Materials and methods for detection of oxalobacter

DESCRIPTION

MATERIALS AND METHODS FOR DETECTION OF OXALOBACTER

0003This invention was made with government support under National Institutes of Heath

0004Grant No. DK 20586. The government has certain rights in this invention.

0005Gross-Reference to a Related Application This application is a continuation-in-part of co-pending patent application Serial No. 08/883,610, filed June 26, 1997; which is a continuation-in-part of co-pending patent application

0006Serial No. 08/717,587, filed September 27, 1996; which is a continuation-in-part of co-pending patent application Serial No. 08/493,197, filed June 20, 1995; which is a continuation-in-part of co-pending patent application Serial No.08/262,424, filed June 20, 1994, now U.S. Patent No. 5,604,111.

0007Field of Invention The present invention relates to novel assay methods and devices for determining the presence or concentration of oxalate in a sample; Oxalobacter genes encoding enzymes required for the catabolism of oxalate; and materials and methods for detecting and identifying Oxalobacter formigenes in a sample.

0008Background of the Invention Oxalic acid (Oxalate) is a highly toxic natural by-product of catabolism in vertebrate animals and many consumable plants. Unfortunately, a significant portion of humans are unable to properly metabolizing oxalate, a condition which may result in the formation of kidney stones in those persons. It is estimated that 70% of all kidney stones are composed of some amount of oxalate. Approximately 12 percent of the U.S. population will suffer from a kidney stone at some time in their lives, and the incidence is rising not only in the United States, but also in Sweden and Japan (Curhan, 1993). Moreover, although a healthy person breaks down or excretes sufficient quantities of oxalate to avoid excessive accumulation of oxalate in the tissues, a number of disease states are known to be associated with malfunctions of oxalate metabolism, including pyridoxine deficiency, renal failure and primary hyperoxaluria, a metabolic genetic disorder that results in the excessive deposition of oxalate in the kidneys. Persons suffering from and at risk for developing kidney stones, as well as patients with lipid malabsorption problems (e.g., sprue, pancreatic insufficiency, inflammatory intestinal disease, bowel resection, etc.), tend to have elevated levels of urinary oxalate, a fact that has been exploited as a means for identifying individuals at risk. While elevated levels of oxalate may be present in urine, detecting elevated levels of oxalate in serum has not been routine due to the difficulty in detecting the low levels of oxalate present in serum.

0009Most previous methods for measuring oxalate in a biological sample first require the isolation of the oxalate by precipitation, solvent extraction, or an ion-exchange absorption (Hodgkinson, 1970). Quantitation of the isolated oxalate may be determined by any one of several methods including colorimetry, fluorometry, gas-liquid chromatography or isotope dilution techniques. Because many of the oxalate isolation techniques used in these analytical methods are not quantitative, it is normally necessary to correct for the low recovery of oxalate by adding a ^l4C-labeled oxalic acid internal standard, which further complicates the analytical method. All these methods are laborious, and consequently expensive because of the amount of skilled laboratory technician time which must be employed. In addition, isolation of the oxalate may require relatively large sample volumes for starting material.

0010Recently, several advances in the detection and quantitation of oxalate have been made through the use of (a) oxalate degrading enzymes and (b) high performance liquid chromatography. One commercially-available enzymatic test (Sigma Chemical Company, St. Louis, MO) employs oxalate oxidase to oxidize oxalate to carbon dioxide and hydrogen peroxide. The hydrogen peroxide produced can then be measured colorimetrically in a second enzymatic reaction in the presence of peroxidase.

0011In another enzymatic method for measuring oxalate, oxalate decarboxylase is used to convert oxalate to carbon dioxide and formate. The resultant carbon dioxide can be measured manometrically, by the pH change in a carbon dioxide trapping buffer or by the color change in a pH indicator buffer. Whatever method of carbon dioxide assay is adopted, the time required for diffusion and equilibration of carbon dioxide is much longer than is desirable for a rapid analytical method.

0012Alternatively, the formate produced by the action of oxalate decarboxylase can be assayed with formate dehydrogenase in an NAD/NADH coupled reaction, as described in

0013Costello, 1976 and Yriberri, 1980. This method is both cumbersome and time-consuming because oxalate decarboxylase and formate dehydrogenase differ in their optimum pH requirements, thus necessitating a pH adjustment during the analysis. Another commercially available enzymatic test (Boehringer Mannheim) cleaves oxalate to formate and carbon dioxide, then oxidizes the formate to bicarbonate by NAD in the presence of the enzyme formate dehydrogenase. The amount of NADH is determined by means of its absorbance at 334, 340, or 365 nm. Another test ("STONE RISK" by Mission Pharmacal) measures oxalate as a part of a battery of tests for kidney stones.

0014Oxalobacter formigenes is a recently discovered, oxalate-degrading obligately anaerobic bacterium residing primarily in the intestines of vertebrate animals, including man (Allison et al, 1986). Although the first isolates of O. formigenes were cultured from sheep rumen (Dawson et al, 1980), additional strains have now been isolated from cecal contents of rats, guinea pigs and pigs (Argenzio et al, 1988, Daniel et al, 1987), fecal samples from man

0015(Allison et al, 1985), and anaerobic aquatic sediments (Smith et al, 1985). This bacterium is unique among oxalate-degrading organisms having evolved a total dependence on oxalate metabolism for energy (Dawson et al, 1980). Recent evidence suggests that Oxalobacter formigenes has an important symbiotic relationship with vertebrate hosts by regulating oxalic acid absorption in the intestine as. well as oxalic acid levels in the plasma (Hatch and Freel,

00161996). Studies by Jensen and Allison (1994) comparing various O. formigenes isolates revealed only limited diversity of their cellular fatty acids, proteins, and nucleic acid fragments. Based on these comparisons, strains of O. formigenes have been divided into two major subgroups. In general, group I strains have shown limited intragroup diversity, while group II strains have shown greater intragroup diversity.

0017Special conditions are required to culture O. formigenes and their detection is based generally on the appearance of zones of clearance of calcium oxalate crystals surrounding colonies (Allison et al, 1986). Assays based on the appearance of zones of clearance of calcium-oxalate crystals surrounding bacterial colonies (Allison et al, 1985) or degradation of oxalate in culture media measured by calcium-chloride precipitation (Dawson et al, 1980) fail to confirm the oxalate-degrading bacteria as Oxalobacter.

0018As illustrated above, the currently existing assays for oxalate suffer from numerous problems, including cost, inaccuracy, reliability, complexity, and lack of sensitivity. Accordingly, it is an object of the subject invention to provide a simple, accurate, and sensitive assay for the detection of low levels of oxalate in a biological sample.

0019The current methods for culturing and identifying the presence of Oxalobacter formigenes are technically demanding and time consuming, and therefore, are not suitable for rapid and specific identification of O. formigenes, particularly for clinical diagnostics. Accordingly, another object of the subject invention is to provide a rapid, accurate polynucleotide probe-based assay for the detection of O. formigenes.

0020Brief Summary of the Invention The subject invention concerns the cloning, sequencing, and expression of the formyl-

0021CoA transferase (frc) and the oxalyl-CoA decarboxylase (oxc) genes of Oxalobacter formigenes, and the use of the enzymes to detect the presence of oxalate in a sample. The assay of the subject invention provides, for the first time, a rapid, sensitive method to detect even very low concentrations of oxalate in biological samples. Advantageously, the biological samples in which oxalate can be detected include both urine and serum samples. The enzyme system used according to the subject invention converts oxalate to carbon dioxide and formate. In a preferred embodiment of the subject invention, the production of formate is then measured colorimetrically. This assay provides a sensitive, accurate and convenient means for detecting oxalate. A further aspect of the subject invention is the discovery of the O. formigenes genes which encode the formyl-CoA transferase and the oxalyl-CoA decarboxylase enzymes. The discovery of these genes makes it possible to efficiently produce large quantities of pure formyl- CoA transferase and oxalyl-CoA decarboxylase for use in the assay of the subject invention or other appropriate application. The subject invention further concerns a dipstick device for the detection and quantitation of oxalate in a sample. The dipstick device comprising comprises the oxalyl-CoA decarboxylase and formyl-CoA transferase enzymes of the present invention immobilized on a carrier matrix. A detectable signal is generated on the dipstick if oxalate is present in the sample. The subject invention also provides a means for detecting the presence of Oxalobacter formigenes organisms in a sample. The method of detection provided for herein involves polynucleotide probes which can be used to identify Oxalobacter formigenes .

0022The subject invention also concerns the polynucleotide primers and the use thereof for polymerase chain reaction (PCR) amplification of Oxalobacter formigenes nucleotide sequences. Amplified Oxalobacter sequences can then be detected using the polynucleotide probes of the subject invention.

0023Brief Description of the Drawings Figures 1A-E show the detection of varying concentrations of oxalate in a sample. Colorimetric absorbance for each sample was plotted over time (minutes). Positive and negative control panels are also shown.

0024Figures 2 A and B show the nucleotide sequence of the formyl-CoA transferase gene and the deduced amino acid sequence of the formyl-CoA transferase polypeptide from

0025Oxalobacter formigenes. Bolded letters represent amino acid residues determined by N-terminal protein sequencing.

0026Figures 3 A and B show the nucleotide sequence of the oxalyl-CoA decarboxylase gene and flanking regions from Oxalobacter formigenes. The consensus ribosome-binding site lies approximately 10 bases upstream (double-underlined letters) from the putative translation initiation codon (positions 1 to 3). A rho-independent termination sequence lies at positions 1758 to 1790 (double-underlined letters). A putative TPP-binding site appears between positions 1351 and 1437.

0027Figures 4A-C show an RFLP analysis of O. formigenes, strain OxB using probes specific for the oxc gene encoding oxalyl-CoA decarboxylase and the frc gene encoding formyl-

0028CoA transferase. Genomic DNA isolated from a 14 day culture of O. formigenes strain OxB was digested with the restriction enzyme HIND ILL The digested DNA was size fractionated by electrophoreses through 0.5% agarose gels, electroblotted to a nylon membrane, then hybridized with either probe API 5 (SEQ LD No. 6-FIG. 4A) or probe AP34 (SEQ O NO. 9-FIG. 4B) to detect oxc or probe AP273 (SEQ LD NO. 10-FIG. 4C) to detect frc.

0029Figures 5A-D show the sensitivity of detecting the oxc and^rc genes in RFLP of O. formigenes strain OxB versus strain HC-l. Genomic DNA from each of the two strains was digested with the restriction enzyme HLND ILT. Two-fold serial dilutions were made of the digested DNA and size fractionated by electrophoresis through 0.5% agarose gels (FIG. 5B and 5D). RFLP analyses were carried out as described in Fig. 4, except the nylon membranes were hybridized with a 1: 1 mixture of probe AP15 (SEQ LO NO. 6) plus probe AP273 (SEQ LD NO. 10) (FIG. 5 A and 5C).

0030Figures 6 shows the detection of the oxc and frc genes in various strains of O. formigenes by RFLP analysis. RFLP was carried out as described in Fig. 5. Figures 7A and B show PCR-based amplification of a genetic region of the oxc gene in various strains of O. formigenes. Using PCR primer API 5 (SEQ ID NO. 6) and primer AP22 (SEQ ID NO. 11) as PCR primers, PCR amplification was performed using genomic DNA isolated from each of the 12 strains of O. formigenes listed in Table 1 as template. PCR products

0031RECTIFIED SHEET (RULE 91) ISA/EP were size fractionated by electrophoresis through 1.2% agarose gels and observed visually usmg ethidium bromide (EtBr) and UV light.

