Polypeptides containing sequences characteristic of pyrrolidone carboxylyl peptidases, polynucleotides containing a sequence coding for such polypeptides, and their use, in particular for diagnostic purposes

Purified polypeptides containing characteristic sequences common to the pyrrolidone carboxylyl peptidases (PYRases) of bacteria, for example of Streptococcus pyogenes, or containing fragments of these sequences; antibodies recognizing these polypeptides; polynucleotides coding for such polypeptides or fragments. Application, in particular, to the overproduction of PYRases by inserting a polynucleotide coding for a PYRase into a vector, and then culturing a host cell transformed using such a vector, or to the production of nucleic acid probes specific for a bacterium having a PYRase gene, expressed or otherwise. These nucleic acid probes may be used as capture or detection probes according to conventional hybridization techniques.

The present invention relates to polypeptides containing sequences 
characteristic of pyrrolidone carboxylyl peptidases, to polynucleotides 
containing a sequence coding for such polypeptides, and to their use, in 
particular for diagnostic purposes. 
Pyrrolidone carboxylyl peptidases are exopeptidasee which specifically 
remove 2-pyrrolidone-5-carboxylic acid (PCA) residues from the NH.sub.2 
-terminal end of polypeptide chains and proteins (Doolittle and 
Armentrout, Biochemistry, 1968, 7, 516-521). These enzymes are also 
referred to as pyrrolidonyl peptidases (Szewczuk and Mulczyk, Eur. J. 
Biochem, 1969, 8, 63-67) or more commonly PYRases (Mitchell et al., Diagn. 
Microbiol. Infect. Dis., 1987, 6, 283-286). These enzymes are specific for 
the L-PCA-L-amino acid optical isomers (Uliana and Doolittle, Arch. 
Biochem. Biophys., 1969, 131, 561-656), and the rate of hydrolysis depends 
on the amino acid adjacent to the PCA residue (Fujiwara et al., Biochim. 
Biophys. Acta, 1979, 570, 140-148). These enzymes also belong to the 
arylamidase group, since they are capable of hydrolysing the peptide bond 
of the synthetic chromogenic substrate L-pyroglutamyl-.beta.-naphthylamide 
(Patterson et al., J. Biol. Chem., 1963, 238, 3611-3620). These enzymes 
were first described in bacteria (Mulczyk and Szewczuk, J. Gen. Microb., 
1970, 61, 9-13), but they are also found to be present in animal and plant 
tissues and in man (Szewczuk and Kwiatkowska, Eur. J. Biochem., 1970, 15, 
92-96). PYRases prove, in addition, to be a specific means for removing 
PCA residues blocking the NH.sub.2 -terminal end of peptides, and to be 
very useful for determining the amino acid sequence of proteins, since 
peptides not possessing a free .alpha.-NH.sub.2 group cannot be sequenced 
according to the usual Edman degradation. The biochemical and 
physicochemical properties of PYRases are quite well known, since they 
have been studied for more than about twenty years. Enzymes having PYRase 
activity have been isolated and partially purified from several 
microorganisms, in particular from bacteria such as Pseudomonas 
fluorescens (Armentrout and Doolittle, Arch. Biochem. Biophys., 1969, 132, 
80-90; Doolittle, Meth. Enzymol. 1970, 19, 555-569), Bacillus subtilis 
(Szewczuk and Mulczyk, Eur. J. Biochem., 1969, 8, 63-67), Bacillus 
amyloliquefaciens (Tsuru etal., J. Biochem, 1978, 84, 467-476), Klebsiella 
cloacas (Kwiatkowska et al., J. Biol. Chem., 1974, 249, 7729-7736) and 
Enterococcus faecium (Sullivan etal., Aust. J. Biol. Sci., 1977, 30, 
543-552). Conventional purification methods have not, to date, enabled the 
pyrrolidone carboxylyl peptidases to be purified to homogeneity from these 
various organisms. Peptide sequencing of them could consequently not be 
performed. It is found, moreover, that the molecular masses of PYRases 
vary greatly according to the microorganisms from which they originate. As 
an example, there may be mentioned Enterococcus faecium PYRase, which 
possesses a molecular mass of approximately 42 kDa, whereas Bacillus 
amyloliquefaciens PYRase possesses a molecular mass of approximately 24 
kDA. 
These enzymes, apart from their known peptidase activity, constitute a 
major criterion for the differentiation of enterobacteria (Mutczyk and 
Szewczuk, J. Gen. Microbiol, 1970, 61, 9-13), from staphylococci (Mulzcyk 
and Szewczuk, J. Gen. Microbiol., 1970, 70, 383-384), as well as an 
important factor in presumption of Group A streptococci and enterococci 
(Ellner et al., J. Clin. Microbiol., 1985, 22, 880-881). 
On studying the genetics of these enzymes, the Applicant discovered, 
surprisingly, that the nucleotide sequences coding for the PYRases are 
very different for each of the species of microorganism studied, and that, 
in addition, the genes coding for PYRases originating from different 
microbial organisms, though having very divergent nucleotide sequences, 
code, in fact, for proteins having very convergent peptide sequences. 
A considerable homology is, in effect, observed between the peptide 
sequences of PYRases extracted, for example, from Streptococcus pyogenes 
and from Bacillus subtilis. This discovery is of twofold interest. On the 
one hand, the production of specific antibodies directed towards the 
highly homologous peptide sequences of PYRases provides reagents capable 
of detecting all microbial species capable of synthesizing a PYRase. On 
the other hand, nucleotide probes comprising fragments of the gene coding 
for the PYRase of a particular microbial species constitute specific 
reagents for the species in question. 
The highly conserved peptide sequences present in the PYRases of various 
microbial species are those which correspond, in particular, to the 
peptides of formulae I to III given below. The subject of the present 
invention is hence a purified polypeptide containing at least one peptide 
sequence of at least six amino acids which is chosen from the peptide 
sequence shown in FIG. 3(SEQ ID NO:1), 5(SEQ ID NO:2), or 6(SEQ ID NO:3) 
possessing at least 40% homology with the peptide sequence shown in FIG. 
3, 5 or 6. 
The invention relates, in particular, to a purified polypeptide as defined 
above, characterized in that it possesses at least 20% identity with the 
peptide sequence shown in FIG. 3, 5 or 6. 
The invention relates especially to a polypeptide as defined above, 
characterized in that it contains at least one peptide sequence of at 
least six amino acids which is chosen from the following sequences: 
__________________________________________________________________________ 
Glu--Arg--Val--Ala--Ile--Asn--X--X--Asp--Ala--Arg--Ile--Pro--Asp--Asn-- 
X--Gly--X--Gln--Pro--Ile--Asp (SEQ ID NO: 4) (I); 
Gly--X--X--Ala--X--Val--Ser--X--Thr--Ala--Gly--Thr--Phe--Val--Cys--Asn-- 
X--X--X--Tyr (SEQ ID NO: 5) (II); 
Leu--X--Thr--Gly--Phe--X--Pro--Phe (SEQ ID NO: 6) (III); 
__________________________________________________________________________ 
in which sequences X is an amino acid. 
The meanings of X in the peptides I, II and III, respectively, are, in 
particular, given below for Z.sub.1, Z.sub.2, Z.sub.3 and Z.sub.4, and 
especially those which are readily identifiable in FIG. 9. 
The invention relates, in particular, to a polypeptide containing a peptide 
sequence of at least six amino acids which is chosen from the following: 
__________________________________________________________________________ 
Gly--Z.sub.1 --Z.sub.1 --Ala--X--Val--Ser--Z.sub.2 --Thr--Ala--Gly--Thr--P 
he--Val--Cys-- 
Asn--Z.sub.4 --Z.sub.1 --Z.sub.1 --Tyr (SEQ ID NO: 7) (IIa), and 
Leu--Z.sub.1 --Thr--Gly--Phe--Z.sub.3 --Pro--Phe (SEQ ID NO: 8) 
__________________________________________________________________________ 
(IIIa), 
in which sequences: 
Z.sub.1, each independently of one another, represents Ala, Val, Leu, Ile, 
Pro, Trp, Phe or Met; 
Z.sub.2, each independently of one another, represents Gly, Ser, Thr, Tyr, 
Gys, Ash or Gln; 
Z.sub.3, each independently of one another, represents Gly, Ser, Thr, Tyr, 
Cys, Ash, Gln, Ala, Asp or Glu; and 
Z.sub.4, each independently of one another, represents Gly, Ser, Thr, Tyr, 
Cys, Ash, Gln, Lys, Arg or His. 
The polypeptides of the invention can possess any number of amino acids 
greater than or equal to 6. The polypeptides of the invention are, in 
particular, the polypeptides having the formulae I to III, especially the 
polypeptides having the formulae Ia to IIIa, as well as the purified 
polypeptides or proteins containing at least the peptide sequence shown in 
FIG. 3, 5 or 6. The invention relates especially to a polypeptide having 
at least 40% homology with the polypeptide shown in FIG. 3, 5 or 6, as 
well as to a polypeptide having at least 20% identity, and in particular 
at least 30% identity, with one of the polypeptides of FIGS. 3, 5 and 6. 
The invention also encompasses peptide-protein conjugates in which a 
polypeptide as defined above is linked to an antigenic protein, either 
directly through a peptide bond or via a spacer arm. Coupling using a 
spacer arm is carried out according to known methods. Such conjugates may 
be used in the preparation of specific antibodies directed towards a 
polypeptide or a protein containing an amino acid sequence chosen from the 
polylpeptide sequences defined above, including those of formulae I to III 
(or IIa and IIIa). 
By administration of such conjugates to a host (mammal or bird), it is 
possible to obtain polyclonal antibodies capable of recognizing peptide 
sequences defined above, chosen, for example, from those of formulae I to 
III, and also to prepare, according to known methods, monoclonal 
antibodies specific for the peptide sequences. 
Such antibodies, which also form part of the invention, make it possible, 
in particular, to detect bacterial proteins having PYRase activity. For 
this detection, the antibodies are used according to conventional 
immunoassay techniques. Specifically, using two monoclonal antibodies each 
recognizing a different peptide chosen, in particular, from the peptide 
sequences of formulae I to III, it is possible to perform an immunological 
test of detection of a protein having PYRase activity according to the 
one-step sandwich technique. 
The subject of the invention is also a purified polynucleotide (nucleic 
acid fragment) coding for a polypeptide as defined above, including a 
polynucleotide coding for a peptide fragment chosen from the sequence of 
the peptides of formulae I to III (in particular Ia to IIIa), as well as a 
polynucleotide coding for a fragment of at least 6 amino acids which is 
chosen from the peptide sequence of FIG. 3, 5 or 6. The invention also 
relates to a fragment of a purified polynucleotide as defined above, this 
fragment having at least 10 nucleotides. The invention encompasses, in 
addition, the polynucleotides or fragments complementary to the 
polynucleotides or fragments which have just been obtained. 
