Nucleotide sequences of actinomycetales, oligonucleotides of said sequences and their use for detecting the presence of actinomycetales

The present invention relates to nucleotide sequences of Actinomycetales, in particular of mycobacteria, to oligonucleotides contained within said nucleotide sequences, to their uses as primers for the synthesis of Actinomycetales DNA and as probes for the detection of DNA and/or the transcription products of Actinomycetales, in particular of mycobacteria, to the products of expression of said sequences, to their uses and to antibodies directed towards the said products, to a method for detecting and identifying Actinomycetales and its uses, as well as to immunogenic compositions comprising the said expression products.

The present invention relates to nucleotide sequences of Actinomycetales, 
in particular of mycobacteria, to oligonucleotides contained within the 
said sequences, to their uses as primers for the synthesis of 
Actinomycetales DNA and as probes for the detection of DNA and/or of the 
transcription products of Actinomycetales, in particular of mycobacteria, 
to the products of expression of the said sequences, to their uses and to 
antibodies directed towards the said products, to a method for detecting 
and identifying Actinomycetales and its uses, as well as to immunogenic 
compositions comprising the said expression products. 
Tuberculosis and leprosy are known to be major public health problems. They 
are currently approximately 60.times.10.sup.6 individuals suffering from 
tuberculosis in the world (with an annual mortality of 3.times.10.sup.6), 
and approximately 15.times.10.sup.6 individuals suffering from leprosy. In 
France, approximately 10.sup.4 new cases of tuberculosis appear every 
year. Vaccination with BCG (Bacillus Calmette-Guerin, an attenuated strain 
of M. bovis) is far from effective in all populations. This efficacy 
varies approximately from 80% in Western countries such as England to 0% 
in India (results of the latest vaccination trial in Chingleput). The 
appearance of strains of M. tuberculosis resistant to the usual 
antituberculosis agents and the existence of mycobacterioses due to other, 
increasingly common mycobacteria such as M. avium, especially in patients 
with immunosuppression (AIDS in the largest number of cases), add to the 
urgency of developing a rapid method of detecting and identifying 
mycobacteria. 
The diagnosis of tuberculosis and other related mycobacterioses is 
difficult to carry out; in effect the microorganisms responsible for these 
diseases are often present in small amounts, and when the amount of them 
is detectable by the methods conventionally used, the disease is already 
progressing and the patients are contagious to those around them. As a 
result of the very long generation time of these bacteria (24 h for M. 
tuberculosis compared with 20 min for E. coli), culturing these organisms 
is difficult. Thus, it requires 6 to 8 weeks to identify the 
microorganisms, and longer to obtain an antibiogram usable for appropriate 
treatment of the patients. The need for a detection test not requiring 
culturing of the microorganisms, and directly usable with the pathological 
samples even when the microorganisms are present therein at low 
concentrations, is hence essential. 
Several techniques are currently used in clinical practice for identifying 
a mycobacterial infection. 
In the first place, direct detection of the microorganisms in the 
microscope should be mentioned; this technique is rapid, but does not 
permit identification of the mycobacterial species observed, and lacks 
sensitivity in as much as a large number of microorganisms have to be 
present in the sample (&gt;10.sup.4 /ml) in order to permit reliable 
detection (RATES J., CHEST, 1979, 76, (suppl.), 757-763). 
Cultures, when positive, have a specificity approaching 100%, and permit 
identification of the mycobacterial species isolated; nevertheless, as 
specified above, growth of mycobacteria in vitro can be achieved only in 
the space of 3 to 6 weeks, and when few mycobacteria are present at the 
site of infection, repeated culturing is necessary in order to ensure a 
positive result (BATES J., 1979 and BATES J. et al., Am. Rev. Respir. 
Dis., 1986, 134, 415-417). 
Serological techniques can prove useful under some conditions, but their 
use is limited by their low sensitivity and/or their low specificity 
(DANIEL T. M. et al., Am. Rev. Respir. Dis., 1987, 135, 1137-1151). 
The presence or absence of mycobacteria may also be determined by 
hybridisation with DNA or RNA using probes specific for DNA sequences 
(KIEHN T. E. et al., J. Clin. Microbiol., 1987, 25, 1551-1552; ROBERTS M. 
C. et al., J. Clin. Microbiol., 1987, 25, 1239-1243; DRAKE T. A. et al., 
J. Clin. Microbiol., 1987, 25, 1442-1445). However, these methods also 
require culturing of the microorganisms. 
Some DNA sequences of various mycobacteria, and in particular some genes 
coding for mycobacterial antigens have been described. There may be 
mentioned, in particular, PCT International Application WO 88/00,974, 
whose inventor is YOUNG R. and the content of which is recapitulated in a 
paper published in Nature, 1985, 316, 450; these publications describe the 
genes coding for five immunodominant antigens of M. leprae and, in 
particular, the gene coding for the 65-kD antigen has been sequenced. 
There may also be mentioned PCT International Application WO 88/05,823, 
whose co-inventors are HUSSON R., YOUNG R. and SHINNICK T. and the content 
of which is recapitulated in the paper published in J. Bact., 1987, 169, 
1080-1088 and which describes the genes of M. tuberculosis coding for 
protein antigens, and in particular for the 65-kD antigen. This 
International Application specifies, in particular, that the genes of M. 
tuberculosis coding for five immunologically active proteins were isolated 
by systematic screening of a recombinant DNA library expressed in a 
bacteriophage lambda gt11, with a collection of monoclonal antibodies 
directed towards the protein antigens of this bacterium. One of the 
antigens of M. tuberculosis, a 65-kD protein possesses determinants common 
to M. tuberculosis and M. leprae. 
PCT International Application WO 88/06,591, a co-inventor of which is, in 
particular, T. SHINNICK, describes a recombinant protein of 540 amino 
acids (65-kD protein) and also the DNA sequence and the vectors for the 
expression of the said protein, as well as the uses of the said 
recombinant protein. This Application also describes peptides 
corresponding to sequences of this protein and their uses. 
Genes coding for proteins of other mycobacteria (M. africanum, M. 
smegmatis, M. bovis BCG and M. avium) have also been isolated. There may 
be mentioned, in particular, THOLE et al. (Infect. Immunol., 1987, 55, 
1466-1475), who have described a 64-kD protein of M. bovis BCG expressed 
in E. coli. 
However, the amounts of mycobacterial DNA present in most biological 
samples are insufficient to give a positive signal; this technique has 
hence proved unsuitable for the identification of mycobacterial DNA 
extracted directly from biological samples. 
A number of studies have also shown some degree of structural homology 
between the different mycobacteria. However, differences in the DNA 
sequence of M. tuberculosis and M. bovis have been described in the 3' 
region of the open reading frame of the 65-kD antigen (SHINNICK et al., 
1987, THOLE et al., 1987), but a homologous region has not been observed 
in the DNA of M. leprae (MEHRA et al., Proc. Nat. Acad. Sci. USA, 1986, 
83, 7013-7017, also PCT 88/000,974). 
There are also publications which describe vaccines against mycobacteria, 
produced by genetic engineering; there may be mentioned, in particular, 
PCT International Application WO 88/02,027, which describes recombinant 
poxviruses capable of expressing mycobacterial antigens and which enable a 
protective immunological response to mycobacteria to be obtained. 
