Polynucleotides encoding a transcriptional response regulator of Streptococcus pneumoniae

The invention provides Response Regulator polypeptides and DNA (RNA) encoding Response Regulator polypetides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing Response Regulator polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS 
This application claims benefit to GB application No. 9609020.4, filed May 
1, 1996. 
FIELD OF THE INVENTION 
This invention relates to newly identified polynucleotides and 
polypeptides, and their production and uses, as well as their variants, 
agonists and antagonists, and their uses. In particular, in these and in 
other regards, the invention relates to novel polynucleotides and 
polypeptides of the response regulator family, hereinafter referred to as 
"Response Regulator". 
BACKGROUND OF THE INVENTION 
The Streptococci make up a medically important genera of microbes known to 
cause several types of disease in humans, including, for example, otitis 
media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, 
pleural empyema and endocarditis, and most particularly meningitis, such 
as for example infection of cerebrospinal fluid. Since its isolation more 
than 100 years ago, Streptococcus pneumoniae has been one of the more 
intensively studied microbes. For example, much of our early understanding 
that DNA is, in fact, the genetic material was predicated on the work of 
Griffith and of Avery, Macleod and McCarty using this microbe. Despite the 
vast amount of research with S. pneumoniae, many questions concerning the 
virulence of this microbe remain. It is particularly preferred to employ 
Streptococcal genes and gene products as targets for the development of 
antibiotics. 
The frequency of Streptococcus pneumoniae infections has risen dramatically 
in the past 20 years. This has been attributed to the emergence of 
multiply antibiotic resistant strains and an increasing population of 
people with weakened immune systems. It is no longer uncommon to isolate 
Streptococcus pneumoniae strains which are resistant to some or all of the 
standard antibiotics. This has created a demand for both new 
anti-microbial agents and diagnostic tests for this organism. 
While certain Streptococcal factors associated with pathogenicity have been 
identified, e.g., capsule polysaccharides, peptidoglycans, pneumolysins, 
PspA Complement factor H binding component, autolysin, neuraminidase, 
peptide permeases, hydrogen peroxide, IgAl protease, the list is certainly 
not complete. Further very little is known concerning the temporal 
expression of such genes during infection and disease progression in a 
mammalian host. Discovering the sets of genes the bacterium is likely to 
be expressing at the different stages of infection, particularly when an 
infection is established, provides critical information for the screening 
and characterization of novel antibacterials which can interrupt 
pathogenesis. In addition to providing a fuller understanding of known 
proteins, such an approach will identify previously unrecognised targets. 
Many two component signal transduction systems (TCSTS) have been identified 
in bacteria (Stock, J. B., Ninfa, A. J. & Stock, A. M.(1989) Microbiol. 
Rev. 53, 450-490). These are involved in the bacterium's ability to 
monitor its surroundings and adapt to changes in its environment. Several 
of these bacterial TCSTS are involved in virulence and bacterial 
pathogenesis within the host. 
Response regulators are components of the TCSTS. These proteins are 
phosphorylated by histidine kinases and in turn once phosphorylated effect 
the response, often through a DNA binding domain becoming activated. The 
response regulators are characterized by a conserved N-termiinal domain of 
approximately 100 amino acids. The N-terminal domains of response 
regulators as well as retaining five functionally important residues, 
corresponding to the residues D12, D13, D57, T87, K109 in CheY (Matsumura, 
P., Rydel, J. J., Linzmeier, R. & Vacante, D. (1984) J. Bacteriol. 160, 
36-41), have conserved structural features (Volz, K. (1993) Biochemistry 
32, 11741-11753). The 3-dimensional structures of CheY from Salmonella 
typhimurium (Stock, A. M., Mottonen, J. M., Stock, J. B. & Schutt, ,C. E. 
(1989) Nature, 337, 745-749) and Escherichia coli (Volz, K. & Matsumura, 
P. (1991) J. Biol. Chem. 266, 15511-15519) and the N-terminal domain of 
nitrogen regulatory protein C from S.typhimurium (Volkman, B. F., Nohaile, 
M. J., Amy, N. K., Kustu, S. & Wemmer, D. E. (1995) Biochemistry, 34 
1413-1424), are available, as well as the secondary structure of SpoOF 
from Bacillus subtilis (Feher, V. A., Zapf, J. W., Hoch, J. A., Dahlquist, 
F. W., Whiteley, J. M. & Cavanagh, J. (1995) Protein Science, 4, 
1801-1814). These structures have a (a/b)5 fold. Several structural 
residues are conserved between different response regulator sequences, 
specifically hydrophobic residues within the .beta.-sheet hydrophobic core 
and sites from the a-helices. This family of response regulators includes 
DegU from Bacillus subtilis which is part of the TCSTS involved in the 
production of extracellular proteases (Henner, D. J., Yang, M. & Ferrari, 
E. (1988) J. Bacteriol. 170,5102-5109). 
Histidine kinases are components of the TCSTS which autophosphorylate a 
histidine residue. The phosphate group is then transferred to the cognate 
response regulator. The Histidine kinases have five short conserved amino 
acid sequences (Stock, J. B., Ninfa, A. J. & Stock, A.M.(1989) Microbiol. 
Rev. 53, 450-490, Swanson, R. V., Alex, L. A. & Simon, M. I.(1994) TIBS 19 
485-491). These are the histidine residue, which is phosphorylated, 
followed after approximately 100 residues by a conserved asparagine 
residue. After another 15 to 45 residues a DXGXG motif is found, followed 
by a FXXF motif after another 10-20 residues. 10-20 residues further on 
another glycine motif, GXG is found. The two glycine motifs are thought to 
be involved in nucleotide binding. 
Among the processes regulated by TCSTS are production of virulence factors, 
motility, antibiotic resistance and cell replication. Inhibitors of TCSTS 
proteins would prevent the bacterium from establishing and maintaining 
infection of the host by preventing it from producing the necessary 
factors for pathogenesis and thereby have utility in anti-bacterial 
therapy. 
Clearly, there is a need for factors, such as the novel compounds of the 
invention, that have a present benefit of being useful to screen compounds 
for antibiotic activity. Such factors are also useful to determine their 
role in pathogenesis of infection, dysfunction and disease. There is also 
a need for identification and characterization of such factors and their 
antagonists and agonists which can play a role in preventing, ameliorating 
or correcting infections, dysfunctions or diseases. 
The polypeptides of the invention have amino acid sequence homology to a 
known Bacillus subtilis DegU protein. 
SUMMARY OF THE INVENTION 
It is an object of the invention to provide polypeptides that have been 
identified as novel Response Regulator polypeptides by homology between 
the amino acid sequence set out in Table 1 SEQ ID NO: 2! and a known 
amino acid sequence or sequences of other proteins such as Bacillus 
subtilis DegU protein. 
It is a further object of the invention to provide polynucleotides that 
encode Response Regulator polypeptides, particularly polynucleotides that 
encode the polypeptide herein designated Response Regulator. 
In a particularly preferred embodiment of the invention the polynucleotide 
comprises a region encoding Response Regulator polypeptides comprising the 
sequence set out in Table 1 SEQ ID NO: 1! which includes a full length 
gene, or a variant thereof. 
In another particularly preferred embodiment of the invention there is a 
novel Response Regulator protein from Streptococcus pneumoniae comprising 
the amino acid sequence of Table 1 SEQ ID NO:2!, or a variant thereof. 
In accordance with another aspect of the invention there is provided an 
isolated nucleic acid molecule encoding a mature polypeptide expressible 
by the Streptococcus pneumoniae 0100993 strain contained in the deposited 
strain. 
A further aspect of the invention there are provided isolated nucleic acid 
molecules encoding Response Regulator, particularly Streptococcus 
pneumoniae Response Regulator, including mRNAs, cDNAs, genomic DNAs. 
Further embodiments of the invention include biologically, diagnostically, 
prophylactically, clinically or therapeutically useful variants thereof, 
and compositions comprising the same. 
In accordance with another aspect of the invention, there is provided the 
use of a polynucleotide of the invention for therapeutic or prophylactic 
purposes, in particular genetic immunization. Among the particularly 
preferred embodiments of the invention are naturally occurring allelic 
variants of Response Regulator and polypeptides encoded thereby. 
Another aspect of the invention there are provided novel polypeptides of 
Streptococcus pneumoniae referred to herein as Response Regulator as well 
as biologically, diagnostically, prophylactically, clinically or 
therapeutically useful variants thereof, and compositions comprising the 
same. 
Among the particularly preferred embodiments of the invention are variants 
of Response Regulator polypeptide encoded by naturally occurring alleles 
of the Response Regulator gene. 
In a preferred embodiment of the invention there are provided methods for 
producing the aforementioned Response Regulator polypeptides. 
In accordance with yet another aspect of the invention, there are provided 
inhibitors to such polypeptides, useful as antibacterial agents, 
including, for example, antibodies. 
In accordance with certain preferred embodiments of the invention, there 
are provided products, compositions and methods for assessing Response 
Regulator expression, treating disease, for example, otitis media, 
conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural 
empyema and endocarditis, and most particularly meningitis, such as for 
example infection of cerebrospinal fluid, assaying genetic variation, and 
administering a Response Regulator polypeptide or polynucleotide to an 
organism to raise an immunological response against a bacteria, especially 
a Streptococcus pneumoniae bacteria. 
In accordance with certain preferred embodiments of this and other aspects 
of the invention there are provided polynucleotides that hybridize to 
Response Regulator polynucleotide sequences, particularly under stringent 
conditions. 
In certain preferred embodiments of the invention there are provided 
antibodies against Response Regulator polypeptides. 