0032Figures 8A and B show a direct analysis of fecal samples for O formigenes. Oxalobacter negative stool sample (A & B) was spiked with 10² (C) and 10⁴ (D) cfu of OxB or 10³ (E) and 10⁴ (F) cfu of OxK per 0.1 gm. DNA from an unspiked O formigenes-posihve stool sample diluted 1:25 (G) and 1:50 (H).

0033Figures 9A and B show the identification of sequence homologies within the oxc gene expressed in representative group I and group LI strains of Oxalobacter formigenes to design oligonucleotide probes. Partial sequences of 5 '-end of the oxc gene generated by PRC amplification of the region bounded by the primer pair, AP34/AP21. A region of high homology shared by all strains (between bp 13 and 43) was selected for the genus-specific oligonucleotide probe, AP286, while regions of high homology shared by only group I strains (between bp 197 and 214) or shared only by group II strains (between bp 133 and 150) were selected for group- specific oligonucleotide probes, HS2 and AP307, respectively. Figures 10A and B show the detection of Oxalobacter formigenes usmg a genus- specific oligonucleotide probe that hybπdizes to the PCR product of the oxc gene. Usmg the pπmer pair AP34/AP21, PCR amplification was performed usmg genomic template DNA isolated from 8 group I and 8 group II strains of O formigenes. The PCR products were size fractionated by electrophoresis through 1 2% agarose gels and the expected 504-508 bp product visualized with EtBr under UV light (upper panel) The PCR products were transblotted to nylon membranes and Southern blotted usmg the genus -specific oligonucleotide probe, AP286 (lower panel)

0034Figures 11A-C show the classification of group I and group II strains of Oxalobacter formigenes using group-specific oligonucleotide probes that hybπdize with PCR products of the oxc gene. Using the pπmer pair AP34/AP21 , PCR amplification was performed usmg genomic template DNA isolated from 8 group I and 8 group II strains of O formigenes The PCR products were size fractionated by electrophoresis through 1 2% agarose gels and the expected 504-508 bp product visualized with EtBr under UV light (upper panel). The PCR products were transblotted to nylon membranes and Southern blotted using HS2, the group I-specific (center panel), or AP307, the group LI-specιfϊc (lower panel), oligonucleotide probes

0035Figures 12A-C show agarose gels containing oxc amplification products from quantitative PCR visualized with UV light (top panel) Figure 12D and E show Log equivalence plots to determine the number of oxc molecules m the sample

0036RECTIFIED SHEET (RULE 91) ISA/EP Brief Description of the Sequences SEQ ID NO. 1 is a nucleotide sequence for the formyl-CoA transferase gene (also shown in Figure 2).

0037SEQ ED NO.2 is a polypeptide encoded by SEQ ID NO. 1, which can be used according to the subject invention.

0038SEQ ID NO.3 is the nucleotide sequence for the oxalyl-CoA decarboxylase gene (also shown in Figures 3A and B).

0039SEQ ED NO.4 is a polypeptide encoded by SEQ ID NO. 3, which can be used according to the subject invention. SEQ ΓD NO. 5 is an oxalyl-CoA decarboxylase sequence, which can be used as a probe according to the subject invention.

0040SEQ ED NO. 6 is an oxalyl-CoA decarboxylase sequence, which can be used as a probe or PCR pπmer according to the subject invention.

0041SEQ ED NO. 7 is an oxalyl-CoA decarboxylase 5 '-pπmer, which can be used according to the subject invention.

0042SEQ ED NO. 8 is an oxalyl-CoA decarboxylase 3 '-pπmer, which can be used according to the subject invention.

0043SEQ ED NO. 9 is an oxalyl-CoA decarboxylase sequence, which can be used as a probe or pπmer according to the subject invention. SEQ ED NO. 10 is a formyl-CoA transferase sequence, which can be used as a probe according to the subject invention.

0044SEQ ED NO. 11 is an oxalyl-CoA decarboxylase sequence, which can be used as a PCR pπmer according to the subject invention.

0045SEQ ED NO. 12 is an oxalyl-CoA decarboxylase sequence, which can be used as a PCR pπmer according to the subject invention.

0046SEQ ED NO. 13 is an oxalyl-CoA decarboxylase sequence, which can be used as a PCR pπmer according to the subject invention.

0047Detailed Descπption of the Invention The subject invention provides an accurate, sensitive assay for oxalate m biological samples such as uπne and serum. Elevated levels of oxalate are correlated with uπnary tract stone formation, as well as other health problems. Early detection of high levels of oxalate makes it possible to prevent, delay or reduce adverse health consequences through appropπate medication and through modulation of diet.

0048RECTIFIED SHEET (RULE 91) ISA/EP In the presently descπbed diagnostic system, two enzymes are used to catabolize oxalate to carbon dioxide and formate. Specifically, any oxalate that may be present in a sample being assayed is converted into formate and carbon dioxide (COj) through the combined action of the enzymes oxalyl-CoA decarboxylase and formyl-CoA transferase. The formate can then be detected usmg a vaπety of techniques known m the art. In a preferred embodiment, the production of formate is measured coloπmetπcally by linking the catabolism of formate with the production of a detectable color change (for example, the formation of a compound that absorbs a particular wavelength of light). The production of formate is directly correlated with the amount of oxalate present in the sample. Therefore, if a known amount of formate is produced usmg the subject enzyme system, then the amount of oxalate present in the sample can be easily quantitated.

0049In a preferred embodiment, the enzymes used m the subject invention are expressed by genes from the bacteπum Oxalobacter formigenes The genes encoding both oxalyl-CoA decarboxylase (Lung et al , 1994) and formyl-CoA transferase enzymes have been cloned and expressed, thus providing a readily-available source of reagent mateπal. The subject assay is capable of detectmg oxalate levels m a range as low as 0 00025-0 0005 mM (Figures 1 A-E). This level of sensitivity makes the subject assay capable of direct detection of oxalate m serum samples consisting of little as 10 μl volume The descπbed system can be easily automated with standard systems known m the art. In a preferred embodiment of the subject assay, the enzymatic reaction can be earned out in the wells of flat-bottomed 96-well microtiter plates and read in an automated plate reader Suitable concentrations of the assay reagents oxalyl-CoA decarboxylase, oxalyl-CoA, β-NAD, formate dehydrogenase, and the sample to be assayed are added to the microtiter wells The reaction is then brought to equihbπum (two mmute incubation at 37 °C in the plate reader) to permit degradation of any residual formate that may be present m the sample The formyl-CoA transferase enzyme is then added to the mixture to start the reaction, and the plate is read at 15 second intervals Formate production is determined by measuπng the reduction m NAD m the presence of formate dehydrogenase by detecting changes in absorbance of the sample at 340 nm (Baetz and Allison, 1989) The quantity of oxalate is determined by compaπson of the unknown samples with standards having a known amount of oxalate.

0050Further, the enzymatic reaction of the subject assay will not be initiated until the formyl- CoA transferase, oxalyl-CoA decarboxylase, and oxalyl-CoA are all present within the reaction mixture Therefore, initiation of the enzymatic reaction can be prevented by withholding one of the above reagents from the reaction mix Preferably, oxalyl-CoA decarboxylase and oxalyl-

0051RECTIFIED SHEET (RULE 91) ISA/EP CoA are added first, and the reaction is initiated by the addition of formyl-CoA transferase to the mix. However, the order of additton of the three reagents is not mateπal to the function of the assay, so long as one of the reagents is withheld until just pπor to the desired initiation point of the assay. The formyl-CoA transferase and oxalyl-CoA decarboxylase enzymes used in the sub_ject invention can be obtained and purified as a natural product of Oxalobacter formigenes (Baetz and Allison, 1989 and 1990). Alternatively, the enzymes can be obtained from host cells expressing the recombinant polynucleotide molecules of the subject invention that encode the enzymes. Other reagents used m the subject assay can be obtained from conventional sources, such as Sigma Chemical Company, St. Louis, MO. Further, a person of ordinary skill in the art can readily determine the optimal concentrations of the reagents to use m the assay descπbed herein.

0052A further aspect of the subject invention concerns the cloning, sequencing and expression of the Oxalobacter formigenes gene which encodes the formyl-CoA transferase used m the assay that is a subject of the invention. The gene was cloned usmg degenerate oligonucleotide probes (based on partial ammo acid sequencing of tryptic peptides) to screen an Oxalobacter genomic DNA library. The gene encodes a polypeptide having a molecular weight of approximately 40 kD. The subject invention further concerns the cloning, sequencing, and expression of the gene which encodes oxalyl-CoA decarboxylase from Oxalobacter formigenes. The nucleotide sequence of the cDNA of formyl-CoA transferase and oxalyl-CoA decarboxylase are shown in Figures 2A-B and 3A-B, respectively (SEQ LD NOS. 1 and 3).

0053Because of the redundancy of the genetic code, a vaπety of different polynucleotide sequences can encode the formyl-CoA transferase polypeptide disclosed herein. It is well within the skill of a person trained m the art to create alternative polynucleotide sequences encoding the same, or essentially the same, polypeptide of the subject invention. These vaπant or alternative polynucleotide sequences are withm the scope of the subject invention. As used herein, references to "essentially the same" sequence refers to sequences which encode ammo acid substitutions, deletions, additions, or insertions which do not mateπally alter the functional enzymatic activity of the encoded polypeptide Further, the subject invention contemplates those polynucleotide molecules having sequences which are sufficiently homologous with the DNA sequences shown m Figures 2A-B and 3A-B (SEQ LD NOS 1 and 3) so as to permit hybπdization with those sequences under standard high-stπngency conditions. Such hybπdizatton conditions are conventional m the art (see, e g , Mamatis et al , 1989).

0054RECTIFIED SHEET (RULE 91) ISA/EP As a person skilled in the art would appreciate, certain amino acid substitutions within the amino acid sequence of the polypeptide can be made without altering the functional activity of the enzyme. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions, whereby an amino acid of one class is replaced with another amino acid of the same class, fall within the scope of the subject invention so long as the substitution does not materially alter the enzymatic reactivity of the polypeptide. Non-conservative substitutions are also contemplated as long as the substitution does not significantly alter the functional activity of the encoded polypeptide.

0055The polynucleotides of the subject invention can be used to express the recombinant formyl-CoA transferase enzyme. They can also be used as a probe to detect related enzymes.

0056The polynucleotides can also be used as DNA sizing standards.

0057The polypeptides encoded by the polynucleotides can be used to raise an immunogenic response to the formyl-CoA transferase enzyme. They can also be used as molecular weight standards, or as inert protein in an assay. The polypeptides can also be used to detect the presence of antibodies immunoreactive with the enzyme.

0058The polynucleotide sequences of the subject invention may be composed of either RNA or DNA. More preferably, the polynucleotide sequences are composed of DNA. The subject invention also encompasses those polynucleotides that are complementary in sequence to the polynucleotide sequences disclosed herein. Another aspect of the subject invention pertains to kits for carrying out the enzyme assay for oxalate. In one embodiment, the kit comprises, in packaged combination and in relative quantities to optimize the sensitivity of the described assay method, (a) the oxalyl-CoA decarboxylase, oxalyl-CoA, β-NAD, and formate dehydrogenase; and (b) formyl-CoA transferase. The kit may optionally include other reagents or solutions, such as buffering and stabilization agents, along with any other reagents that may be required for a particular signal generation system. Other reagents such as positive and negative controls can be included in the kit to provide for convenience and standardization of the assay method.