The various detection methods of the prior art do not permit, on the one 
hand the detection and rapid identification of an Actinomycetales 
infection directly from a biological sample, and on the other hand the 
specific identification of groups, species or strains, which may even be 
present in small amounts. 
The additional references which follow also constitute the state of the art 
prior to the present invention. 
BAESS I., Acta Path. Microbiol. Scand., 1979, 87, 221-226; BEAUCAGE S. L. 
et al., Tetrahedron Lett., 1981, 22, 1859-1862; EISENACH K. D. et al., Am. 
Rev. Respir. Dis., 1986, 133, 1065-1068; GHEORGHIU M. et al., J. Biol. 
Standardization, 1988, 16, 15-26; GLASSROTH J. et al., N. Engl. J. Med., 
1980, 302, 1441-1450; HAWKINS C. C. et al., Ann. Intern. Med., 1985, 105, 
184-188; IMAEDA T., Int. J. Systematic Bacteriol., 1985, 35, 147-150; 
IMAEDA T. et al., Int. J. Systematic Bacteriol., 1988, 38, 151-156; KOGAN 
S. C. et al., N. Engl. J. Med., 1987, 317, 985-990; LI H. et al., Nature 
(Lond.), 1988, 335, 414-417; LU M. C. et al., Infect. Immun., 1987, 55, 
2378-2382; MANIATIS T. et al., 1982, Cold Spring Harbor, New York; 
McFADDEN J. J. et al., Mol. Microbiol., 1987, 1, 283-291; PAO C. C. et 
al., Tubercle, 1988, 69, 27-36; PATEL R., J. Gen. Microbiol., 1986, 132, 
541-551; SAIKI R. K. et al., Science, 1988, 239, 487-491; SANGER F. et 
al., Proc. Natl. Acad. Sci. USA, 1977, 74, 5463-5467; SMIDA J. et al., 
Int. J. Leprosy, 1988, 56, 449-454; THEIN S. L. et al., in Human Genetic 
Diseases, 1986, IRL Press, 33-50; THOLE J. E. R. et al., Infect. Immun., 
1985, 50, 800-806; WATSON E. A., Canad. J. Pub. Health, 1935, 26, 268-275; 
WOLINSKY E., Am. Rev. Respir. Dis, 1979, 119, 107-159.

The aim of the present invention is accordingly to provide a detection and 
identification method enabling small amounts of DNA extracted from 
microorganisms, themselves present in limited numbers, to be detected, the 
method being rapid and enabling Actinomycetales infection, and in 
particular a mycobacterial infection, a Nocardia infection or a 
Rhodococcus infection, to be identified directly in pathological samples 
without having to carry out culturing. 
The subject of the present invention is a nucleotide sequence derived from 
Actinomycetales, characterised in that it consists of a homologous 
sequence of a gene common to Actinomycetales chosen from the group 
comprising mycobacteria, Nocardia and Rhodococcus, within which sequence 
there are conserved regions and variable regions, and in that it comprises 
between 250 and 500 base pairs. 
Nucleotide sequence is understood, in the present invention, to mean both a 
double-stranded DNA sequence, a single-stranded DNA sequence and the 
products of transcription of the said DNA sequences. 
Actinomycetales is understood, in the sense used in the present invention, 
to mean both Actinomycetaceae such as Nocardia and Mycobacteriaceae or 
Rhodococcus. 
There are at least 50 species of mycobacteria divided into several groups. 
In the present invention, the group comprising M. bovis BCG, M. bovis, M. 
tuberculosis, M. africanum and M. microti is referred to as the 
tuberculosis bacillus group; and the group comprising M. avium, M. 
intracellulare and M. paratuberculosis is referred to as the MAIP group. 
Comparison of the nucleotide sequences of the different groups and/or 
species has enabled identical or similar fragments to be demonstrated 
within a gene common to the different groups, and a homology to be defined 
between the different sequences. 
According to an advantageous embodiment of the invention, the said sequence 
possesses an at least 80% homology with the gene coding for the 65-kD 
mycobacterial antigen. 
According to an advantageous variant of this embodiment, the said sequence 
comprise 383 base pairs homologous in at least 8 species of mycobacteria, 
namely M. tuberculosis, M. avium, M. fortuitum, M. paratuberculosis, BCG, 
M. kansasii, M. malmoense and M. marinum. 
The 383 base pairs correspond to the expression product having an amino 
acid sequence of the following formula (I): SEQ ID NO:1) 
##STR1## 
in which: X.sub.1 is non-existent or represents the sequence ASP-PRO, 
X.sub.2 represents LYS or GLU, 
X.sub.3 represents GLY or ALA, 
X.sub.4 represents GLY or ARG, 
X.sub.5 represents ALA or VAL, 
X.sub.6 represents ALA or ARG, 
X.sub.7 represents ARG or LYS, 
X.sub.8 represents PRO or LEU, 
X.sub.9 represents GLY or SER, 
X.sub.10 represents LEU or PHE, 
X.sub.11 represents ARG or CYS, 
X.sub.12 represents LYS or ALA, 
X.sub.13 represents GLU or ALA, 
X.sub.14 represents THR or LYS, 
X.sub.15 represents LYS or ASP, 
X.sub.16 represents SER, GLY, PRO or THR 
X.sub.17 represents ASP or GLU, 
X.sub.18 represents ALA, GLY or VAL, 
X.sub.19 represents ALA or VAL, 
X.sub.20 represents GLN or ALA, 
X.sub.21 represents ASP or GLU, 
X.sub.22 represents LEU or PRO, 
X.sub.23 represents ALA or VAL, 
X.sub.24 represents GLU or ASP, 
X.sub.25 represents ALA or GLY, 
X.sub.26 represents ASN or LYS, 
X.sub.27 represents VAL or SER, 
X.sub.28 represents GLU or GLY, 
X.sub.29 is non-existent or represents the sequence 
ASN-THR-PHE-GLY-LEU-GLN. 
According to another advantageous variant of this embodiment, the said 
sequence comprises 343 base pairs and corresponds to the formula (II) (SEQ 
ID NO:2) below: 
##STR2## 
and corresponds to a fragment of M. fortuitum, similar to the sequence of 
the mycobacterial gene coding for the 65-kD antigen. 
The said fragment comprises, in particular, the following restriction 
sites: 
AccII, AhaII, BanI, BanII, BbvI, Bsp1286, BstXI, CfoI, DdeI, EcoRII, EspI, 
Fnu4HI, HaeII, HaeIII, HapII, HgaI, HinfI, HphI, MaeIII, MboII, MnlI, 
NarI, NcoI, NlaIII, SacI, SacII, Sau3A, Sau96A, ScrFI, StyI, TaqI, YmaIII. 
According to another advantageous variant of this embodiment, the said 
sequence comprises 343 base pairs and corresponds to the formula (III)(SEQ 
ID NO:3) below: 
##STR3## 
and corresponds to a fragment common to the MAIP group similar to the 
sequence of the mycobacterial gene coding for the 65-kD antigen. 
The said fragment comprises, in particular, the 5 following restriction 
sites: 
AccII, AflI, AhaII, BanI, BbvI, BglI, Bsp1286, BstEII, BstXI, CfoI, EaeI, 
HaeII, HaeIII, HphI, MaeIII, MnlI, NarI, PvuI, SacII, Sau3A, Sau96A, TaqI. 