In other embodiments of the invention there are provided methods for 
identifying compounds which bind to or otherwise interact with and inhibit 
or activate an activity of a polypeptide or polynucleotide of the 
invention comprising: contacting a polypeptide or polynucleotide of the 
invention with a compound to be screened under conditions to permit 
binding to or other interaction between the compound and the polypeptide 
or polynucleotide to assess the binding to or other interaction with the 
compound, such binding or interaction being associated with a second 
component capable of providing a detectable signal in response to the 
binding or interaction of the polypeptide or polynucleotide with the 
compound; and determining whether the compound binds to or otherwise 
interacts with and activates or inhibits an activity of the polypetide or 
polynucleotide by detecting the presence or absence of a signal generated 
from the binding or interaction of the compound with the polypeptide or 
polynucleotide. 
In accordance with yet another aspect of the invention, there are provided 
Response Regulator agonists and antagonists, preferably bacteriostatic or 
bacteriocidal agonists and antagonists. 
In a further aspect of the invention there are provided compositions 
comprising a Response Regulator polynucleotide or a Response Regulator 
polypeptide for administration to a cell or to a multicellular organism. 
Various changes and modifications within the spirit and scope of the 
disclosed invention will become readily apparent to those skilled in the 
art from reading the following descriptions and from reading the other 
parts of the present disclosure. 
GLOSSARY 
The following definitions are provided to facilitate understanding of 
certain terms used frequently herein. 
"Host cell" is a cell which has been transformed or transfected, or is 
capable of transformation or transfection by an exogenous polynucleotide 
sequence. 
"Identity," as known in the art, is a relationship between two or more 
polypeptide sequences or two or more polynucleotide sequences, as 
determined by comparing the sequences. In the art, "identity" also means 
the degree of sequence relatedness between polypeptide or polynucleotide 
sequences, as the case may be, as determined by the match between strings 
of such sequences. "Identity" and "similarity" can be readily calculated 
by known methods, including but not limited to those described in 
(Computational Molecular Biology, Lesk, A. M., ed., Oxford University 
Press, New York, 1988; Biocomputing: Informatics and Genome Projects, 
Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of 
Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana 
Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von 
Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, 
M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, 
H., and Lipman, D., SLAM J. Applied Math., 48: 1073 (1988). Preferred 
methods to determine identity are designed to give the largest match 
between the sequences tested. Methods to determine identity and similarity 
are codified in publicly available computer programs. Preferred computer 
program methods to determine identity and similarity between two sequences 
include, but are not limited to, the GCG program package (Devereux, J., et 
al., Nucleic Acids Research 12(l): 387 (1984)), BLASTP, BLASTN, and FASTA 
(Atschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X 
program is publicly available from NCBI and other sources (BLAST Manual, 
Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et 
al, J. Mol. Biol. 215: 403-410 (1990). As an illustration, by a 
polynucleotide having a nucleotide sequence having at least, for example, 
95% "identity" to a reference nucleotide sequence of SEQ ID NO: 1 it is 
intended that the nucleotide sequence of the polynucleotide is identical 
to the reference sequence except that the polynucleotide sequence may 
include up to five point mutations per each 100 nucleotides of the 
reference nucleotide sequence of SEQ ID NO: 1. In other words, to obtain a 
polynucleotide having a nucleotide sequence at least 95% identical to a 
reference nucleotide sequence, up to 5% of the nucleotides in the 
reference sequence may be deleted or substituted with another nucleotide, 
or a number of nucleotides up to 5% of the total nucleotides in the 
reference sequence may be inserted into the reference sequence. These 
mutations of the reference sequence may occur at the 5' or 3' terminal 
positions of the reference nucleotide sequence or anywhere between those 
terminal positions, interspersed either individually among nucleotides in 
the reference sequence or in one or more contiguous groups within the 
reference sequence. Analogously, by a polypeptide having an amino acid 
sequence having at least, for example, 95% identity to a reference amino 
acid sequence of SEQ ID NO:2 is intended that the amino acid sequence of 
the polypeptide is identical to the reference sequence except that the 
polypeptide sequence may include up to five amino acid alterations per 
each 100 amino acids of the reference amino acid of SEQ ID NO: 2. In other 
words, to obtain a polypeptide having an amino acid sequence at least 95% 
identical to a reference amino acid sequence, up to 5% of the amino acid 
residues in the reference sequence may be deleted or substituted with 
another amino acid, or a number of amino acids up to 5% of the total amino 
acid residues in the reference sequence may be inserted into the reference 
sequence. These alterations of the reference sequence may occur at the 
amino or carboxy terminal positions of the reference amino acid sequence 
or anywhere between those terminal positions, interspersed either 
individually among residues in the reference sequence or in one or more 
contiguous groups within the reference sequence. 
"Isolated" means altered "by the hand of man" from its natural state, i.e., 
if it occurs in nature, it has been changed or removed from its original 
environment, or both. For example, a polynucleotide or a polypeptide 
naturally present in a living organism is not "isolated," but the same 
polynucleotide or polypeptide separated from the coexisting materials of 
its natural state is "isolated", as the term is employed herein. 
"Polynucleotide(s)" generally refers to any polyribonucleotide or 
polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA 
or DNA. "Polynucleotide(s)" include, without limitation, single- and 
double-stranded DNA, DNA that is a mixture of single- and double-stranded 
regions or single-, double- and triple-stranded regions, single- and 
double-stranded RNA, and RNA that is mixture of single- and 
double-stranded regions, hybrid molecules comprising DNA and RNA that may 
be single-stranded or, more typically, double-stranded, or triple-stranded 
regions, or a mixture of single- and double-stranded regions. In addition, 
"polynucleotide" as used herein refers to triple-stranded regions 
comprising RNA or DNA or both RNA and DNA. The strands in such regions may 
be from the same molecule or from different molecules. The regions may 
include all of one or more of the molecules, but more typically involve 
only a region of some of the molecules. One of the molecules of a 
triple-helical region often is an oligonucleotide. As used herein, the 
term "polynucleotide(s)" also includes DNAs or RNAs as described above 
that contain one or more modified bases. Thus, DNAs or RNAs with backbones 
modified for stability or for other reasons are "polynucleotide(s)" as 
that term is intended herein. Moreover, DNAs or RNAs comprising unusual 
bases, such as inosine, or modified bases, such as tritylated bases, to 
name just two examples, are polynucleotides as the term is used herein. It 
will be appreciated that a great variety of modifications have been made 
to DNA and RNA that serve many useful purposes known to those of skill in 
the art. The term "polynucleotide(s)" as it is employed herein embraces 
such chemically, enzymatically or metabolically modified forms of 
polynucleotides, as well as the chemical forms of DNA and RNA 
characteristic of viruses and cells, including, for example, simple and 
complex cells. "Polynucleotide(s)" also embraces short polynucleotides 
often referred to as oligonucleotide(s). 
"Polypeptide(s)" refers to any peptide or protein comprising two or more 
amino acids joined to each other by peptide bonds or modified peptide 
bonds. "Polypeptide(s)" refers to both short chains, commonly referred to 
as peptides, oligopeptides and oligomers and to longer chains generally 
referred to as proteins. Polypeptides may contain amino acids other than 
the 20 gene encoded amino acids. "Polypeptide(s)" include those modified 
either by natural processes, such as processing and other 
post-translational modifications, but also by chemical modification 
techniques. Such modifications are well described in basic texts and in 
more detailed monographs, as well as in a voluminous research literature, 
and they are well known to those of skill in the art. It will be 
appreciated that the same type of modification may be present in the same 
or varying degree at several sites in a given polypeptide. Also, a given 
polypeptide may contain many types of modifications. Modifications can 
occur anywhere in a polypeptide, including the peptide backbone, the amino 
acid side-chains, and the amino or carboxyl termini. Modifications 
include, for example, acetylation, acylation, ADP-ribosylation, amidation, 
covalent attachment of flavin, covalent attachment of a heme moiety, 
covalent attachment of a nucleotide or nucleotide derivative, covalent 
attachment of a lipid or lipid derivative, covalent attachment of 
phosphotidylinositol, cross-linking, cyclization, disulfide bond 
formation, demethylation, formation of covalent cross-links, formation of 
cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, 
glycosylation, GPI anchor formation, hydroxylation, iodination, 
methylation, myristoylation, oxidation, proteolytic processing, 
phosphorylation, prenylation, racemization, glycosylation, lipid 
attachment, sulfation, gammacarboxylation of glutamic acid residues, 
hydroxylation and ADP-ribosylation, selenoylation, sulfation, transfer-RNA 
mediated addition of amino acids to proteins, such as arginylation, and 
ubiquitination. See, for instance, PROTEINS--STRUCTURE AND MOLECULAR 
PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York 
(1993) and Wold, F., Posttranslational Protein Modifications: Perspectives 
and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF 
PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter et 
al., Meth. Enzymol. 182:626-646 (1990) and Rattan et al., Protein 
Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 
663: 48-62 (1992). Polypeptides may be branched or cyclic, with or without 
branching. Cyclic, branched and branched circular polypeptides may result 
from post-translational natural processes and may be made by entirely 
synthetic methods, as well. 
"Variant(s)" as the term is used herein, is a polynucleotide or polypeptide 
that differs from a reference polynucleotide or polypeptide respectively, 
but retains essential properties. A typical variant of a polynucleotide 
differs in nucleotide sequence from another, reference polynucleotide. 
Changes in the nucleotide sequence of the variant may or may not alter the 
amino acid sequence of a polypeptide encoded by the reference 
polynucleotide. Nucleotide changes may result in amino acid substitutions, 
additions, deletions, fusions and truncations in the polypeptide encoded 
by the reference sequence, as discussed below. A typical variant of a 
polypeptide differs in amino acid sequence from another, reference 
polypeptide. Generally, differences are limited so that the sequences of 
the reference polypeptide and the variant are closely similar overall and, 
in many regions, identical. A variant and reference polypeptide may differ 
in amino acid sequence by one or more substitutions, additions, deletions 
in any combination. A substituted or inserted amino acid residue may or 
may not be one encoded by the genetic code. A variant of a polynucleotide 
or polypeptide may be a naturally occurring such as an allelic variant, or 
it may be a variant that is not known to occur naturally. Non-naturally 
occurring variants of polynucleotides and polypeptides may be made by 
mutagenesis techniques, by direct synthesis, and by other recombinant 
methods known to skilled artisans. 
