentrations of the antigen, begins to fall. See,
for example, Ouchterlony, O. et al., Chap. 19 in: Handbook of
Experimental Immunology, Wier, D., ed, Blackwell (1973). Plateau
concentration of antibody is usually in the range of 0.1 to 0.2
mg/ml of serum (about 12M). Affinity of the antisera for the
antigen is determined by preparing competitive binding curves, as
described, for example, by Fisher, D., Chap. 42 in: Manual of
Clinical Immunology, second edition, Rose and Friedman, eds., Amer.
Soc. For Microbiology, Washington, D.C. (1980).
[0272] Antibody preparations prepared according to either protocol
are useful in quantitative immunoassays which determine
concentrations of antigen-bearing substances in biological samples;
they are also used semi-quantitatively or qualitatively to identify
the presence of antigen in a biological sample. In addition, they
are useful in various animal models of Staphylococcal disease known
to those of skill in the art as a means of evaluating the protein
used to make the antibody as a potential vaccine target or as a
means of evaluating the antibody as a potential immunothereapeutic
reagent.
[0273] Preparation of PCR Primers and Amplification of DNA
[0274] Various fragments of the Staphylococcus aureus genome, such
as those of Tables 1-3 and SEQ ID NOS:1-5,191 can be used, in
accordance with the present invention, to prepare PCR primers for a
variety of uses. The PCR primers are preferably at least 15 bases,
and more preferably at least 18 bases in length. When selecting a
primer sequence, it is preferred that the primer pairs have
approximately the same G/C ratio, so that melting temperatures are
approximately the same. The PCR primers and amplified DNA of this
Example find use in the Examples that follow.
[0275] Gene Expression from DNA Sequences Corresponding to ORFs
[0276] A fragment of the Staphylococcus aureus genome provided in
Tables 1-3 is introduced into an expression vector using
conventional technology. Techniques to transfer cloned sequences
into expression vectors that direct protein translation in
mammalian, yeast, insect or bacterial expression systems are well
known in the art. Commercially available vectors and expression
systems are available from a variety of suppliers including
Stratagene (La Jolla, Calif.), Promega (Madison, Wis.), and
Invitrogen (San Diego, Calif.). If desired, to enhance expression
and facilitate proper protein folding, the codon context and codon
pairing of the sequence may be optimized for the particular
expression organism, as explained by Hatfield et al., U.S. Pat. No.
5,082,767, incorporated herein by this reference.
[0277] The following is provided as one exemplary method to
generate polypeptide(s) from cloned ORFs of the Staphylococcus
aureus genome fragment. Bacterial ORFs generally lack a poly A
addition signal. The addition signal sequence can be added to the
construct by, for example, splicing out the poly A addition
sequence from pSG5 (Stratagene) using BglI and SalI restriction
endonuclease enzymes and incorporating it into the mammalian
expression vector pXT1 (Stratagene) for use in eukaryotic
expression systems. pXT1 contains the LTRs and a portion of the gag
gene of Moloney Murine Leukemia Virus. The positions of the LTRs in
the construct allow efficient stable transfection. The vector
includes the Herpes Simplex thymidine kinase promoter and the
selectable neomycin gene. The Staphylococcus aureus DNA is obtained
by PCR from the bacterial vector using oligonucleotide primers
complementary to the Staphylococcus aureus DNA and containing
restriction endonuclease sequences for PstI incorporated into the
5' primer and BglII at the 5' end of the corresponding
Staphylococcus aureus DNA 3' primer, taking care to ensure that the
Staphylococcus aureus DNA is positioned such that its followed with
the poly A addition sequence. The purified fragment obtained from
the resulting PCR reaction is digested with PstI, blunt ended with
an exonuclease, digested with BglII, purified and ligated to pXT1,
now containing a poly A addition sequence and digested BglII.
[0278] The ligated product is transfected into mouse NIH 3T3 cells
using Lipofectin (Life Technologies, Inc., Grand Island, N.Y.)
under conditions outlined in the product specification. Positive
transfectants are selected after growing the transfected cells in
600 ug/ml G418 (Sigma, St. Louis, Mo.). The protein is preferably
released into the supernatant. However if the protein has membrane
binding domains, the protein may additionally be retained within
the cell or expression may be restricted to the cell surface. Since
it may be necessary to purify and locate the transfected product,
synthetic 15-mer peptides synthesized from the predicted
Staphylococcus aureus DNA sequence are injected into mice to
generate antibody to the polypeptide encoded by the Staphylococcus
aureus DNA.
[0279] Alternativly and if antibody production is not possible, the
Staphylococcus aureus DNA sequence is additionally incorporated
into eukaryotic expression vectors and expressed as, for example, a
globin fusion. Antibody to the globin moiety then is used to purify
the chimeric protein. Corresponding protease cleavage sites are
engineered between the globin moiety and the polypeptide encoded by
the Staphylococcus aureus DNA so that the latter may be freed from
the formed by simple protease digestion. One useful expression
vector for generating globin chimerics is pSG5 (Stratagene). This
vector encodes a rabbit globin. Intron II of the rabbit globin gene
facilitates splicing of the expressed transcript, and the
polyadenylation signal incorporated into the construct increases
the level of expression. These techniques are well known to those
skilled in the art of molecular biology. Standard methods are
published in methods texts such as Davis et al., cited elsewhere
herein, and many of the methods are available from the technical
assistance representatives from Stratagene, Life Technologies,
Inc., or Promega. Polypeptides of the invention also may be
produced using in vitro translation systems such as in vitro
Express.TM. Translation Kit (Stratagene).
[0280] While the present invention has been described in some
detail for purposes of clarity and understanding, one skilled in
the art will appreciate that various changes in form and detail can
be made without departing from the true scope of the invention. All
patents, patent applications and publications referred to above are
hereby incorporated by reference. TABLE-US-00001 LENGTHY TABLE
REFERENCED HERE US20070020746A1-20070125-T00001 Please refer to the
end of the specification for access instructions.
TABLE-US-00002 LENGTHY TABLE REFERENCED HERE
US20070020746A1-20070125-T00002 Please refer to the end of the
specification for access instructions.
TABLE-US-00003 LENGTHY TABLE REFERENCED HERE
US20070020746A1-20070125-T00003 Please refer to the end of the
specification for access instructions.
TABLE-US-00004 LENGTHY TABLE REFERENCED HERE
US20070020746A1-20070125-T00004 Please refer to the end of the
specification for access instructions.
TABLE-US-00005 LENGTHY TABLE The patent application contains a
lengthy table section. A copy of the table is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070020746A1)
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070020746A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070020746A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References