0059The subject invention further concerns a method for detecting the presence of Oxalobacter formigenes organisms in a sample. Specific polynucleotide probes can be prepared based on the nucleotide sequence of either the oxalyl-CoA decarboxylase or the formyl-CoA transferase gene sequence of Oxalobacter formigenes . The polynucleotide probes of the subject invention can be used to identify Oxalobacter formigenes in a sample, and to classify the strain of Oxalobacter formigenes detected. The polynucleotide probes of the subject invention can be used according to standard procedures and conditions to specifically and selectively detect polynucleotide sequences that have sufficient homology to hybridize with the probe. DNA can be isolated from bacterial microorganisms in a biological specimen (e.g. , biopsy, fecal matter, tissue scrapings, etc.) using standard techniques known in the art and the isolated DNA screened for hybridization with Oxalobacter oxalyl-CoA decarboxylase-specific and or formyl-CoA transferase-specific polynucleotide probes. Various degrees of stringency can be employed during the hybridization, depending on the amount of probe used for hybridization, the level of complementarity (i.e., homology) between the probe and target DNA fragment to be detected. The degree of stringency can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing. Hybridization methods and conditions are known in the art and are generally described in Nucleic Acid

0060Hybridization: A Practical Approach (Hames, B.D., S.J. Higgins, eds.), IRL Press (1985).

0061The polynucleotide probes of the subject invention include, for example, the oxalyl-CoA decarboxylase probe A (SEQ ID NO. 5), probe API 5 (SEQ ID NO. 6), and probe AP34 (SEQ ID NO. 9), probe AP 286 (SEQ ID NO. 14), probe AP307 (SEQ ID NO. 15), and probe HS-2 (SEQ ID NO. 16), specifically exemplified herein. Probes for formyl-CoA transferase include, for example, probe AP273 (SEQ ID NO. 10) specifically exemplified herein. The nucleotide sequences of the exemplified probes are shown below:

0062Probe A 5 '- GAGCGATACCGATTGGA -3 ' (SEQ ID NO. 5)

0063Probe AP 15 5 ' - GCACAATGCGACGACGA -3 ' (SEQ ID NO. 6) Probe AP34 5 ' - ATACTCGGAATTGACGT -3 ' (SEQ ID NO. 9)

0064Probe AP273 5 '-TTCATGTCCAGTTCAATCGAACG-3 ' (SEQ ID NO. 10) Probe AP286 5 ' -GACAATGTAGAGTTGACTGATGGCTTTCATG-3 '

0065(SEQ ID NO. 14) Probe AP307 5 '-CAGGATGGTCAGAAGTTC-3 ' (SEQ ID NO. 15) Probe HS-2 5'-CCGGTTACATCGAAGGA-3' (SEQ ID NO. 16)

0066The polynucleotide probes contemplated in the subject invention also include any polynucleotide molecule comprising a nucleotide sequence capable of specifically hybridizing with oxalyl-CoA decarboxylase or formyl-CoA transferase polynucleotide sequence of the present invention. As used herein, reference to "substantial homology" or "substantially complementary" refers not only to polynucleotide probes of the subject invention having 100% homology with the nucleotide sequence of the target polynucleotide, or fragments thereof, but also to those sequences with sufficient homology to hybridize with the target polynucleotide. Preferably, the degree of homology will be 100%; however, the degree of homology required for detectable hybridization will vary in accordance with the level of stringency employed in the hybridization and washes. Thus, probes having less than 100% homology to the oxalyl-CoA decarboxylase or formyl-CoA transferase polynucleotide sequences can be used in the subject method under appropriate conditions of stringency. In a preferred embodiment, high stringency conditions are used. In addition, analogs of nucleosides may be substituted for naturally occurring nucleosides within the polynucleotide probes. Such probes having less than 100% homology or containing nucleoside analogs are within the scope of the subject invention. The skilled artisan, having the benefit of the disclosure contained herein, can readily prepare probes encompassed by the subject invention.

0067In addition, the subject invention also concerns polynucleotide primers that can be used for polymerase chain reaction (PCR) amplification of Oxalobacter formigenes nucleotide sequences. PCR amplification methods are well known in the art and are described in U.S.

0068Patent Nos. 4,683,195; 4,683,202; and 4,800,159. The polynucleotide primers and probes of the present invention can also be used to amplify nucleotide sequences encoding oxalyl-CoA decarboxylase and formyl-CoA transferase by non-PCR based amplification methods. The subject primers and probes in any method where amplification of specific sequences is desired.

0069In a preferred embodiment, the polynucleotide primers are based on the oxalyl-CoA decarboxylase or formyl-CoA transferase gene sequence and can be used to amplify the full length or a portion of the target gene. The amplified Oxalobacter sequences can be detected using the probes of the subject invention according to standard procedures known in the art. The polynucleotide primers of the subject invention include, for example, oxalyl-CoA decarboxylase PCR primer 1 (SEQ ID NO. 7), PCR primer 2 (SEQ ID NO. 8), PCR primer AP 15

0070(SEQ ID NO. 6), PCR primer AP22 (SEQ ID NO. 11), PCR primer AP34 (SEQ ID NO. 9), PCR primer AP21 (SEQ ID NO. 17), OXF6 (SEQ ID NO. 12), and OXF7 (SEQ ID NO. 13), specifically exemplified herein. The nucleotide sequences of the exemplified PCR primers are shown below:

0071PCR primer 1 5 '- CAGGTTATGCAGCTTCT -3 ' (SEQ ID NO. 7)

0072PCR primer 2 5 '- GGATGGTTGTCAGGCAG -3 ' (SEQ ID NO. 8)

0073PCR primer AP15 5'-GCACAATGCGACGACGA-3' (SEQ ID NO. 6)

0074PCR primer AP22 5 '-GTAGTTCATCATTCCGG-3 ' (SEQ ID NO. 11 ) PCR primer AP34 5 '-ATACTCGGAATTGACGT-3 ' (SEQ ID NO. 9)

0075PCR primer AP21 5 ' -TCCAATCGGTATCGCTC-3 ' (SEQ ID NO. 17)

OXF6 5'-AATGTAGAGTTGACTGA-3' (SEQ ID NO. 12)

0077OXF7 S'-TTGATGCTGTTGATACG-S' (SEQ ID NO. 13) The primer pair AP34 and AP21 (derived from oxc sequences between bp -59 to -41 and by 451 to 435, respectively), consistently amplifies a 500 bp segment of oxc from all O. formigenes strains and isolates tested. PCR application of whole fecal DNA with this genus- specific primer pair, in conjunction with Southern Blotting using genus and group specific probes, now provides a rapid diagnostic tool to detect and speciate O. formigenes. Time- consuming steps, e.g., agarose-gel electrophoresis and Souther blot hybridizations, can be substituted with newer technologies such as microtiter-plate based colorimetric or fluorogenic assays (Jordan et al, 1996).

0078Polynucleotide primers contemplated by the subject invention also include any polynucleotide molecule comprising a nucleotide sequence capable of specifically priming amplification of oxalyl-CoA decarboxylase or formyl-CoA transferase polynucleotide sequences disclosed herein. As used herein, reference to "substantial homology" or "substantially complementary" refers not only to polynucleotide primers of the subject invention having 100% homology with the nucleotide sequence of the target polynucleotide, or fragments thereof, but also to those sequences with sufficient homology to hybridize with and prime the amplification of a target polynucleotide. Preferably, the degree of homology will be equal to or about 100%. The skilled artisan, having the benefit of the disclosure contained herein, can readily prepare other primers of varying nucleotide length and sequence that can be used to amplify all or portions of the oxalyl-CoA decarboxylase and/or the formyl-CoA transferase gene. The polynucleotide probes and primers of the subject invention can be chemically synthesized or prepared through recombinant means using standard methods and equipment. The polynucleotide probes and primers can be in either single- or double-stranded form. If the probe or primer is double-stranded, then single-stranded forms can be prepared from the double- stranded form. The polynucleotide probes and primers may be comprised of natural nucleotide bases or known analogs of the natural nucleotide bases. The probes and primers of the subject invention may also comprise nucleotides that have been modified to bind labeling moieties for detecting the probe or primer or amplified gene fragment.

0079The polynucleotide molecules of the subject invention can be labeled using methods that are known in the art. The polynucleotides may be radioactively labeled with an isotope such as ³H, ³⁵S, ^l4C, or ³²P. The polynucleotides can also be labeled with fluorophores, chemiluminescent compounds, or enzymes. For example, a polynucleotide molecule could be conjugated with fluorescein or rhodamine, or luciferin or luminol. Similarly, the polynucleotide molecule can be conjugated with an enzyme such as horseradish peroxidase or alkaline phosphatase. Polynucleotide molecules can also be detected by indirect means. For example, the polynucleotide may be conjugated with ligands, haptens, or antigenic determinants. The conjugated polynucleotide is then contacted with the ligand receptor, with an anti-ligand molecule that binds to the ligands, or with an antibody that binds to the hapten/antigenic determinant, respectively. For example, the polynucleotide can be labeled with digoxygenin and detected with labeled anti-digoxygenin antibodies. The ligand receptor, anti-ligand molecule, or antibody may be directly labeled with a detectable signal system, such as a fluorophore, chemiluminescent molecule, radioisotope, or enzyme. Methods for preparing and detecting labeled moieties are known in the art.

0080The subject invention also concerns methods for quantitatively detecting oxalyl-CoA decarboxylase and formyl-CoA transferase nucleotide sequences and, thereby, determining the number of Oxalobacter formigenes in a sample. General methods and techniques for performing quantitative PCR are known in the art (Tarmuzzer et al, 1996)

0081In one embodiment of the present detection method, samples to be tested for the presence of Oxalobacter formigenes are obtained from a person or animal, and DNA is isolated from the specimen using standard techniques known in the art. For example, cells can be lysed in an alkali solution, the nucleic acid extracted in phenol: chloroform, and then precipitated with ethanol. The DNA is then fragmented into various sizes using restriction endonuclease enzymes or other means known in the art. The DNA fragments are then electrophoretically separated by size on an agarose gel. In an alternative embodiment, the DNA fragments are subjected to PCR amplification using PCR primers of the present invention prior to gel electrophoresis in order to specifically amplify portions of the formyl-CoA transferase and oxalyl-CoA decarboxylase genes.

0082After the DNA fragments are separated on the gel, the size-fractionated DNA fragments are transferred to a membrane matrix, such as nitrocellulose, nylon, or polyvinylidene difluoride (PVDF), by Southern blotting. The DNA immobilized on the membrane matrix is single- stranded. Polynucleotide probes of the subject invention are then contacted with the membrane and allowed to hybridize with the DNA immobilized on the membrane. A probe of the present invention can be labeled with a detectable signal, such as a radioisotope, or the probe can be labeled with a hapten or antigen such as digoxigenin. The hybridization can be performed under conditions known in the art. After hybridization of the probe with the DNA fragments on the membrane, the membrane is washed to remove non-hybridized probe. Standard wash conditions are known in the art, and the stringency and number of washes employed can vary.