The invention relates especially to such a polynucleotide, chosen from the 
nucleotide sequences shown in FIGS. 3, 5 and 6, coding, respectively, for 
S.pyogenes, B.subtilis and S.fluorescens PYRase. 
The invention encompasses all native or recombinant genes coding for an 
enzyme possessing PYRase activity whose protein homology is at least 40% 
and/or identity at least 20% with S.pyrogenes, S.fluorescens or B.subtilis 
PYRase. 
The subject of the invention is also a recombinant cloning or expression 
vector containing a polynucleotide as defined above, and a transformed 
living cell containing said recombinant vector, especially a cell which 
can be cultured in vitro, for example a bacterial cell. 
The subject of the present invention is also the use of a nucleic acid 
fragment (polynucleotide) coding for an enzyme having pyrrolidone 
carboxylyl peptidase activity, possessing at least one of the peptide 
sequences I to III (or IIa and IIIa), for the overproduction of this 
enzyme according to genetic techniques, using any suitable recombinant 
expression vector, in particular a plasmid, inserted into a host cell, by 
culturing the host cell thus transformed, or alternatively for any genetic 
construction, such as the construction of a "reporter" gene, in a cloning 
vector of any kind, using at least a portion of the nucleic acid fragment 
coding for this enzyme. The invention also relates to a DNA or RNA nucleic 
acid probe, characterized in that it contains a sequence of at least 10 
nucleotides (or at least 12 or at least 15 nucleotides) belonging to a 
polynucleotide according to the invention as has just been defined, or a 
sequence complementary to said sequence. Special mention may be made of 
the oligonucleotides referred to in the experimental part below. Such 
probes may be used as capture probes (for example bound to a solid support 
in a known manner, in particular by adsorbtion or covalently, or using a 
coupling agent), or may be linked to a label (or tracer agent), in a known 
manner, to constitute a detection probe, according to conventional 
hybridization techniques. Specifically, the probes containing nucleotide 
sequences chosen from those shown in FIGS. 3, 5 and 6 constitute specific 
detection probes for S.pyogenes, B.subtilis and S.fluorescens, 
respectively. 
These nucleic acid probes are used in a known manner in bacterial 
detection, identification or epidemiology. 
The subject of the invention is also a method for the detection of a gene 
coding for a pyrrolidone carboxylyl peptidase in a microorganism, using at 
least one nucleic acid probe as defined above. Such a method may be 
carried out according to conventional techniques, for example by sandwich 
hybridization. A nucleic acid probe bound to a support may be used as a 
capture probe, and a labeled probe may be used as a detection probe. 
As stated above, the cloning of a pcp gene coding for a PYRase is hence, in 
particular, of interest in making possible any form of overproduction of 
this enzyme, and exportation into the culture medium of said enzyme, onto 
which, for example, a signal sequence favoring protein excretion has been 
grafted by genetic engineering in a known manner. Recourse to the 
production of cloned enzymes thus makes it possible to decrease very 
considerably the operating costs of the processes of purification of the 
enzyme of interest. The invention hence enables pcp genes of various 
bacterial organisms to be cloned and sequenced, in particular in order to 
be able to overproduce a PYRase. This cloning may, for example, be carried 
out on the organisms for which the Applicant has demonstrated PYRase 
activity, namely: Enterococcus faecalis, Enterococcus faecium, 
Enterococcus durans, Enterococcus avium, Enterococcus gallinarum, 
Enterococcus malodoratus, Enterococcus suis, Enterococcus sp., Lactococcus 
lactis, Lactococcus sp., Streptococcus equisimilis, Streptococcus sanguis, 
Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus 
porcinus, Streptococcus salivarius, Streptococcus uberis, Streptococcus 
acidominimus, Streptococcus boris, Streptococcus sp., Aerococcus viridans, 
Aerococcus sp., Micrococcus kristinae, Micrococcus luteus, Micrococcus 
lylae, Micrococcus varians, Micrococcus sp., Stomatococcus mucilaginosus, 
Stomatococcus sp., Gemella haemolysans, Gemella morbillorum, Gemella sp., 
Pseudomonas fluorescens, Pseudomonas sp., Citrobacter freundii, 
Citrobacter sp., Enterobacter cloacae, Enterobacter amnigenus, 
Enterobacter aerogenes, Enterobacter liquefaciens Enterobacter sp., 
Klebsiella aerogenes, Klebsiella pneumoniae, Klebsiella edwardsii, 
Klebsiella ozaenae, Klebsiella rhinoscleromatis, Klebsiella oxytoca, 
Klebsiella cloacae, Klebsiella sp., Serratia marcescens, Serratia 
grimesii, Serratia sp., Staphylococcus aureus, Staphylococcus capitis, 
Staphylococcus chromogenes, Staphylococcus gallinarum, Staphylococcus 
epidermis, Staphylococcus hominis, Staphylococcus hyicus, Staphylococcus 
lentus, Staphylococcus saprophyticus, Staphylococcus schleiferi, 
Staphylococcus warneri, Staphylocococcus xylosus Staphylococcus 
lugdunensis, Staphylococcus simulans, Staphylococcus intermedius, 
Staphylococcus haemolyticus, Staphylococcus caprae, Staphylococcus 
carnosus, Staphylococcus cohnii, Staphylococcus kloosii, Staphylococcus 
sp., Neisseria mucosa, Neisseria sp., Bacillus megaterium, Bacillus 
subtilis, Bacillus cereus, Bacillus sp., Corynebacterium aquaticum, 
Corynebacterium bovis, Corynebacterium group A, Corynebacterium group ANF, 
Corynebacterium group B, Corynebacterium group G1, Corynebacterium group 
G2, Corynebacterium jeikeium, Corynebacterium kutscheri, Corynebacterium 
minutissimum, Corynebacterium pseudodiphteriticum, Corynebacterium 
striatum, Corynebacterium xerosis, Actinomyces pyogenes, Arcanobacterium 
haemolyticum, Brevibacterium sp., Erysipelothrix rhusiopathiae, 
Gardnerella vaginalis, Oerskovia spp., Rhodococcus equi. 
A few definitions of terms used in the present application are given below: 
PYRase means a native or recombinant protein possessing pyrrolidone 
carboxylyl peptidase activity which is expressed before or after 
post-translational modifications; pcp gene(s) means native or recombinant 
gene coding for pyrrolidone carboxylyl peptidase (PYRase) originating from 
bacterial organisms; 
"Reporter" gene corresponds to a coding unit whose expression product is 
readily assayable or detectable. The expression of a reporter gene makes 
it possible, in particular, to study the function or content of a nucleic 
acid sequence placed in an expression vector upstream of the gene, for 
example those of a promoter or operator; 
Consensus sequence corresponds to the ideal sequence in which each position 
represents the base most often encountered on comparison of several 
sequences; 
Underlined bases are the bases which correspond to the consensus sequences; 
Canonical promoter denotes the consensus sequence of the promoter; 
Transcripts are the products of DNA transcription; 
Nucleic acid fragment means DNA or RNA fragment; 
Homology relates to homologous amino acids, that is to say amino acids 
which have the same chemical properties such as polarity and/or 
hydrophobicity and/or basicity and/or acidity and/or neutrality. An amino 
acid is also considered to be homologous to another if their respective 
codons for the amino acid differ by only one base, termed degenerate; 
Identity refers to strictly identical amino acids; 
Cosmids refers to plasmids into which the Cos sites of phage lambda have 
been inserted; the resulting plasmid DNA can be encapsidated in vitro in 
phage particles; 
Plasmid means extrachromosomal circular DNA capable of replicating 
autonomously; Phagemid means plasmid into which an origin of replication 
of a phage has been inserted; the resulting DNA can be encapsidated in 
vivo in phage particles; inclusivity results means the set of bacterial 
strains leading to the obtaining of a PCR amplification product using the 
oligonucleotides employed, or of a hybridization signal with the pcp probe 
for S.pyogenes; 
Exclusivity results means the set of bacterial strains not leading to the 
obtaining of a PCR amplification product using the oligonucleotides 
employed, or of a hybridization signal with the pcp probe for S.pyogenes. 
According to the invention, the cloning vectors can be plasmids such as: 
pBR322, pUC18/pUC19, pUC118/pUC119, pSP64/pSP65, pGEM-3/pGEM-4, pGEM-37, 
AN3, pBluescript M13 or the like. The cloning vectors according to the 
present invention may also be chosen from cosmids such as pJB8, c2RB, 
pCos1EMb1, pHC79, pTM, pMCS, pNNL, pHSG274, pWE15, Charomid9 or the like. 
The cloning vectors of the present invention can, in addition, be 
prokaryotic expression vectors such as bacterial expression vectors, or 
yeast expression vectors, or eukaryotic expression vectors such as 
mammalian expression vectors. The host cells can be eukaryotic or 
prokaryotic cells. Preferably, the host cells are prokaryotic cells; such 
as bacteria. Advantageously, bacterial host cells which do not express 
PYRase activity, such as E. coli cells, are chosen, but it is also 
possible to use bacterial host cells originally possessing PYRase 
activity, after carrying out a mutogenisis resulting in the disappearance 
of the PYRase activity in these bacteria. It is thus possible to clone 
Pseudomonas fluorescens PYRase in a mutant of this bacterium lacking 
PYRase activity. Preferably, according to the invention, E. coli is used 
as host cell.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
A shows the electrophoretic separation on denaturing polyacrylemide gel of 
the proteins synthesized in vivo by E.col K38/pGP1-2 possessing plasmid 
pT7-5 (lane 1), pPC39 (lane 2), pT7-6 (lane 3) and pPC40 (lane 4). 
Staining of the proteins is carried out with Coomassie blue; the arrow 
indicates the expression product of approximately 25 kDa relative to the 
molecular weight markers (M) (Bethesda Research Laboratories) (trade 
mark). B represents the corresponding autoradiograph of the gel. The 
exclusive labeling of the overexpressed protein of interest of pPC39 
confirms the direction of transcription of the pcp gene shown in FIG. 1. 
Unless specified, all the methods relating to the experiments which are 
presented below were carried out according to Sambrook et al. (Molecular 
cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring 
Harbor, N.Y.). 
Example 1: cloning of the pcp gene coding for Streptococcus pyogenes PYRase 
A library of genes of Streptococcus pyogenes strain D471 (Group A of 
Lancefield, type 6 protein M) (Scott and Fischetti, Science, 1983, 221, 
758-760), which possesses PYRase activity, was constructed in Escherichia 
coli strain C600NR (.lambda.cI857) (Scott and Fischetti, Science, 1983, 
221, 758-760). S. pyogenes chromosomal DNA, extracted according to the 
method of Chassy (Biochem. Biophys. Res. Comm., 1976, 68, 603-608), was 
partially digested with the restriction enzyme Mbo I in order to obtain a 
random series of fragments ranging from 30 to 50 kilobases (kb). The 
latter were inserted into cosmid pJB8 (Ish-Horowicz and Burke, Nucl. Acids 
Res., 1981, 9, 2989-2998) linearized by complete digestion using the 
endonuclease Bam HI. These recombinant vectors were introduced in vitro 
inside phage .lambda. capsids (Hohn and Collins, Gene, 1980, 11, 291-298) 
using a Gigapack plus kit (Stratagene, trade mark). The resulting phages 
were used to transduce the strain C600NR, and bacterial colonies 
possessing the ampicillin resistance conferred by the cosmid were selected 
at 30.degree. C. 
