Materials and methods for detection of oxalate

Description of equivalent WO 9535377 A2

DESCRIPTION

MATERIALS AND METHODS FOR DETECTION OF OXALATE

This invention was made with government support under National Institutes of

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

Cross-Reference to a Related Application This application is a continuation-in-part of co-pending application Serial No. 08/262,424, filed June 20, 1994.

Background of the Invention The present invention relates to novel assay methods for determining the presence or concentration of oxalate in a sample. The present invention further relates to the cloning, sequencing and expression of formyl-CoA transferase, an enzyme used in the novel assay for the detection of oxalate.

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. Most 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 ¹⁴C-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.

Recently, 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.

In 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.

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

As 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.

Brief Summary of the Invention

The subject invention concerns the cloning, sequencing, and expression of the formyl-CoA transferase and the oxalyl-CoA decarboxylase 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 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 specific Oxalobacter organisms.

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

Brief Description of the Drawings Figures 1A through IE 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.

Figure 2 shows a partial nucleotide sequence of the formyl-CoA transferase gene and a partial amino acid sequence of the formyl-CoA transferase polypeptide. Bolded letters represent amino acid residues determined by N-terminal protein sequencing. Figure 3 shows the nucleotide sequence of the oxalyl-CoA decarboxylase gene and flanking regions. 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.

Brief Description of the Sequences SEQ ID NOS. 1-3 are the known parts of the nucleotide sequence for the formyl- CoA transferase gene (also shown in Figure 2). SEQ ID NO. 4 is a polypeptide encoded by SEQ LD NO. 1, which can be used according to the subject invention.

SEQ ID NO. 5 is a polypeptide encoded by SEQ LD NO. 2, which can be used according to the subject invention.

SEQ ID NO.6 is the nucleotide sequence for the oxalyl-CoA decarboxylase gene (also shown in Figure 3). SEQ ID NO. 7 is a polypeptide encoded by SEQ LD NO. 6, which can be used according to the subject invention.

SEQ ID NO. 8 is an oxalyl-CoA decarboxylase probe sequence, which can be used according to the subject invention. SEQ ID NO. 9 is an oxalyl-CoA decarboxylase probe sequence, which can be used according to the subject invention.

SEQ ID NO. 10 is an oxalyl-CoA decarboxylase 5 '-primer, which can be used according to the subject invention.

SEQ ID NO. 11 is an oxalyl-CoA decarboxylase 3 '-primer, which can be used according to the subject invention.

Detailed Description of the Invention The subject invention provides an accurate, sensitive assay for oxalate in biological samples such as urine and serum. Elevated levels of oxalate are correlated with urinary 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 appropriate medication and through modulation of diet.

In the presently described 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

(CO₂) .through the combined action of the enzymes oxalyl-CoA decarboxylase and formyl-CoA transferase. The formate can then be detected using a variety of techniques known in the art. In a preferred embodiment, the production of formate is measured colorimetrically 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 using the subject enzyme system, then the amount of oxalate present in the sample can be easily quantitated. In a preferred embodiment, the enzymes used in the subject invention are expressed by genes from the bacterium Oxalobacter formigenes. The genes encoding both oxalyl-CoA decarboxylase (Lung, 1994) and formyl-CoA transferase enzymes been cloned and expressed, thus providing a readily-available source of reagent material. The subject assay is capable of detecting oxalate levels in a range as low as 0.00025- 0.0005 mM (Figures 1A-1E). This level of sensitivity makes the subject assay capable of direct detection of oxalate in serum samples consisting of little as 10 μl volume. The described system can be easily automated with standard systems known in the art.

In a preferred embodiment of the subject assay, the enzymatic reaction can be carried 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 equilibrium

(two minute incubation at 37° C in the plate reader) to permit degradation of any residual formate that may be present in 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 measuring the reduction in NAD in 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 comparison of the unknown samples with standards having a known amount of oxalate.

Further, 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 πiixture. 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-CoA are added first, and the reaction is initiated by the addition of formyl-CoA transferase to the mix. However, the order of addition of the three reagents is not material to the function of the assay, so long as one of the reagents is withheld until just prior to the desired initiation point of the assay.