0083The membrane is then tested or observed for the presence of hybridized probe. For example, if the hybridized probe was labeled with a hapten or antigen, then it can be detected using an antibody that binds to the conjugated hapten or antigen on the probe. The antibody can be directly labeled with a detectable fluorophore, chemiluminescent molecule, radioisotope, enzyme, or other signal generating system known in the art. Alternatively, the antibody can be detected using a secondary reagent that binds to the antibody, such as anti-immunoglobulin, protein A, protein G, and other antibody binding compositions known in the art. The secondary reagent can be labeled with a detectable fluorophore, chemiluminescent molecule, radioisotope, or enzyme. The presence of a detectable hybridization signal on the membrane indicates the presence of Oxalobacter formigenes in a test sample.

0084The subject invention also concerns a kit for the detection of Oxalobacter formigenes in a sample. A kit contemplated by the subject invention may include in one or more containers: polynucleotide probes, positive and negative control reagents, and reagents for detecting the probes. The kit may also include polynucleotide primers for performing PCR amplification of specific Oxalobacter formigenes genes. In a preferred embodiment, the polynucleotide probes and primers are specific for the oxalyl-CoA decarboxylase and formyl-CoA transferase genes of O. formigenes.

0085The subject invention also concerns a dipstick device comprising the enzymes of the subject invention and dyes and or substrates immobilized on a carrier matrix. Any dye or substrate that yields a detectable product upon exposure to the reaction products that are produced by the enzymatic reaction of oxalate with oxalyl-CoA decarboxylase and formyl-CoA transferase as described herein is contemplated for use with the subject dipstick device. The carrier matrix of the assay device can be composed of any substance capable of being impregnated with the enzyme and dye components of the subject invention, as long as the matrix is substantially inert with respect to the analyte being assayed for. For example, the carrier matrix may be composed of paper, nitrocellulose, PVDF, or plastic materials and the like. Incorporation of the enzymes, dye and other components on the carrier matrix can be accomplished by any method such as dipping, spreading or spraying. A preferred method is impregnation of the carrier matrix material by dipping in a reagent solution and drying to remove solvent. Drying can be accomplished by any means which will not deleteriously affect the reagents incoφorated, and typically is by means of an air drying oven. The dipstick device of the subject invention is dipped in or contacted with a sample to be tested for the presence or amount of oxalate. Positive and negative controls can be used in conjunction with the dipstick device. An appropriate amount of time is allowed to pass and then the dipstick is assessed for a positive reaction by visual inspection. If oxalate is present in the sample then a detectable signal, usually in the form of a color, can be observed on the dipstick. Typically, the intensity of the color developed in a fixed time period is proportional to the concentration of oxalate present in the sample.

0086All publications cited herein are incorporated reference.

0087Following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

0088Example 1 - Determination of Level of Sensitivity of Enzvme Assay System Samples containing oxalate at concentrations ranging from 0.004 mM to 0.00025 mM were prepared in 10 μl volumes. The samples were then assayed using the enzyme system of the subject invention in 96- well microtiter plates. Reagents were then added at the following concentrations: KH₂P0₄ (pH 6.7), 50 mM; MgCl₂, 5 mM; thiamine PPi (TPP), 2 mM; oxalyl- CoA, 0.375 mM; β-NAD, 1.0 mM; formate dehydrogenase, 0.25 IU; and oxalyl-CoA decarboxylase, 0.03 U. The reaction mixture was then incubated at 37°C for 2 minutes in order to permit the degradation of any residual formate that may be present in the sample mixture. The reaction was then initiated by the addition of formyl-CoA transferase to the sample mixture. Changes in A₃₄₀ were measured every 15 seconds at 37°C (Figures 1A-E). Appropriate positive and negative controls were run simultaneously with the assay.

0089Example 2 - Detection of Oxalobacter formigenes in a Sample

0090Strains of Oxalobacter formigenes used in the following methods are listed in Table 1 below.

0091RECTIFIED SHEET (RULE 91) ISA/EP TABLE 1. Description of the Oxalobacter formigenes strains

0092Group Classification of O. Source of formigenes strains" Strain Isolate

0093Group I OxB Sheep rumen

0094OxWR Wild rat cecum

0095SOx-4 Freshwater lake sediment

0096SOx-6 Freshwater lake sediment

0097POxC Pig cecum

0098HC-l Human feces

0099Group II BA-1 Human feces

0100OxK Human feces

0101HOxBLS Human feces

0102HOxRW Human feces

0103OxCR Lab rat cecum

0104OxGP Guinea pig cecum

0105^a From Jensen and Allison (1994).

0106All Oxalobacter formigenes strains were grown in medium B containing 30 mM oxalate, as described in Allison et al. (1985). Human fecal samples (approximately 60 mg) were inoculated anaerobically into vials containing 9 ml of media B, then sequentially transferred through 10⁸ dilutions. Cultures were incubated at 37° C for 10 days and biochemically tested for the catabolic consumption of oxalate by CaCl₂ precipitation (50 μl media, 100 μl 1% CaCl₂, and 2.7 ml dH₂0) and spectrophotometric analyses (600 nm).

0107Cultures (10-15 ml) of O. formigenes were centrifuged at 10,000 x g, the bacterial pellet was resuspended in 567 μl TE buffer (10 mM Tris-Cl, pH 7.5 plus 1 mM EDTA, pH 8.0), 30 μl

010810% sodium dodecyl sulfate (SDS) and 3 μl of proteinase K (20 mg/ml), and the mixture incubated 5 hr at 37 °C to ensure bacterial cell lysis. Nucleic acids were extracted from the lysates using phenol/chloroform/isoamylalcohol (25 :24: 1). Chromosomal DNA was precipitated from the aqueous phase by adding 'Λ volume of 7.5 M ammonium acetate and 2 volumes of 100% ethanol. DNA was recovered by centrifugation (12,000 x g), washed once with 70% ethanol, and the pellet resuspended in 15-20 μl H₂0. Bacterial DNA was also isolated directly from fresh human stool samples following lysis with chaotropic salt and guanidine thiocyanate, then binding to glass matrix (GlasPac, National Scientific Supply, San Rafael, CA) (Stacy-Phips et al, 1995).

0109Bacterial DNA was digested with the restriction endonuclease Hind III (Life Technologies, Inc., Gaithersburg, MD). The restriction-enzyme generated fragments were size separated by gel electrophoresis through 0.5% agarose, stained with ethidium bromide (EtBr), illuminated with UV light, and photographed to document proper digestion. Digested DNA was then transferred from the agarose gels to positively-charged nylon membranes (Boehringer- Mannheim GmBH, Indianapolis, IN) by positive pressure blotting and UV cross-linking (Stratagene, LaJolla, CA). Hybridizations were carried out using internal sequence oligonucleotide probes. Oligonucleotides were synthesized in the University of Florida ICBR

0110Oligonucleotide Synthesis Laboratory (Gainesville, FL) and have the sequences:

AP15 5'-GCACAATGCGACGACGA-3' (SEQ ID NO. 6)

AP22 5 '-GTAGTTCATCATTCCGG-3 ' (SEQ ID NO. 11)

0113AP34 5ΑTACTCGGAATTGACGT-3' (SEQ ID NO. 9) AP273 5'-TTCATGTCCAGTTCAATCGAACG-3' (SEQ ID NO. 10).

0114Each oligonucleotide was end-labeled with digoxigenin in a reaction using terminal transferase. The digoxigenin-labeled oligonucleotide probes were hybridized to the immobilized DNA fragments and hybridization detected colorimetrically by enzyme-linked immunoassay (ELISA) using an anti-digoxigenin alkaline phosphatase conjugate according to the manufacturer's protocol provided with the GENIUS III detection system (Boehringer-Mannheim).

0115All PCRs were performed according to protocols described in Anderson et al. (1993). Briefly, 50 μl reactions contained 1.5 mM MgCl₂, 200 μM dNTP, 1.25 U Taq polymerase (GIBCO-BRL, Bethesda, MD), 1 μg template DNA and 1 μM each of a 5' and 3' primer. A preferred reaction profile proved to be 94 °C for 5 min, then 45 cycles of 94 °C for 1 min of denaturation, 55°C for 2 min of annealing and 72°C for 3 min of primer extension. PCR products were size separated by gel electrophoresis in 1.2% agarose containing EtBr and photographed in UV light. PCR primer AP15 (SEQ ID NO. 6) and primer AP22 (SEQ ID NO. 11) were used as primers.

0116Previous studies by Lung et al. (1994) showed that genomic DNA of O. formigenes, strain OxB, could be digested with the restriction enzyme Hind III and that a limited number of enzyme cleavage sites existed near or within the oxalyl-CoA decarboxylase (oxc) gene. A RFLP analysis of Hind III digested OxB genomic DNA using either probe API 5 (SEQ ID NO. 6), a probe homologous to an internal sequence of the oxc gene, probe AP34 (SEQ ID NO. 9), a probe homologous to a 5' -end sequence of the oxc gene but separated from the probe API 5 (SEQ ID NO. 6) sequence by a Hind EH site, or probe AP273 (SEQ ID NO. 10), a probe homologous to an internal sequence of the formyl-CoA transferase (frc) gene, is shown in Figures 4A-C. Using probe API 5 (SEQ ED NO. 6), a fragment of approximately 7 kb containing a portion of the oxc gene was detected, while fragments of approximately 3 kb were detected using either probe AP34 (SEQ ED NO. 9) or probe AP273 (SEQ ED NO. 10). The 3 kb fragment identified by probe AP34 (SEQ ED NO. 9) is distinct from the 3 kb fragment detected by probe AP273 (SEQ ED NO. 10).

0117As shown in Figures 5A-D, the oxalyl-CoA decarboxylase and formyl-CoA transferase genes were consistently detected in samples containing as little as 0.06 to 0.20 μg of O. formigenes, strain OxB, DNA or approximately 0.20 to 0.40 μg of O. formigenes DNA from other group I strains, such as HC-l. The 23-bρ probe AP273 (SEQ ED NO. 10) can detect the frc gene in DNA samples containing only one-fourth the amount of DNA required for the 13 bp probe API 5 (SEQ ED NO. 6) to detect the oxc gene (Fig. 5 A and B). These probes are highly specific for O. formigenes since they fail to bind to other bacterial DNA, including Escherichia coli, Alcaligenes oxalaticus, and fecal bacteroides.

0118Protein, lipid and genetic studies of several isolates of O. formigenes have provided the basis for dividing this genus into two major subgroupings (Jensen et al, 1994). When RFLP analyses were performed on genomic DNA isolated from various Oxalobacter formigenes strains, probes API 5 (SEQ LD NO. 6) and AP273 (SEQ ED NO. 10) were able to distinguish group I strains from group El strains on the Southern blot hybridizations (Fig. 6). All strains of

0119O. formigenes belonging to group I (to which OxB is assigned) hybridized with both probe API 5 (SEQ D NO. 6) and probe AP273 (SEQ ED NO. 10). Due to a characteristic slow growth of strain HC-l, only faint bands appeared in this experiment. In contrast, none of the O. formigenes strains assigned to group II hybridized with probe AP273 (SEQ ED NO. 10) and only BA-1 hybridized with probe AP 15 (SEQ D NO. 6). These data indicate a highly conserved homology of oxc and frc within group I strains and a less conserved homology within group II strains.