DESCRIPTION OF THE INVENTION 
The invention relates to novel Response Regulator polypeptides and 
polynucleotides as described in greater detail below. In particular, the 
invention relates to polypeptides and polynucleotides of a novel Response 
Regulator of Streptococcus pneumoniae, which is related by amino acid 
sequence homology to Bacillus subtilis DegU polypeptide. The invention 
relates especially to Response Regulator having the nucleotide and amino 
acid sequences set out in Table 1 SEQ ID NO: 1! and Table 1 SEQ ID NO: 
2! respectively, and to the Response Regulator nucleotide sequences of the 
DNA in the deposited strain and amino acid sequences encoded thereby. 
TABLE 1 
__________________________________________________________________________ 
Response Regulator Polynucleotide and Polypeptide Sequences 
__________________________________________________________________________ 
(A) Sequences from Streptococcus pneumoniae Response Regulator 
polynucleotide 
sequence SEQ ID NO:1!. 
5'-1 GCTAGCTTGA CCCAGGATGT GGAGTCGACG GCTGCTATGA TTTTGCTTGA 
51 GTTGGTGACC AATATCATCA AACATGCCAA AGCGTTTAAA GCTTACTTAA 
101 AATTAGAACG GACAGAGAAG GAACTCATTT TAACAGTAAG TGATGATGGC 
151 TGCGGCTTTG CTTTTCTAAA AGGAGATGAG CTCCATACAG TCCGAGATCG 
201 TGTTTTTCCA TTTTCAGGAG AAGTAAGTGT AATCAGTCAG AAACATCCAA 
251 CGGAAGTGCA AGTTCGACTA CCTTATAAGG AGAGAAACTA AGATGAAAGT 
301 ATTAGTCGCA GAAGATCAAA GTATGTTGCG AGATGCCATG TGTCAATTGC 
351 TCACGCTTCA ACCAGATGTG GAGTCTGTCC TTCAAACCAA GAATGGGCAA 
401 GAAGCAATCC AACTATTAGA AAAGGAGTCT GTAGATATCG CCATCCTTGA 
451 CGTAGAAATG CCTGTTAAGA CAGGTCTTGA AGTCTTGGAG TGGATACGAT 
501 CAGAAAAGCT TGAAACAAAG GTGGTTGTGG TGACGACCTT CAAGCGTGCT 
551 GGGTATTTTG AACGTGCGGT CAAGGCTGGA GTGGATGCTT ATGTATTAAA 
601 GGAACGAAGC ATTGCAGACC TCATGCAAAC CTTGCACACC GTCCTCGAAG 
651 GACGCAAGGA GTATTCGCCT GAATTGATGG AAGTGGTGAT GACCCGTCCC 
701 AATCCGTTGA CAGAACAAGA GATTGCTGTC TTAAAGGGAA TCGCCCGGGG 
751 CTTATCCAAC CAAGAAATCG CAGATCAGCT TTACCTCTCA AACGGAACTA 
801 TTCGAAACTA TGTCACCAAT ATTCTTTCAA AACTGGATGC TGGTAATCGA 
851 ACAGAGGCAG CTAATATCGC AAAAGAATCT GGTTGGTTAT GATGATATAA 
901 TATTTTCATG AAGACTATGT GCTAA-3' 
(B) Response Regulator polypeptide sequence deduced from the 
polynucleotide 
sequence in this table SEQ ID NO:2!. 
NH.sub.2 -1 
MKVLVAEDQS MLRDAMCQLL TLQPDVESVL QTKNGQEAIQ LLEKESVDIA 
51 ILDVEMPVKT GLEVLEWIRS EKLETKVVVV TTFKRAGYFE RAVKAGVDAY 
101 VLKERSIADL MQTLHTVLEG RKEYSPELME WMTRPNPLT EQEIAVLKGI 
151 ARGLSNQEIA DQLYLSNGTI RNYVTNILSK LDAGNRTEAA NIAKESGWL-COOH 
(C) Polynucleotide sequence embodiments SEQ ID NO:1!. 
X-(R.sub.1).sub.n -1 
GCTAGCTTGA CCCAGGATGT GGAGTCGACG GCTGCTATGA TTTTGCTTGA 
51 GTTGGTGACC AATATCATCA AACATGCCAA AGCGTTTAAA GCTTACTTAA 
101 AATTAGAACG GACAGAGAAG GAACTCATTT TAACAGTAAG TGATGATGGC 
151 TGCGGCTTTG CTTTTCTAAA AGGAGATGAG CTCCATACAG TCCGAGATCG 
201 TGTTTTTCCA TTTTCAGGAG AAGTAAGTGT AATCAGTCAG AAACATCCAA 
251 CGGAAGTGCA AGTTCGACTA CCTTATAAGG AGAGAAACTA AGATGAAAGT 
301 ATTAGTCGCA GAAGATCAAA GTATGTTGCG AGATGCCATG TGTCAATTGC 
351 TCACGCTTCA ACCAGATGTG GAGTCTGTCC TTCAAACCAA GAATGGGCAA 
401 GAAGCAATCC AACTATTAGA AAAGGAGTCT GTAGATATCG CCATCCTTGA 
451 CGTAGAAATG CCTGTTAAGA CAGGTCTTGA AGTCTTGGAG TGGATACGAT 
501 CAGAAAAGCT TGAAACAAAG GTGGTTGTGG TGACGACCTT CAAGCGTGCT 
551 GGGTATTTTG AACGTGCGGT CAAGGCTGGA GTGGATGCTT ATGTATTAAA 
601 GGAACGAAGC ATTGCAGACC TCATGCAAAC CTTGCACACC GTCCTCGAAG 
651 GACGCAAGGA GTATTCGCCT GAATTGATGG AAGTGGTGAT GACCCGTCCC 
701 AATCCGTTGA CAGAACAAGA GATTGCTGTC TTAAAGGGAA TCGCCCGGGG 
751 CTTATCCAAC CAAGAAATCG CAGATCAGCT TTACCTCTCA AACGGAACTA 
801 TTCGAAACTA TGTCACCAAT ATTCTTTCAA AACTGGATGC TGGTAATCGA 
851 ACAGAGGCAG CTAATATCGC AAAAGAATCT GGTTGGTTAT GATGATATAA 
901 TATTTTCATG AAGACTATGT GCTAA-(R.sub.2)n-Y 
(D) Polypeptide sequence embodiments SEQ ID NO:2!. 
X-(R.sub.1).sub.n -1 
MKVLVAEDQS MLRDAMCQLL TLQPDVESVL QTKNGQEAIQ LLEKESVDIA 
51 ILDVEMPVKT GLEVLEWIRS EKLETKWVV TTFKRAGYFE RAVKAGVDAY 
101 VLKERSIADL MQTLHTVLEG RKEYSPELME VVMTRPNPLT EQEIAVLKGI 
151 ARGLSNQEIA DQLYLSNGTI RNYNTNILSK LDAGNRTEAA NIAKESGWL-(R.sub.2).sub.n 
-Y 
(E) Sequences from Streptococcus pneumoniae 
Response Regulator polynucleotide ORF sequence SEQ ID NO:3!. 
5'-1 CTGATCTGCG ATTTCTTGGT TGGATAAGCC CCGGGCGATT CCCTTTAAGA 
51 CAGCAATCTC TTGTTCTGTC AACGGATTGG GACGGGTCAT CACCACTTCC 
101 ATCAATTCAG GCGAATACTC CTTGCGTCCT TCGAGGACGG TGTGCAAGGT 
151 TTGCATGAGG TCTGCAATGC TTCGTTCCTT TAATACATAA GCATCCACTC 
201 CAGCCTTGAC CGCACGTTCA AAATACCCAG CACGCTTGAA GGTCGTCACC 
251 ACAACCACCT TTGTTTCAAGCTTTTCTGAT CGTATCCACT CCAAGACTTC 
301 AAGACCTGTC TTAACAGGCA TTTCTACGTC AAGGATGGCG ATATCTACAG 
351 ACTCCTTTTC TAATAGTTGG ATTGCTTCTT GCCCATTCTT GGTTTGAAGG 
401 ACAGACTCCA CATCTGGTTG AAGCGTGAGC AATTGACACA TGGCATCTCG 
451 CAACATACTT TGATCTTCTG CGACTAATAC TTTCATCTTA GTTTCTCTCC 
501 TTATAAGGTA GTCGAACTTG CACTTCCGTT GGATGTTTCT GACTGATTAC 
551 ACTTACTTCT CCTGAAAATG GAAAAACACG ATCTCCGACT GTATGGA-3' 
(F) Response Regulator polypeptide sequence deduced from the 
polynucleotide ORF 
sequence in this table SEQ ID NO:4!. 
NH.sub.2 -1 
MKVLVAEDQS MLRDAMCQLL TLQPDVESVL QTKNGQEAIQ LLEKESVDIA 
51 ILDVEMPVKT GLEVLEWIRS EKLETKVVVV TTFKRAGYFE RAVKAGVDAY 
101 VLKERSIADL MQTLHTVLEG RKEYSPELME VVMTRPNPLT EQEIAVLKGI 
151 ARGLSNQEIA DQ-COOH 
__________________________________________________________________________ 
Deposited materials 
A deposit containing a Streptococcus pneumoniae 0100993 strain has been 
deposited with the National Collections of Industrial and Marine Bacteria 
Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 IRY, Scotland on 
11 Apr. 1996 and assigned deposit number 40794. The deposit was described 
as Streptococcus pneumoniae 0100993 on deposit. On 17 Apr. 1996 a 
Streptococcus pneumoniae 0100993 DNA library in E. coli was similarly 
deposited with the NCIMB and assigned deposit number 40800. The 
Streptococcus pneumoniae strain deposit is referred to herein as "the 
deposited strain" or as "the DNA of the deposited strain." 