According to another advantageous variant of this embodiment, the said 
sequence comprises 343 nucleotides and corresponds to the formula (IV) 
(SEQ ID NO:4) below: 
##STR4## 
and corresponds to a fragment of the sequence of the gene coding for the. 
65-kD antigen of the tuberculosis bacillus group. 
The said fragment comprises, in particular, the following restriction 
sites: 
AccII, AhaII, BanI, BbvI, BstXI, CfoI, EaeI, HaeIII, HphI, MaeIII, MnlI, 
NarI, NrvI, SacII, Sau3A, Sau96A, TaqI. 
According to yet another advantageous variant of this embodiment, the said 
sequence comprises 343 nucleotides and corresponds to the formula (V) (SEQ 
ID NO: 5) below: 
##STR5## 
and corresponds to a fragment of Mycobacterium kansasii similar to the 
sequence of the mycobacterial gene coding for the 65-kD antigen. 
The said fragment comprises, in particular, the following restriction 
sites: 
AccII, AhaII, AluI, AosI, BanI, BglI, Bsp1286, BstEII, CfoI, EaeI, HapII, 
HgaI, HphI, MboII, MhlI, NaeI, NlaIII, RsaI, Sau3A, Sau96A, SfaNI, StyI, 
TaqI, Tth111I. 
According to another advantageous variant of this embodiment, the said 
sequence comprises 343 nucleotides and corresponds to the formula (VI) 
(SEQ ID NO:6) below: 
##STR6## 
and corresponds to a fragment of Mycobacterium malmoense similar to the 
sequence of the mycobacterial gene coding for the 65-KD antigen. 
The said fragment comprises, in particular, the following restriction 
sites: 
AccII, AhaII, AluI, AosI, BanI, BstEII, EaeI EspI, Fnu4HI, HaeII, HinfI, 
HphI, MboII, MnlI, NaeI, Sau3A, StyI, TaqI, 
According to yet another advantageous variant of this embodiment, the said 
sequence comprises 343 nucleotides and corresponds to the formula (VII) 
(SEQ ID NO:7) below: 
##STR7## 
and corresponds to a fragment of Mycobacterium marinum, similar to the 
sequence of the mycobacterial gene coding for the 65-kD antigen. 
The said fragment comprises, in particular, the following restriction 
sites: 
AatI, AosI, AhaII, AluI, BbvI, BstEII, CfoI, EaeI, Fnu4HI, HaeII, HapII, 
HinfI, MboII, MnlI, NaeI, PvuI, Sau3A, StyI, TaqI, Tth111I. 
According to another advantageous variant of this embodiment, the said 
sequence comprises 343 nucleotides and corresponds to the formula (VIII) 
(SEQ ID NO: 8) below: 
##STR8## 
and corresponds to a fragment of Nocardia asteroides, similar to the 
sequence of the mycobacterial gene coding for the 65-kD antigen. 
The said fragment comprises, in particular, the following restriction 
sites: 
AccII, AhaII, AluI, AosI, BanI, Bsp1286, CfoI, EaeI, Fnu4HI, HaeIII, HgaI, 
HphI, MaeIII, MboII, MnlI, NlaIII, SacII, Sau3A, Sau96A, SfaNI, StyI, 
TaqI, Tth111I. 
The subject of the present invention is also oligonucleotides, 
characterised in that they consist of a fragment of a nucleotide sequence 
according to the invention. 
Among these fragment, special mention may be made of the following: 
an oligonucleotides, characterised in that it possesses the following 
sequence of formula (IX) (SEQ ID NO:9): 
EQU 5'GAGATCGAGCTGGAGGATCC (IX). 
Such a sequence corresponds, in particular, to the base sequence 397-416 
after the start codon of the "+" strand of the gene coding for the 65-kD 
antigen of the tuberculosis bacillus group; this sequence is hereinafter 
designated TB-b 1; 
an oligonucleotide, characterised in that it possesses the following 
sequence of formula (X) (SEQ ID NO:10): 
EQU 5'AGCTGCAGCCCAAAGGTGTT (X). 
Such a sequence is complementary to the base sequence 535-554 after the 
start codon of the "+" strand of the gene coding for the 65-kD antigen of 
the tuberculosis bacillus group; this sequence is hereinafter designated 
TB-2; 
an oligonucleotide, characterised in that it possesses the following 
sequence of formula (XI) (SEQ ID NO:11): 
EQU 5'GCGGCATCGAAAAGGCCGTG (XI) 
which sequence permits recognition of tuberculosis bacilli and is 
hereinafter designated TB-3; 
an oligonucleotide, characterised in that it possesses the following 
sequence of formula (XII) (SEQ ID NO:12): 
EQU 5'CGAAATCGCTGCGGTGGCCG (XII) 
which sequence permits recognition of tuberculosis bacilli and is 
hereinafter designated TB-4; 
an oligonucleotide, characterised in that it possesses the following 
sequence of formula (XIII) (SEQ ID NO:13): 
EQU 5'CTGCCACCGCGGCCATCTCC (XIII) 
which sequence permits recognition of MAIP group bacilli and is hereinafter 
designated TB-5; this oligonucleotide advantageously comprises a single 
BglI restriction site; 
an oligonucleotide, characterised in that it possesses the following 
sequence of formula (XIV) (SEQ ID NO:14): 
EQU 5'CTGCCACCGCCGGTATCTCC (XIV) 
which sequence permits recognition of M. fortuitum and is hereinafter 
designated TB-6; 
an oligonucleotide, characterised in that it possesses the following 
sequence of formula (XV) (SEQ ID NO:15): 
EQU 5'AACGTCGCGGCCGGCGCCAA 3' (XV) 
which sequence is hereinafter designated TB-7; 
an oligonucleotide, characterised in that it possesses the following 
sequence of formula (XVI) (SEQ ID NO:16); 
EQU 5'GACTCCTCGACGGTGATGAC 3' (XVI) 
which sequence is hereinafter designated TB-8; 
an oligonucleotide, characterised in that it possesses the following 
sequence of formula (XVII) (SEQ ID NO:17): 
EQU 5'CCTGCTCAAGGGCGCCAAG 3' (XVII) 
which sequence is hereinafter designated TB-9; this oligonucleotide TB-9 
advantageously comprises a single BanI restriction site. 
an oligonucleotide, characterised in that it possesses the following 
sequence of formula (XVIII) (SEQ ID NO:18): 
EQU 3'CGAAATCGCTGCGGTGGCCGCAATCTGCTC 5' (XVIII), 
which sequence permits recognition of tuberculosis group bacilli and is 
hereinafter designated TB-10. 
an oligonucleotide, characterised in that it possesses the following 
sequence of formula (XIX) (SEQ ID NO:19): 
EQU 5'GGTGCTCGCCCAGGCGTTGGTCCGC 3' (XIX) 
which sequence permits recognition of MAIP group bacilli and is hereinafter 
designated TB-11. 
an oligonucleotide, characterised in that it possesses the following 
sequence of formula (XX) (SEQ ID NO:20): 
EQU 5'TGTGCTCGCGCAGGCGCTGGTCAAA 3' (XX), 
which sequence permits specific recognition of M. kansasii and is 
hereinafter designated TB-12. 
According to yet another embodiment, the said oligonucleotides are obtained 
synthetically using, in particular, an apparatus marketed by APPLIED 
BIOSYSTEMS (USA). 