0120To increase the sensitivity of detecting O. formigenes, PCR was used to amplify that portion of oxc which by RFLP appeared to differentiate the group I and group II strains. Using primer AP15 (SEQ ED NO. 6) and primer AP22 (SEQ ED NO. 11) as PCR primers to amplify a DNA segment in the carboxy-terminal region of oxc, strains assigned to group I (i.e., OxB,

0121HC-l, OxWR, POxC, SOx-4 and SOx-6) exhibited a common band at 452 bp (Fig. 7A-B). In contrast, the other six strains, all belonging to group π, showed variable amplification patterns, but all showed a dominant PCR band of approximately 630 bp, with a weaker 452 bp band. Sequence analysis of this 630 bp band from strain OxK has revealed the presence of the 452 bp

0122RECTIFIED SHEET (RULE 91) ISA/EP sequence present in the 630 bp PCR product. Close analysis of the group II strains suggest that their PCR amplification profiles are highly reproducible, suggesting group π strains may fall into three (sub)groupings: HOxBLS and HOxRW (subgroup 1), OxCR and OxGP (subgroup 2), and BA-1 and OxK (subgroup 3). The use of PCR-based detection of the oxc gene to identify O. formigenes in clinical specimens was examined by comparing PCR and biochemical methods of detection. Specimen 1, known to be positive for O. formigenes, gave ambiguous results in biochemical testing for oxalate depletion, but exhibited the presence of the 450 bp PCR product indicative of an O. formigenes group I strain. Specimen 2, known to be negative for O. formigenes, proved negative using both PCR-based and biochemical testing. Specimen 3, known to be positive for O. formigenes, showed depletion of oxalate in all dilutions and revealed a PCR pattern suggestive of an O. formigenes group El strain. PCR amplification was not observed in the original culture or the first dilution due to the presence of inhibitors of PCR e.g., bile salts, bilirubin, etc.) which copurify with DNA. To circumvent the inhibition of the PCR by factors co-purifying with the bacterial DNA,

0123DNA isolation was performed by lysing fresh stool samples with guanidine thiocyanate followed by adsorption to and elution from glass matrices. Using this method, PCR-based detection of O. formigenes can be performed using fecal DNA diluted only 1:25 to 1:50 to eliminate PCR inhibitors. Sensitivity experiments using different stool samples spiked with strains OxB or OxK in the range of 10' to 10⁷ cfu per 0.1 g of sample showed that as few as 10² to 10³ cfu of

0124O. formigenes per 0.1 g sample could be detected (Figure 8A-B). Again, PCR-based analyses of DNA isolated directly from a stool sample known to be positive for O. formigenes by culture methods, showed amplification patterns indicative of a group II strain (Figure 8B, lanes F & G).

0125Example 3 - Detection and Classification of Oxalobacter formigenes

0126Bacterial Strains

0127O. formigenes strains used included OxB (isolated from sheep rumen) and HC1, OxK, BA1, HOxBLS, HOxRW, HOxRA, HOxCC . and HOxHMlδ (isolated from human feces). In addition, several new purified cultures, including HOxUK5, HOxUK88, HOxUK90, and HOxHS (grown from human feces), were also used. All strains and isolates were grown in media B containing 30 mM potassium oxalate, as described elsewhere (Allison et al, 1985), and maintained under strict anaerobic conditions until used.

0128Preparation of Genomic DNA from O. formigenes Cultures

0129RECTIFIED SHEET (RULE 91) ISA/EP Fifteen ml cultures of O. formigenes were centrifuged at 10,000 x g, the bacterial pellet resuspended in 567 μl of TE buffer (10 mM Tris-HCl, pH 7.5, plus 1 mM EDTA, pH 8.0), 30 μl of 10% sodium dodecyl sulfate plus 3 μl of proteinase K (20 mg/ml), and this mixture incubated for 5 hours at 37°C to ensure bacterial cell lysis. Nucleic acids were extracted from the lysates with phenol:chloroform:isoamyl alcohol (25:24:1). Chromosomal DNA was precipitated by adding ! volume of 7.5 M ammonium acetate and 2 volumes of 100% ethanol. DNA was recovered by centrifugation (12,000 x g) and washed once in 70% ethanol. The final DNA precipitation was resuspended in 20 μl H₂0.

0130Sequence Analysis of the oxc Genes

0131The primer pair,

01325'-ATACTCGGAATTGACGT-3' (a 5 '-primer designated AP34) and

01335'-TCCAATCGGTATCGCTC-3' (a 3'-primer designated AP21) homologous to sequences within the 5 '-end of the oxc gene present in strain OxB (Lung et al,

01341994), was used to amplify a 500 bp DNA fragment from genomic DNA isolated from each of twelve human O. formigenes strains. Amplifications were performed in 50 μl PCR reactions containing 1.5 mM MgCl₂, 200 μM deoxynucleoside triphosphate, 1.25 U of Taq polymerase (Gibco-BRL, Bethesda, MD), 1 μg of genomic DNA and 1 μM each of 5'- and 3 '-primer. PCR were carried out for 35 cycles and included an initial 5 minute denaturation step at 94 °C, 1 minute annealing (with a temperature stepdown from 60°C to 55°C), 1 minute extension at 72 °C and a final 8 minute extension at 72 °C. The PCR products were size fractionated by electrophoreses through 1.2% agarose gels containing ethidium bromide for visualization of the bands in UV light. Each 500 bp PCR product was cloned into the TA cloning system, pCR-2.1 (Invitrogen, Inc., San Diego, CA). Competent DH5_°= E. coli bacteria were transfected with the recombinant plasmid and transformed bacteria selected on LB agar plates containing 10 μl/ml of ampicillin and 20 mg/ml of X-Gal. DNA from appropriate clones was isolated, checked for the presence of an insert of correct size by digestion with the restriction enzyme, Eco RI. Inserts of recombinant plasmids were sequenced using M13-forward and M13-reverse primers.

0135Clinical Samples

0136Fecal samples of 100 generally healthy children of either sex ranging in age from 0 to 12 years were examined for the presence of O. formigenes. All fecal samples were collected in Dzerzhinsk, a city in the Donetsk region of the Ukraine. This particular population was selected due to the fact that these children have had limited use of antibiotics, that might influence bacterial colonization of the intestinal tract, in treatment of childhood diseases. Approximately 25 mg sample of fresh stool (within 3-4 hours of collection), was inoculated into vials containing 10 ml of anaerobically sealed media B supplemented to 30 mM with potassium-oxalate. The vials were analyzed at the University of Florida, Gainesville, FL. After incubation at 37°C for one week, the loss of oxalate from each fecal culture was determined using a calcium-chloride precipitation method in which 50 μl culture media is mixed with lOOμl 0.1% CaCl₂ plus 3.0 ml dH₂0 and the absorbance of each mixture determined spectrophotometrically (600nm). The calcium precipitation test for loss of oxalate has been repeatedly verified as reliable by other methods (e.g., gas chromatography and butyl esters) for detection of oxalate. Typically, cultures not showing catabolism of oxalate generally have O.D. readings of about 0.1, whereas cultures with oxalate degradation have O.D. readings less than about 0.02.

0137PCR-based Detection and Identification of O. formigenes DNA was isolated from individual fecal cultures by the method of Phipps et al. (Stacy-

0138Phipps et al, 1995) using guanidine thiocyanate as a chaotropic agent and glass-matrix for DNA binding. One μl of each DNA sample was used as template in a 50 μl PCR reaction as described above. The amplified PCR products were size separated by electrophoresis through 1.2% agarose gels containing ethidium bromide and visualized with UV light. Each reaction was controlled using a reaction containing all the components of the PCR with the exception of template DNA.

0139Southern Blot Analysis

0140Southern blots were carried out as previously detailed in Example 2. Briefly, the size separated PCR products were transferred to positively charged nylon membranes (Boehringer

0141Mannheim GmBH, Indianapolis, IN) by positive pressure blotting and UV-crosslinking. The oxc derived genus specific (AP286), group I specific (HS-2) and group II specific (AP307) oligonucleotides were synthesized in the University of Florida ICBR DNA Synthesis Laboratory (University of Florida, Gainesville, FL) and end-labeled with digoxigenin in a reaction using terminal transferase. The digoxigenin labeled oligonucleotides were hybridized to the immobilized PCR products under conditions of high stringency (5X SSC and 68 °C). Hybridization was detected colorimetrically by enzyme-linked immunosorbent assay (ELISA) with an anti-digoxigenin alkaline phosphatase conjugate according to the manufacturer's protocol provided with the GENIUS III kit (Boehringer Mannheim GmBH). Generation of Genus-specific and Group-specific Probes

0142Preliminary studies looking at the efficacy of various oligonucleotide pairs to amplify portions of the oxc gene present in various O. formigenes strains revealed that the PCR primer pair AP34 (5'-primer)/Ap21 (3'-primer) amplified a 500 bp DNA fragment in both group I and group II strains. To determine the degree of sequence homology within the 5 '-end of the oxc gene between various strains of O. formigenes, genomic DNA was prepared from 5 group I and 7 group II strains isolated from human fecal samples for use as template in PCR with AP34 and AP21. Each PCR amplified an expected 500 bp product that was subsequently cloned into the pCR-2.1 vector system and sequenced. A comparison of the 5 '-end sequences of the oxc gene from these 12 human isolates with the OxB gene is shown in part in Figures 9A-B. The 5'-end of the oxc gene appears to be relatively conserved for a bacterial gene, with most bp changes occurring in the wobble base such that the codon translation is not altered. Nevertheless, there were enough sequence differences to demarcate group I strains from group El strains, thus permitting selection of regions that are conserved within strains of a specific group, but differ significantly from strains of the other group. Based on these conserved regions, genus-specific oligonucleotide probes (for example, probe AP286, homologous to the region between bp 13 and 43 of the open-reading frame), as well as group I-specific (for example, probe HS2, homologous to the region between bp 197 and 214 of the open-reading frame) and group El-specific (for example, probe AP307, homologous to the region between bp 133 and 150 of the open-reading frame) probes were prepared.

0143Specificity of the Genus-specific and Group-specific Oligonucleotide Probes

0144The specificity of probes AP286, AP307, and HS2 in detecting and classifying O. formigenes was examined using genomic DNA prepared from number of known strains and isolates. PCR amplifications with the genus-specific primer pair AP34 and AP21 resulted in the

0145500 bp amplification product in all cultures tested (Figure 10 A, top panel). On Southern blotting, this 500 bp fragment hybridized with a genus-specific probe, AP286 (Figure 10B, bottom panel).

0146In a separate experiment, the amplified 500 bp PCR product was hybridized with either the group I-specific probe, HS2, (Figure 1 IB, middle panel) or the group Il-specific probe,

0147AP307, (Figure 11C, bottom panel). Results clearly show a group specificity in the binding of these group-specific probes and their ability to identify subgroups of O. formigenes.

0148RECTIFIED SHEET (RULE 91) ISA EP Application of a PCR-based Detection System for O. formigenes

0149In a double-blinded study, 100 fecal samples were collected from children ranging in age from newborn to 12 years and tested for the presence of O. formigenes using both an oxalate degradation system and our PCR-based assay system. The aim of this study was to determine the age at which children become naturally colonized with this intestinal anaerobic bacterium.