These bacterial colonies were screened in situ on agar medium using the 
pyrrolidone carboxylyl peptidase test (Mulczyk and Szewczuk, J. Gen. 
Microb., 1970, 61, 9-13). Extraction of the recombinant plasmids from 
Escherichia coli clones possessing PYRase activity enabled plasmid pPC10 
as shown FIG. 1, containing a 5-kb chromosomal DNA fragment of S. 
pyogenes, to be isolated. Introduction of pPC10 into E. coli JM83 bacteria 
(Yannish-Perron et aI., Gene, 1985, 33,103-109) led to the production of 
transformants systematically possessing PYRase activity, confirming that 
the isolated DNA segment actually codes for this enzyme (Cleuziat et al., 
Mol. Microbiol., 1992, 6, 2051-2063). 
Example 2: subcloning of the pcp gene coding for Streptococcus pyogenes 
PYRase 
Different endonucleases were used to establish a restriction map of the DNA 
fragment of interest contained in pPC10 (FIG. 1). This made it possible to 
localize more accurately the DNA region coding for the PYRase. The main 
steps in this subcloning are summarized in FIG. 1. Isolation and insertion 
of the Eco RI-Ava I fragment of pPC10 into the Eco RI-Sal I sites of the 
vector pBluescript KS.sup.+ (trade mark, Stratagene), leading to plasmid 
pPC20, permits detection of the PYRase activity in E. coli strain NM522 
(Gough and Murray, J. Mol. Biol., 1983, 166, 1-19) transformed with this 
plasmid. According to the diagram of FIG. 1, plasmids pPC20.01, pPC20.02, 
pPC20.03, pPC30 and pPC36 were prepared and studied successively. The 
smallest DNA region conferring PYRase activity consists of a 1.3-kb Spe 
I-Hpa I restriction fragment (pPC36), the restriction map of which is 
detailed in FIG. 2. 
Example 3: sequencing of the Spe I--Hpa I restriction fragment of pPC36 
The complete nucleotide sequence of the two complementary strands of the 
Spe I--Hpa I fragment of plasmid pPC36 was determined by the chain 
termination method (Sanger et al., Proc. Natl. Acad. Sci. USA, 1977, 74, 
5463-5467), according to the strategy described in FIG. 2, on 
double-stranded DNA templates. This sequence, described in FIG. 3, 
discloses the presence of a single open reading frame of 645 nucleotides. 
This open reading frame, the beginning of which is situated at the ATG 
initiation codon at position 285 and which terminates at the TAAstop codon 
located at position 930, codes for a 23135-Da protein composed of 215 
amino acids and possessing a statistical isoelectric point of 7.3. 
Example 4: analysis of the nucleotide sequence 
This gene coding for pyrrolidone carboxylyl peptidase was designated pcp. 
It is preceded by a ribosome binding site (AAAGGA), the location and 
sequence of which are comparable to those described in other Gram-positive 
organisms (Moran et al., Mol. Gen. Genet., 1982, 186, 339-386). Two 
promoters (Pa and Pb) preceding the coding frame of pcp (FIG. 3) have been 
identified (Cleuziat, Doctorate es Sciences Thesis: Institut National des 
Sciences Appliquees de Lyon, 1992, 157 p). 
Example 5: analysis of the RNA transcripts 
By carrying out primer extension experiments, it was possible to localize 
the 5' end of the pcp transcript, corresponding to the transcription 
start-site of this gene in the recombinant strain E. coli NM522/pPC30. For 
this purpose, the total RNA of this bacterium was extracted according to a 
method described by Shimotsu et al., (J. Bacteriol., 1986, 166, 466-471). 
The oligonucleotide YM1 (.sup.5' dAATAGCTTCGCCGCCAAAGGGATCAAAGCC.sup.3') 
(SEQ ID NO:15), complementary to nucleotides 303 to 332 (FIG. 3), was 
labeled at its 5' end with [.gamma.-.sup.32 P]-ATP (6000 Ci/mmol) with T4 
polynucleotide kinase and hybridizedunder stringent conditions with the 
total RNA. Its extension with reverse transcriptase (FIG. 2) enabled two 
cDNAs to be detected, differing from one another by only one nucleotide 
and corresponding to a start of transcription situated on the G at 
position 257 or the T at position 258 (FIG. 3). The first position proves 
to be more in agreement with the fact that transcription generally starts 
on a purine base (A, G) and occasionally on a pyrimidine base (C, T) 
(Rosenberg and Court, Ann. Rev. Genet., 1979, 13, 319-353). 
Two inverted repeat sequences were identified on the nucleotide sequence 
(FIG. 3). The shorter, situated within the A/T-rich region and partially 
overlapping the promoter sequences, constitutes a potential binding site 
for a regulatory protein (Gicquel-Sanzey and Cossart, EMBO J., 1982, 1, 
591-595). The longer, situated 25 bases downstream of the TGA stop codon 
of pcp, is involved in the termination of transcription of pcp (Rosenberg 
and Court, Ann. Rev. Genet., 1979, 13, 319-353). Determination of the 3' 
end of the pcp transcript by the S1 nuclease mapping technique enabled the 
stem-loop structure responsible for the termination of transcription to be 
identified within this inverted repeat sequence. An Afl III-Ssp I 
restriction fragment of plasmid pPC36 (FIG. 2) was labeled at its 3' end 
using [.alpha.-.sup.32 P]-dCTP (3000 Ci/mmol) with the Klenow fragment of 
Escherichia coli DNA polymerase I. This probe of 353 nucleotides, 
overlapping the coding region over a zone of 21 bases (FIG. 3), was 
hybridized with the total RNA extracted from the recombinant E. coli 
bacterium obtained by transformation using plasmid pPC30. The whole was 
then subjected to the action of S1 nuclease according to defined 
conditions (Brakhage et al., Biochimie, 1990, 72, 725-734) in order to 
digest any single-stranded DNA fragment which is not protected by 
hybridization with the mRNA. Determination of the size of the fragments 
obtained consecutively under these conditions enabled it to be 
demonstrated that transcription stops between nucleotides 981 and 986 
included in the nucleotide sequence (FIG. 3). 
Example 6: Determination of the copy number of the pcp gene within the 
Streptococcus pyogenes genome 
Hybridization experiments according to Southern's method showed that the S. 
pyogenes genome comprises only one copy of the pcp gene. To do this, the 
DNA of this organism, extracted according to the method of Chassy 
(Biophys. Res. Comm., 1976, 68, 603-608), was digested with the 
restriction endonucleases Hpa I and Bgl II, which do not cut the pcp gene, 
and subjected to agarose gel electrophoresis. Transfer of the genetic 
material was carried out by capillarity onto a nylon membrane before 
hybridization with a double-stranded DNA probe, synthesized by 
polymerization chain reaction (PCR), comprising the larger part of the 
coding region of the pcp gene. PCR was carried out as described by Saiki 
et al., (Science, 1988, 239, 487-491) on a double-stranded plasmid DNA 
template (pPC30) using AmpliTaq (Perkin Elmer Cetus, trade mark), 
employing the oligonucleotides YR2 (.sup.5' dACAGGCTTTGATCCCTTTGG.sup.3') 
(SEQ ID NO:16) and YU5 corresponding to positions 300 to 319 and 
complementary to 759 to 778 of the sequence, respectively (FIG. 3). This 
479-base pair fragment was purified on agarose gel and radioactively 
labeled with [.alpha.-.sup.32 P]-dCTP (3000 Ci/mmol), using the "random 
priming" kit (Boehringer, trade mark). The hybridization and washing 
conditions, termed stringent, were carried out in a known manner in order 
to permit hybridization of the pcp probe only with a completely homologous 
DNA target. To this end, a control was set up by double digestion of 
plasmid pPC20 with the endonucleases Hpa I and Bgl II. FIG. 4 shows that a 
positive hybridization signal was obtained for the 1.6-kb fragment 
containing the pcp gene, while the 4-kb fragment corresponding to the 
cloning vector and to the DNA regions of S. pyogenes flanking pcp did not 
disclose any signal. The presence of a single hybridization signal in the 
case of each digestion of DNA confirms that pcp is a single-copy gene in 
the S. pyogenes genome. The size of the bands observed (6.8 kb and 4.1 kb) 
is in complete agreement with the restriction map of the DNA region coding 
for the PYRase. 
The pcp gene did not disclose any homology with the nucleotide and protein 
sequences present in the databases (Genbank, version No. 68 of Jun. 15, 
1991). 
Example 7: analysis and comparison of pcp genes originating for different 
bacterial species 
The work performed on the Streptococcus pyogenes pcp gene was extrapolated 
to other bacterial organisms possessing PYRase activity. 
From a library of Bacillus subtilis genes constructed in Escherichia coli 
in plasmid pMK4 (Debarbouille et al., FEMS Microbiol. Lett. 1987, 41, 
137-140), the cloning and molecular characterization of the Bacillus 
subtilis pcp gene were carried out according to the techniques described 
above for S. pyogenes. The sequence of the coding portion of this gene 
together with that of the protein for which it codes are indicated in FIG. 
5. The B. subtilis pcp gene possesses a length of 645 nucleotides and 
codes for a protein of 215 amino acids of theoretical molecular weight 
equal to 23777 Da (Awade et al., FEBS Lett., 1992, 305, 67-73). 
No homology of nucleotide or protein sequence could be determined by 
comparison with the databases of existing sequences. Similarly, there is 
very little homology between the S. pyogenes pcp gene and that of B. 
subtilis, although these genes are both composed of exactly 645 
nucleotides. FIG. 7 shows that, despite a complete absence of homology 
from the nucleotide standpoint (B), the corresponding proteins are 
extremely conserved (A). The primary structure of the PYRases of S. 
pyogenes and of B. subtilis were compared (FIG. 8): the alignment of these 
two proteins is based on the "BestFit" program (University of Wisconsin) 
using the algorithm of Devereux et al., (Nucl. Acid Res., 1984, 12, 
387-395). The two proteins contain the same number of amino acids (215 
residues). Their total sequences possess 47.5% identity and greater than 
66% homology, indicating a very high degree of conservation of these 
proteins. Some zones can have up to 80% identity and greater than 87% 
homology, as is the case with the unit of 41 amino acids 
Gln-Pro-Asp-Ala-Val-Leu-Cys-Ile-Gly-Gln-Ala-Gly-Gly-Arg-Thr-Gly-Leu-Thr-Pr 
o-Glu-Arg-Val-Ala-Ile-Asn-Gln-AsP-AsP-Ala-Arg-Ile-Pro-Asp-Asn-Glu- 
Gly-Asn-Gln-Pro-Ile-Asp (SEQ ID NO:17) (residues 59 to 99 inclusive) of S. 
pyogenes PYRase. The regions situated in the vicinity of the cysteine 
residues (S. pyogenes Cys 65, Cys 141 and Cys 156) are also highly 
conserved, in particular the units 
Gly-Leu-Pro-Ala-Ser-Val-Ser-Asn-Thr-Ala-Gly-Thr-Phe-Val-Cys-Asn-His-Leu-Me 
t-Tyr (SEQ ID NO:18) (residues 129 to 146 inclusive) (75% identity, 85% 
homology) and Cys-pro-Asn-Ala-Lys-Ala-Gly-Phe-Met-His-Ile-Pro-Phe-Met (SEQ 
ID NO:19) (residues 156 to 169 inclusive) (42% identity and 93% homology). 