The subject of the present invention is also pairs of primers for the 
synthesis of an Actinomycetales DNA or RNA, characterised in that each 
primer comprises a nucleotide sequence or a fragment of a nucleotide 
sequence as defined above. 
Such primers permit the synthesis of a DNA or RNA sequence or a fragment of 
the latter present in a gene coding for an antigen present in all 
Actinomycetales, and in particular the gene coding for the 65-kD antigen. 
According to an embodiment of the said pairs of primers, they 
advantageously consist of an oligonucleotide of formula (IX) (SEQ ID NO:9) 
(TB-1) paired with an oligonucleotide of formula (X) (SEQ ID NO:10) 
(TB-2). 
According to another embodiment of the said pairs of primers, they 
advantageously consist of an oligonucleotide of the formula (XV) (SEQ ID 
NO:15) (TB-7) paired with an oligonucleotide of formula (XV) (SEQ ID 
NO:16) (TB-8). 
The primers TB-1 (SEQ ID NO:9) and TB-2 (SEQ ID NO:10) permit the synthesis 
of a DNA or RNA sequence present in mycobacteria or related bacteria such 
as Nocardia or Rhodococcus. 
The subject of the present invention is also nucleotide probes, 
characterised in that they comprise a nucleotide sequence or a fragment of 
the latter as defined above, where appropriate labelled using a label such 
as a radioactive isotope, a suitable enzyme, a fluorochrome, an antibody 
or a base analogue such as that described in French Patent No. 81/24,131. 
According to an advantageous embodiment of this invention, the said probe 
is chosen from the group comprising the oligonucleotides of formulae XI 
(SEQ ID NO:11) (TB-3), XII (SEQ ID NO:12) (TB-4), XIII (SEQ ID NO:13) 
(TB-5), XIV (SEQ ID NO:14) (TB-6), XVII (SEQ ID NO:17) (TB-9), XVIII (SEQ 
ID NO:18) (TB-10), XIX (SEQ ID NO:19) (TB-11) and XX (SEQ ID NO:20) 
(TB-12). 
The probe TB-6 (SEQ ID NO:14) enables M. fortuitum to be detected in 
particular; the probes TB-5 (SEQ ID NO:13) and TB-11 (SEQ ID NO:19) enable 
MAIP group mycobacteria to be detected; the probes TB-3 (SEQ ID NO:11), 
TB-4 (SEQ ID NO:12) TB-9 (SEQ ID NO:17) and TB-10 (SEQ ID NO:18) enable 
mycobacteria of the tuberculosis bacillus group to be detected; and the 
probe TB-12 enables M. kansasii to be detected. 
The subject of the present invention is also the peptides or peptide 
fragments encoded by any one of the sequences defined above. The following 
may be mentioned in particular: 
a peptide and/or peptide fragment, characterised in that it is encoded by 
the sequence of 343 nucleotides of formula (II) above and corresponds to 
the formula (XXI) (SEQ ID NO:21) below: 
##STR9## 
a peptide and/or peptide fragment, characterised in that it is encoded by 
the sequence of 343 nucleotides of formula (III) above and corresponds to 
the formula (XXII) (SEQ ID NO:22) below: 
##STR10## 
a peptide and/or peptide fragment, characterised in that it is encoded by 
the sequence of 343 nucleotides of formula (V) and corresponds to the 
formula (XXIII) (SEQ ID NO:23) below: 
##STR11## 
a peptide and/or peptide fragment, characterised in that it is encoded by 
the sequence of 343 nucleotides of formula (VI) and corresponds to the 
formula (XXIV) (SEQ ID NO:24) below: 
##STR12## 
a peptide and/or peptide fragment, characterised in that it is encoded by 
the sequence of 343 nucleotides of formula (VII) and corresponds to the 
formula (XXV) (SEQ ID NO:25) below: 
##STR13## 
a peptide and/or peptide fragment, characterised in that it is encoded by 
the sequence of 343 nucleotides of formula (VIII) and corresponds to the 
formula (XXVI) (SEQ ID NO:26) below: 
##STR14## 
The subject of the present invention is also a composition having 
immunogenic capability, characterised in that it comprises at least one 
peptide and/or peptide fragment as defined above, optionally combined with 
at least one pharmaceutically acceptable vehicle. 
The subject of the present invention is also polyclonal or monoclonal 
antibodies, characterised in that they are obtained by immunisation of a 
suitable animal with a peptide or peptide fragment according to the 
invention. 
Such antibodies can, in particular, find application for demonstrating the 
presence of mycobacteria in a suitable sample obtained from a patient to 
be tested, according to a known method of the ELISA or RIA type. 
The subject of the present invention is also a method for the detection and 
rapid identification, by amplification and hybridisation, of small amounts 
of Actinomycetales chosen from the group comprising mycobacteria, Nocardia 
and Rhodococcus, possibly present in a biological sample suitably treated 
to extract the DNA and/or the transcription products of the said 
Actinomycetales, which method is characterised in that the said sample: 
(1) is brought into contact with a pair of primers according to the 
invention, to amplify at least one fragment of the said DNA or RNA, 
(2) after which the amplified DNA or RNA sequence is detected by at least 
one nucleotide probe according to the invention. 
The method carried out in (1) is, in particular, one of the techniques of 
genetic amplification such as the so-called Q .beta. replicase method 
(LIZARDI P. M. et al., Biotechnol., 1988, 6) or the so-called PCR 
(polymerase chain reaction) method described in European Patent 
Applications No. 200,363, No. 201,184 and No. 229,701 filed by CETUS CO. 
Such a method has the advantage of enabling a specific, direct and rapid 
test distinguishing the different groups of Actinomycetales, and in 
particular of mycobacteria, to be carried out, on the one hand using 
non-specific primers which amplify a DNA or RNA fragment, and on the other 
hand using group- or genus-specific probes. 
According to an advantageous embodiment of the said method, it comprises in 
addition: 
(3) cleavage of any probe which has hybridised during the above step (2), 
using a suitable restriction enzyme; 
(4) detection of any probe fragment obtained. 
According to a variant of this embodiment, the restriction enzyme is 
advantageously chosen from the group comprising BanI and BglI. 
Such an embodiment has the advantage of enabling a genus or a group of 
Actinomycetales to be detected. 
According to another advantageous embodiment of the said method, the 
detection of the amplified DNA or RNA sequence is carried out using two 
suitable nucleotide probes, the said method comprising in addition: 
(3) enzymatic coupling of the two hybridised probes; 
(4) detection of any fragment obtained containing the two combined probes. 
According to another advantageous embodiment of the said method, the DNA is 
isolated from the biological sample during a step prior to the detection 
and identification steps, by suspending the centrifugation pellet from the 
said biological sample in a suitable lysis solution, followed by an 
incubation at approximately 95.degree. C. for a suitable time, the 
incubation itself being followed by the addition of a buffer solution to 
the medium, after which the DNA is extracted by suitable means of 
extraction. 
According to an advantageous variant of this embodiment, the lysis solution 
employed is a solution comprising 0.1N NaOH, 2M NaCl and 0.5% SDS. 
According to another advantageous variant of this embodiment, the 
incubation is carried out at a temperature of approximately 95.degree. C. 
for approximately 15 minutes. 