The deposited strain contains the full length Response Regulator gene. The 
sequence of the polynucleotides contained in the deposited strain, as well 
as the amino acid sequence of the polypeptide encoded thereby, are 
controlling in the event of any conflict with any description of sequences 
herein. 
The deposit of the deposited strain has been made under the terms of the 
Budapest Treaty on the International Recognition of the Deposit of 
Micro-organisms for Purposes of Patent Procedure. The strain will be 
irrevocably and without restriction or condition released to the public 
upon the issuance of a patent. The deposited strain is provided merely as 
convenience to those of skill in the art and is not an admission that a 
deposit is required for enablement, such as that required under 35 U.S.C. 
.sctn.112. 
A license may be required to make, use or sell the deposited strain, and 
compounds derived therefrom, and no such license is hereby granted. 
Polypeptides 
The polypeptides of the invention include the polypeptide of Table 1 SEQ 
ID NO:2! (in particular the mature polypeptide) as well as polypeptides 
and fragments, particularly those which have the biological activity of 
Response Regulator, and also those which have at least 70% identity to a 
polypeptide of Table 1 SEQ ID NOS:2 and 4! or the relevant portion, 
preferably at least 80% identity to a polypeptide of Table 1 SEQ ID NOS:2 
and 4!, and more preferably at least 90% similarity (more preferably at 
least 90% identity) to a polypeptide of Table 1 SEQ ID NOS:2 and 4! and 
still more preferably at least 95% similarity (still more preferably at 
least 95% identity) to a polypeptide of Table 1 SEQ ID NOS:2 and 4! and 
also include portions of such polypeptides with such portion of the 
polypeptide generally containing at least 30 amino acids and more 
preferably at least 50 amino acids. 
The invention also includes polypeptides of the formula set forth in Table 
1 (D) SEQ ID NO:2! wherein, at the amino terminus, X is hydrogen, and at 
the carboxyl terminus, Y is hydrogen or a metal, R.sub.1 and R.sub.2 is 
any amino acid residue, and n is an integer between 1 and 1000. Any 
stretch of amino acid residues denoted by either R group, where R is 
greater than 1, may be either a heteropolymer or a homopolymer, preferably 
a heteropolymer. 
A fragment is a variant polypeptide having an amino acid sequence that 
entirely is the same as part but not all of the amino acid sequence of the 
aforementioned polypeptides. As with Response Regulator polypeptides 
fragments may be "free-standing," or comprised within a larger polypeptide 
of which they form a part or region, most preferably as a single 
continuous region, a single larger polypeptide. 
Preferred fragments include, for example, truncation polypeptides having a 
portion of an amino acid sequence of Table 1 SEQ ID NOS:2 and 4!, or of 
variants thereof, such as a continuous series of residues that includes 
the amino terminus, or a continuous series of residues that includes the 
carboxyl terminus. Degradation forms of the polypeptides of the invention 
in a host cell, particularly a Streptococcus pneumoniae, are also 
preferred. Further preferred are fragments characterized by structural or 
functional attributes such as fragments that comprise alpha-helix and 
alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, 
turn and turn-forming regions, coil and coil-forming regions, hydrophilic 
regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic 
regions, flexible regions, surface-forming regions, substrate binding 
region, and high antigenic index regions. 
Also preferred are biologically active fragments which are those fragments 
that mediate activities of Response Regulator, including those with a 
similar activity or an improved activity, or with a decreased undesirable 
activity. Also included are those fragments that are antigenic or 
immunogenic in an animal, especially in a human. Particularly preferred 
are fragments comprising receptors or domains of enzymes that confer a 
function essential for viability of Streptococcus pneumoniae or the 
ability to initiate, or maintain cause disease in an individual, 
particularly a human. 
Variants that are fragments of the polypeptides of the invention may be 
employed for producing the corresponding full-length polypeptide by 
peptide synthesis; therefore, these variants may be employed as 
intermediates for producing the full-length polypeptides of the invention. 
Polynucleotides Another aspect of the invention relates to isolated 
polynucleotides, including the full length gene, that encode the Response 
Regulator polypeptide having a deduced amino acid sequence of Table 1 SEQ 
ID NOS:2 and 4! and polynucleotides closely related thereto and variants 
thereof. 
Using the information provided herein, such as a polynucleotide sequence 
set out in Table 1 SEQ ID NOS:1 and 3!, a polynucleotide of the invention 
encoding Response Regulator polypeptide may be obtained using standard 
cloning and screening methods, such as those for cloning and sequencing 
chromosomal DNA fragments from bacteria using Streptococcus pneumoniae 
0100993 cells as starting material, followed by obtaining a full length 
clone. For example, to obtain a polynucleotide sequence of the invention, 
such as a sequence given in Table 1 SEQ ID NOS: 1 and 3!, typically a 
library of clones of chromosomal DNA of Streptococcus pneumoniae 0100993 
in E.coli or some other suitable host is probed with a radiolabeled 
oligonucleotide, preferably a 17-mer or longer, derived from a partial 
sequence. Clones carrying DNA identical to that of the probe can then be 
distinguished using stringent conditions. By sequencing the individual 
clones thus identified with sequencing primers designed from the original 
sequence it is then possible to extend the sequence in both directions to 
determine the full gene sequence. Conveniently, such sequencing is 
performed using denatured double stranded DNA prepared from a plasmid 
clone. Suitable techniques are described by Maniatis, T., Fritsch, E. F. 
and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold 
Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). (see 
in particular Screening By Hybridization 1.90 and Sequencing Denatured 
Double-Stranded DNA Templates 13.70). Illustrative of the invention, the 
polynucleotide set out in Table 1 SEQ ID NO: 1! was discovered in a DNA 
library derived from Streptococcus pneumoniae 0100993. 
The DNA sequence set out in Table 1 SEQ ID NOS: 1! contains an open 
reading frame encoding a protein having about the number of amino acid 
residues set forth in Table 1 SEQ ID NOS:2! with a deduced molecular 
weight that can be calculated using amino acid residue molecular weight 
values well known in the art. The start codon of the DNA in Table 1 is 
nucleotide number 293 and last codon that encodes an amino acid is number 
889, the stop codon being the next codon following this last codon 
encoding an amino acid (890). 
Response Regulator of the invention is structurally related to other 
proteins of the response regulator family, as shown by the results of 
sequencing the DNA encoding Response Regulator of the deposited strain. 
The protein exhibits greatest homology to Bacillus subtilis DegU protein 
among known proteins. Response Regulator of Table 1 SEQ ID NO:2! has 
about 30% identity over its entire length and about 56% similarity over 
its entire length with the amino acid sequence of Bacillus subtilis DegU 
polypeptide. 
The invention provides a polynucleotide sequence identical over its entire 
length to the coding sequence in Table 1 SEQ ID NO:1!. Also provided by 
the invention is the coding sequence for the mature polypeptide or a 
fragment thereof, by itself as well as the coding sequence for the mature 
polypeptide or a fragment in reading frame with other coding sequence, 
such as those encoding a leader or secretory sequence, a pre-, or pro- or 
prepro- protein sequence. The polynucleotide may also contain non-coding 
sequences, including for example, but not limited to non-coding 5' and 3' 
sequences, such as the transcribed, non-translated sequences, termination 
signals, ribosome binding sites, sequences that stabilize mRNA, introns, 
polyadenylation signals, and additional coding sequence which encode 
additional amino acids. For example, a marker sequence that facilitates 
purification of the fused polypeptide can be encoded. In certain 
embodiments of the invention, the marker sequence is a hexa-histidine 
peptide, as provided in the pQE vector (Qiagen, Inc.) and described in 
Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA tag 
(Wilson et al, Cell 37: 767 (1984). Polynucleotides of the invention also 
include, but are not limited to, polynucleotides comprising a structural 
gene and its naturally associated sequences that control gene expression. 
A preferred embodiment of the invention is the polynucleotide of comprising 
nucleotide 293 to 889 set forth in SEQ ID NO:1 of Table 1 which encodes 
the Response Regulator polypeptide. 
The invention also includes polynucleotides of the formula set forth in 
Table 1 (C) SEQ ID NO:1! wherein, at the 5' end of the molecule, X is 
hydrogen, and at the 3' end of the molecule, Y is hydrogen or a metal, 
R.sub.1 and R.sub.2 is any nucleic acid residue, and n is an integer 
between 1 and 1000. Any stretch of nucleic acid residues denoted by either 
R group, where R is greater 10 than 1, may be either a heteropolymer or a 
homopolymer, preferably a heteropolymer. 
The term "polynucleotide encoding a polypeptide" as used herein encompasses 
polynucleotides that include a sequence encoding a polypeptide of the 
invention, particularly a bacterial polypeptide and more particularly a 
polypeptide of the Streptococcus pneumoniae Response Regulator having the 
amino acid sequence set out in Table 1 SEQ ID NO:2!. The term also 
encompasses polynucleotides that include a single continuous region or 
discontinuous regions encoding the polypeptide (for example, interrupted 
by integrated phage or an insertion sequence or editing) together with 
additional regions, that also may contain coding and/or noncoding 
sequences. 
The invention further relates to variants of the polynucleotides described 
herein that encode for variants of the polypeptide having the deduced 
amino acid sequence of Table 1 SEQ ID NO:2!. Variants that are fragments 
of the polynucleotides of the invention may be used to synthesize 
full-length polynucleotides of the invention. 
Further particularly preferred embodiments are polynucleotides encoding 
Response Regulator variants, that have the amino acid sequence of Response 
Regulator polypeptide of Table 1 SEQ ID NO:2! in which several, a few, 5 
to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, 
deleted or added, in any combination. Especially preferred among these are 
silent substitutions, additions and deletions, that do not alter the 
properties and activities of Response Regulator. 