Other, shorter regions possess large homologies: the fragments of 11 amino 
acids Lys-Ile-Leu-Val-Thr-Gly-Phe-Asp-Pro-Phe-GlY and 
Glu-Val-Pro-Thr-Val-Phe (SEQ ID NO:20) (residues 2 to 12 inclusive) (72% 
identity, 100% homology) and of 6 amino acids (residues 39 to 44 
inclusive) (66% identity, 100% homology). 
Moreover, following the construction of a library of genes of Pseudomonas 
fluorescens strain A32 (Gugi et al., J. Bacteriol., 1991, 173, 3814-3820) 
in the host Escherichia coli DH5.alpha. in plasmid pUC19, the cloning and 
molecular characterization of the Pseudomonas fluorescens pcp gene were 
also carried out according to the techniques described above for S. 
pyogenes. The sequence of the coding portion of this gene together with 
that of the protein for which it codes are indicated in FIG. 6. The 
Pseudomonas fluorescens pcp gene possesses a length of 639 nucleotides and 
codes for a protein of 213 amino acids of 22436 Da. 
No homology of nucleotide or protein sequence could be determined by 
comparison with the databases of existing sequences. Similarly, there is 
very little homology between the Pseudomonas fluorescens pcp gene and 
those of S. pyogenes and B. subtilis, although these genes are composed of 
an almost identical number of nucleotides. FIG. 7 shows that, despite a 
complete absence of homology from the nucleotide standpoint (B), the 
corresponding proteins are extremely homologous (A). The PYRase of P. 
fluoroscens possesses globally 47% identity and 66% homology with that of 
B. subtilis, and 39% identity and 62% homology with that of S. pyogenes. 
The structure of the bacterial PYRases hence appears to be highly 
conserved from the peptide standpoint, although the nucleotide sequences 
coding for the corresponding peptides are very divergent. 
Determination of the optimal alignment using the program mentioned above 
from the three PYRases characterized makes it possible to bring out the 
major peptide units of these enzymes (FIG. 9). Three highly conserved 
units, included in those determined above between B. subtilis and S. 
pyogenes (FIG. 8), are clearly seen (FIG. 9), from which the consensus of 
identity (bold character) may be deduced. With respect to the numbering of 
S. pyogenes PYRase, the units involved are Leu-Val-Thr-Gly-Phe-Asp-Pro-Phe 
(residues 4 to 11 inclusive) (75% identity and 100% homology), 
Glu-Arg-Val-Ala-Ile-Asn-Gln-Asp-Asp-Ala-Arg-Ile-Pro-Asp-Asn-Glu-Gly-Asn-Gl 
n-Pro-Ile-Asp (SEQ ID NO:21) (residues 78 to 99 inclusive) (82% identity 
and 86% homology) and Val-Ser-Asn-Thr-Ala-Gly-Thr-Phe-Val-Cys-Asn (SEQ ID 
NO:22) (residues 132 to 142 inclusive) (91% identity and 100% homology). 
This unit, the most highly conserved of all, contains the cystein residue 
common to the three PYRases (Streptococcus pyogenes Cys 141). It is 
probably the one involved in the catalytic site of this family of enzymes. 
Example 8: detection of pyrrolidone carboxylyl peptidase by antibodies 
Polyclonal antibodies specifically directed towards one of the peptides 
Gln-Pro-Asp-Ala-Val-Leu-Cys-Ile-Gly-Gln-Ala-Gly-Gly-Arg-Thr-Gly-Leu-Thr-Pr 
o-Glu-Arg-Val-Ala-Ile-Asn-Gln-AsP-AsP-Ala-Arg-Ile-Pro-Asp-Asn-Glu- 
Gly-Asn-Gln-Pro-Ile-Asp (SEQ ID NO:23), 
Gly-Leu-Pro-Ala-Ser-Val-Ser-Asn-Thr-Ala-Gly-Thr-Phe-Val-Cys-Asn-His-Leu-Me 
t-Tyr (SEQ ID NO:24), 
Cys-Pro-Asn-Ala-Lys-Ala-Gly-Phe-Met-His-Ile-Pro-Phe-Met (SEQ ID NO:25), 
Lys-Ile-Leu-Val-Thr-Gly-Phe-Asp-Pro-Phe-Gly (SEQ ID NO:26) and 
Glu-Val-Pro-Thr-Val-Phe described above in Example 7 were produced. To do 
this, these peptides, previously synthesized and purified, were separately 
grafted onto the protein KLH (high molecular weight immunogenic molecule): 
5 mg of protein and 5 mg of peptide are brought into contact in a volume 
of 2 ml of 50 mM phosphate buffer pH 7.5, and coupling is carried out by 
adding 1 ml of 20 mM glutaraldehyde. The conjugates obtained were dialyzed 
overnight against PBS buffer. Rabbits were then immunized by intramuscular 
injection of 1 ml of the solution obtained above, added to 1 ml of 
Freund's complete adjuvant. A booster was performed one month after the 
initial injection, and a volume of blood of 20 ml was withdrawn one week 
after this booster. After coagulation, the serum fraction is filtered and 
immunoglobulins are purified on a Sepharose protein A affinity column 
(Pharmacia, trade mark) according to the protocol recommended by the 
supplier. The specificity of the antibodies obtained was tested by 
conventional immunological detection methods (ELISA). For this purpose, 
each peptide described above, the immunogenic molecule KLH and bovine 
serumalbumin (BSA) were adsorbed individually on the wells of 
microtitration plates (Nunc, trade mark). By bringing each category of 
purified polyclonal antibodies into contact, and detecting the latter with 
goat anti-rabbit antibodies coupled to peroxidase (Sigma, trade mark), it 
was shown that the antibodies obtained recognize specifically and 
exclusively the peptides with which the immunization was performed. No 
cross-reaction was observed between, on the one hand each category of 
antibodies, and on the other hand the other peptides which were not used 
for obtaining them. The antibodies obtained also made it possible to 
detect pyrrolidone carboxylyl peptidase in Streptococcus pyogenes, 
Bacillus subtilis and also other bacteria possessing such an enzymatic 
activity. Since this enzyme is located in the intracellular compartment, 
its detection in the microorganisms involved prior lysis of the cells by 
physical or chemical means. In this case, the lysis was carried out by 
disintegration of the cell membranes under the action of ultrasound 
(sonication). By taking up and washing the sonicate in 50 mM phosphate 
buffer pH 7.5, the presence of the enzyme could be determined by the 
immunological method described above. No positive reaction could be 
observed using organisms not possessing enzymatic activity, confirming the 
specificity of the purified antibodies. These results hence confirm the 
conservation and specificity of the peptide units studied within the 
pyrrolidone carboxylyl peptidases (PYRases) originating from 
microorganisms. 
Example 9: detection of Streptococcus pyogenes (Group A .beta.-hemolytic 
streptococci) 
The detection of Streptococcus pyogenes is of very great importance in 
clinical bacteriology, since this organism is the source of numerous and 
varied pathologies in man. Group A .beta.-hemolytic streptococci, which 
correspond to the species Streptococcus pyogenes, can be responsible for 
many disorders such as pharyngitis, sore throat, sinusitis, osteomyelitis, 
cellulitis and various skin disorders, endocarditis, meningitis, etc. They 
are known above all to be responsible for scarlet fever, the two main 
secondary manifestations of which are acute rheumatic fever (ARF) and 
acute glomerulonephritis (AGN) (Delmas and Freney, Lyon Pharmaceutique, 
1989, 40, 353-369). Some of these pathologies can be lethal (Bartter et 
al., Arch. Intern. Med., 1987, 148, 1421-1424). 
At present, the identification of Group A streptococci in medical 
microbiology involves study of hemolysis on blood agar, testing for a 
Lancefield Group A antigen or for physiological and biochemical characters 
(API 20 STREP identification gallery, marketed by the company bioMerieux). 
The need to identify the streptococci responsible for acute infections 
rapidly has led to the development of agglutination techniques (Malbrunot 
et al., Pathologie Biologie, 1990, 35, 665-668) permitting the direct 
recognition of Group A antigens (Slidex-strepto kit, BioMerieux) from the 
primary culture colonies. These methods can nevertheless make it 
obligatory to perform enrichment and isolation steps sometimes 
necessitating up to 48 hours, and they possess limits from the standpoint 
of sensitivity. 
The use of DNA probes decreases the time needed for establishment of the 
diagnosis by direct and sensitive detection of microbes from biological 
samples. In effect, DNA probes may be used for the detection of particular 
organisms in biological samples as described in U.S. Pat. No. 4,358,535 in 
the name of Falkow et al. Probes for Group A streptococci have already 
been proposed, such as oligonucleotides for the type 1 protein M gene of 
Streptococcus pyogenes (Podbielski et al., Med. Microbiol. Immunol. 1990, 
179, 255-262) or the type A exotoxin gene (Yu and Ferretti, Infect. 
Immun., 1989, 57, 3715-3719). These probes are, however, specific for 
targets which define several subgroups within Group A streptococci, and 
hence do not make it possible to detect the whole of the species; hence 
there does not exist at present a DNA probe for the detection of 
Streptococcus pyogenes. 
The feasability and specificity of detection or of identification of Group 
A streptococci has been demonstrated below using an experimental protocol 
employing PCR or Southern hybridization. 
a) study by PCR 
Different pairs of primers covering the region of the pcp gene were tested 
in respect of the specificity of obtaining an amplified fragment from 
genomic DNA of various bacteria. These pairs of primers are shown in Table 
1 below. 
