Materials and methods for detection of oxalobacter

The subject invention concerns the novel use of formyl-CoA transferase enzyme together with oxalyl-CoA decarboxylase enzyme for the detection and measurement of oxalate in biological samples. The use of the enzyme system according to the subject invention results in the conversion of oxalate into carbon dioxide and formate. Because the production of formate is directly correlated to the concentration of oxalate present in a sample, the determination of the resulting formate concentration provides an accurate, sensitive and rapid means for detecting even low levels of oxalate. The subject invention further concerns the cloning, sequencing and expression of the genes that encode the formyl-CoA transferase enzyme and the oxalyl-CoA decarboxylase enzyme of Oxalobacter formigenes. The subject invention also concerns methods for detecting the presence of Oxalobacter formigenes organisms in a sample, and the polynucleotide probes and primers used in the detection method.

BACKGROUND OF THE INVENTION 
The present invention relates to novel assay methods and devices 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. The present invention also relates to novel 
materials and methods for the detection of Oxalobacter formigenes in a 
sample. 
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 .sup.14 C-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. 
Oxalobacter formigenes is a recently discovered, oxalate-degrading 
obligately anaerobic bacterium residing primarily in the intestines of 
vertebrate animals, including man (Allison et al., 1986). 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, 1996). 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. Special 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). 
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. 
The 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. 
BRIEF SUMMARY OF THE INVENTION 
The subject invention concerns the cloning, sequencing, and expression of 
the formyl-CoA 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. 
The 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.

BRIEF DESCRIPTION OF THE SEQUENCES 
SEQ ID NO. 1 is a nucleotide sequence for the formyl-CoA transferase gene 
(also shown in FIGS. 2A-2B). 
SEQ ID NO. 2 is a polypeptide encoded by SEQ ID NO. 1, which can be used 
according to the subject invention. 
SEQ ID NO. 3 is the nucleotide sequence for the oxalyl-CoA decarboxylase 
gene (also shown in FIGS. 3A-3B). 
SEQ ID NO. 4 is a polypeptide encoded by SEQ ID NO. 3, which can be used 
according to the subject invention. 
SEQ ID NO. 5 is an oxalyl-CoA decarboxylase sequence, which can be used as 
a probe according to the subject invention. 
SEQ ID NO. 6 is an oxalyl-CoA decarboxylase sequence, which can be used as 
a probe or PCR primer according to the subject invention. 
SEQ ID NO. 7 is an oxalyl-CoA decarboxylase 5'-primer, which can be used 
according to the subject invention. 
SEQ ID NO. 8 is an oxalyl-CoA decarboxylase 3'-primer, which can be used 
according to the subject invention. 
SEQ ID NO. 9 is an oxalyl-CoA decarboxylase sequence, which can be used as 
a probe according to the subject invention. 
SEQ ID NO. 10 is a formyl-CoA transferase sequence, which can be used as a 
probe according to the subject invention. 
SEQ ID NO. 11 is an oxalyl-CoA decarboxylase sequence, which can be used as 
a PCR primer 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 (CO.sub.2) 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 have 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 (FIGS. 
1A-1E). This level of sensitivity makes the subject assay capable of 
direct detection of oxalate in serum samples consisting of little as 10 
.mu.l 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, .beta.-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.degree. 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 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-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 enzymes 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, 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 FIGS. 2A-2B and 3A-3B, respectively (SEQ ID 
NOS. 1 and 3). 
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 FIGS. 2A-2B and 3A-3B (SEQ ID NOS. 1 and 3) 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. 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, .beta.-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. 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 Hybridization: A Practical Approach (Hames, B. D., S. J. Higgins, 
eds.), IRL Press (1985). 
The polynucleotide probes of the subject invention include, for example, 
the oxalyl-CoA decarboxylase probe A (SEQ ID NO. 5), probe AP15 (SEQ ID 
NO. 6), and probe AP34 (SEQ ID NO. 9) 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: 
Probe A 5'-GAGCGATACCGATTGGA-3' (SEQ ID NO. 5) 
Probe AP15 5'-GCACAATGCGACGACGA-3' (SEQ ID NO. 6) 
Probe AP34 5'-ATACTCGGAATTGACGT-3' (SEQ ID NO. 9) 
Probe AP273 5'-TTCATGTCCAGTTCAATCGAACG-3' (SEQ ID NO. 10) 
The 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 sequences 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 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. 
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. Pat. 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 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 AP15 (SEQ ID NO. 6), and PCR primer AP22 (SEQ ID NO. 
11), specifically exemplified herein. The nucleotide sequences of the 
exemplified PCR primers are shown below: 
PCR primer 1 5'-CAGGTTATGCAGTTCT-3' (SEQ ID NO.7) 
PCR primer 2 5'-GGATGGTTGTCAGGCAG-3' (SEQ ID NO. 8) 
PCR primer AP15 5'-GCACAATGCGACGACGA-3' (SEQ ID NO. 6) 
PCR primer AP22 5'-GTAGTTCATCATTCCGG-3' (SEQ ID 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 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. 