Further preferred embodiments of the invention are polynucleotides that are 
at least 70% identical over their entire length to a polynucleotide 
encoding Response Regulator polypeptide having an amino acid sequence set 
out in Table 1 SEQ ID NOS:2 and 4!, and polynucleotides that are 
complementary to such polynucleotides. Alternatively, most highly 
preferred are polynucleotides that comprise a region that is at least 80% 
identical over its entire length to a polynucleotide encoding Response 
Regulator polypeptide of the deposited strain and polynucleotides 
complementary thereto. In this regard, polynucleotides at least 90% 
identical over their entire length to the same are particularly preferred, 
and among these particularly preferred polynucleotides, those with at 
least 95% are especially preferred. Furthermore, those with at least 97% 
are highly preferred among those with at least 95%, and among these those 
with at least 98% and at least 99% are particularly highly preferred, with 
at least 99% being the more preferred. 
Preferred embodiments are polynucleotides that encode polypeptides that 
retain substantially the same biological function or activity as the 
mature polypeptide encoded by the DNA of Table 1 SEQ ID NO: 1!. 
The invention further relates to polynucleotides that hybridize to the 
herein abovedescribed sequences. In this regard, the invention especially 
relates to polynucleotides that hybridize under stringent conditions to 
the herein above-described polynucleotides. As herein used, the terms 
"stringent conditions" and "stringent hybridization conditions" mean 
hybridization will occur only if there is at least 95% and preferably at 
least 97% identity between the sequences. An example of stringent 
hybridization conditions is overnight incubation at 42.degree. C. in a 
solution comprising: 50% formamide, 5x SSC (105 mM NaCl, 15 mM trisodium 
citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% 
dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, 
followed by washing the hybridization support in 0.1x SSC at about 
65.degree. C. Hybridization and wash conditions are well known and 
exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 
Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 
therein. 
The invention also provides a polynucleotide consisting essentially of a 
polynucleotide sequence obtainable by screening an appropriate library 
containing the complete gene for a polynucleotide sequence set forth in 
SEQ ID NO:1 or SEQ ID NO:3 under stringent hybridization conditions with a 
probe having the sequence of said polynucleotide sequence set forth in SEQ 
ID NO:1 or a fragment thereof; and isolating said DNA sequence. Fragments 
useful for obtaining such a polynucleotide include, for example, probes 
and primers described elsewhere herein. 
As discussed additionally herein regarding polynucleotide assays of the 
invention, for instance, polynucleotides of the invention as discussed 
above, may be used as a hybridization probe for RNA, cDNA and genomic DNA 
to isolate full-length cDNAs and genomic clones encoding Response 
Regulator and to isolate cDNA and genomnic clones of other genes that have 
a high sequence similarity to the Response Regulator gene. Such probes 
generally will comprise at least 15 bases. Preferably, such probes will 
have at least 30 bases and may have at least 50 bases. Particularly 
preferred probes will have at least 30 bases and will have 50 bases or 
less. 
For example, the coding region of the Response Regulator gene may be 
isolated by screening using the known DNA sequence provided in SEQ ID NO: 
1 to synthesize an oligonucleotide probe. A labeled oligonucleotide having 
a sequence complementary to that of a gene of the invention is then used 
to screen a library of cDNA, genomic DNA or mRNA to determine which 
members of the library the probe hybridizes to. 
The polynucleotides and polypeptides of the invention may be employed, for 
example, as research reagents and materials for discovery of treatments of 
and diagnostics for disease, particularly human disease, as further 
discussed herein relating to polynucleotide assays. 
Polynucleotides of the invention that are oligonucleotides derived from the 
sequences of SEQ ID NOS:1 and/or 2 may be used in the processes herein as 
described, but preferably for PCR, to determine whether or not the 
polynucleotides identified herein in whole or in part are transcribed in 
bacteria in infected tissue. It is recognized that such sequences will 
also have utility in diagnosis of the stage of infection and type of 
infection the pathogen has attained. 
The invention also provides polynucleotides that may encode a polypeptide 
that is the mature protein plus additional amino or carboxyl-terminal 
amino acids, or amino acids interior to the mature polypeptide (when the 
mature form has more than one polypeptide chain, for instance). Such 
sequences may play a role in processing of a protein from precursor to a 
mature form, may allow protein transport, may lengthen or shorten protein 
half-life or may facilitate manipulation of a protein for assay or 
production, among other things. As generally is the case in vivo, the 
additional amino acids may be processed away from the mature protein by 
cellular enzymes. 
A precursor protein, having the mature form of the polypeptide fused to one 
or more prosequences may be an inactive form of the polypeptide. When 
prosequences are removed such inactive precursors generally are activated. 
Some or all of the prosequences may be removed before activation. 
Generally, such precursors are called proproteins. 
In sum, a polynucleotide of the invention may encode a mature protein, a 
mature protein plus a leader sequence (which may be referred to as a 
preprotein), a precursor of a mature protein having one or more 
prosequences that are not the leader sequences of a preprotein, or a 
preproprotein, which is a precursor to a proprotein, having a leader 
sequence and one or more prosequences, which generally are removed during 
processing steps that produce active and mature forms of the polypeptide. 
Vectors, host cells, expression 
The invention also relates to vectors that comprise a polynucleotide or 
polynucleotides of the invention, host cells that are genetically 
engineered with vectors of the invention and the production of 
polypeptides of the invention by recombinant techniques. Cell-free 
translation systems can also be employed to produce such proteins using 
RNAs derived from the DNA constructs of the invention. 
For recombinant production, host cells can be genetically engineered to 
incorporate expression systems or portions thereof or polynucleotides of 
the invention. Introduction of a polynucleotide into the host cell can be 
effected by methods described in many standard laboratory manuals, such as 
Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et 
al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor 
Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium 
phosphate transfection, DEAE-dextran mediated transfection, transvection, 
microinjection, cationic lipid-mediated transfection, electroporation, 
transduction, scrape loading, ballistic introduction and infection. 
Representative examples of appropriate hosts include bacterial cells, such 
as streptococci, staphylococci, enterococci E. coli, streptomyces and 
Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus 
cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal 
cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma 
cells; and plant cells. 
A great variety of expression systems can be used to produce the 
polypeptides of the invention. Such vectors include, among others, 
chromosomal, episomal and virus-derived vectors, e.g., vectors derived 
from bacterial plasmids, from bacteriophage, from transposons, from yeast 
episomes, from insertion elements, from yeast chromosomal elements, from 
viruses such as baculoviruses, papova viruses, such as SV40, vaccinia 
viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and 
retroviruses, and vectors derived from combinations thereof, such as those 
derived from plasmid and bacteriophage genetic elements, such as cosmids 
and phagemids. The expression system constructs may contain control 
regions that regulate as well as engender expression. Generally, any 
system or vector suitable to maintain, propagate or express 
polynucleotides and/or to express a polypeptide in a host may be used for 
expression in this regard. The appropriate DNA sequence may be inserted 
into the expression system by any of a variety of well-known and routine 
techniques, such as, for example, those set forth in Sambrook et al., 
MOLECULAR CLONING, A LABORATORY MANUAL, (supra). 
For secretion of the translated protein into the lumen of the endoplasmic 
reticulum, into the periplasmic space or into the extracellular 
environment, appropriate secretion signals may be incorporated into the 
expressed polypeptide. These signals may be endogenous to the polypeptide 
or they may be heterologous signals. 
Polypeptides of the invention can be recovered and purified from 
recombinant cell cultures by well-known methods including ammonium sulfate 
or ethanol precipitation, acid extraction, anion or cation exchange 
chromatography, phosphocellulose chromatography, hydrophobic interaction 
chromatography, affinity chromatography, hydroxylapatite chromatography, 
and lectin chromatography. Most preferably, high performance liquid 
chromatography is employed for purification. Well known techniques for 
refolding protein may be employed to regenerate active conformation when 
the polypeptide is denatured during isolation and or purification. 
Diagnostic Assays 
This invention is also related to the use of the Response Regulator 
polynucleotides of the invention for use as diagnostic reagents. Detection 
of Response Regulator in a eukaryote, particularly a mammal, and 
especially a human, will provide a diagnostic method for diagnosis of a 
disease. Eukaryotes (herein also "individual(s)"), particularly mammals, 
and especially humans, infected with an organism comprising the Response 
Regulator gene may be detected at the nucleic acid level by a variety of 
techniques. 
Nucleic acids for diagnosis may be obtained from an infected individual's 
cells and tissues, such as bone, blood, muscle, cartilage, and skin. 
Genomic DNA may be used directly for detection or may be amplified 
enzymatically by using PCR or other amplification technique prior to 
analysis. RNA or cDNA may also be used in the same ways. Using 
amplification, characterization of the species and strain of prokaryote 
present in an individual, may be made by an analysis of the genotype of 
the prokaryote gene. Deletions and insertions can be detected by a change 
in size of the amplified product in comparison to the genotype of a 
reference sequence. Point mutations can be identified by hybridizing 
amplified DNA to labeled Response Regulator polynucleotide sequences. 
Perfectly matched sequences can be distinguished from mismatched duplexes 
by RNase digestion or by differences in melting temperatures. DNA sequence 
differences may also be detected by alterations in the electrophoretic 
mobility of the DNA fragments in gels, with or without denaturing agents, 
or by direct DNA sequencing. See, e.g., Myers et al., Science, 230: 1242 
(1985). Sequence changes at specific locations also may be revealed by 
nuclease protection assays, such as RNase and S1 protection or a chemical 
cleavage method. See, e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 
85:4397-4401 (1985). 
Cells carrying mutations or polymorphisms in the gene of the invention may 
also be detected at the DNA level by a variety of techniques, to allow for 
serotyping, for example. For example, RT-PCR can be used to detect 
mutations. It is particularly preferred to used RT-PCR in conjunction with 
automated detection systems, such as, for example, GeneScan. RNA or cDNA 
may also be used for the same purpose, PCR or RT-PCR. As an example, PCR 
primers complementary to a nucleic acid encoding Response Regulator can be 
used to identify and analyze mutations. Examples of representative primers 
are shown below in Table 2. 