TABLE 1 
__________________________________________________________________________ 
Pairs of oligonucleotides used in PCR 
Pair.sup.a Name 
Position.sup.b 
Size of the product.sup.c 
__________________________________________________________________________ 
1: ACAGGCTTTGATCCCTTTGG (SEQ ID NO: 16) 
YR2 300-319 
2: TTCTGGCGTTAGTCCAGTCC (SEQ ID NO: 27) 
YU3 499-518 
219 
1: ACAGGCTTTGATCCCTTTGG (SEQ ID NO: 16) 
YR2 300-319 
2: TATCAGGAATGCGAGCATCG (SEQ ID NO: 24) 
YU8 539-558 
259 
1: ACAGGCTTTGATCCCTTTGG (SEQ ID NO: 16) 
YR2 300-319 
2: TGCATAAACCCAGCTTTGGC (SEQ ID NO: 29) 
YU5 759-778 
479 
1: TTGCCAGCAACCATTCATGG (SEQ ID NO: 30) 
YR4 360-379 
2: AGAAACAGAAGCAGGAAGCC (SEQ ID NO: 31) 
YU9 664-683 
324 
1: TTGCCAGCAACCATTCATGG (SEQ ID NO: 30) 
YR4 360-379 
2: TGCATAAACCCAGCTTTGGC (SEQ ID NO: 29) 
YU5 759-778 
419 
1: AAAATCTGCCGATGTGCTCC (SEQ ID NO: 32) 
YR5 419-438 
2: AGAAACAGAAGCAGGAAGCC (SEQ ID NO: 31) 
YU9 664-683 
265 
1: AAAATCTGCCGATGTGCTCC (SEQ ID NO: 32) 
YR5 419-438 
2: TGCATAAACCCAGCTTTGGC (SEQ ID NO: 29) 
YU5 759-778 
360 
1: CTTTGTATTGGGCAAGCTGG (SEQ ID NO: 33) 
YR6 474-4 93 
2: AGAAACAGAAGCAGGAAGCC (SEQ ID NO: 31) 
YU9 664-683 
210 
1: CTTTGTATTGGGCAAGCTGG (SEQ ID NO: 33) 
YR6 474-493 
2: TACTGGCAAACTATACCCGC (SEQ ID NO: 34) 
YU4 1075-1094 
621 
1: TGCTCGCATTCCTGATAACG (SEQ ID NO: 35) 
YR7 542-561 
2: TGCATAAACCCAGCTTTGGC (SEQ ID NO: 29) 
YU5 759-778 
237 
1: TGCTCGCATTCCTGATAACG (SEQ ID NO: 35) 
YR7 542-561 
2: TACTGGCAAACTATACCCGC (SEQ ID NO: 34) 
YU4 1075-1094 
553 
1: ATCAAAGCGATGGTTGCTGC (SEQ ID NO: 36) 
YR8 630-649 
2: TACTGGCAAACTATACCCGC (SEQ ID NO: 34) 
YU4 1075-1094 
465 
1: ATCAAAGCGATGGTTGCTGC (SEQ ID NO: 36) 
YR8 630-649 
2: CCCTATCTCAATGCTTAACG (SEQ ID NO: 37) 
YU1 1297-1316 
687 
1: TCAAAAATCTGCCGATGTGC (SEQ ID NO: 38) 
YF1 416-435 
2: AGGAAGCCCAGCCTGATGAA (SEQ ID NO: 39) 
YF2 652-671 
256 
__________________________________________________________________________ 
.sup.a sense oligonucleotide (corresponding to the sequence); 2: antisens 
oligonucleotide (reverse and complementary to the sequence). The 
nucleotide sequence is indicated from the 5' end to the 3' end 
.sup.b the numbering corresponds to that of the sequence described in FIG 
3. 
.sup.c the size of the amplification product is indicated in base pairs. 
The bacteria studied (international collection strains and strains of 
clinical origin) were isolated and cultured on suitable agar medium. 
A 5 ml ampoule of heart-brain broth is inoculated with a bacterial clone 
originating from a 12-hour culture on agar medium, and is incubated at 
37.degree. C. under conditions appropriate to each organism. The 
concentration of the bacteria in the exponential growth phase is 
determined by measuring the optical density at 590 nm, and a volume 
corresponding to a quantity of 10.sup.8 bacteria (approximately 100 .mu.l) 
is centrifuged for 5 minutes at 14,000 rpm in a sterile microtube. The 
bacterial pellet is washed with 200 .mu.l of physiological saline (0.9% 
NaCl), centrifuged again and resuspended in 100 .mu.l of Tween 20 buffer 
(0.45% in sterile distilled H.sub.2 O). The sample is then heated to 
100.degree. C. for 5 minutes) in order to lyse the bacteria, and then 
cooled on ice or frozen for subsequent use. 10 .mu.l of bacterial lysate, 
equivalent to the DNA originating from approximately 10.sup.7 bacteria, 
are used per PCR test. 
The composition of the reaction mixture used for the PCR is strictly that 
described by Saiki et al., (Science, 1988, 239, 487-491). The reaction was 
carried out using AmpliTaq (Perkin Elmer Cetus, trade mark) on a Thermal 
Reactor apparatus (Hybaid, trade mark) according to the following 
temperature cycle: 
(92.degree. C., 5 min) 
[(60.degree. C., 30 sec), (75.degree. C., 30 sec), (95.degree. C., 15 sec)] 
40 times 
(60.degree. C., 30 sac) 
(75.degree. C., 10 min) 
1/10th of the amplification product (10 .mu.l) is analyzed by 
electrophoresis on 2% agarose gel relative to molecular weight markers. 
No difference could be observed regarding the specificity of the 
oligonucleotide pairs used for the amplification of a DNA fragment from 
the bacterial genome: the specificity results obtained for each bacterial 
strain studied were the same irrespective of the pair used. 
The quantitative results nevertheless vary slightly according to the pair 
studied. The oligo-nucleotide pair YF1-YF2 is the one which enabled the 
best amplification yields to be obtained. These oligonucleotides prove 
especially advantageous from the standpoint of sensitivity of detection of 
the microorganisms by molecular hybridization techniques. The qualitative 
results described for YF1-YF2 in Tables 2 and 3 below, collating, 
respectively, the bacterial species which did or did not give a positive 
amplification signal, can be fully extrapolated to the other pairs of 
oligonucleotides. 
TABLE 2 
__________________________________________________________________________ 
Inclusivity results 
Hybrid- 
Inter- ization VIDAS 
LRA national 
Bacterial PYRase 
pcp PCR ampli- 
detec- 
Collection 
collection 
species activity 
probe* 
fication** 
tion*** 
__________________________________________________________________________ 
JRS4 Streptococcus pyogenes M6 
+ + + 2306 
77-01-085 
NCTC 8191 
Streptocoque groupe A 
+ + + 693 
84-05-026 Streptocoque groupe A 
+ + + 519 
77-01-083 
NCTC 10879 
Streptococcus pyogenes 
+ + + 981 
89-11-070 Streptococcus pyogenes 
+ + + 704 
89-11-071 Streptococcus pyogenes 
+ + + 3153 
83-02-114 Streptocoque groupe A 
+ + + 5707 
89-11-070 Streptocoque groupe A 
+ + + 6287 
78-06-155 Streptococcus pyogenes 
+ + + 2682 
76-11-002 
ATCC 12202 
Streptococcus pyogenes 
+ + + 1929 
77-01-028 
ATCC 12203 
Streptococcus pyogenes 
+ + + 2013 
77-01-029 
NCTC 10085 
Streptococcus pyogenes 
+ + + 2527 
77-01-030 
NCTC 8306 
Streptococcus pyogenes 
+ + + 1991 
__________________________________________________________________________ 
*Hybridization results obtained using the doublestranded DNA probe 
corresponding to the coding frame of the Streptococcus pyrogenes pcp gene 
**Results of PCR amplification using the pair of primers YF1YF2 
***Results of enzymatic (alkaline phosphatase) activity following capture 
and detection of the amplification products 
TABLE 3 
__________________________________________________________________________ 
Exclusivity results 
Hybrid- 
Inter- ization VIDAS 
LRA national 
Bacterial PYRase 
pcp PCR ampli- 
detec- 
Collection 
collection 
species activity 
probe* 
fication** 
tion*** 
__________________________________________________________________________ 
CG110 Enterococcus faecalis 
+ - - 5 
83-10-064 Enterococcus faecalis 
+ - - 5 
89-10-032 Enterococcus faecium 
+ - - 4 
84-07-114 Enterococcus durans 
+ - - 6 
89-10-033 Enterococcus avium 
+ - - 7 
87-12-068 Enterococcus gallinarum 
+ - - 6 
84-10-087 Enterococcus malodoratus 
+ - - 8 
89-09-060 Enterococcus suis I 
+ - - 6 
90-02-014 Streptococcus uberis 
- - - 7 
86-03-031 Streptococcus equisimilis 
+ - - 9 
76-11-006 
ATCC 35666 
Streptococcus equisimilis 
- - - 5 
89-04-053 
ATCC 12401 
Streptococcus agalactiae 
- - - 8 
78-11-148 
NCTC 10234 
Streptococcus suis II 
- - - 7 
80-02-036 
NCTC 7864 
Streptococcus sanguis II 
+ - - 5 
77-01-036 
NCTC 6177 
Streptococcus zooepidemicus 
- - - 5 
84-11-031 Streptococcus salivarius 
- - - 9 
85-10-113 Streptococcus pneumoniae 
+ - - 8 
78-04-060 
NCTC 7465T 
Streptococcus pneumoniae 
- - - 5 
89-04-053 Group B streptocoque 
- - - 7 
77-01-032 
NCTC 9828 
Group B streptocoque 
- - - 5 
77-09-006 
NCTC 10228 
Group E streptocoque 
- - - 6 
86-12-029 Group G streptocoque 
- - - 9 
77-01-039 Group G streptocoque 
- - - 8 
77-01-038 
NCTC 9603 
Group G streptocoque 
- - - 8 
Aerococcus viridans 
+ - - 9 
89-10-001 Aerococcus viridans 
+ - - 11 
78-11-159 Aerococcus viridans 
+ - - 8 
75-15-060 
ATCC 25571 
Micrococcus kristinae 
+ - - 10 
89-08-091 Lactococus lactis ssp. cremoris 
- - - 12 
89-09-022 Lactococcus lactis ssp. lactis 
- - - 7 
89-06-176 Stomatococcus mucilaginosus 
+ - - 8 
89-01-032 Listeria sp. - - - 15 
C304 Corynebacterium glutamicum 
- - - 
89-04-074 Pseudomonas fluorescens 
+ - - 10 
89-06-024 Psuedomonas fluorescens 
+ - - 7 
89-06-025 Pseudomonas fluorescens 
- - - 9 
88-06-067 Shigella sp. - - - 10 
P4X Escherichia coli K-12 
- - - 8 
83-09-123 Escherichia vulneris 
- - - 10 
73-08-010 
ATCC 8090 
Citrobacter freundii 
+ - - 4 
76-03-117 Citrobacter freundii 
+ - - 6 
73-08-013 
ATCC 13047 
Enterobacter cloacae 
+ - - 6 
75-08-036 
ATCC 13048 
Enterobacter aerogenes 
+ - - 6 
89-09-015 Enterobacter aerogenes 
+ - - 8 
81-09-011 
ATCC 33072 
Enterobacter amnigenus 
+ - - 7 
85-05-027 Klebsiella pneumoniae 
+ - - 7 
75-01-109 Klebsiella oxytoca 
+ - - 10 
75-09-007 
ATCC 810 
Serratia marcescens 
+ - - 10 
85-01-018 
ATCC 29909 
Serratia grimesii 
+ - - 10 
75-09-008 
ATCC 25923 
Staphylococcus aureus 
+ - - 9 
87-12-083 
ATCC 43809 
Staphylococcus lugdenensis 
+ - - 7 
75-15-313 
ATCC 27848 
Staphylococcus simulans 
+ - - 8 
82-02-100 
ATCC 29663 
Staphylococcus intermedius 
+ - - 9 
75-15-306 
ATCC 29970 
Staphylococcus haemolyticus 
+ - - 10 
90-04-012 
ATCC 13102 
Neisseria meningitidis 
- - - 8 
76-11-014 Neisseria mucosa 
+ - - 8 
86-06-009 
ATCC 19695 
Neisseria mucosa 
- - - 10 
87-12-074 Bacillus megaterium 
+ - - 8 
78-02-084 
NCTC 10400 
Bacillus subtilis 
+ - - 6 
78-02-085 
NCTC 10320 
Bacillus cereus 
+ - - 15 
__________________________________________________________________________ 
*Hybridization results obtained using the doublestranded DNA probe 
corresponding to the coding frame of the Streptococcus pyogenes pcp gene 
****results of PCR amplification using the pair of primers YF1YF2 
***Results of enzymatic (alkaline phosphatase) activity following capture 
and detection of the amplification products 
Tables 2 and 3 giving, respectively, the inclusivity and exclusivity 
results show that the proposed oligonucleotides systematically and 
exclusively detect Group A streptococci (Streptococcus pyogenes). The 
diversity and location of the oligonucleotides on the coding portion of 
the gene and on its flanking regions prove that the region of the pcp gene 
is a region of the genome which is very specific to this bacterial species 
and hence favorable to the development of DNA probes for its detection in 
biological samples. 