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 .sup.3 H, .sup.35 S, .sup.14 
C, or .sup.32 P. 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 labelled with digoxygenin and detected with labelled 
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. 
In 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. 
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 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. 
The 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. 
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 and formyl-CoA transferase genes of O. formigenes. 
The 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 incorporated, 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. 
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 .mu.l 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: KH.sub.2 PO.sub.4 (pH 6.7), 50 mM; MgCl.sub.2, 5 mM; 
thiamine PPi (TPP), 2 mM; oxalyl-CoA, 0.375 mM; .beta.-NAD, 1.0 mM; 
formate dehydrogenase, 0.25 IU; and oxalyl-CoA decarboxylase, 0.03 U. The 
reaction mixture was then incubated at 37.degree. 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 
A.sub.340 were measured every 15 seconds at 37.degree. C. (FIGS. 1A-1E). 
Appropriate positive and negative controls were run simultaneously with 
the assay. 
EXAMPLE 2 
Detection of Oxalobacter formigenes in a Sample 
Strains of Oxalobacter formigenes used in the following methods are listed 
in Table 1 below. 
TABLE 1 
______________________________________ 
Description of the Oxalobacter formigenes strains 
Group Classification of 
O. formigenes strains.sup.a 
Strain Source of Isolate 
______________________________________ 
Group I OxB Sheep rumen 
OxWR Wild rat cecum 
SOx-4 Freshwater lake sediment 
SOx-6 Freshwater lake sediment 
POxC Pig cecum 
HC-1 Human feces 
Group II BA-1 Human feces 
OxK Human feces 
HOxBLS Human feces 
HOxRW Human feces 
OxCR Lab rat cecum 
OxGP Guinea pig cecum 
______________________________________ 
.sup.a From Jensen and Allison (1994). 
All 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 10.sup.-8 
dilutions. Cultures were incubated at 37.degree. C. for 10 days and 
biochemically tested for the catabolic consumption of oxalate by 
CaCl.sub.2 precipitation (50 .mu.l media, 100 .mu.l 1% CaCl.sub.2, and 2.7 
ml dH.sub.2 O) and spectrophotometric analyses (600 nm). 
Cultures (10-15 ml) of O. formigenes were centrifuged at 10,000.times.g, 
the bacterial pellet was resuspended in 567 .mu.l TE buffer (10 mM 
Tris-Cl, pH 7.5 plus 1 mM EDTA, pH 8.0), 30 .mu.l 10% sodium dodecyl 
sulfate (SDS) and 3 .mu.l of proteinase K (20 mg/ml), and the mixture 
incubated 5 hr at 37.degree. 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 1/2 volume of 7.5 M ammonium 
acetate and 2 volumes of 100% ethanol. DNA was recovered by centrifugation 
(12,000.times.g), washed once with 70% ethanol, and the pellet resuspended 
in 15-20 .mu.l H.sub.2 O. 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, Calif.) (Stacy-Phips et al., 1995). 
Bacterial 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, Ind.) by positive pressure 
blotting and UV cross-linking (Stratagene, LaJolla, Calif.). 
Hybridizations were carried out using internal sequence oligonucleotide 
probes. Oligonucleotides were synthesized in the University of Florida 
ICBR Oligonucleotide Synthesis Laboratory (Gainesville, Fla.) and have the 
sequences: 
AP15 5'-GCACAATGCGACGACGA-3' (SEQ ID NO. 6) 
AP22 5'-GTAGTTCATCATTCCGG-3' (SEQ ID NO. 11) 
AP34 5'ATACTCGGAATTGACGT-3' (SEQ ID NO. 9) 
AP273 5'-TTCATGTCCAGTTCAATCGAACG-3' (SEQ ID NO. 10). 
Each 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). 
All PCRs were performed according to protocols described in Anderson et al. 
(1993). Briefly, 50 .mu.l reactions contained 1.5 mM MgCl.sub.2, 200 .mu.M 
dNTP, 1.25 U Taq polymerase (GIBCO-BRL, Bethesda, Md.), 1 .mu.g template 
DNA and 1 .mu.M each of a 5' and 3' primer. A preferred reaction profile 
proved to be 94.degree. C. for 5 min, then 45 cycles of 94.degree. C. for 
1 min of denaturation, 55.degree. C. for 2 min of annealing and 72.degree. 
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. 
Previous 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 AP15 (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 AP15 (SEQ ID NO. 6) sequence by a Hind III 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 FIGS. 4A-4C. Using 
probe AP15 (SEQ ID 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 ID NO. 9) or probe AP273 
(SEQ ID NO. 10). The 3 kb fragment identified by probe AP34 (SEQ ID NO. 9) 
is distinct from the 3 kb fragment detected by probe AP273 (SEQ ID NO. 