The subject of the present invention is, in addition, a ready-to-use kit, 
outfit or coordinated set for carrying put the method for detecting at 
least one Actinomycetales bacterium, in particular at least one 
mycobacterium, characterised in that it comprises, apart from the 
appropriate amounts of suitable buffers and reagents for carrying out the 
said detection: 
suitable doses of a pair of primers according to the invention; 
suitable doses of at least one nucleotide probe or probe fragment according 
to the invention. 
Apart from the foregoing variants, the invention comprises yet other 
variants which will become apparent from the description which follows, 
which relates to examples of implementation of the method which is the 
subject of the present invention. 
It should, however, be clearly understood that these examples are given 
only by way of illustration of the subject of the invention and in no way 
constitute a limitation of the latter. 
Example 1 
Detection and comparative identification of M. tuberculosis, M. bovis, M. 
avium and M. fortuitum. 
a) Isolation of mycobacterial DNA. 
The biological extracts are treated in a suitable manner and then 
centrifuged. To extract the DNA, the centrifugation pellet is resuspended 
in 50 .mu.l of 0.1N NaOH containing 2M NaCl and 0.5% SDS and incubated at 
95.degree. C. for 15 min (occasional gentle shaking), and then, after the 
addition of 0.4 ml of 0.1M Tris-HCl, pH 7, the DNA is extracted by three 
treatments with a phenol/chloroform mixture, precipitated with ethanol and 
dissolved in 50 .mu.l of 10 mM Tris-HCl, pH 8, containing 0.1 mM EDTA. 
b) DNA amplification. 
Amplification is carried out as described in SAIKI et al. (Science, 1988, 
vol. 239, 487-491) and also in European Patent Application No. 200,362: 
0.1 ml of a reaction mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 
8.3), 1.5-2.4 mM MgCl.sub.2, 100 .mu.g/ml of gelatin, 300 .mu.M 
deoxyribonucleotides (mixture of the 4 deoxyribonucleotides dA, dG, dC and 
dI), 50 pM of the primers according to the invention designated TB-1 and 
TB-2, two units of Taq polymerase and 10-50 .mu.l of an extract of a 
mixture of human cells and mycobacteria or 50 ng of DNA extracted from 
mycobacteria is maintained at 94.degree. C. (1.5 min), 50.degree. C. (2 
min) and 72.degree. C. (2 min) for approximately 40 cycles. After the last 
cycle, the samples are maintained at 37.degree. C. for 10 min and then 
stored at 4.degree. C. 
c. Analysis of the amplified samples by Southern blot analysis. 
10 .mu.l aliquots are removed from the amplified samples and subjected to 
electrophoresis on 2% agarose gel. The DNA is then transferred onto nylon 
filters (standard technique: REED K. L. et al., Nucleic Acid Research, 
1985, 13, 7207). The filters with DNA are then washed in 2.times.SSPE 
solution (20.times.SSPE solution corresponds to 3.0M NaCl, 200 mM 
NaH.sub.2 PO.sub.4 and 20 mM EDTA), then treated with a prehybridisation 
mixture at 63.degree. C. in a solution comprising 5.times.SSPE and 
5.times.Denhardt (1.times.Denhardt solution corresponds to 0.02% of 
Ficoll, 0.02% of polyvinylpyrrolidone and 0.02% of bovine serum albumin) 
for 2 hours and then hybridised in the same solution containing three 
probes according to the invention, TB-4, TB-5 and TB-6, labelled at their 
5' end with .sup.32 P (2.times.10.sup.5 cpm/ml, specific activity 1-3 
.mu.Ci/pmol) overnight at 63.degree. C. The blots or deposits obtained are 
washed for 2 hours at room temperature in 0.1.times.SSC solution 
(1.times.SSC corresponds to 0.15M NaCl and 0.015M Na citrate) containing 
0.5% SDS for 2 to 4 minutes at 67.degree.-72.degree. C. in 5.times.SSPE 
solution containing 0.5% SDS and 2 hours at room temperature in 
0.1.times.SSC containing 0.5% SDS. The deposits obtained are dried and any 
hybrids present are visualised by exposure to an XAR-5 film. 
In dot-blot analysis, 10 .mu.l of aliquots of amplified samples are 
denatured by heating to 95.degree. C. for 2 min in 0.2 ml of 0.4M NaOH 
containing 25 mM EDTA. The samples are cooled rapidly and loaded into the 
wells of a manifold (BIORAD, (USA)) or minifold (CERA LABO (France)) 
fitted with a nylon membrane. Each well is washed twice with 0.4 ml of 
20.times.SSPE, the membrane is heated to 80.degree. C. for one hour and 
hybridisation is carried out as described above. The results as seen in 
FIG. 1 are obtained. 
FIG. 1 shows the hybridisation of amplified DNA of M. bovis (B), M. avium 
(A) and M. fortuitum (F) with the specific probes TB-4 (SEQ ID NO:12), 
TB-5 (SEQ ID NO:13) and TB-6 (SEQ ID NO:14), respectively. 
Example 2 
Comparison of sequences obtained in Example 1 with the DNA sequences as 
described in the literature. 
a) Sequencing of the amplified mycobacterial sequences obtained. 
The DNA is extracted with phenol, precipitated with ethanol and redissolved 
in 10 mM Tris-HCl (pH 8) containing 1 mM EDTA. 
The DNA is then digested with the restriction endonucleases PstI and BamHI, 
cloned into the phages M13mp18 and M13mp19 and sequenced according to 
SANGER's method using T7 polymerase or Taq polymerase in the presence of 
d-azaGTP in place of dGTP. 
The amplified DNA corresponds to the expected region of the gene coding for 
the 65-kD mycobacterial antigen, as shown in FIG. 2 (SEQ ID NO:27), in 
which the DNA sequences of the amplified fragments obtained from the gene 
coding for the 65-kD antigen of M. bovis, M. avium, M. paratuberculosis 
and M. fortuitum are specified. 
The sequence of the amplified DNA of M. bovis is identical to the 
corresponding region of the sequence coding for the 65-kD antigen of M. 
bovis (THOLE et al. 1987) and M. tuberculosis (SHINNICK et al. 1987). 
The sequences of the amplified DNA from M. avium, M. paratuberculosis and 
M. fortuitum are very similar to those of M. bovis and M. tuberculosis, 
and the deduced translation products corresponding to these sequences are 
also very similar to the 65-kD antigen of M. bovis/M. tuberculosis as 
shown in FIG. 2 (SEQ ID NO:27). 
FIG. 3 shows a number of restriction sites of the fragments of 343 
nucleotides contained within the gene coding for the 65-kD antigen of M. 
avium (FIG. 3a) (SEQ ID NO:28), M. fortuitum (FIG. 3b) (SEQ ID NO:29), M. 
paratuberculosis (FIG. 3c) (SEQ ID NO:30) and M. bovis BCG (FIG. 3d) (SEQ 
ID NO:31). 
Example 3 
Development of the sensitivity of the method. 
The sensitivity of the method was tested using BCG diluted in a biological 
medium, pleural fluid. It was possible to detect approximately 10 bacilli 
per ml of fluid; this represents a considerable improvement on the direct 
examination tests, which require 10.sup.3 to 10.sup.4 bacilli/ml for the 
detection of mycobacteria, and without identification. 
Furthermore, this test may be accomplished much more rapidly than the 
detection and identification of mycobacteria after enrichment and 
culturing. 