These tables demonstrate that the presence of a hybridization signal is 
independent of that of a PYRase phenotype, since bacteria other than S. 
pyogenes possessing this activity do not interfere in the detection of 
this microbe. 
This amplification product of 256 base pairs obtained using the 
oligonucleotide pair YF1-YF2 may be visualized extremely rapidly and 
sensitively by an automated non-radioactive detection system based on 
sandwich hybridization (Dunn and Hassel, Cell, 1977, 12, 23-36). This 
system employs two non-overlapping oligonucleotides which correspond to 
the same DNA strand of the amplified fragment; the first, bound to a solid 
phase, is a capture probe for the amplification product, and the second, 
linked to alkaline phosphatase, serves as a detection probe (Jablonski et 
al., Nucl. Acids Res., 1986, 14, 6115-6128). After denaturation, 
hybridization and washing, detection is carried out by fluorescence using 
the substrate umbelliferyl phosphate. Tables 2 and 3 present the results 
of measurement of relative enzymatic activity, obtained using the 
oligonucleotide pair YF3 (.sup.5' dCAGCTTATTTTTCAACCTTGCC.sup.3') (SEQ ID 
NO:40 ) and YF4 (.sup.5' dCAAAGCGATGGTTG-CTGCCA.sup.3') (SEQ ID NO:41) 
corresponding to positions 607 to 628 and 632 to 651 of the pcp sequence 
(FIG. 3), used, respectively, as detection and capture probe, on a VIDAS 
(registered trade mark, marketed by the company bioMerieux) automated 
laboratory apparatus. Any activity less than or equal to 50 units 
corresponds to a negative result, comparable to the background of a 
control assay not including amplified DNA, while an activity above 500 
units denotes the presence of the desired amplification product. The 
figures observed corroborate the qualitative results of testing for the 
amplification product after electrophoretic separation of a fraction of 
the reaction medium, and confirm the specificity of detection of Group A 
streptococci with these probes (Cleuziat et al., Proceedings of the 
Conference on Taxonomy and Automated Indentification of Bacteria, Prague, 
1992, 70-73). 
b) study by hybridization with genomic DNA 
The genomic DNA of the bacteria studied above (international collection 
strains and strains of clinical origin) and listed in Tables 2 and 3 was 
extracted according to the method of Chassy cited above following their 
culture in a suitable liquid medium. Each DNA (quantity equivalent to 
approximately 10.sup.12 bacteria) was deposited on a nylon membrane using 
a vacuum filtration apparatus, then denatured and fixed on a membrane as 
described by Sambrook et al. (Molecular cloning. A Laboratory Manual. Cold 
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). These DNAs were then 
hybridized with a double-stranded DNA probe corresponding exactly to the 
coding frame of the Streptococcus pyogenes pcp gene under conditions 
permitting its hybridization only to a completely homologous target 
sequence, as described above. This probe was synthesized by PCR as 
described above, using the oligonucleotide primers .sup.5' 
dATGAAAATTCTTGTAACAGGC.sup.3' (SEQ ID NO:42) corresponding to positions 
285 to 305, and .sup.5' dGTGAGTAGCGCCCCCTAC.sup.3' (SEQ ID NO:43) 
complementary to positions 912 to 929 of the sequence of the Streptococcus 
pyogenes pcp gene (FIG. 3). The hybridization results are indicated in 
Tables 2 and 3. They demonstrate that the probe consisting of the whole of 
the Streptococcus pyogenes pcp gene permits the specific and exclusive 
detection of all strains of Streptococcus pyogenes (Group A 
.beta.-hemolytic streptococci). No non-specific cross-reaction is observed 
with any of the other bacterial species, in particular those possessing 
PYRase activity, which demonstrates that the whole of the Streptococcus 
pyogenes pcp gene is a very specific genetic marker for this species. All 
or part of the sequence of the Streptococcus pyogenes pcp gene hence 
constitutes a specific detection probe for this species. 
c) study by Southern hybridization 
The genomic DNA of the bacteria S. pyogenes JRS4 (spontaneous 
streptomycin-resistant mutant of D471), E. faecalis CG110 (Gawron-Burke 
and Clewell, Nature, 1982, 300, 281-284) and B. subtilis 168 (Lepesant et 
al., Mol. Gen. Genet., 1972, 118, 135-160) is extracted according to the 
Chassy method cited above. These DNAs are subjected, respectively, to 
complete digestion with the restriction endonucleases Hpa I or Bgl II, 
Hind III and Rsa I. The resulting fragments are separated by agarose gel 
electrophoresis and transferred onto a nylon membrane according to 
Southern's method. This membrane is then hybridized with the PCR probe 
covering the larger part of the pcp gene, under conditions permitting its 
hybridization only with a completely homologous target sequence, as 
described above. 
No hybridization signal is observed (FIG. 4) with the genome of the 
bacteria Enterococcus faecalis and Bacillus subtilis, in contrast to 
Streptococcus pyogenes which discloses the presence of a single 
hybridization band, the size of which confirms the established restriction 
map of the DNA region coding for the PYRase. This result hence shows that 
the whole of the pcp gene may be used as a DNA probe for the specific 
detection of Group A streptococci, and that any nucleotide sequence of 
sufficient length and of any location originating from this gene may also 
be used for this purpose. 
The hybridization results corroborate those obtained by amplification. DNA 
extracted from the bacteria E. faecalis and B. subtilis does not give rise 
to cross-hybridization phenomena, although these organisms both possess 
PYRase activity. This demonstrates that there is no homology between the 
gene coding for the PYRase of these bacteria and the S. pyogenes pcp gene. 
Example 10: overproduction of PYRase 
PYRase is of paramount importance in the field of chemistry and 
biochemistry. Its activity makes it possible, in effect, to liberate 
terminal NH.sub.2 groups which are blocked by a pyroglutamic residue, 
thereby making it possible to carry out the peptide sequencing procedure 
by the Edman sequential degradation method (Acta Chem. Scan, 1950, 4, 
283-293). 
The 1.6-kbHpa I-Bgl II fragment originating from plasmid pPC20 (FIG. 1) was 
inserted into the expression vectors pT7-5 and pT7-6 (Tabor and 
Richardson, Proc. Natl. Acad. Sci. USA, 1985, 82, 1074-1078), which differ 
from one another in the orientation of the multiple cloning site situated 
downstream of the bacteria phage T7 promoter. The resulting plasmids, 
pPC39 and pPC40, respectively, were introduced into E. coli strain K38 
(Russel and Model, J. Bacteriol., 1985, 159, 1034-1039) containing the 
compatible plasmid pGP1-2 (Tabor and Richardson, Proc. Natl. Acad. Sci. 
USA, 1985, 82, 1074-1078). After growth of the bacteria at 30.degree. C. 
in a selective medium, the synthesis of RNA polymerase of phage T7 from 
pGP1-2 is induced by thermal shock at 42.degree. C for 20 min. The 
bacterial RNA polymerase is then inhibited by adding rifampicin (200 
.mu.g/ml), leading to exclusive transcription of the pcp gene placed 
downstream of the phage T7 promoter. The product resulting from 
translation of the specifically transcribed RNA is visualized by means of 
the in vivo incorporation of L-[.sup.35 S]methionine (1000 Ci/mmol) added 
at the required time. The cells are thereafter lysed and their protein 
content is analyzed on denaturing 12.5% polyacrylamide gel (FIG. 10). 
Expression of the pcp gene under its own promoter from plasmids pPC39 and 
pPC40 makes it possible, as a result of the high copy number of these 
plasmids per bacterium, to obtain a significant quantity of PYRase, the 
apparent molecular weight of which is estimated at 26 kDa. The expressed 
enzyme proves to be greatly preponderant among the total cell proteins 
(FIG. 10). The expression of a labeled 26 kDa protein corresponding to 
PYRase from pPC39 shows that insertion of the pcp gene in the 
transcription direction imposed by the phage T7 promoter enables the 
product of this gene to be overproduced specifically. This overproduction 
permits the purification to homogeneity of a large quantity of PYRase per 
unit bacterial dry weight, by means of a chromatographic process involving 
only one step (Awadeet al., FEBS Lett., 1992, 308, 70-74). Likewise, it 
was possible to overexpress the Bacillus subtilis pcp gene which has been 
characterized, in order to overproduce the PYRase of this organism in E. 