0150Of the 100 fecal samples examined, 72 samples tested positive for O. formigenes by PCR, 59 of which also exhibited oxalate degradation in an oxalate degradation assay. Interestingly, of the 72 positive samples, 68 were group II strains while only 4 were group I strains. All fecal cultures exhibiting degradation of oxalate tested positive for O. formigenes by PCR. Although there were 13 cultures that failed to degrade oxalate that proved positive for O. formigenes by

0151PCR, the majority of the samples that failed to degrade oxalate also failed to exhibit amplification of a product in the PCR-reaction. These data show that the PCR-based assay is probably more sensitive than the biochemical (calcium chloride precipitation) test, yet highly specific. When the data were unblinded, a clear pattern for the natural colonization of children became evident. O. formigenes could not be detected in infants less than 6-9 months of age. O. formigenes began appearing in the intestinal tracts of children around 1 year of age, and by 3-4 years of age, all children showed signs of being colonized. Although the sample size is small, the number of children colonized with O. formigenes declined between 8-12 years of age, reaching the colonization frequency of 70-80% estimated for adult populations (Doane et al,

01521989, Kleinschmidt et al, 1993, Allison et al, 1986, and Goldkin et al, 1985).

0153Example 4 - Quantitative DNA-based detection and identification of Oxalobacter formigenes Because the original PCR primer pair AP34/AP21 was based on the nucleotide sequence encoding oxalyl-CoA decarboxylase in strain OxB (isolated from sheep), a PCR primer pair based on conserved sequences found in human strains was used. This primer pair, OXF6 and OXF7, amplifies a 416 bp product. The nucleotide sequences of OXF6 and OXF7 are shown below:

OXF6 5'-AATGTAGAGTTGACTGA-3'

OXF7 5'-TTGATGCTGTTGATACG-3'

0156To develop a competitive DNA template for use as an internal control for QC-PCR, a 227 bp fragment of the oxalyl-CoA decarboxylase gene (oxc) flanked by sequences homologous for the OXF6/OXF7 pπmer pair and containing the genus-specific, group I-specific and group El-specific probe sites was generated. To accomplish this, a PCR reaction was performed usmg the OXF6 5'-pπmer plus a modified OXF7 3'-pπmer. The modified 3'-pπmer consisted of two portions: a 5'-end which contained the 3'-pπmer sequence within the oxc gene plus a 3'-end which annealed at a site located approximately 200 bp downstream of the 5'-pπmer site. The

0157PCR usmg the pπmer pair OXF6/modιfied-OXF7 amplified the 210 bp segment and synthesized the 17 bp OXF7 pπmer site at the 3 '-end. This PCR fragment was puπfied and hgated into pCR- 2.1 (Envitrogen, Inc., San Diego, CA). A recombinant pCR-2.1 plasmid with the proper insert (confirmed by sequencing) was selected for use as the internal competitive template. Quantitative PCR (QC-PCR) were performed as descπbed below. Competitive template diluted from 1 x 10¹⁰ to 1 x 10² copies/PCR were used to establish standard curves. Expeπmental PCRs were established containing both expeπmental O formigenes DNA and dilutions of the synthetic template. The PCR products were size separated by electrophoresis through 1.5% agarose gels visualized with UV light and photographed (Figure 12A-C). Photographs were scanned for relative band intensities, normalized for differences m molecular mass, and plotted against the log of the copy number of synthetic template added per reaction.

0158Quantitation of the number of oxc genes, thereby the number of bacteπa m a sample, revealed the accuracy of this QC-PCR detection system. Assuming the genome of O formigenes is similar m size to that of E coli (4.7 x 10³ Kb), then 1 μg of genomic DNA would contain 1.8 x 10⁸ molecules (or gene copies). Genomic DNA prepared from O formigenes OxB had a concentration by spectrophotometπc analysis of 1 126 μg DNA μl or 2 x 10⁸ molecules/μl. Two dilutions, 10"¹ (20,000 molecules) and 10⁶ (200 molecules) of this DNA were used as template m the QC-PCR with dilutions of competitive template ranging from 50 to 250,000 molecules. As shown in Figure 12D and E, the Log equivalence revealed that the number of molecules of O formigenes OxB in the reaction were estimated between 19,900-25,100 and 126-158.

0159It should be understood that the examples and embodiments descπbed herein are for illustrative purposes only and that vaπous modifications or changes m light thereof will be suggested to persons skilled in the art and are to be included within the spiπt and purview of this application and the scope of the appended claims

0160RECTIFIED SHEET (RULE 91) ISA EP References

0161Allison, M.J., H.M. Cook, D.B. Milne, S. Gallaher, R.V. Clayman (1986) "Oxalate degradation by gastrointestinal bacteria from humans," JNutr 116:455-460.

0162Allison, M.J., K.A. Dawson, W.R. Mayberry, J.G. Foss (1985) "Oxalobacter formigenes gen. nov., sp. nov.: oxalate degrading bacteria that inhabit the gastrointestinal tract," Arch Microbiol 141:1-7. Anderson, J.T., J.G. Comellius, A.J. Jarpe, W.E. Winter, A.B. Peck (1993) "Insulin-dependent diabetes in the NOD mouse model. II. β cell destruction in autoimmune diabetes is a T_H1 mediated event," Autoimmunity 15: 113-122.

0163Argenzio, R.A., J.A. Liacos, M.J. Allison (1988) "Intestinal oxalate degrading bacteria reduce oxalate absorption and toxicity in guinea pigs," JNutr 118:787-791.

0164Baetz, A.L., M.J. Allison (1989) "Purification and Characterization of Oxalyl-Coenzyme A Decarboxylase from Oxalobacter formigenes," J. Bacteriol. 171 :2605-2608. Baetz, A.L., M.J. Allison (1990) "Purification and Characterization of Formyl-Coenzyme A

0165Transferase from Oxalobacter formigenes " J. Bacteriol. 172:3537-3540.

0166Costello, J., M. Hatch, E. Bourke (1976) "An enzymic method for the spectrophotometric determination of oxalic acid," /. Lab. Clin. Med. 87(5):903-908.

0167Curhan, et al. (1993) "A Prospective study of dietary calcium and other nutrients and the risk of symptomatic kidney stones," N.E.J. Med. 328:833-838.

0168Daniel, S.L., P.A. Hartman, M.J. Allison (1987) "Microbial degradation of oxalate in the gastrointestinal tracts of rats," Appl Environ Microbiol 53:957-964.

0169Dawson, K.A., M.J. Allison, P.A. Hartman (1980) "Characteristics of anerobic oxalate- degrading bacteria from the rumen," Applied Microbiol. 22:522-529. Dawson, K.A., M.J.Allison, P.A. Hartman (1980) "Isolation and some characteristics of anaerobic oxalate-degrading bacteria from ruman" Appl. Environ. Microbiol 40:833- 839.

0170Doane, L.A., M. Liebman, D.R. Caldwell (1989) "Microbial oxalata degradation: effects on oxalate and calcium balance in humans," Nutrition Res 9:957-964.

0171Goldkin, L., D.R. Cave, B. Jaffm, W. Robinson, CM. Bliss (1985) "A new factor in enteric hyperoxaluria: Oxalobacter formigenes" AM J Gastro 80:860. Hatch, M., R.W. Freel (1996) "Oxalate transport across intestinal and renal epithelia" Calcium

0172Oxalate in Biological Systems, pages 217-238, CRC Press, Boca Raton, FL.

0173Hodgkinson, A. (1970) "Determination of Oxalic acid in Biological Material," Clin. Chem. 16(7):547-557. Jensen, N.S., M.J. Allison (1994) "Studies on the diversity among anaerobic oxalate-degrading bacteria now in the species Oxalobacter formigenes" Abst. Ann. Mtg. Amer. Soc. Microbial, pages 1-29. Jordan, J.A., M.B. Durso (1996) "Rapid speciation of the five most medically relevant Candida species using PCR amplification and a microtitre plate-based detection system," Mol Diagnosis 1:51-58.

0174Kleinschmidt K., A. Mahlmann, R. Hautmann (1993) "Anaerobic oxalate-degrading bacteria in the gut decrease faecal and urinary oxalate concentrations in stone formers," In R. Ryall,

0175R. Bais, V.R. Marshall, A.M. Rofe, L.H. Smith, V.R. Walker Urolithiasis 2, Plenum Press, New York, pp. 439-441.

0176Lung, H., A.L. Baetz, A.B. Peck (1994) "Molecular Cloning, DNA Sequence and Gene Expression of the Oxalyl-CoA Decarboxylase Gene, oxc, from the Bacterium

0177Oxalobacter formigenes ," J. Bacteriol 176(8):2468-2472.

0178Maniatis, T., E.F. Fritsch, J. Sambrook (1989) Molecular Cloning: A Laboratory Manual, 2d Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

0179Smith, R.L., F.E. Strohmaier, R.S. Oremland (1985) "Isolation of anaerobic oxalate-degrading bacteria from fresh water lake sediments," Arch Microbiol 141:8-13.

0180Stacy-Phips, S., J.J. Mecca, J.B. Weiss (1995) "Multiplex PCR assay and simple preparation method for stool specimens detect enterotoxigenic E. coli DNA during course of infection," /. Clin. Microbiol. 33:1054-1059.

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0182M.B. Grant, M.G. Humphreys-Beher, L. Franzen, A.B. Peck, and G.S. Schultz (1996) "Competitive RNA templates for detection and quantitation of growth factors, cytokines, extracellular matrix components and matrix metalloproteinases by RT-PCR,"

0183Biotechnology

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SEQUENCE LISTING

(1) GENERAL INFORMATION:

0187(i) APPLICANT INFORMATION:

0188Applicant Name(s) University of Florida

0189Street Address 223 Grinter Hall

0190City Gainesville

0191State/Province Florida

0192Country US

0193Postal Code/Zip 32611

0194Phone number (352) 392-8929 Fax: (352) 392-6600

0195(ii) TITLE OF INVENTION: Materials and Methods for Detection of Oxalobacter

0196(iii) NUMBER OF SEQUENCES: 17

0197(iv) CORRESPONDENCE ADDRESS:

0198(A) ADDRESSEE: Saliwanchik & Saliwanchik

0199(B) STREET: 2421 N.W. 41st Street, Suite A-1

0200(C) CITY: Gainesville

(D) STATE: FL

(E) COUNTRY: US

(F) ZIP: 32606

0204(v) COMPUTER READABLE FORM:

0205(A) MEDIUM TYPE: Floppy disk

0206(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS -DOS

0208(D) SOFTWARE: Patentln Release #1.0, Version #1.25

0209(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

0213(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/883,610

(B) FILING DATE: 26-JUN-1997

(C) CLASSIFICATION:

0217(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/717,587

(B) FILING DATE: 27-SEP-1996

(C) CLASSIFICATION:

0221(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/493,197

(B) FILING DATE: 20-JUN-1995

(C) CLASSIFICATION:

0225(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/262,424 (B) FILING DATE: 20-JUN-1994

(C) CLASSIFICATION:

0228(viii) ATTORNEY/AGENT INFORMATION:

0229(A) NAME: Saliwanchik, Doran R.