The formyl-CoA transferase and oxalyl-CoA decarboxylase enzyme used in the subject 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 in the subject assay can be obtained from conventional sources, such as Sigma Chemical Company, St. Louis, subject invention that encode the enzymes. Other reagents used in 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 in the assay described herein. A further aspect of the subject invention concerns the cloning, sequencing and expression of the Oxalobacter formigenes gene which encodes the formyl-CoA transferase used in the assay that is a subject of the invention. The gene was cloned using degenerate oligonucleotide probes (based on partial amino 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 2 and 3, respectively (SEQ ID NOS. 1-3 and 6). Because of the redundancy of the genetic code, a variety of different polynucleotide sequences can encode the formyl-CoA transferase polypeptide disclosed herein. It is well within the skill of a person trained in the art to create alternative polynucleotide sequences encoding the same, or essentially the same, polypeptide of the subject invention. These variant or alternative polynucleotide sequences are within the scope of the subject invention. As used herein, references to "essentially the same" sequence refers to sequences which encode amino acid substitutions, deletions, additions, or insertions which do not materially 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 in Figures 2 and 3 (SEQ ID NOS. 1-3 and 6) so as to permit hybridization with those sequences under standard high-stringency conditions. Such hybridization conditions are conventional in the art (see, e.g., Maniatis et al., 1989).

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.

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

The polynucleotide sequences of the subject invention may be composed of either RNA or DNA. More preferably, the polynucleotide sequences are composed of DNA.

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.

The 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. In a preferred embodiment, the polynucleotide probes are based on the oxalyl-CoA decarboxylase gene sequence. The polynucleotide probes of the subject invention can be used according to standard procedures and conditions to specifically and selectively detect other polynucleotide sequences that have sufficient homology to hybridize with the probe. In one embodiment of the subject invention, DNA is isolated from bacterial microorganisms in a biological specimen {e.g., biopsy, fecal matter, scrapings, etc.) using standard techniques known in the art. The isolated DNA is then screened for hybridization with an Oxalobacter oxalyl-CoA decarboxylase-specific polynucleotide probe. It is not essential to the practice of the subject invention that any particular hybridization technique be employed. For example, 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 Hybridization: A

Practical Approach (Hames, B.D., S.J. Higgins, eds.), LRL Press (1985).

The polynucleotide probes of the subject invention include, for example, the oxalyl-CoA decarboxylase probe A (SEQ LD NO. 8) and probe B (SEQ LD NO. 9) specifically exemplified herein. The nucleotide sequences of probe A and probe B are shown below:

Probe A 5'- GAGCGATACCGATTGGA -3' (SEQ LD NO. 8)

Probe B 5'- GCACAATGCGACGACGA -3' (SEQ LD NO. 9)

The polynucleotide probes contemplated in the subject invention also include any polynucleotide molecule comprising a nucleotide sequence capable of specifically hybridizing with the oxalyl-CoA decarboxylase or formyl-CoA transferase genes disclosed herein. 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 gene, or fragments thereof,' but also to those sequences with sufficient homology to hybridize with the target gene. 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 nucleotide 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.

In 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. Patent Nos. 4,683,195; 4,683,202; and 4,800,159. In a preferred embodiment, the polynucleotide primers are based on the oxalyl-CoA decarboxylase gene sequence and can be used to amplify all or portions of the 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. 10) and PCR primer 2 (SEQ ID NO. 11), specifically exemplified herein. The nucleotide sequences of PCR primers 1 and 2 are shown below:

PCR primer 1 5'- CAGGTTATGCAGCTTCT -3' (SEQ ID NO. 10)

PCR primer 2 5'- GGATGGTTGTCAGGCAG -3' (SEQ LD NO. 11)

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 the oxalyl-CoA decarboxylase 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.

The 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, ¹⁴C, 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. 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.

The 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 gene of O. formigenes.

Following 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. Example 1 - Determination of Level of Sensitivity of Enzyme 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: KH₂PO₄ (pH 6.7), 50 mM; MgCl₂, 5 mM; tliiamine 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 1 A-1E). Appropriate positive and negative controls were run simultaneously with the assay.

Example 2 - Detection of Oxalobacter formigenes in a Sample Samples to be tested for the presence of Oxalobacter formigenes are obtained from a patient {e.g., biopsy, fecal matter, scrapings), 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. The DNA fragments are then electrophoretically separated by size on a 0.7-1.0% agarose gel. In an alternative embodiment, the DNA fragments are subjected to PCR prior to gel electrophoresis in order to specifically amplify the oxalyl- CoA decarboxylase gene. Using polynucleotide primers 1 (SEQ LD NO. 10) and 2 (SEQ ID NO. 11) in the PCR method generates a 585 bp amplification product. After 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 hybridized with the DNA immobilized on the membrane. In one embodiment, Probe A (SEQ ID NO. 8) or Probe B (SEQ LD NO. 9), labeled with a hapten or antigen such as digoxigenin, is used in the hybridization process. The hybridization can be performed 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 may vary. The membrane is then tested for the presence of hybridized probe. The hybridized probe 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, or enzyme. Alternatively, the antibody can be detected using a secondary reagent that binds to the antibody, such as anti-immunoglobulin, protein A, protein G, etc. The secondary reagent can be labeled with a detectable fluorophore, chemiluminescent molecule, radioisotope, or enzyme. The presence of a detectable hybridization signal indicates the presence of Oxalobacter formigenes in a test sample.