coli using the phage T7 promoter/polymerase system described above. This 
approach also permits the pure protein to be obtained by a one step 
chromatographic purification process (Gonzales and Awade, J. Chromatogr., 
1992, 584, in the press). 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 43 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1336 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 284..928 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AACAAAATAAAAGAACTTACCTATTTTCCATCCAAAATGTTTAGCAATCATCATCTGCAA60 
GGCAACGTATTGCATGGCATTGATGTGATGAGCAACTAATATGTCATTAGAACGTTGCGT120 
CAAACTAGCATCTAAATAAAGATCGAAATGCAGTTATCAAAAATGCAAGCTCCTATCGGC180 
CCTTGTTTTAATTATTACTCACATGCCTTAATGTATTTACTTGCTTATTATTAACTTTTT240 
TGCTAAGTTAGTAGCGTCAGTTATTCATTGAAAGGACATTATTATGAAAATTCTT295 
MetLysIleLeu 
GTAACAGGCTTTGATCCCTTTGGCGGCGAAGCTATTAATCCTGCCCTT343 
ValThrGlyPheAspProPheGlyGlyGluAlaIleAsnProAlaLeu 
5101520 
GAAGCTATCAAGAAATTGCCAGCAACCATTCATGGAGCAGAAATCAAA391 
GluAlaIleLysLysLeuProAlaThrIleHisGlyAlaGluIleLys 
253035 
TGTATTGAAGTTCCAACGGTTTTTCAAAAATCTGCCGATGTGCTCCAG439 
CysIleGluValProThrValPheGlnLysSerAlaAspValLeuGln 
404550 
CAGCATATCGAAAGCTTTCAACCTGATGCAGTCCTTTGTATTGGGCAA487 
GlnHisIleGluSerPheGlnProAspAlaValLeuCysIleGlyGln 
556065 
GCTGGTGGCCGGACTGGACTAACGCCAGAACGCGTTGCCATTAATCAA535 
AlaGlyGlyArgThrGlyLeuThrProGluArgValAlaIleAsnGln 
707580 
GACGATGCTCGCATTCCTGATAACGAAGGGAATCAGCCTATTGATACA583 
AspAspAlaArgIleProAspAsnGluGlyAsnGlnProIleAspThr 
859095100 
CCTATTCGTGCAGATGGTAAAGCAGCTTATTTTTCAACCTTGCCAATC631 
ProIleArgAlaAspGlyLysAlaAlaTyrPheSerThrLeuProIle 
105110115 
AAAGCGATGGTTGCTGCCATTCATCAGGCTGGGCTTCCTGCTTCTGTT679 
LysAlaMetValAlaAlaIleHisGlnAlaGlyLeuProAlaSerVal 
120125130 
TCTAATACAGCTGGTACCTTTGTTTGCAATCATTTGATGTATCAAGCC727 
SerAsnThrAlaGlyThrPheValCysAsnHisLeuMetTyrGlnAla 
135140145 
CTTTACTTAGTGGATAAATATTGTCCAAATGCCAAAGCTGGGTTTATG775 
LeuTyrLeuValAspLysTyrCysProAsnAlaLysAlaGlyPheMet 
150155160 
CATATTCCCTTTATGATGGAACAGGTTGTTGATAAACCTAATACAGCT823 
HisIleProPheMetMetGluGlnValValAspLysProAsnThrAla 
165170175180 
GCCATGAACCTCGATGATATTACAAGAGGAATTGAGGCTGCTATTTTT871 
AlaMetAsnLeuAspAspIleThrArgGlyIleGluAlaAlaIlePhe 
185190195 
GCCATTGTCGATTTCAAAGATCGTTCCGATTTAAAACGTGTAGGGGGC919 
AlaIleValAspPheLysAspArgSerAspLeuLysArgValGlyGly 
200205210 
GCTACTCACTGACTGTGACGCTACTAAACCTATTTTAAAAAAACAGAGA968 
AlaThrHis 
215 
TATGAACTAACTCTGTTTTTTTTGTGCTAAAAATGAAAGACCTAGGGAAACTTTTCATCG1028 
GTCTTTCTCAATTGTCATCTTAATCTAATACTACTTCTAACATCAGCGGGTATAGTTTGC1088 
CAGTAATTAAGAAACGTTGTTGATCTAAATGAGCAATCCCATTCAAAACATTAAGGTCAG1148 
GGTAATGGGACTTATCAAGATTTAAGGCTTTTAACAAAGGACTAATATCATAGGTGGCTA1208 
CCACCTTTCCAGAATCAGGTTGGAGTTTGACAATAGTATTGGTTTGCCAAATATTGGCAT1268 
AGAGATAACCATCTACATACTCTAATTCGTTAAGCATTGAGATAGGGACACTTTCTATAG1328 
CAACTAGT1336 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 645 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..645 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ATGAGAAAAAAAGTGCTGATCACAGGCTTTGACCCTTTTGACAAAGAA48 
MetArgLysLysValLeuIleThrGlyPheAspProPheAspLysGlu 
151015 
ACCGTCAATCCATCATGGGAAGCGGCGAAACGGCTTAATGGCTTCGAG96 
ThrValAsnProSerTrpGluAlaAlaLysArgLeuAsnGlyPheGlu 
202530 
ACAGAAGAAGCCATTATTACAGCGGAACAAATTCCAACCGTCTTTAGA144 
ThrGluGluAlaIleIleThrAlaGluGlnIleProThrValPheArg 
354045 
TCCGCTCTGGACACTCTGCGCCAAGCCATTCAAAAACATCAGCCAGAT192 
SerAlaLeuAspThrLeuArgGlnAlaIleGlnLysHisGlnProAsp 
505560 
ATCGTAATTTGTGTCGGCCAAGCAGGAGGACGGATGCAGATTACACCG240 
IleValIleCysValGlyGlnAlaGlyGlyArgMetGlnIleThrPro 
65707580 
GAACGAGTGGCAATCAACCTTGCAGATGCGCGAATCCCCGATAACGAA288 
GluArgValAlaIleAsnLeuAlaAspAlaArgIleProAspAsnGlu 
859095 
GGACATCAGCCGATTGATGAAGAGATTTCTCCAGATGGGCCCGCCGCT336 
GlyHisGlnProIleAspGluGluIleSerProAspGlyProAlaAla 
100105110 
TACTGGACAAGGCTTCCCGTGAAACGAATGACTGCTAAGATGAAGGAA384 
TyrTrpThrArgLeuProValLysArgMetThrAlaLysMetLysGlu 
115120125 
CACGGCATTCCAGCTGCGGTTTCCTACACAGCGGGGACCTTTGTATGC432 
HisGlyIleProAlaAlaValSerTyrThrAlaGlyThrPheValCys 
130135140 
AACTATTTGTTCTACGGGTTAATGGATCACATTAGCCGGACATCCCCA480 
AsnTyrLeuPheTyrGlyLeuMetAspHisIleSerArgThrSerPro 
145150155160 
CACATTCGCGGCGGTTTTATTCATATTCCTTACATTCCGCAGCAAACA528 
HisIleArgGlyGlyPheIleHisIleProTyrIleProGlnGlnThr 
165170175 
ATCGACAAAACAGCGCCGAGCCTCAGCCTGGACACGATTGTCCGGGCA576 
IleAspLysThrAlaProSerLeuSerLeuAspThrIleValArgAla 
180185190 
TTGAGAATCGCCGCTGTTACGGCCGCACAATATGATGAGGATGTGAAG624 
LeuArgIleAlaAlaValThrAlaAlaGlnTyrAspGluAspValLys 
195200205 
TCACCGGGTGGTACGCTGCAC645 
SerProGlyGlyThrLeuHis 
210215 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 639 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..639 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
ATGCGAATTGTACTGCTGACGGGTTTCGAACCCTTTGATCAAGACCCG48 
MetArgIleValLeuLeuThrGlyPheGluProPheAspGlnAspPro 
151015 
GTGAACCCCTCCTGGGAAGCTGTGCGCCAACTGGATGGCGTGCAGTTG96 
ValAsnProSerTrpGluAlaValArgGlnLeuAspGlyValGlnLeu 
202530 
GGAAGCGACGTGAAGATTGTTGCGCGCCGGCTGCCTTGTGCATTTGCC144 
GlySerAspValLysIleValAlaArgArgLeuProCysAlaPheAla 
354045 
ACGGCGGGTGAATGCCTGACCCGGCTGATCGACGAGTTGCACCCGGCG192 
ThrAlaGlyGluCysLeuThrArgLeuIleAspGluLeuHisProAla 
505560 
ATGGTGATCGCCACCGGATTGGGGCCGGGGCGTAGCGATATCTCAGTC240 
MetValIleAlaThrGlyLeuGlyProGlyArgSerAspIleSerVal 
65707580 
GAACGGGTGGCGATCAACATCAATGATGCACGCATCCCCGATAATCTG288 
GluArgValAlaIleAsnIleAsnAspAlaArgIleProAspAsnLeu 
859095 
GGTGAGCAGCCTATCGATACGGCAGTCGTGGCTGACGGCCCGGCGGCT336 
GlyGluGlnProIleAspThrAlaValValAlaAspGlyProAlaAla 
100105110 
TTTTTCACGACGCTGCCGATCAAGGCGATGGTCAAGGCCGTGCGTGAA384 
PhePheThrThrLeuProIleLysAlaMetValLysAlaValArgGlu 
115120125 
GCGGGAATCGCGGCCTCGGTATCGCAGACGGCAGGGACGTTCGTGTGT432 
AlaGlyIleAlaAlaSerValSerGlnThrAlaGlyThrPheValCys 
130135140 
AATCAGGTTTTTTATCTGCTGCAGCATGCGCTCGCAGGGTCTGGGGTA480 
AsnGlnValPheTyrLeuLeuGlnHisAlaLeuAlaGlySerGlyVal 
145150155160 
CGCACTGGGTTTATCCACGTGCCGTTTCTGCCGGAGCAGGTGGCGGGT528 
ArgThrGlyPheIleHisValProPheLeuProGluGlnValAlaGly 
165170175 
TCGCAGCGGCCCTCGATGGCACTGGATGCAATGGTTGCGGGATTGCAG576 
SerGlnArgProSerMetAlaLeuAspAlaMetValAlaGlyLeuGln 
180185190 
GCGGCTGTACTGACAGCTTGGCATACACCGGTGGATGTCAAAGAAGCG624 
AlaAlaValLeuThrAlaTrpHisThrProValAspValLysGluAla 
195200205 
GGCGGGCAGGTCAGC639 
GlyGlyGlnValSer 
210 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GluArgValAlaIleAsnXaaXaaAspAlaArgIleProAspAsnXaa 
151015 
GlyXaaGlnProIleAsp 
20 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GlyXaaXaaAlaXaaValSerXaaThrAlaGlyThrPheValCysAsn 
151015 
XaaXaaXaaTyr 
20 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
LeuXaaThrGlyPheXaaProPhe 
15 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 2..3 
(D) OTHER INFORMATION: /label=Z 
/note="Each independently of one another 
represents Ala, Val, Leu, Ile, Pro, Trp, Phe or 
Met" 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 8 
(D) OTHER INFORMATION: /label=Z 
/note="Each independently of one another, 
represents Gly, Ser, Thr, Tyr, Gys, Asn or Gln" 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 17 
(D) OTHER INFORMATION: /label=Z 
/note="Each independently of one another, 
represents Gly, Ser, Thr, Tyr, Cys, Asn, Gln, Lys, 
Arg or His" 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 18..19 
(D) OTHER INFORMATION: /label=Z 
/note="Each independently of one another 
represents Ala, Val, Leu, Ile, Pro, Trp, Phe or 
Met" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
GlyGlxGlxAlaXaaValSerGlxThrAlaGlyThrPheValCysAsn 
151015 
GlxGlxGlxTyr 
20 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 2 
(D) OTHER INFORMATION: /label=Z 
/note="Each independently of one another, 
represents Ala, Val, Leu, Ile, Pro, Trp, Phe or 
Met" 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /label=Z 
/note="Each independently of one another, 
represents Gly, Ser, Thr, Tyr, Cys, Asn, Gln, Ala, 
Asp or Glu" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
LeuGlxThrGlyPheGlxProPhe 
15 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 215 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