(B) REGISTRATION NUMBER: 38,261

(C) REFERENCE/DOCKET NUMBER: UF145C4

0232(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 352-375-8100

(B) TELEFAX: 352-372-5800

(2) INFORMATION FOR SEQ ID NO : 1 :

0236(i) SEQUENCE CHARACTERISTICS:

0237(A) LENGTH: 1577 base pairs

0238(B) TYPE: nucleic acid

0239(C) STRANDEDNESS : single

0240(D) TOPOLOGY: linear

0241(ii) MOLECULE TYPE: DNA (genomic)

0242(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

AAGCTTGCTT CATTTTGAGA TGTTATGCGA AGTGTTAGCA ACCCAAGTTA GTACCCTTCA 60

GCCCTTTGGG CGAAGTTTTT CTTTCTTGGC AGTTCCTTTC GGGGAAACAG CACAGAGAAT 120

AAAAACCAAA AGTTGTACCA ACGACAAGGA AATGAGAAAT TATGACTAAA CCATTAGATG 180

GAATTAATGT GCTTGACTTT ACCCACGTCC AGGCAGGTCC TGCCTGTACA CAGATGATGG 240

GTTTCTTGGG CGCAAACGTC ATCAAGATTG AAAGACGTGG TTCCGGAGAT ATGACTCGTG 300

GATGGCTGCA GGACAAACCA AATGTTGATT CCCTGTATTT CACGATGTTC AACTGTAACA 360

AACGTTCGAT TGAACTGGAC ATGAAAACCC CGGAAGGCAA AGAGCTTCTG GAACAGATGA 420

TCAAGAAAGC CGACGTCATG GTCGAAAACT TCGGACCAGG CGCACTGGAC CGTATGGGCT 480

TTACTTGGGA ATACATTCAG GAACTGAATC CACGCGTCAT TCTGGCTTCC GTTAAAGGCT 540

ATGCAGAAGG CCACGCCAAC GAACACCTGA AAGTTTATGA AAACGTTGCA CAGTGTTCCG 600

GCGGTGCTGC AGCTACCACC GGTTTCTGGG ATGGTCCTCC AACCGTTTCC GGCGCTGCTC 660

TGGGTGACTC CAACTCCGGT ATGCACCTGA TGATCGGTAT TCTGGCCGCT CTGGAAATGC 720

GTCACAAAAC CGGCCGTGGT CAGAAAGTTG CCGTCGCTAT GCAGGACGCT GTTCTGAATC 780

TGGTTCGTAT CAAACTGCGT GACCAGCAAC GTCTGGAAAG AACCGGCATT CTGGCTGAAT 840

0257ACCCACAGGC TCAGCCTAAC TTTGCCTTCG ACAGAGACGG TAACCCACTG TCCTTCGACA 900 ACATCACTTC CGTTCCACGT GGTGGTAACG CAGGTGGCGG CGGCCAGCCA GGCTGGATGC 960

TGAAATGTAA AGGTTGGGAA ACCGATGCGG ACTCCTACGT TTACTTCACC ATCGCTGCAA 1020

ACATGTGGCC ACAGATCTGC GACATGATCG ACAAGCCAGA ATGGAAAGAC GACCCAGCCT 1080

ACAACACATT CGAAGGTCGT GTTGACAAGC TGATGGACAT CTTCTCCTTC ATCGAAACCA 1140

AGTTCGCTGA CAAGGACAAA TTCGAAGTTA CCGAATGGGC TGCCCAGTAC GGCATTCCTT 1200

GCGGTCCGGT CATGTCCATG AAAGAACTGG CTCACGATCC TTCCCTGCAG AAAGTTGGTA 1260

CCGTCGTTGA AGTTGTCGAC GAAATTCGTG GTAACCACCT GACCGTTGGC GCACCGTTCA 1320

AATTCTCCGG ATTCCAGCCG GAAATTACCC GTGCTCCGCT GTTGGGCGAA CATACCGACG 1380

AAGTTCTGAA AGAACTGGGT CTTGACGATG CCAAGATCAA GGAACTGCAT GCAAAACAGG 1440

TAGTTTGATC CGTCAGACTT TCTGGGCAAA ACGGCACTCT CCGGAGTGCC GTTTTTTGTC 1500

ACACGAAACC TAATCAAACA AGCACGTGCA ATGATTCCAC ATCATTGCGG CCACATTCAT 1560

CCTTCGGGTC ATTACTG 1577

(2) INFORMATION FOR SEQ ID NO : 2 :

0270(i) SEQUENCE CHARACTERISTICS:

0271(A) LENGTH: 428 amino acids

0272(B) TYPE: amino acid

0273(C) STRANDEDNESS: single

0274(D) TOPOLOGY: linear

0275(ii) MOLECULE TYPE: protein

0276(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

0277Met Thr Lys Pro Leu Asp Gly lie Asn Val Leu Asp Phe Thr His Val 1 5 10 15

0278Gin Ala Gly Pro Ala Cys Thr Gin Met Met Gly Phe Leu Gly Ala Asn 20 25 30

0279Val lie Lys lie Glu Arg Arg Gly Ser Gly Asn Met Thr Arg Gly Trp 35 40 45

0280Leu Gin Asp Lys Pro Asn Val Asp Ser Leu Tyr Phe Thr Met Phe Asn 50 55 60

0281Cys Asn Lys Arg Ser lie Glu Leu Asp Met Lys Thr Pro Glu Gly Lys 65 70 75 80

0282Glu Leu Leu Glu Gin Met lie Lys Lys Ala Asp Val Met Val Glu Asn 85 90 95 Phe Gly Pro Gly Ala Leu Asp Arg Met Gly Phe Thr Trp Glu Tyr lie 100 105 110

0283Gin Glu Leu Asn Pro Arg Val lie Leu Ala Ser Val Lys Gly Tyr Ala 115 120 125

0284Glu Gly His Ala Asn Glu His Leu Lys Val Tyr Glu Asn Val Ala Gin 130 135 140

0285Cys Ser Gly Gly Ala Ala Ala Thr Thr Gly Phe Trp Asp Gly Pro Pro 145 150 155 160

0286Thr Val Ser Gly Ala Ala Leu Gly Asp Ser Asn Ser Gly Met His Leu 165 170 175

0287Met He Gly He Leu Ala Ala Leu Glu Met Arg His Lys Thr Gly Arg 180 185 190

0288Gly Gin Lys Val Ala Val Ala Met Gin Asp Ala Val Leu Asn Leu Val 195 200 205

0289Arg He Lys Leu Arg Asp Gin Gin Arg Leu Glu Arg Thr Gly He Leu 210 215 220

0290Ala Glu Tyr Pro Gin Ala Gin Pro Asn Phe Ala Phe Asp Arg Asp Gly 225 230 235 240

0291Asn Pro Leu Ser Phe Asn Asn He Thr Ser Val Pro Arg Gly Gly Asn 245 250 255

0292Ala Gly Gly Gly Gly Glu Pro Gly Trp Met Leu Lys Cys Lys Gly Trp 260 265 270

0293Glu Thr Asp Ala Asp Ser Tyr Val Tyr Phe Thr He Ala Ala Asn Met 275 280 285

0294Trp Pro Gin He Cys Asn Met He Asp Lys Pro Glu Trp Lys Asp Asp 290 295 300

0295Pro Ala Tyr Asn Thr Phe Glu Gly Arg Val Asp Lys Leu Met Asp He 305 310 315 320

0296Phe Ser Phe He Glu Thr Lys Phe Ala Asp Lys Asp Lys Phe Glu Val 325 330 335

0297Thr Glu Trp Ala Ala Gin Tyr Gly He Pro Cys Gly Pro Val Met Ser 340 345 350

0298Met Lys Glu Leu Ala His Asp Pro Ser Leu Gin Lys Val Gly Thr Val 355 360 365

0299Val Glu Val Val Asp Glu He Arg Gly Asn His Leu Thr Val Gly Ala 370 375 380 Pro Phe Lys Phe Ser Gly Phe Gin Pro Glu He Thr Arg Ala Pro Leu

0300385 390 395 400

0301Leu Gly Glu His Thr Asp Glu Val Leu Lys Glu Leu Gly Leu Asp Asp

0302405 410 415

0303Ala Lys He Lys Glu Leu His Ala Lys Gin Val Val 420 425

(2) INFORMATION FOR SEQ ID NO : 3 :

0305(i) SEQUENCE CHARACTERISTICS:

0306(A) LENGTH: 2088 base pairs

0307(B) TYPE: nucleic acid

0308(C) STRANDEDNESS: single

0309(D) TOPOLOGY: linear

0310(ii) MOLECULE TYPE: DNA (genomic)

0311(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

ATTTGTTTAA ATTGACCTGA ATCAATATTG CCGGATTGAT CTAGGTCAAT GAATGCAAAT 60

TGACTTATGT CAATGGTGCC AAATTGACCT AGGTCAACGG GATTTTTAAA GGGTATGCGG 120

CATACTCGGA ATTGACGTTA AACAACGTTT ATCAAAACCA ACCAAAGAAA GGTATTACTC 180

ATGAGTAACG ACGACAATGT AGAGTTGACT GATGGCTTTC ATGTTTTGAT CGATGCCCTG 240

AAAATGAATG ACATCGATAC CATGTATGGT GTTGTCGGCA TTCCTATCAC GAACCTGGCT 300

CGTATGTGGC AAGATGACGG TCAGCGTTTT TACAGCTTCC GTCACGAACA ACACGCAGGT 360

TATGCAGCTT CTATCGCCGG TTACATCGAA GGAAAACCTG GCGTTTGCTT GACCGTTTCC 420

GCCCCTGGCT TCCTGAACGG CGTGACTTCC CTGGCTCATG CAACCACCAA CTGCTTCCCA 480

ATGATCCTGT TGAGCGGTTC CAGTGAACGT GAAATCGTCG ATTTCCAAGA CGGCGATTAC 540

GAAGAAATGG ATCAGATGAA TGTTGCACGT CCACACTGCA AAGCTTCTTT CCGTATCAAC 600

AGCATCAAAG ACATTCCAAT CGGTATCGCT CGTGCAGTTC GCACCGCTGT ATCCGGACGT 660

CCAGGTGGTG TTTACGTTGA CTTCCCAGCA AAACTGTTCG GTCAGACCAT TTCTGTAGAA 720

GAAGCTAACA AACTGCTCTT CAAACCAATC GATCCAGCTC CGGCACAGAT TCTTGCTGAA 780

GACGCTATCG CTCGCGCTGC TGACCTGATC AAGAACGCCA AACGTCCAGT TATCATGCTG 840

GGTAAAGGCG CTGCATACGC ACAATGCGAC GACGAAATCC GCGCACTGGT TGAAGAAACC 900

GGCATCCCAT TCCTGCCAAT GGGTATGGCT AAAGGCCTGC TGCCTGACAA CCATCCACAA 960

0328TCCGCTGCTG CAACCCGTGC TTTCGCACTG GCACAGTGTG ACGTTTGCGT ACTGATCGGC 1020 GCTCGTCTGA ACTGGCTGAT GCAGCACGGT AAAGGCAAAA CCTGGGGCGA CGAACTGAAG 1080