TABLE 2 
______________________________________ 
Primers for amplification of Response Regulator polynucleotides 
SEQ ID NO PRIMER SEQUENCE 
______________________________________ 
5 5'-ATGAAAGTATTAGTCGCAGAAGATCAAA-3' 
6 5'-TAACCAACCAGATTCTTTTGCGATATTAG-3' 
______________________________________ 
The invention further provides these primers with 1, 2, 3 or 4 nucleotides 
removed from the 5' and/or the 3' end. These primers may be used for, 
among other things, amplifying Response Regulator DNA isolated from a 
sample derived from an individual. The primers may be used to amplify the 
gene isolated from an infected individual such that the gene may then be 
subject to various techniques for elucidation of the DNA sequence. In this 
way, mutations in the DNA sequence may be detected and used to diagnose 
infection and to serotype and/or classify the infectious agent. 
The invention further provides a process for diagnosing, disease, 
preferably bacterial infections, more preferably infections by 
Streptococcus pneumoniae, and most preferably otitis media, 
conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural 
empyema and endocarditis, and most particularly meningitis, such as for 
example infection of cerebrospinal fluid, comprising determining from a 
sample derived from an individual a increased level of expression of 
polynucleotide having the sequence of Table 1 SEQ ID NO:1!. Increased or 
decreased expression of Response Regulator polynucleotide can be measured 
using any on of the methods well known in the art for the quantitation of 
polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase 
protection, Northern blotting and other hybridization methods. 
In addition, a diagnostic assay in accordance with the invention for 
detecting over-expression of Response Regulator protein compared to normal 
control tissue samples may be used to detect the presence of an infection, 
for example. Assay techniques that can be used to determine levels of a 
Response Regulator protein, in a sample derived from a host are well-known 
to those of skill in the art. Such assay methods include 
radioimmunoassays, competitive-binding assays, Western Blot analysis and 
ELISA assays. 
Antibodies 
The polypeptides of the invention or variants thereof, or cells expressing 
them can be used as an immunogen to produce antibodies immunospecific for 
such polypeptides. "Antibodies" as used herein includes monoclonal and 
polyclonal antibodies, chimeric, single chain, simianized antibodies and 
humanized antibodies, as well as Fab fragments, including the products of 
an Fab immunolglobulin expression library. 
Antibodies generated against the polypeptides of the invention can be 
obtained by administering the polypeptides or epitope-bearing fragments, 
analogues or cells to an animal, preferably a nonhuman, using routine 
protocols. For preparation of monoclonal antibodies, any technique known 
in the art that provides antibodies produced by continuous cell line 
cultures can be used. Examples include various techniques, such as those 
in Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al., 
Immunology Today 4:72 (1983); Cole et al., pg. 77-96 in MONOCLONAL 
ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985). 
Techniques for the production of single chain antibodies (U.S. Pat. No. 
4,946,778) can be adapted to produce single chain antibodies to 
polypeptides of this invention. Also, transgenic mice, or other organisms 
such as other mammals, may be used to express humanized antibodies. 
Alternatively phage display technology may be utilized to select antibody 
genes with binding activities towards the polypeptide either from 
repertoires of PCR amplified v-genes of lymphocytes from humans screened 
for possessing anti-Response Regulator or from naive libraries 
(McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al., 
(1992) Biotechnology 10, 779-783). The affinity of these antibodies can 
also be improved by chain shuffling (Clackson, T. et al., (1991) Nature 
352, 624-628). 
If two antigen binding domains are present each domain may be directed 
against a different epitope--termed `bispecific` antibodies. 
The above-described antibodies may be employed to isolate or to identify 
clones expressing the polypeptides to purify the polypeptides by affinity 
chromatography. 
Thus, among others, antibodies against Response Regulator- polypeptide may 
be employed to treat infections, particularly bacterial infections and 
especially otitis media, conjunctivitis, pneumonia, bacteremia, 
meningitis, sinusitis, pleural empyema and endocarditis, and most 
particularly meningitis, such as for example infection of cerebrospinal 
fluid. 
Polypeptide variants include antigenically, epitopically or immunologically 
equivalent variants that form a particular aspect of this invention. The 
term "antigenically equivalent derivative" as used herein encompasses a 
polypeptide or its equivalent which will be specifically recognized by 
certain antibodies which, when raised to the protein or polypeptide 
according to the invention, interfere with the immediate physical 
interaction between pathogen and mammalian host. The term "immunologically 
equivalent derivative" as used herein encompasses a peptide or its 
equivalent which when used in a suitable formulation to raise antibodies 
in a vertebrate, the antibodies act to interfere with the immediate 
physical interaction between pathogen and mammalian host. 
The polypeptide, such as an antigenically or immunologically equivalent 
derivative or a fusion protein thereof is used as an antigen to immunize a 
mouse or other animal such as a rat or chicken. The fusion protein may 
provide stability to the polypeptide. The antigen may be associated, for 
example by conjugation, with an immunogenic carrier protein for example 
bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH). 
Alternatively a multiple antigenic peptide comprising multiple copies of 
the protein or polypeptide, or an antigenically or immunologically 
equivalent polypeptide thereof may be sufficiently antigenic to improve 
immunogenicity so as to obviate the use of a carrier. 
Preferably, the antibody or variant thereof is modified to make it less 
immunogenic in the individual. For example, if the individual is human the 
antibody may most preferably be "humanized"; where the complimentarity 
determining region(s) of the hybridoma-derived antibody has been 
transplanted into a human monoclonal antibody, for example as described in 
Jones, P. et al. (1986), Nature 321, 522-525 or Tempest et al.,(1991) 
Biotechnology 9, 266-273. 
The use of a polynucleotide of the invention in genetic immunization will 
preferably employ a suitable delivery method such as direct injection of 
plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992, 1:363, 
Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNA complexed 
with specific protein carriers (Wu et al., J Biol Chem. 1989:264,16985), 
coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, PNAS, 
1986:83,9551), encapsulation of DNA in various forms of liposomes (Kaneda 
et al., Science 1989:243,375), particle bombardment (Tang et al., Nature 
1992, 356:152, Eisenbraun et al., DNA Cell Biol 1993, 12:791) and in vivo 
infection using cloned retroviral vectors (Seeger et al., PNAS 
1984:81,5849). 
Antagonists and agonists--assays and molecules 
Polypeptides of the invention may also be used to assess the binding of 
small molecule substrates and ligands in, for example, cells, cell-free 
preparations, chemical libraries, and natural product mixtures. These 
substrates and ligands may be natural substrates and ligands or may be 
structural or functional mimetics. See, e.g., Coligan et al., Current 
Protocols in Immunology 1(2): Chapter 5 (1991). 
The invention also provides a method of screening compounds to identify 
those which enhance (agonist) or block (antagonist) the action of Response 
Regulator polypeptides or polynucleotides, particularly those compounds 
that are bacteriostatic and/or bacteriocidal. The method of screening may 
involve high-throughput techniques. For example, to screen for agonists or 
antagonists, a synthetic reaction mix, a cellular compartment, such as a 
membrane, cell envelope or cell wall, or a preparation of any thereof, 
comprising Response Regulator polypeptide and a labeled substrate or 
ligand of such polypeptide is incubated in the absence or the presence of 
a candidate molecule that may be a Response Regulator agonist or 
antagonist. The ability of the candidate molecule to agonize or antagonize 
the Response Regulator polypeptide is reflected in decreased binding of 
the labeled ligand or decreased production of product from such substrate. 
Molecules that bind gratuitously, i.e., without inducing the effects of 
Response Regulator polypeptide are most likely to be good antagonists. 
Molecules that bind well and increase the rate of product production from 
substrate are agonists. Detection of the rate or level of production of 
product from substrate may be enhanced by using a reporter system. 
Reporter systems that may be useful in this regard include but are not 
limited to calorimetric labeled substrate converted into product, a 
reporter gene that is responsive to changes in Response Regulator 
polynucleotide or polypeptide activity, and binding assays known in the 
art. 
Another example of an assay for Response Regulator antagonists is a 
competitive assay that combines Response Regulator and a potential 
antagonist with Response Regulator-binding molecules, recombinant Response 
Regulator binding molecules, natural substrates or ligands, or substrate 
or ligand mimetics, under appropriate conditions for a competitive 
inhibition assay. Response Regulator can be labeled, such as by 
radioactivity or a colorimetric compound, such that the number of Response 
Regulator molecules bound to a binding molecule or converted to product 
can be determined accurately to assess the effectiveness of the potential 
antagonist. 
Potential antagonists include small organic molecules, peptides, 
polypeptides and antibodies that bind to a polynucleotide or polypeptide 
of the invention and thereby inhibit or extinguish its activity. Potential 
antagonists also may be small organic molecules, a peptide, a polypeptide 
such as a closely related protein or antibody that binds the same sites on 
a binding molecule, such as a binding molecule, without inducing Response 
Regulator-induced activities, thereby preventing the action of Response 
Regulator by excluding Response Regulator from binding. 
Potential antagonists include a small molecule that binds to and occupies 
the binding site of the polypeptide thereby preventing binding to cellular 
binding molecules, such that normal biological activity is prevented. 
Examples of small molecules include but are not limited to small organic 
molecules, peptides or peptide-like molecules. Other potential antagonists 
include antisense molecules (see Okano, J. Neurochem. 56: 560 (1991); 
OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC 
Press, Boca Raton, Fla. (1988), for a description of these molecules). 
Preferred potential antagonists include compounds related to and variants 
of Response Regulator. 
Each of the DNA sequences provided herein may be used in the discovery and 
development of antibacterial compounds. The encoded protein, upon 
expression, can be used as a target for the screening of antibacterial 
drugs. Additionally, the DNA sequences encoding the amino terminal regions 
of the encoded protein or Shine-Delgarno or other translation facilitating 
sequences of the respective mRNA can be used to construct antisense 
sequences to control the expression of the coding sequence of interest. 