MetArgLysLysValLeuIleThrGlyPheAspProPheAspLysGlu 
151015 
ThrValAsnProSerTrpGluAlaAlaLysArgLeuAsnGlyPheGlu 
202530 
ThrGluGluAlaIleIleThrAlaGluGlnIleProThrValPheArg 
354045 
SerAlaLeuAspThrLeuArgGlnAlaIleGlnLysHisGlnProAsp 
505560 
IleValIleCysValGlyGlnAlaGlyGlyArgMetGlnIleThrPro 
65707580 
GluArgValAlaIleAsnLeuAlaAspAlaArgIleProAspAsnGlu 
859095 
GlyHisGlnProIleAspGluGluIleSerProAspGlyProAlaAla 
100105110 
TyrTrpThrArgLeuProValLysArgMetThrAlaLysMetLysGlu 
115120125 
HisGlyIleProAlaAlaValSerTyrThrAlaGlyThrPheValCys 
130135140 
AsnTyrLeuPheTyrGlyLeuMetAspHisIleSerArgThrSerPro 
145150155160 
HisIleArgGlyGlyPheIleHisIleProTyrIleProGlnGlnThr 
165170175 
IleAspLysThrAlaProSerLeuSerLeuAspThrIleValArgAla 
180185190 
LeuArgIleAlaAlaValThrAlaAlaGlnTyrAspGluAspValLys 
195200205 
SerProGlyGlyThrLeuHis 
210215 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 215 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
MetLysIleLeuValThrGlyPheAspProPheGlyGlyGluAlaIle 
151015 
AsnProAlaLeuGluAlaIleLysLysLeuProAlaThrIleHisGly 
202530 
AlaGluIleLysCysIleGluValProThrValPheGlnLysSerAla 
354045 
AspValLeuGlnGlnHisIleGluSerPheGlnProAspAlaValLeu 
505560 
CysIleGlyGlnAlaGlyGlyArgThrGlyLeuThrProGluArgVal 
65707580 
AlaIleAsnGlnAspAspAlaArgIleProAspAsnGluGlyAsnGln 
859095 
ProIleAspThrProIleArgAlaAspGlyLysAlaAlaTyrPheSer 
100105110 
ThrLeuProIleLysAlaMetValAlaAlaIleHisGlnAlaGlyLeu 
115120125 
ProAlaSerValSerAsnThrAlaGlyThrPheValCysAsnHisLeu 
130135140 
MetTyrGlnAlaLeuTyrLeuValAspLysTyrCysProAsnAlaLys 
145150155160 
AlaGlyPheMetHisIleProPheMetMetGluGlnValValAspLys 
165170175 
ProAsnThrAlaAlaMetAsnLeuAspAspIleThrArgGlyIleGlu 
180185190 
AlaAlaIlePheAlaIleValAspPheLysAspArgSerAspLeuLys 
195200205 
ArgValGlyGlyAlaThrHis 
210215 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 215 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
MetLysIleLeuValThrGlyPheAspProPheGlyGlyGluAlaIle 
151015 
AsnProAlaLeuGluAlaIleLysLysLeuProAlaThrIleHisGly 
202530 
AlaGluIleLysCysIleGluValProThrValProGlnLysSerAla 
354045 
AspValLeuGlnGlnHisIleGluSerPheGlnProAspAlaValLeu 
505560 
CysIleGlyGlnAlaGlyGlyArgThrGlyLeuThrProGluArgVal 
65707580 
AlaIleAsnGlnAspAspAlaArgIleProAspAsnGluGlyAsnGln 
859095 
ProIleAspThrProIleArgAlaAspGlyLysAlaAlaTyrPheSer 
100105110 
ThrLeuProIleLysAlaMetValAlaAlaIleHisGlnAlaGlyLeu 
115120125 
ProAlaSerValSerAsnThrAlaGlyThrPheValCysAsnHisLeu 
130135140 
MetTyrGlnAlaLeuTyrLeuValAspLysTyrCysProAsnAlaLys 
145150155160 
AlaGlyPheMetHisIleProPheMetMetGluGlnValValAspLys 
165170175 
ProAsnThrAlaAlaMetAsnLeuAspAspIleThrArgGlyIleGlu 
180185190 
AlaAlaIlePheAlaIleValAspPheLysAspArgSerAspLeuLys 
195200205 
ArgValGlyGlyAlaThrHis 
210215 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 213 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
MetArgIleValLeuLeuThrGlyPheGluProPheAspGlnAspPro 
151015 
ValAsnProSerTrpGluAlaValArgGlnLeuAspGlyValGlnLeu 
202530 
GlySerAspValLysIleValAlaArgArgLeuProCysAlaPheAla 
354045 
ThrAlaGlyGluCysLeuThrArgLeuIleAspGluLeuHisProAla 
505560 
MetValIleAlaThrGlyLeuGlyProGlyArgSerAspIleSerVal 
65707580 
GluArgValAlaIleAsnIleAsnAspAlaArgIleProAspAsnLeu 
859095 
GlyGluGlnProIleAspThrAlaValValAlaAspGlyProAlaAla 
100105110 
PhePheThrThrLeuProIleLysAlaMetValLysAlaValArgGlu 
115120125 
AlaGlyIleAlaAlaSerValSerGlnThrAlaGlyThrPheValCys 
130135140 
AsnGlnValPheTyrLeuLeuGlnHisAlaLeuAlaGlySerGlyVal 
145150155160 
ArgSerGlyPheIleHisValProPheLeuProGluGlnValAlaGly 
165170175 
SerGlnArgProSerMetAlaLeuAspAlaMetValAlaGlyLeuGln 
180185190 
AlaAlaValLeuThrAlaTrpHisThrProValAspValLysGluAla 
195200205 
GlyGlyGlnValSer 
210 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 215 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
MetArgLysLysValLeuIleThrGlyPheAspProPheAspLysGlu 
151015 
ThrValAsnProSerTrpGluAlaAlaLysArgLeuAsnGlyPheGlu 
202530 
ThrGluGluAlaIleIleThrAlaGluGlnIleProThrValPheArg 
354045 
SerAlaLeuAspThrLeuArgGlnAlaIleGlnLysHisGlnProAsp 
505560 
IleValIleCysValGlyGlnAlaGlyGlyArgMetGlnIleThrPro 
65707580 
GluArgValAlaIleAsnLeuAlaAspAlaArgIleProAspAsnGlu 
859095 
GlyHisGlnProIleAspGluGluIleSerProAspGlyProAlaAla 
100105110 
TyrTrpThrArgLeuProValLysArgMetThrAlaLysMetLysGlu 
115120125 
HisGlyIleProAlaAlaValSerTyrThrAlaGlyThrPheValCys 
130135140 
AsnTyrLeuPheTyrGlyLeuMetAspHisIleSerArgThrSerPro 
145150155160 
HisIleArgGlyGlyPheIleHisIleProTyrIleProGlnGlnThr 
165170175 
IleAspLysThrAlaProSerLeuSerLeuAspThrIleValArgAla 
180185190 
LeuArgIleAlaAlaValThrAlaAlaGlnTyrAspGluAspValLys 
195200205 
SerProGlyGlyThrLeuHis 
210215 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
LeuXaaThrGlyPheXaaProPheGluArgValAlaIleAsnXaaXaa 
151015 
AspAlaArgIleProAspAsnXaaGlyXaaGlnProIleAspValSer 
202530 
XaaThrAlaGlyThrPheValCysAsn 
3540 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
AATAGCTTCGCCGCCAAAGGGATCAAAGCC30 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
ACAGGCTTTGATCCCTTTGG20 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
GlnProAspAlaValLeuCysIleGlyGlnAlaGlyGlyArgThrGly 
151015 
LeuThrProGluArgValAlaIleAsnGlnAspAspAlaArgIlePro 
202530 
AspAsnGluGlyAsnGlnProIleAsp 
3540 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
GlyLeuProAlaSerValSerAsnThrAlaGlyThrPheValCysAsn 
151015 
HisLeuMetTyr 
20 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
CysProAsnAlaLysAlaGlyPheMetHisIleProPheMet 
1510 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
LysIleLeuValThrGlyPheAspProPheGly 
1510 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
GluArgValAlaIleAsnGlnAspAspAlaArgIleProAspAsnGlu 
151015 
GlyAsnGlnProIleAsp 
20 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
ValSerAsnThrAlaGlyThrPheValCysAsn 
1510 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 41 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
GlnProAspAlaValLeuCysIleGlyGlnAlaGlyGlyArgThrGly 
151015 
LeuThrProGluArgValAlaIleAsnGlnAspAspAlaArgIlePro 
202530 
AspAsnGluGlyAsnGlnProIleAsp 
3540 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
GlyLeuProAlaSerValSerAsnThrAlaGlyThrPheValCysAsn 
151015 
HisLeuMetTyr 
20 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
CysProAsnAlaLysAlaGlyPheMetHisIleProPheMet 
1510 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
LysIleLeuValThrGlyPheAspProPheGly 
1510 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
TTCTGGCGTTAGTCCAGTCC20 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
TATCAGGAATGCGAGCATCG20 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
TGCATAAACCCAGCTTTGGC20 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
TTGCCAGCAACCATTCATGG20 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
AGAAACAGAAGCAGGAAGCC20 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
AAAATCTGCCGATGTGCTCC20 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
CTTTGTATTGGGCAAGCTGG20 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
TACTGGCAAACTATACCCGC20 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
TGCTCGCATTCCTGATAACG20 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
ATCAAAGCGATGGTTGCTGC20 
(2) INFORMATION FOR SEQ ID NO:37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
CCCTATCTCAATGCTTAACG20 
(2) INFORMATION FOR SEQ ID NO:38: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
TCAAAAATCTGCCGATGTGC20 
(2) INFORMATION FOR SEQ ID NO:39: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
AGGAAGCCCAGCCTGATGAA20 
(2) INFORMATION FOR SEQ ID NO:40: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
CAGCTTATTTTTCAACCTTGCC22 
(2) INFORMATION FOR SEQ ID NO:41: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
CAAAGCGATGGTTGCTGCCA20 
(2) INFORMATION FOR SEQ ID NO:42: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 
ATGAAAATTCTTGTAACAGGC21 
(2) INFORMATION FOR SEQ ID NO:43: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: 
GTGAGTAGCGCCCCCTAC18 
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