FIG. 4 shows the results obtained for the DNA extracted from samples 
containing 10.sup.6 human mononuclear blood cells and 6.times.10.sup.5 
(column 1), 6.times.10.sup.4 (column 2), 6.times.10.sup.3 (column 3), 600 
(column 4), 60 (column 5) and 6 (column 6) M. bovis bacilli, and amplified 
using Taq polymerase and the oligonucleotide primers TB-1 (SEQ ID NO:9) 
and TB-2 (SEQ ID NO:10) (FIG. 4a: gel; FIG. 4b: dot blot). 
Example 4 
Detection of amplified sequences of the tuberculosis bacillus group by the 
oligonucleotide restriction test. 
To detect the presence of amplified sequences of mycobacteria belonging to 
the tuberculosis bacillus group by the restriction test, 4.times.10.sup.4 
cpm of oligonucleotide TB-9 (SEQ ID NO:17) labelled with .sup.32 P at its 
5' end is mixed with 2 .mu.l of a 10.times.buffer (40 mM Tris-HCl pH 7.0, 
60 mM MgCl.sub.2 and 60 mM 2-mercaptoethanol) in a final volume of 15 
.mu.l. 4 .mu.l of amplified product are added and the tube is incubated at 
95.degree. C. for 5 minutes, transferred to ice and then incubated at 
52.degree. C. for 2 hours. 1 .mu.l of BanI (25 units) is added and the 
tubes are incubated at 37.degree. C. for 1-2 hours. 4 .mu.l of 95% 
formamide containing 20% of Ficoll, 25 mM EDTA, 25 .mu.g/ml of bromophenol 
blue and 25 .mu.g/ml of xylene cyanol are added, the tubes are heated to 
65.degree. C. for 10 minutes and 12 .mu.l are subjected to electrophoresis 
on a 30% polyacrylamide gel (340 V for 1-2 hours). The gel is then exposed 
to an X-ray film for 3-18 hours and the positive samples are identified by 
the presence of a band corresponding to the fragment of 11 nucleotides at 
the 5' end of the oligonucleotide TB-9, produced by the cleavage of TB-9 
by the restriction enzyme. 
Example 5 
Synthesis of the oligonucleotides according to the invention. 
The oligonucleotides are synthesised using the phosphoramidite method 
(BEAUCAGE, 1985, loc.cit.) with a 380 D DNA synthesiser (APPLIED 
BIOSYSTEMS, CA). TB-1 (SEQ ID NO:9), TB-2 (SEQ ID NO:10), TB-3 (SEQ ID 
NO:11), TB-4 (SEQ ID NO:12), TB-5 (SEQ ID NO:13 ), TB-6 (SEQ ID NO:14 ), 
TB-7 (SEQ ID NO:15), TB-8 (SEQ ID NO:16 ), and TB-9 (SEQ ID NO:17) are 
thereby obtained. 
Example 6 
Detection of amplified sequences of MAIP group by the oligonucleotide 
restriction test. 
The protocol is identical to that of Example 4; however, a number of 
reagents are different: 
the oligonucleotide TB-5 (SEQ ID NO:13), is used; 
the 1.times. buffer contains 500 mM Tris-HCl pH 8.0, 100 mM MgCl.sub.2 and 
400 mM NaCl; 
the restriction enzyme used is BglI (10 units). 
As in Example 4, the positive samples are identified by the presence of a 
band corresponding to a fragment of 9 nucleotides resulting from cleavage 
of the oligonucleotide TB-5 by the restriction enzyme BglI. 
FIG. 5 shows the detection of amplified mycobacterial DNA sequences by the 
oligonucleotide restriction test. 
The purified mycobacterial DNA is amplified, and equivalent amounts of the 
amplified product from M. avium (column 1-5), M. bovis BCG (column 6) and 
M. fortuitum (column 7) are evaluated as described above, using the 
oligonucleotide TB-5 labelled with .sup.32 P and the restriction enzyme 
BglI. An amount of enzyme corresponding to the following enzymatic 
activities is added: 1 unit (samples 1, 6 and 7); 5 units (sample 2); 10 
units (sample 3); 20 units (sample 4); and 50 units (sample 5). 
The autoradiogram is exposed for 3 hours with a single intensification 
screen. FIG. 5 shows clearly that only columns 1-5 enable the cleaved 
oligomer to be demonstrated, hence permitting identification of M. avium. 
Example 7 
Detection of mycobacterial DNA in a sputum specimen. 
DNA purified from M. tuberculosis (FIG. 6, T), from M. avium (FIG. 6, A) 
and from M. fortuitum (FIG. 6, F) and DNA extracted from sputum samples 
which yielded a negative culture (FIG. 6, columns 1-6) or from sputum 
samples which yielded a positive culture for M. tuberculosis (FIG. 6, 
columns 7 and 8) was amplified using Taq polymerase in the case of the PCR 
or using another polymerase and the oligonucleotides TB-1 (SEQ ID NO:9) 
and TB-2 (SEQ ID NO:10). Samples of the amplified sequences are bound to 
filters (dot blots) and hybridised with the oligonucleotides TB-4 (SEQ ID 
NO:12), TB-5 (SEQ ID NO:13) and TB-6 (SEQ ID NO:14) labelled at their 5' 
end with .sup.32 P. 
It is seen that the DNA of M. tuberculosis hybridises with TB-4 (SEQ ID 
NO:12). 