The invention also provides the use of the polypeptide, polynucleotide or 
inhibitor of the invention to interfere with the initial physical 
interaction between a pathogen and mammalian host responsible for sequelae 
of infection. In particular the molecules of the invention may be used: in 
the prevention of adhesion of bacteria, in particular gram positive 
bacteria, to mammalian extracellular matrix proteins on in-dwelling 
devices or to extracellular matrix proteins in wounds; to block Response 
Regulator protein-mediated mammalian cell invasion by, for example, 
initiating phosphorylation of mammalian tyrosine kinases (Rosenshine et 
al., Infect. Immun. 60:2211 (1992); to block bacterial adhesion between 
mammalian extracellular matrix proteins and bacterial Response Regulator 
proteins that mediate tissue damage and; to block the normal progression 
of pathogenesis in infections initiated other than by the implantation of 
in-dwelling devices or by other surgical techniques. 
This invention provides a method of screening drugs to identify those which 
i) interfere with the interaction of the response regulator with a 
histidine kinase, the method comprising incubating the response regulator 
with histidine kinase in the presence of the drug and measuring the 
ability of the drug to block this interaction; 
ii) interfere with the ability of the response regulator to catalyse the 
transfer of phosphate group from the histidine kinase to itself, the 
method comprising incubating the response regulator with drug and 
measuring the ability of the response regulator to catalyse the removal of 
phosphate from phosphorylated histidine kinase; and/or 
iii) interfere with the ability of the molecule to autodephosphorylate 
itself once the phosphate had been transferred, the method comprising 
incubating the phosphorylated response regulator with drug and measuring 
the ability of the response regulator to catalyse the 
autodephosphorylation. 
The histidine kinase is preferably the cognate histidine kinase of the 
response regulator, or another histidine kinase which is capable of acting 
as a substrate for the response regulator, and may be from Streptococcus 
pneumoniae or another microorganism e.g. Bacillus. Generally the genes for 
a histidine kinase and its cognate response regulator are found close 
together on the chromosome so a suitable histidine kinase may conveniently 
be identified by further sequencing along the chromosome. The invention 
also relates to inhibitors identfied thereby. 
The antagonists and agonists of the invention may be employed, for 
instance, to inhibit and treat otitis media, conjunctivitis, pneumonia, 
bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and 
most particularly meningitis, such as for example infection of 
cerebrospinal fluid. 
Vaccines 
Another aspect of the invention relates to a method for inducing an 
immunological response in an individual, particularly a mammal which 
comprises inoculating the individual with Response Regulator, or a 
fragment or variant thereof, adequate to produce antibody and/ or T cell 
immune response to protect said individual from infection, particularly 
bacterial infection and most particularly Streptococcus pneumoniae 
infection. Also provided are methods whereby such immunological response 
slows bacterial replication. Yet another aspect of the invention relates 
to a method of inducing immunological response in an individual which 
comprises delivering to such individual a nucleic acid vector to direct 
expression of Response Regulator, or a fragment or a variant thereof, for 
expressing Response Regulator, or a fragment or a variant thereof in vivo 
in order to induce an immunological response, such as, to produce antibody 
and/ or T cell immune response, including, for example, cytokine-producing 
T cells or cytotoxic T cells, to protect said individual from disease, 
whether that disease is already established within the individual or not. 
One way of administering the gene is by accelerating it into the desired 
cells as a coating on particles or otherwise. 
Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or 
a DNA/RNA hybrid. 
A further aspect of the invention relates to an immunological composition 
which, when introduced into an individual capable or having induced within 
it an immunological response, induces an immunological response in such 
individual to a Response Regulator or protein coded therefrom, wherein the 
composition comprises a recombinant Response Regulator or protein coded 
therefrom comprising DNA which codes for and expresses an antigen of said 
Response Regulator or protein coded therefrom. The immunological response 
may be used therapeutically or prophylactically and may take the form of 
antibody immunity or cellular immunity such as that arising from CTL or 
CD4+T cells. 
A Response Regulator polypeptide or a fragment thereof may be fused with 
co-protein which may not by itself produce antibodies, but is capable of 
stabilizing the first protein and producing a fused protein which will 
have immunogenic and protective properties. Thus fused recombinant 
protein, preferably further comprises an antigenic co-protein, such as 
lipoprotein D from Hemophilus influenzae, Glutathione-S-transferase (GSI) 
or beta-galactosidase, relatively large co-proteins which solubilize the 
protein and facilitate production and purification thereof. Moreover, the 
co-protein may act as an adjuvant in the sense of providing a generalized 
stimulation of the immune system. The co-protein may be attached to either 
the amino or carboxy terminus of the first protein. 
Provided by this invention are compositions, particularly vaccine 
compositions, and methods comprising the polypeptides or polynucleotides 
of the invention and immunostimulatory DNA sequences, such as those 
described in Sato, Y. et al. Science 273: 352 (1996). 
Also, provided by this invention are methods using the described 
polynucleotide or particular fragments thereof which have been shown to 
encode non-variable regions of bacterial cell surface proteins in DNA 
constructs used in such genetic immunization experiments in animal models 
of infection with Streptococcus pneumoniae will be particularly useful for 
identifying protein epitopes able to provoke a prophylactic or therapeutic 
immune response. It is believed that this approach will allow for the 
subsequent preparation of monoclonal antibodies of particular value from 
the requisite organ of the animal successfully resisting or clearing 
infection for the development of prophylactic agents or therapeutic 
treatments of bacterial infection, particularly Streptococcus pneumoniae 
infection, in mammals, particularly humans. 
The polypeptide may be used as an antigen for vaccination of a host to 
produce specific antibodies which protect against invasion of bacteria, 
for example by blocking adherence of bacteria to damaged tissue. Examples 
of tissue damage include wounds in skin or connective tissue caused, e.g., 
by mechanical, chemical or thermal damage or by implantation of indwelling 
devices, or wounds in the mucous membranes, such as the mouth, mammary 
glands, urethra or vagina. 
The invention also includes a vaccine formulation which comprises an 
immunogenic recombinant protein of the invention together with a suitable 
carrier. Since the protein may be broken down in the stomach, it is 
preferably administered parenterally, including, for example, 
administration that is subcutaneous, intramuscular, intravenous, or 
intradermal. Formulations suitable for parenteral administration include 
aqueous and nonaqueous sterile injection solutions which may contain 
anti-oxidants, buffers, bacteriostats and solutes which render the 
formulation insotonic with the bodily fluid, preferably the blood, of the 
individual; and aqueous and non-aqueous sterile suspensions which may 
include suspending agents or thickening agents. The formulations may be 
presented in unit-dose or multi-dose containers, for example, sealed 
ampules and vials and may be stored in a freeze-dried condition requiring 
only the addition of the sterile liquid carrier immediately prior to use. 
The vaccine formulation may also include adjuvant systems for enhancing 
the immunogenicity of the formulation, such as oil-in water systems and 
other systems known in the art. The dosage will depend on the specific 
activity of the vaccine and can be readily determined by routine 
experimentation. 
While the invention has been described with reference to certain Response 
Regulator protein, it is to be understood that this covers fragments of 
the naturally occurring protein and similar proteins with additions, 
deletions or substitutions which do not substantially affect the 
immunogenic properties of the recombinant protein. 
Compositions, kits and administration 
The invention also relates to compositions comprising the polynucleotide or 
the polypeptides discussed above or their agonists or antagonists. The 
polypeptides of the invention may be employed in combination with a 
non-sterile or sterile carrier or carriers for use with cells, tissues or 
organisms, such as a pharmaceutical carrier suitable for administration to 
a subject. Such compositions comprise, for instance, a media additive or a 
therapeutically effective amount of a polypeptide of the invention and a 
pharmaceutically acceptable carrier or excipient. Such carriers may 
include, but are not limited to, saline, buffered saline, dextrose, water, 
glycerol, ethanol and combinations thereof. The formulation should suit 
the mode of administration. The invention further relates to diagnostic 
and pharmaceutical packs and kits comprising one or more containers filled 
with one or more of the ingredients of the aforementioned compositions of 
the invention. 
Polypeptides and other compounds of the invention may be employed alone or 
in conjunction with other compounds, such as therapeutic compounds. 
The pharmaceutical compositions may be administered in any effective, 
convenient manner including, for instance, administration by topical, 
oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, 
subcutaneous, intranasal or intradermal routes among others. 
In therapy or as a prophylactic, the active agent may be administered to an 
individual as an injectable composition, for example as a sterile aqueous 
dispersion, preferably isotonic. 
Alternatively the composition may be formulated for topical application for 
example in the form of ointments, creams, lotions, eye ointments, eye 
drops, ear drops, mouthwash, impregnated dressings and sutures and 
aerosols, and may contain appropriate conventional additives, including, 
for example, preservatives, solvents to assist drug penetration, and 
emollients in ointments and creams. Such topical formulations may also 
contain compatible conventional carriers, for example cream or ointment 
bases, and ethanol or oleyl alcohol for lotions. Such carriers may 
constitute from about 1% to about 98% by weight of the formulation; more 
usually they will constitute up to about 80% by weight of the formulation. 
For administration to mammals, and particularly humans, it is expected that 
the daily dosage level of the active agent will be from 0.01 mg/kg to 10 
mg/kg, typically around 1 mg/kg. The physician in any event will determine 
the actual dosage which will be most suitable for an individual and will 
vary with the age, weight and response of the particular individual. The 
above dosages are exemplary of the average case. There can, of course, be 
individual instances where higher or lower dosage ranges are merited, and 
such are within the scope of this invention. 