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

References

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

Curhan, 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.

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

Hodgkinson, A. (1970) "Determination of Oxalic acid in Biological Material," Clin. Chem. 16(7):547-557.

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

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

Yriberri, J., L.S. Posten (1980) "A semi-automatic enzymic method for estimating urinary oxalate," Clin. Chem. 26(7):881-884.

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

(iii) NUMBER OF SEQUENCES: 11

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Saliwanchik & Saliwanchik

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

(Vi) CURRENT APPLICATION DATA:

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(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/262,424

(B) FILING DATE: 20-JUN-1994

(C) CLASSIFICATION:

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Saliwanchik, David R.

(B) REGISTRATION NUMBER: 31,794

(C) REFERENCE/DOCKET NUMBER: UF145

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 904-375-8100

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(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 63 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l: ATGACNAANC CNNNNGANGG NATNAANGTN NNNGANTTNA CNCANGTNCA NGCNGGNCCN 60 GCN 63

(2) INFORMATION FOR SEQ ID Nθ:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 870 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

CATCTCCCCA AGAAATTGCG TCAATGCTCA ATGGATACAT AGACGAGGGC AAGCCGGCGT 60

CAGCCAAGCT AATCGATCAA CCCTGACCGA TGCATTCCGA GAGGCAATAG CTGAAGGCCA 120

TATAACAACA ACCATGTCTC TGCCATTCGC GCACCAAACT CAGAGGTAAG GAGATCAAGA 180

CTTACGGCTG GCGAATGCCT GACTATTTAT CAAGCATCAG AATCATCCCC ATGTTTGCTG 240

ACTCATACAT TGGATTTGCC TGTTGTTACC GGCCTACGAG TTGGTGATTT ATTCGATAAT 300

GAAGTGCGCT TATCTGTAGA TGGATATCTT TATGTCGAGC AAACCAAAAC AGGCGTAAAA 360

ATTGCCATCC CAACAGCATT GCATATTGAT GCTCTCGGAA TAACAATGAA GGAAACACTT 420

GATAAATGCA AAGAGATTCT TGGCGGAGAA ACCATAATTG CATCTACTCG TCGCGAACCG 480

CTTTCATCCG GCACAGTATC AAGGTATTTT ATGCGCGCAC GAAAAGCATC AGGTCTTTCC 540

TTCGAAGGGG ATACTCTAGA ACCTTGTCGA CGTCCTGCCT GTACACAGAT GATGGGTTTC 600

TTGGGCGCAA ACGTCATCAA GATTGAAAGA CGTGGTTCCG GAGATATGAC TCGTGGATGN 660

CTGCAGGACA AACCAAATGT TGATTCCCTG TATTTCACGA TGTTCAACTG TAACAAACGT 720

TCGATTGAAC TGGACATGAA AACCCCGGAA GGCAAAGAGC TTCTGGAACA GATGATCAAG 780

AAAGCCGACG TCATGGTCGA AAACTTCGGA CCAGGNGCNN NNGANNNNAT GGGNTTNACN 840

TGGGANTANA TNCANGANNT NAANCCNCGT 870

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 259 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:3:

GGCTGGCATT TCAATGAAAA CAACAAACCC GTCATTATCC GGACTGCCGG CAATCTCGAC 60

TGCAACGACG GAGAACTCCT GCACCGCTGG TGTCTGGAAG GATATGGGCT GGCCTGGCGT 120

TCGACATGGG AAATACAATC CGAACTCGCT TCCGGTAAAC TGGTCACCGT ACTGGACGAT 180

TACGCCCTTC CCAACTACGA CATCATGGGA GTTTACCCCC AGCAAAGATA CCTCCCCGTG 240

AAAACCCGTC TGTACAGTG 259 (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

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

Gin Ala Gly Pro Ala 20

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 226 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Glu Cys Leu Thr lie Tyr Gin Ala Ser Glu Ser Ser Pro Cys Leu Leu 1 5 10 15

His Thr Leu Asp Leu Pro Val Val Thr Gly Leu Arg Val Val Gly Asp 20 25 30

Leu Phe Asp Asn Glu Val Arg Leu Ser Val Asp Gly Tyr Leu Tyr Val 35 40 45

Glu Gin Thr Lys Thr Gly Val Lys ie Ala lie Pro Thr Ala Leu His 50 55 60 lie Asp Ala Leu Gly lie Thr Met Lys Glu Thr Leu Asp Lys Cys Lys 65 70 75 80