As is apparent from the foregoing, the invention is in no way limited to 
those of its methods of implementation, embodiments and methods of use 
which have just been described more explicitly; on the contrary, it 
encompasses all variants which may occur to the specialist in the field 
without departing from the scope or range of the present invention. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 31 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 116 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
XaaTyrGluLysIleGlyAlaGluLeuValXaaGluValAlaLysLys 
151015 
ThrAspAspValAlaXaaAspXaaThrThrThrAlaThrValLeuXaa 
202530 
GlnXaaLeuValXaaGluGlyLeuArgAsnValAlaAlaGlyAlaAsn 
354045 
XaaLeuXaaXaaLysXaaGlyIleGluLysAlaValGluXaaValThr 
505560 
XaaXaaLeuLeuXaaXaaAlaLysGluValGluThrLysXaaGlnIle 
65707580 
AlaAlaThrAlaXaaIleSerXaaGlyAspXaaSerIleGlyXaaXaa 
859095 
IleXaaXaaXaaMetAspLysValGlyXaaGluGlyValIleThrXaa 
100105110 
XaaGluSerXaa 
115 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 342 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
TACGAGAAGATCGGCGCTGAGCTCGTCAAGGAAGTCGCCAAGAAGACCGACGACGTCGCG60 
GGCGACGGCACCACCACCGCCACCGTTCTGGCACAGGCCCTGGTTCGTGAAGGTCTGCGC120 
AACGTCGCTGCCGGCGCCAACCCGCTCGGCCTGAAGCGCGGCATCGAGAAGGCCGTCGAG180 
AAGGTCACCGAGACGCTGCTGAAGAGCGCCAAGGAGGTGGAGACCAAGGAGCAGATCGCT240 
GCCACCGCCGGTATCTCCGCCGGTGACCAGTCCATCGGTGACCTGATCGCCGAGGCCATG300 
GACAAGGTCGGCAACGAGGGTGTCATCACCGTCGAGGAGAGC342 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 342 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
TACGAGAAGATCGGCGCCGAGCTGGTCAAGGAAGTCGCCAAGAAGACCGACGACGTCGCC60 
GGTGACGGCACGACGACGGCCACGGTGCTCGCCCAGGCGTTGGTCCGCGAGGGCCTGCGC120 
AACGTCGCGGCCGGCGCCAACCCGCTGGGTCTCAAGCGCGGCATCGAGAAGGCCGTCGAG180 
AAGGTCACCGAGACCCTGCTCAAGTCGGCCAAGGAGGTCGAGACCAAGGACCAGATCGCT240 
GCCACCGCGGCCATCTCCGCGGGCGACCAGTCGATCGGCGACCTGATCGCCGAGGCGATG300 
GACAAGGTCGGCAACGAGGGCGTCATCACCGTCGAGGAGTCC342 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 342 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
TACGAGAAGATCGGCGCCGAGCTGGTCAAAGAGGTAGCCAAGAAGACCGATGACGTCGCC60 
GGTGACGGCACCACGACGGCCACCGTGCTGGCCCAGGCGTTGGTTCGCGAGGGCCTGCGC120 
AACGTCGCGGCCGGCGCCAACCCGCTCGGTCTCAAACGCGGCATCGAAAAGGCCGTGGAG180 
AAGGTCACCGAGACCCTGCTCAAGGGCGCCAAGGAGGTCGAGACCAAGGAGCAGATTGCG240 
GCCACCGCAGCGATTTCGGCGGGTGACCAGTCCATCGGTGACCTGATCGCCGAGGCGATG300 
GACAAGGTGGGCAACGAGGGCGTCATCACCGTCGAGGAGTCC342 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 343 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GTACGAGAAGATCGGCGCCGAGCTGGTCGAGGAAGTCGCCAAGAAGACCGACGACGTCGC60 
CGGCGACGGCACCACCACGGCCACTGTGCTCGCGCAGGCGTTGGTCAAAGAGGGCCTGCG120 
CAACGTCGCGGCCGGCGCCAACCCACTGGGCCTGAAGCGCGGCATCGAGAAGGCAGTCGA180 
GAAGGTCACCGAGACGCTGCTCAAGGGCGCCAAGGAGGTCGAGACCAAGGAGCAGATCGC240 
TGCCACCGCGGCCATCTCCGCCGGTGACCAGTCGATCGGCGACCTGATCGCCGATGGCAT300 
GGACAAGGTCGGCAACGAGGGTGTCATCACCGTTGAGGAGTCC343 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 343 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
CTACGAGAAGATCGGCGCCGAGCTGGTCAAGGAAGTCGCCAAGAAGACCGACGACGTGGC60 
CGGTGACCGGACGACGACGGCCACCGTGCTGGTGCAGGCGCTGGTCAAAGAGGGCCTGCG120 
CAACGTCGCGGCCGGTGCCAACCTGCTCAGCTTCAAGTGCGGCATCGAGAAGGCGGTCGA180 
GAAGGTCACCGAGACCCTGCTCAAGCCGGCCAAGGAGGTCGAGACCAAGGAGCAGATCGC240 
CGCGACCGCCGTGATCTCGGTGGGCGACCAGTCGATCGGTGACCTGATCGCCGAGGCGAT300 
GGACAAGGTTGGCAACGAGGGCGTCATCACCGTCGAGGAGTCC343 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 343 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
CTACGAGAAGATCGGCGCCGAGCTGGTCAAAGAGGTCGCCAAGAAGACCGACGATGTCGC60 
CGGTGACCGGACCACCACGGCCACCGTGCTGGCACAGGCGCTGGTCAAGGAAGGCCTGCG120 
CAACGTTGCGGCCGGTGCCAACCCGCTCGGTCTGAAGCGCGGCATTGAGAAGGCAGTCGA180 
GAAGGTCACCGAGACCTTGCTCAAGTCGGCCAAAGAGGTCGAGACCAAGGAGCAGATCGC240 
GGCGACCGCAGCCATCTCCGCCGGCGACCAGTCGATCGGCGACCCGATCGTCGAGGCGAT300 
GGACAAGGTCGGCAACGAGGGCGTCATTACCGTCGAGGAGTCC343 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 343 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
CTACGAGAAGATCGGCGCCGAGCTGGTCAAGGAAGTCGCCAAGAAGACCGACGACGTCGC60 
GGCTGACGGCACCACCACCGCCACCGTGCTCGCCCAGCGGCTGGTGCGCGAGGGTCTGCG120 
CAACGTGGCCGCGGGCGCGAACCCGCTGGGCCTCAAGCGCGGCATCGAGAAGGCCGTCGA180 
GGCCGTGACCGCCAAGCTGCTCGACACCGCCAAGGAGGTCGAGACCAAGGAGCAGATCGC240 
CGCCACCGCGGGCATCTCCGCGGGCGACGCGTCCATCGGTGAGCTGATCGCCGAGGCCAT300 
GGACAAGGTCGGCAAGGAAGGCGTCATCACCGTCGAGGAGAGC343 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
GAGATCGAGCTGGAGGATCC20 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
AGCTGCAGCCCAAAGGTGTT20 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
GCGGCATCGAAAAGGCCGTG20 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
CGAAATCGCTGCGGTGGCCG20 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
CTGCCACCGCGGCCATCTCC20 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
CTGCCACCGCCGGTATCTCC20 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
AACGTCGCGGCCGGCGCCAA20 
(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: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
GACTCCTCGACGGTGATGAC20 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
CCTGCTCAAGGGCGCCAAG19 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
CGAAATCGCTGCGGTGGCCGCAATCTGCTC30 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
GGTGCTCGCCCAGGCGTTGGTCCGC25 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
TGTGCTCGCGCAGGCGCTGGTCAAA25 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 114 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
TyrGluLysIleGlyAlaGluLeuValLysGluValAlaLysLysThr 
151015 
AspAspValAlaGlyAspGlyThrThrThrAlaThrValLeuAlaGln 