In-dwelling devices include surgical implants, prosthetic devices and 
catheters, i.e., devices that are introduced to the body of an individual 
and remain in position for an extended time. Such devices include, for 
example, artificial joints, heart valves, pacemakers, vascular grafts, 
vascular catheters, cerebrospinal fluid shunts, urinary catheters, 
continuous ambulatory peritoneal dialysis (CAPD) catheters. 
The composition of the invention may be administered by injection to 
achieve a systemic effect against relevant bacteria shortly before 
insertion of an in-dwelling device. Treatment may be continued after 
surgery during the in-body time of the device. In addition, the 
composition could also be used to broaden perioperative cover for any 
surgical technique to prevent bacterial wound infections, especially 
Streptococcus pneumoniae wound infections. 
Many orthopaedic surgeons consider that humans with prosthetic joints 
should be considered for antibiotic prophylaxis before dental treatment 
that could produce a bacteremia. Late deep infection is a serious 
complication sometimes leading to loss of the prosthetic joint and is 
accompanied by significant morbidity and mortality. It may therefore be 
possible to extend the use of the active agent as a replacement for 
prophylactic antibiotics in this situation. 
In addition to the therapy described above, the compositions of this 
invention may be used generally as a wound treatment agent to prevent 
adhesion of bacteria to matrix proteins exposed in wound tissue and for 
prophylactic use in dental treatment as an alternative to, or in 
conjunction with, antibiotic prophylaxis. 
Alternatively, the composition of the invention may be used to bathe an 
indwelling device immediately before insertion. The active agent will 
preferably be present at a concentration of 1 g/ml to 10 mg/ml for bathing 
of wounds or indwelling devices. 
A vaccine composition is conveniently in injectable form. Conventional 
adjuvants may be employed to enhance the immune response. A suitable unit 
dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is 
preferably administered 1-3 times and with an interval of 1-3 weeks. With 
the indicated dose range, no adverse toxicological effects will be 
observed with the compounds of the invention which would preclude their 
administration to suitable individuals. 
Each reference disclosed herein is incorporated by reference herein in its 
entirety. Any patent application to which this application claims priority 
is also incorporated by reference herein in its entirety.

EXAMPLES 
The examples below are carried out using standard techniques, which are 
well known and routine to those of skill in the art, except where 
otherwise described in detail. The examples are illustrative, but do not 
limit the invention. 
Example 1 
Strain selection, Library Production and Sequencing 
The polynucleotide having the DNA sequence given in SEQ ID NO:1 was 
obtained from a library of clones of chromosomal DNA of Streptococcus 
pneumoniae in E. coli. The sequencing data from two or more clones 
containing overlapping Streptococcus pneumoniae DNAs was used to construct 
the contiguous DNA sequence in SEQ ID NO:1. Libraries may be prepared by 
routine methods, for example: 
Methods 1 and 2 below. 
Total cellular DNA is isolated from Streptococcus pneumoniae 0100993 
according to standard procedures and size-fractionated by either of two 
methods. 
Method 1 
Total cellular DNA is mechanically sheared by passage through a needle in 
order to size-fractionate according to standard procedures. DNA fragments 
of up to 1 lkbp in size are rendered blunt by treatment with exonuclease 
and DNA polymerase, and EcoRI linkers added. Fragments are ligated into 
the vector Lambda ZapII that has been cut with EcoRI, the library packaged 
by standard procedures and E.coli infected with the packaged library. The 
library is amplified by standard procedures. 
Method 2 
Total cellular DNA is partially hydrolyzed with a one or a combination of 
restriction enzymes appropriate to generate a series of fragments for 
cloning into library vectors (e.g., RsaI, PalI, AluI, Bshl2351), and such 
fragments are size-fractionated according to standard procedures. EcoRI 
linkers are ligated to the DNA and the fragments then ligated into the 
vector Lambda ZapII that have been cut with EcoRI, the library packaged by 
standard procedures, and E.coli infected with the packaged library. The 
library is amplified by standard procedures. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 6 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 925 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Genomic DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GCTAGCTTGACCCAGGATGTGGAGTCGACGGCTGCTATGATTTTGCTTGAGTTGGTGACC60 
AATATCATCAAACATGCCAAAGCGTTTAAAGCTTACTTAAAATTAGAACGGACAGAGAAG120 
GAACTCATTTTAACAGTAAGTGATGATGGCTGCGGCTTTGCTTTTCTAAAAGGAGATGAG180 
CTCCATACAGTCCGAGATCGTGTTTTTCCATTTTCAGGAGAAGTAAGTGTAATCAGTCAG240 
AAACATCCAACGGAAGTGCAAGTTCGACTACCTTATAAGGAGAGAAACTAAGATGAAAGT300 
ATTAGTCGCAGAAGATCAAAGTATGTTGCGAGATGCCATGTGTCAATTGCTCACGCTTCA360 
ACCAGATGTGGAGTCTGTCCTTCAAACCAAGAATGGGCAAGAAGCAATCCAACTATTAGA420 
AAAGGAGTCTGTAGATATCGCCATCCTTGACGTAGAAATGCCTGTTAAGACAGGTCTTGA480 
AGTCTTGGAGTGGATACGATCAGAAAAGCTTGAAACAAAGGTGGTTGTGGTGACGACCTT540 
CAAGCGTGCTGGGTATTTTGAACGTGCGGTCAAGGCTGGAGTGGATGCTTATGTATTAAA600 
GGAACGAAGCATTGCAGACCTCATGCAAACCTTGCACACCGTCCTCGAAGGACGCAAGGA660 
GTATTCGCCTGAATTGATGGAAGTGGTGATGACCCGTCCCAATCCGTTGACAGAACAAGA720 
GATTGCTGTCTTAAAGGGAATCGCCCGGGGCTTATCCAACCAAGAAATCGCAGATCAGCT780 
TTACCTCTCAAACGGAACTATTCGAAACTATGTCACCAATATTCTTTCAAAACTGGATGC840 
TGGTAATCGAACAGAGGCAGCTAATATCGCAAAAGAATCTGGTTGGTTATGATGATATAA900 
TATTTTCATGAAGACTATGTGCTAA925 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 199 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetLysValLeuValAlaGluAspGlnSerMetLeuArgAspAlaMet 
151015 
CysGlnLeuLeuThrLeuGlnProAspValGluSerValLeuGlnThr 
202530 
LysAsnGlyGlnGluAlaIleGlnLeuLeuGluLysGluSerValAsp 
354045 
IleAlaIleLeuAspValGluMetProValLysThrGlyLeuGluVal 
505560 
LeuGluTrpIleArgSerGluLysLeuGluThrLysValValValVal 
65707580 
ThrThrPheLysArgAlaGlyTyrPheGluArgAlaValLysAlaGly 
859095 
ValAspAlaTyrValLeuLysGluArgSerIleAlaAspLeuMetGln 
100105110 
ThrLeuHisThrValLeuGluGlyArgLysGluTyrSerProGluLeu 
115120125 
MetGluValValMetThrArgProAsnProLeuThrGluGlnGluIle 
130135140 
AlaValLeuLysGlyIleAlaArgGlyLeuSerAsnGlnGluIleAla 
145150155160 
AspGlnLeuTyrLeuSerAsnGlyThrIleArgAsnTyrValThrAsn 
165170175 
IleLeuSerLysLeuAspAlaGlyAsnArgThrGluAlaAlaAsnIle 
180185190 
AlaLysGluSerGlyTrpLeu 
195 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 597 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Genomic DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CTGATCTGCGATTTCTTGGTTGGATAAGCCCCGGGCGATTCCCTTTAAGACAGCAATCTC60 
TTGTTCTGTCAACGGATTGGGACGGGTCATCACCACTTCCATCAATTCAGGCGAATACTC120 
CTTGCGTCCTTCGAGGACGGTGTGCAAGGTTTGCATGAGGTCTGCAATGCTTCGTTCCTT180 
TAATACATAAGCATCCACTCCAGCCTTGACCGCACGTTCAAAATACCCAGCACGCTTGAA240 
GGTCGTCACCACAACCACCTTTGTTTCAAGCTTTTCTGATCGTATCCACTCCAAGACTTC300 
AAGACCTGTCTTAACAGGCATTTCTACGTCAAGGATGGCGATATCTACAGACTCCTTTTC360 
TAATAGTTGGATTGCTTCTTGCCCATTCTTGGTTTGAAGGACAGACTCCACATCTGGTTG420 
AAGCGTGAGCAATTGACACATGGCATCTCGCAACATACTTTGATCTTCTGCGACTAATAC480 
TTTCATCTTAGTTTCTCTCCTTATAAGGTAGTCGAACTTGCACTTCCGTTGGATGTTTCT540 
GACTGATTACACTTACTTCTCCTGAAAATGGAAAAACACGATCTCCGACTGTATGGA597 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 162 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetLysValLeuValAlaGluAspGlnSerMetLeuArgAspAlaMet 
151015 
CysGlnLeuLeuThrLeuGlnProAspValGluSerValLeuGlnThr 
202530 
LysAsnGlyGlnGluAlaIleGlnLeuLeuGluLysGluSerValAsp 
354045 
IleAlaIleLeuAspValGluMetProValLysThrGlyLeuGluVal 
505560 
LeuGluTrpIleArgSerGluLysLeuGluThrLysValValValVal 
65707580 
ThrThrPheLysArgAlaGlyTyrPheGluArgAlaValLysAlaGly 
859095 
ValAspAlaTyrValLeuLysGluArgSerIleAlaAspLeuMetGln 
100105110 
ThrLeuHisThrValLeuGluGlyArgLysGluTyrSerProGluLeu 
115120125 
MetGluValValMetThrArgProAsnProLeuThrGluGlnGluIle 
130135140 
AlaValLeuLysGlyIleAlaArgGlyLeuSerAsnGlnGluIleAla 
145150155160 
AspGln 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Genomic DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
ATGAAAGTATTAGTCGCAGAAGATCAAA28 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: Genomic DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
TAACCAACCAGATTCTTTTGCGATATTAG29 
__________________________________________________________________________