10). 
As shown in FIGS. 5A-5D, the oxalyl-CoA decarboxylase and formyl-CoA 
transferase genes were consistently detected in samples containing as 
little as 0.06 to 0.20 .mu.g of O. formigenes, strain OxB, DNA or 
approximately 0.20 to 0.40 .mu.g of O. formigenes DNA from other group I 
strains, such as HC-1. The 23-bp probe AP273 (SEQ ID NO. 10) can detect 
the frc gene in DNA samples containing only one-fourth the amount of DNA 
required for the 13 bp probe AP15 (SEQ ID NO. 6) to detect the oxc gene 
(FIGS. 5A-5B, upper panel). 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. 
Protein, 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 
AP15 (SEQ ID NO. 6) and AP273 (SEQ ID NO. 10) were able to distinguish 
group I strains from group II strains on the Southern blot hybridizations 
(FIG. 6). All strains of O. formigenes belonging to group I (to which OxB 
is assigned) hybridized with both probe AP15 (SEQ ID NO. 6) and probe 
AP273 (SEQ ID NO. 10). Due to a characteristic slow growth of strain HC-1, 
only faint bands appeared in this experiment. In contrast, none of the O. 
formigenes strains assigned to group II hybridized with probe AP273 (SEQ 
ID NO. 10) and only BA-1 hybridized with probe AP15 (SEQ ID 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. 
To 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 ID NO. 6) and primer 
AP22 (SEQ ID 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, 
HC-1, OxWR, POxC, SOx-4 and SOx-6) exhibited a common band at 452 bp 
(FIGS. 7A-7B). In contrast, the other six strains, all belonging to group 
II, 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 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 II 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 II 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 copurifying with the 
bacterial DNA, DNA 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.sup.1 to 10.sup.7 cfu 
per 0.1 g of sample showed that as few as 10.sup.2 to 10.sup.3 cfu of O. 
formigenes per 0.1 g sample could be detected (FIGS. 8A-8B). 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 (FIGS. 8A-8B, lanes F & G). 
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 in the art and are 
to be included within the spirit and purview of this application and the 
scope of the appended claims. 
REFERENCES 
Allison, M. J., K. S. 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. Cornellius, A. J. Jarpe, W. E. Winter, A. B. Peck 
(1993) "Insulin-dependent diabetes in the NOD mouse model. II. .beta. cell 
destruction in autoimmune diabetes is a T.sub.H1 mediated event," 
Autoimmunity 15:113-122. 
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. 
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. 
Hatch, M., R. W. Freel (1996) "Oxalate transport across intestinal and 
renal epithelia" Calcium Oxalate in Biological Systems, pages 217-238, CRC 
Press, Boca Raton, Fla. 
Hodgkinson, 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 divirsity among 
anaerobic oxalate-degrading bacteria now in the species Oxalobacter 
formigenes" Abst. Ann. Mtg. Amer. Soc. Microbial., pages 1-29. 
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, N.Y. 
Stacy-Phips, S., J. J. Mecca, J. B. Weiss (1995) J. Clin. Microbiol. 
33:1054. 
Yriberri, J., L. S. Posten (1980) "A semi-automatic enzymic method for 
estimating urinary oxalate," Clin. Chem. 26(7):881-884. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
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60 
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120 
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180 
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480 
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540 
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600 
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660 
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720 
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780 
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840 
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900 
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1020 
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1080 
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1140 
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1200 
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1260 
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# 15 
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# 125 
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# 365 
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180 
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240 
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300 
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480 
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540 
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600 
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660 
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720 
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780 
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840 
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900 
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960 
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1020 
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1080 
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1140 
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1200 
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1260 
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1320 
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1380 
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1440 
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1500 
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1560 
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1620 
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1680 
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1740 
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1800 
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1860 
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1920 
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1980 
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# 2088TGCC ATAAACACAT TTTTAAAGCT GGCTTTTT 
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# 365 
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# 380 
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# 415 
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# 445 
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# 460 
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# 480 
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# 495 
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# 510 
#Thr Pro Ala Glu Leu Lys Ala Alaa Asn 
# 525 
#Lys Pro Cys Leu Ile Asn Ala Metr Gly 
# 540 
#Ser Gly Arg Ile Lys Ser Leu Asnl Gly 
# 560 
- Val Val Ser Lys Val Gly Lys Lys 
# 565 
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#pairs (A) LENGTH: 17 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
# 17 T 
- (2) INFORMATION FOR SEQ ID NO:10: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 23 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
# 23TCGA ACG 
- (2) INFORMATION FOR SEQ ID NO:11: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 17 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
# 17 G 
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