202530 
AlaLeuValArgGluGlyLeuArgAsnValAlaAlaGlyAlaAsnPro 
354045 
LeuGlyLeuLysArgGlyIleGluLysAlaValGluLysValThrGlu 
505560 
ThrLeuLeuLysSerAlaLysGluValGluThrLysGluGlnIleAla 
65707580 
AlaThrAlaGlyIleSerAlaGlyAspGlnSerIleGlyAspLeuIle 
859095 
AlaGluAlaMetAspLysValGlyAsnGluGlyValIleThrValGlu 
100105110 
GluSer 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 114 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
TyrGluLysIleGlyAlaGluLeuValLysGluValAlaLysLysThr 
151015 
AspAspValAlaGlyAspGlyThrThrThrAlaThrValLeuAlaGln 
202530 
AlaLeuValArgGluGlyLeuArgAsnValAlaAlaGlyAlaAsnPro 
354045 
LeuGlyLeuLysArgGlyIleGluLysAlaValGluLysValThrGlu 
505560 
ThrLeuLeuLysSerAlaLysGluValGluThrLysAspGlnIleAla 
65707580 
AlaThrAlaAlaIleSerAlaGlyAspGlnSerIleGlyAspLeuIle 
859095 
AlaGluAlaMetAspLysValGlyAsnGluGlyValIleThrValGlu 
100105110 
GluSer 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 114 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
TyrGluLysIleGlyAlaGluLeuValGluGluValAlaLysLysThr 
151015 
AspAspValAlaGlyAspGlyThrThrThrAlaThrValLeuAlaGln 
202530 
AlaLeuValLysGluGlyLeuArgAsnValAlaAlaGlyAlaAsnPro 
354045 
LeuGlyLeuLysArgGlyIleGluLysAlaValGluLysValThrGlu 
505560 
ThrLeuLeuLysGlyAlaLysGluValGluThrLysGluGlnIleAla 
65707580 
AlaThrAlaAlaIleSerAlaGlyAspGlnSerIleGlyAspLeuIle 
859095 
AlaAspGlyMetAspLysValGlyAsnGluGlyValIleThrSerGly 
100105110 
GluSer 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 114 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
TyrGluLysIleGlyAlaGluLeuValLysGluValAlaLysLysThr 
151015 
AspAspValAlaGlyAspArgThrThrThrAlaThrValLeuValGln 
202530 
AlaLeuValLysGluGlyLeuArgAsnValAlaAlaGlyAlaAsnLeu 
354045 
LeuSerPheLysCysGlyIleGluLysAlaValGluLysValThrGlu 
505560 
ThrLeuLeuLysProAlaLysGluValGluThrLysGluGlnIleAla 
65707580 
AlaThrAlaValIleSerValGlyAspGlnSerIleGlyAspLeuIle 
859095 
AlaGluAlaMetAspLysValGlyAsnGluGlyValIleThrValGlu 
100105110 
GluSer 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 122 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
AspProTyrGluLysIleGlyAlaGluLeuValLysGluValAlaLys 
151015 
LysThrAspAspValAlaGlyAspArgThrThrThrAlaThrValLeu 
202530 
AlaGlnAlaLeuValLysGluGlyLeuArgAsnValAlaAlaGlyAla 
354045 
AsnProLeuGlyLeuLysArgGlyIleGluLysAlaValGluLysVal 
505560 
ThrGluThrLeuLeuLysSerAlaLysGluValGluThrLysGluGln 
65707580 
IleAlaAlaThrAlaAlaIleSerAlaGlyAspGlnSerIleGlyAsp 
859095 
ProIleValGluAlaMetAspLysValGlyAsnGluGlyValIleThr 
100105110 
ValGluGluSerAsnThrPheGlyLeuGln 
115120 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 114 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
TyrGluLysIleGlyAlaGluLeuValLysGluValAlaLysLysThr 
151015 
AspAspValAlaAlaAspGlyThrThrThrAlaThrValLeuAlaGln 
202530 
ArgLeuValArgGluGlyLeuArgAsnValAlaAlaGlyAlaAsnPro 
354045 
LeuGlyLeuLysArgGlyIleGluLysAlaValGluAlaValThrAla 
505560 
LysLeuLeuAspThrAlaLysGluValGluThrLysGluGlnIleAla 
65707580 
AlaThrAlaGlyIleSerAlaGlyAspAlaSerIleGlyGluLeuIle 
859095 
AlaGluAlaMetAspLysValGlyLysGluGlyValIleThrValGlu 
100105110 
GluSer 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 114 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
TyrGluLysIleGlyAlaGluLeuValLysGluValAlaLysLysThr 
151015 
AspAspValAlaGlyAspGlyThrThrThrAlaThrValLeuAlaGln 
202530 
AlaLeuValArgGluGlyLeuArgAsnValAlaAlaGlyAlaAsnPro 
354045 
LeuGlyLeuLysArgGlyIleGluLysAlaValGluLysValThrGlu 
505560 
ThrLeuLeuLysGlyAlaLysGluValGluThrLysGluGlnIleAla 
65707580 
AlaThrAlaAlaIleSerAlaGlyAspGlnSerIleGlyAspLeuIle 
859095 
AlaGluAlaMetAspLysValGlyAsnGluGlyValIleThrValGlu 
100105110 
GluSer 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 342 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
TACGAGAAGATCGGCGCCGAGCTGGTCAAGGAAGTCGCCAAGAAGACCGACGACGTCGCC60 
GGTGACGGCACGACGACGGCCACGGTGCTCCCCCAGGCGTTGGTCCGCGAGGGCCTGCGC120 
AACGTCGCGGCCGGCGCCAACCCGCTGGGTCTCAAGCGCGGCATCGAGAAGGCCGTCGAG180 
AAGGTCACCGACACCCTGCTCAAGTCGGCCAAGGAGGTCGAGACCAAGGACCAGATCGCT240 
GCCACCGCGGCCATCTCCGCGGGCGACCAGTCGATCGGCGACCTGATCGCCGAGGCGATG300 
GACAAGGTCGGCAACGAGGGCGTCATCACCGTCGAGGAGTCC342 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 342 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
TACGAGAAGATCGGCGCTGAGCTCGTCAAGGAAGTCGCCAAGAAGACCGACGACGTCGCG60 
GGCGACGGCACCACCACCGCCACCGTTCTGGCACAGGCCCTGGTTCGTGAAGGTCTGCGC120 
AACGTCGCTGCCGGCGCCAACCCGCTCGGCCTGAAGCGCGGCATCGAGAAGCCCGTCGAG180 
AAGGTCACCGAGACGCTGCTGAAGAGCGCCAAGGAGGTGGAGACCAAGGAGCAGATCGCT240 
GCCACCGCCGGTATCTCCGCCGGTGACCAGTCCATCGGTGACCTGATCCCCGAGGCCATG300 
GACAAGGTCGGCAACGAGGGTGTCATCACCGTCGAGGAGAGC342 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 342 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
TACGAGAAGATCGGCGCCGAGCTGGTCAAGGAAGTCGCCAAGAAGACCGACGACGTCGCC60 
GGTGACGGCACGACGACGGCCACGGTCCTCGCCCAGGCGTTGGTCCGCGAGGGCCTGCGC120 
AACGTCGCGGCCGGCGCCAACCCGCTGGGTCTCAAGCGCGGCATCGAGAAGGCCGTCGAG180 
AAGGTCACCGAGACCCTGCTCAAGTCGGCCAAGGAGGTCGAGACCAAGGACCAGATCGCT240 
GCCACCGCGGCCATCTCCGCGGGCGACCAGTCGATCGGCGACCTGATCGCCGAGGCGATG300 
GACAAGGTCGGCAACGAGGGCGTCATCACCGTCGAGGAGTCC342 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 342 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
TACGAGAAGATCGGCGCCGAGCTGGTCAAAGAGGTAGCCAAGAAGACCGATGACGTCGCC60 
GGTGACGGCACCACGACGGCCACCGTGCTGGCCCAGGCGTTGGTTCGCGAGGGCCTGCGC120 
AACGTCGCGGCCGGCGCCAACCCGCTCGGTCTCAAACGCGGCATCGAAAAGGCCGTGGAG180 
AAGGTCACCGAGACCCTGCTCAAGGGCGCCAAGGAGGTCGAGACCAAGGAGCAGATTGCG240 
GCCACCGCAGCGATTTCGGCGGGTGACCAGTCCATCGGTGACCTGATCGCCGAGGCGATG300 
GACAAGGTGGGCAACGAGGGCGTCATCACCGTCGAGGAGTCC342 
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