AAATACGTTC AGATCGACAT CCAGGCTAAC GAAATGGACA GCAACCAGCC TATCGCTGCA 1140

CCAGTTGTTG GTGACATCAA GTCCGCCGTT TCCCTGCTCC GCAAAGCACT GAAAGGCGCT 1200

CCAAAAGCTG ACGCTGAATG GACCGGCGCT CTGAAAGCCA AAGTTGACGG CAACAAAGCC 1260

AAACTGGCTG GCAAGATGAC TGCCGAAACC CCATCCGGAA TGATGAACTA CTCCAATTCC 1320

CTGGGCGTTG TTCGTGACTT CATGCTGGCA AATCCGGATA TTTCCCTGGT TAACGAAGGC 1380

GCTAATGCAC TCGACAACAC TCGTATGATT GTTGACATGC TGAAACCACG CAAACGTCTT 1440

GACTCCGGTA CCTGGGGTGT TATGGGTATT GGTATGGGCT ACTGCGTTGC TGCAGCTGCT 1500

GTTACCGGCA AACCGGTTAT CGCTGTTGAA GGCGATAGCG CATTCGGTTT CTCCGGTATG 1560

GAACTGGAAA CCATCTGCCG TTACAACCTG CCAGTTACCG TTATCATCAT GAACAATGGT 1620

GGTATCTATA AAGGTAACGA AGCAGATCCA CAACCAGGCG TTATCTCCTG TACCCGTCTG 1680

ACCCGTGGTC GTTACGACAT GATGATGGAA GCATTTGGCG GTAAAGGTTA TGTTGCCAAT 1740

ACTCCAGCAG AACTGAAAGC TGCTCTGGAA GAAGCTGTTG CTTCCGGCAA ACCATGCCTG 1800

ATCAACGCGA TGATCGATCC AGACGCTGGT GTCGAATCTG GCCGTATCAA GAGCCTGAAC 1860

GTTGTAAGTA AAGTTGGCAA GAAATAATTA GCCCAACTTT GATGACCGGT TACGACCGGT 1920

CACATAAAGT GTTCGAATGC CCTTCAAGTT TACTTGAAGG GCATTTTTTT ACCTTGCAGT 1980

TTATAAACAG GAAAAATTGT ATTCAGAGCG GAAAAGCAGA TTTAAGCCAC GAGAAACATT 2040

CTTTTTTATT GAAAATTGCC ATAAACACAT TTTTAAAGCT GGCTTTTT 2088

(2) INFORMATION FOR SEQ ID NO : 4 :

0347(i) SEQUENCE CHARACTERISTICS:

0348(A) LENGTH: 568 amino acids

0349(B) TYPE: amino acid

0350(C) STRANDEDNESS: single

0351(D) TOPOLOGY: linear

0352(ii) MOLECULE TYPE: peptide

0353(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :

0354Met Ser Asn Asp Asp Asn Val Glu Leu Thr Asp Gly Phe His Val Leu 1 5 10 15

0355He Asp Ala Leu Lys Met Asn Asp He Asp Thr Met Tyr Gly Val Val 20 25 30 Gly He Pro He Thr Asn Leu Ala Arg Met Trp Gin Asp Asp Gly Gin 35 40 45

0356Arg Phe Tyr Ser Phe Arg His Glu Gin His Ala Gly Tyr Ala Ala Ser 50 55 60

0357He Ala Gly Tyr He Glu Gly Lys Pro Gly Val Cys Leu Thr Val Ser 65 70 75 80

0358Ala Pro Gly Phe Leu Asn Gly Val Thr Ser Leu Ala His Ala Thr Thr 85 90 95

0359Asn Cys Phe Pro Met He Leu Leu Ser Gly Ser Ser Glu Arg Glu He 100 105 110

0360Val Asp Leu Gin Gin Gly Asp Tyr Glu Glu Met Asp Gin Met Asn Val 115 120 125

0361Ala Arg Pro His Cys Lys Ala Ser Phe Arg He Asn Ser He Lys Asp 130 135 140

0362He Pro He Gly He Ala Arg Ala Val Arg Thr Ala Val Ser Gly Arg 145 150 155 160

0363Pro Gly Gly Val Tyr Val Asp Leu Pro Ala Lys Leu Phe Gly Gin Thr 165 170 175

0364He Ser Val Glu Glu Ala Asn Lys Leu Leu Phe Lys Pro He Asp Pro 180 185 190

0365Ala Pro Ala Gin He Pro Ala Glu Asp Ala He Ala Arg Ala Ala Asp 195 200 205

0366Leu He Lys Asn Ala Lys Arg Pro Val He Met Leu Gly Lys Gly Ala 210 215 220

0367Ala Tyr Ala Gin Cys Asp Asp Glu He Arg Ala Leu Val Glu Glu Thr 225 230 235 240

0368Gly He Pro Phe Leu Pro Met Gly Met Ala Lys Gly Leu Leu Pro Asp 245 250 255

0369Asn His Pro Gin Ser Ala Ala Ala Thr Arg Ala Phe Ala Leu Ala Gin 260 265 270

0370Cys Asp Val Cys Val Leu He Gly Ala Arg Leu Asn Trp Leu Met Gin 275 280 285

0371His Gly Lys Gly Lys Thr Trp Gly Asp Glu Leu Lys Lys Tyr Val Gin 290 295 300

0372He Asp He Gin Ala Asn Glu Met Asp Ser Asn Gin Pro He Ala Ala 305 310 315 320 Pro Val Val Gly Asp He Lys Ser Ala Val Ser Leu Leu Arg Lys Ala 325 330 335

0373Leu Lys Gly Ala Pro Lys Ala Asp Ala Glu Trp Thr Gly Ala Leu Lys 340 345 350

0374Ala Lys Val Asp Gly Asn Lys Ala Lys Leu Ala Gly Lys Met Thr Ala 355 360 365

0375Glu Thr Pro Ser Gly Met Met Asn Tyr Ser Asn Ser Leu Gly Val Val 370 375 380

0376Arg Asp Phe Met Leu Ala Asn Pro Asp He Ser Leu Val Asn Glu Gly 385 390 395 400

0377Ala Asn Ala Leu Asp Asn Thr Arg Met He Val Asp Met Leu Lys Pro 405 410 415

0378Arg Lys Arg Leu Asp Ser Gly Thr Trp Gly Val Met Gly He Gly Met 420 425 430

0379Gly Tyr Cys Val Ala Ala Ala Ala Val Thr Gly Lys Pro Val He Ala 435 440 445

0380Val Glu Gly Asp Ser Ala Phe Gly Phe Ser Gly Met Glu Leu Glu Thr 450 455 460

0381He Cys Arg Tyr Asn Leu Pro Val Thr Val He He Met Asn Asn Gly 465 470 475 480

0382Gly He Tyr Lys Gly Asn Glu Ala Asp Pro Gin Pro Gly Val He Ser 485 490 495

0383Cys Thr Arg Leu Thr Arg Gly Arg Tyr Asp Met Met Met Glu Ala Phe 500 505 510

0384Gly Gly Lys Gly Tyr Val Ala Asn Thr Pro Ala Glu Leu Lys Ala Ala 515 520 525

0385Leu Glu Glu Ala Val Ala Ser Gly Lys Pro Cys Leu He Asn Ala Met 530 535 540

0386He Asp Pro Asp Ala Gly Val Gly Ser Gly Arg He Lys Ser Leu Asn 545 550 555 560

0387Val Val Ser Lys Val Gly Lys Lys 565

(2) INFORMATION FOR SEQ ID NO : 5 :

0389(i) SEQUENCE CHARACTERISTICS:

0390(A) LENGTH: 17 base pairs

0391(B) TYPE: nucleic acid (C) STRANDEDNESS: single

0392(D) TOPOLOGY: linear

0393(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 : GAGCGATACC GATTGGA 17

(2) INFORMATION FOR SEQ ID NO : 6 :

0395(i) SEQUENCE CHARACTERISTICS:

0396(A) LENGTH: 17 base pairs

0397(B) TYPE: nucleic acid

0398(C) STRANDEDNESS: single

0399(D) TOPOLOGY: linear

0400(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 : GCACAATGCG ACGACGA 17

(2) INFORMATION FOR SEQ ID NO : 7 :

0402(i) SEQUENCE CHARACTERISTICS:

0403(A) LENGTH: 17 base pairs

0404(B) TYPE: nucleic acid

0405(C) STRANDEDNESS: single

0406(D) TOPOLOGY: linear

0407(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 : CAGGTTATGC AGCTTCT 17

(2) INFORMATION FOR SEQ ID NO : 8 :

0409(i) SEQUENCE CHARACTERISTICS:

0410(A) LENGTH: 17 base pairs

0411(B) TYPE: nucleic acid

0412(C) STRANDEDNESS: single

0413(D) TOPOLOGY: linear

0414(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 : GGATGGTTGT CAGGCAG 17 (2) INFORMATION FOR SEQ ID NO : 9 :

0415(i) SEQUENCE CHARACTERISTICS:

0416(A) LENGTH: 17 base pairs

0417(B) TYPE: nucleic acid

0418(C) STRANDEDNESS: single

0419(D) TOPOLOGY: linear

0420(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 : ATACTCGGAA TTGACGT 17

(2) INFORMATION FOR SEQ ID NO: 10

0422(i) SEQUENCE CHARACTERISTICS:

0423(A) LENGTH: 23 base pairs

0424(B) TYPE: nucleic acid

0425(C) STRANDEDNESS: single

0426(D) TOPOLOGY: linear

0427(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: TTCATGTCCA GTTCAATCGA ACG 23

(2) INFORMATION FOR SEQ ID NO: 11

0429(i) SEQUENCE CHARACTERISTICS:

0430(A) LENGTH: 17 base pairs

0431(B) TYPE: nucleic acid

0432(C) STRANDEDNESS: single

0433(D) TOPOLOGY: linear

0434(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GTAGTTCATC ATTCCGG 17

(2) INFORMATION FOR SEQ ID NO : 12

0436(i) SEQUENCE CHARACTERISTICS:

0437(A) LENGTH: 17 base pairs

0438(B) TYPE: nucleic acid

0439(C) STRANDEDNESS: single

0440(D) TOPOLOGY: linear

0441(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: AATGTAGAGT TGACTGA 17

(2) INFORMATION FOR SEQ ID NO: 13

0443(i) SEQUENCE CHARACTERISTICS:

0444(A) LENGTH: 17 base pairs

0445(B) TYPE: nucleic acid

0446(C) STRANDEDNESS: single

0447(D) TOPOLOGY: linear

0448(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TTGATGCTGT TGATACG 17

(2) INFORMATION FOR SEQ ID NO : 14

0450(i) SEQUENCE CHARACTERISTICS:

0451(A) LENGTH: 31 base pairs

0452(B) TYPE: nucleic acid

0453(C) STRANDEDNESS: single

0454(D) TOPOLOGY: linear

0455(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 14 : GACAATGTAG AGTTGACTGA TGGCTTTCAT G 31

(2) INFORMATION FOR SEQ ID NO : 15

0457(i) SEQUENCE CHARACTERISTICS:

0458(A) LENGTH: 18 base pairs

0459(B) TYPE: nucleic acid

0460(C) STRANDEDNESS: single

0461(D) TOPOLOGY: linear

0462(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: CAGGATGGTC AGAAGTTC 18

(2) INFORMATION FOR SEQ ID NO: 16

0464(i) SEQUENCE CHARACTERISTICS:

0465(A) LENGTH: 17 base pairs

0466(B) TYPE: nucleic acid (C) STRANDEDNESS: single

0467(D) TOPOLOGY: linear

0468(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: CCGGTTACAT CGAAGGA 17

(2) INFORMATION FOR SEQ ID NO: 17

0470(i) SEQUENCE CHARACTERISTICS:

0471(A) LENGTH: 17 base pairs

0472(B) TYPE: nucleic acid

0473(C) STRANDEDNESS: single

0474(D) TOPOLOGY: linear

0475(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: TCCAATCGGT ATCGCTC 17