Glu lie Leu Gly Gly Glu Thr lie lie Ala Ser Thr Arg Arg Glu Pro 85 90 95

Leu Ser ser Gly Thr Val ser Arg Tyr Phe Met Arg Ala Arg Lys Ala 100 105 110

Ser Gly Leu Ser Phe Glu Gly Asp Thr Leu Glu Pro Cys Arg Arg Pro 115 120 125

Ala Cys Thr Gin Met Met Gly Phe Leu Gly Ala Asn Val lie Lys lie 130 135 140

Glu Arg Arg Gly ser Gly Asp Met Thr Arg Gly Xaa Leu Gin Asp Lys 145 150 155 160

Pro Asn Val Asp Ser Leu Tyr Phe Thr Met Phe Asn Cys Asn Lys Arg 165 170 175

Ser ie Glu Leu Asp Met Lys Thr Pro Glu Gly Lys Glu Leu Leu Glu 180 185 190 Gin Met lie Lys Lys Ala Asp Val Met Val Glu Asn Phe Gly Pro Gly 195 200 205

Ala Leu Asp Arg Met Gly Phe Thr Trp Glu Tyr lie Gin Glu Leu Asn 210 215 220

Pro Arg 225

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2088 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

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

TCCGCTGCTG 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:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 568 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Met Ser Asn Asp Asp Asn Val Glu Leu Thr Asp Gly Phe His Val Leu 1 5 10 15 lie Asp Ala Leu Lys Met Asn Asp lie Asp Thr Met Tyr Gly Val Val 20 25 30

Gly ie Pro lie Thr Asn Leu Ala Arg Met Trp Gin Asp Asp Gly Gin 35 40 45

Arg Phe Tyr Ser Phe Arg His Glu Gin His Ala Gly Tyr Ala Ala Ser 50 55 60 lie Ala Gly Tyr lie Glu Gly Lys Pro Gly Val Cys Leu Thr Val ser 65 70 75 80

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

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

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

Ala Arg Pro His Cys Lys Ala Ser Phe Arg lie Asn Ser lie Lys Asp 130 135 140 lie Pro ie Gly lie Ala Arg Ala Val Arg Thr Ala val Ser Gly Arg 145 150 155 160 Pro Gly Gly Val Tyr Val Asp Leu Pro Ala Lys Leu Phe Gly Gin Thr 165 170 175 lie Ser Val Glu Glu Ala Asn Lys Leu Leu Phe Lys Pro lie Asp Pro 180 185 190

Ala Pro Ala Gin lie Pro Ala Glu Asp Ala ie Ala Arg Ala Ala Asp 195 200 205

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

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

Gly lie Pro Phe Leu Pro Met Gly Met Ala Lys Gly Leu Leu Pro Asp 245 250 255

Asn His Pro Gin ser Ala Ala Ala Thr Arg Ala Phe Ala Leu Ala Gin 260 265 270 cys Asp Val Cys Val Leu lie Gly Ala Arg Leu Asn Trp Leu Met Gin 275 280 285

His Gly Lys Gly Lys Thr Trp Gly Asp Glu Leu Lys Lys Tyr Val Gin 290 295 300 lie Asp lie Gin Ala Asn Glu Met Asp Ser Asn Gin Pro lie Ala Ala 305 310 315 320

Pro Val Val Gly Asp ie Lys Ser Ala Val Ser Leu Leu Arg Lys Ala 325 330 335

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

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

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

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

Ala Asn Ala Leu Asp Asn Thr Arg Met lie val Asp Met Leu Lys Pro 405 410 415

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

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

Val Glu Gly Asp Ser Ala Phe Gly Phe Ser Gly Met Glu Leu Glu Thr 450 455 460 lie Cys Arg Tyr Asn Leu Pro Val Thr Val He He Met Asn Asn Gly 465 470 475 480

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

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

Gly Gly Lys Gly Tyr Val Ala Asn Thr Pro Ala Glu Leu Lys Ala Ala 515 520 525 Leu Glu Glu Ala Val Ala Ser Gly Lys Pro Cys Leu He Asn Ala Met 530 535 540

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

Val Val Ser Lys Val Gly Lys Lys 565

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GAGCGATACC GATTGGA 17

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GCACAATGCG ACGACGA 17

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CAGGTTATGC AGCTTCT 17

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GGATGGTTGT CAGGCAG 17