Antibodies and assay for detecting mutant APC proteins

TECHNICAL FIELD OF THE INVENTION
This invention relates to the field of medical diagnostics, in particular to diagnoses of an inherited predisposition to develop cancer.
BACKGROUND OF THE INVENTION
Familial Adenomatous Polyposis (FAP) is an autosomal dominant disease in which affected individuals develop hundreds to thousands of benign colorectal tumors (adenomas). Some of these tumors, if not removed, invariably progress to malignancy (carcinomas). Recently, a candidate tumor suppressor gene from chromosome 5q21, APC.sup.1, was isolated and implicated in the development of FAP (Kinzler, K. W. et al., Science 253:661-665 (1991); Nishisho, I., et al., Science 253:665-669 (1991); Groden, J., et al., Cell 66:589-600 (1991); Joslyn, G., et al., Cell 66:601-613 (1991)). Further analyses of the APC gene in 150 kindreds indicate that APC mutations can account for most if not all cases of FAP (Miyoshi, Y., et al., Proc. Natl. Acad. Sci. USA 89:4452-4456 (1992)).
Carcinomas in FAP patients, however, account for less than 1% of all colorectal cancers. Most colorectal cancers do not have a well recognized inherited basis and therefore are classified as sporadic. Recent studies have indicated that the majority of these sporadic tumors have somatic mutations of the APC gene (Miyoshi, Y., et al., Human Molecular Genetics 1: 229-233 (1992); Powell, S. M., et al., Nature 17:235-237 (1992)). Furthermore, these mutations appear to occur early during colorectal tumorigenesis and have been detected in tumors as small as 0.5 cm in diameter (Powell, S. M., et al., Nature 17:235-237 (1992)). The nature of the mutations identified in sporadic tumors and in FAP patients is striking, with greater than 94% of the mutations predicted to result in truncation of the APC gene product. Taken together, the above studies indicate that APC plays an important and early role in the development of the major forms of colorectal neoplasia.
The APC gene contains an 8,538 bp open reading frame (ORF) and is predicted to encode a 2,843 amino acid polypeptide with few homologies to other proteins (Kinzler, K. W., et al., Science 253:661-665 (1991); Groden, J., et al., Cell 66:589-600 (1991)). The large size of the APC coding region makes identification of mutations in patients labor intensive and costly. Therefore, there is a need in the art for a quicker, less expensive method for identifying patients who carry a mutant APC allele.
SUMMARY OF THE INVENTION
It is an object of the invention to provide an antibody which can detect full-length and truncated forms of the human APC protein.
It is another object of the invention to provide an antibody which can detect full-length but not truncated forms of the human APC protein.
It is still another object of the invention to provide a method for determining the presence of mutations in a human APC gene by determining the size of the APC proteins produced in a tissue.
It is yet another object of the invention to provide an immunohistochemical method for determining the presence of APC mutations in a body sample by comparing the amount of binding of two antibodies which bind to APC in different portions of the protein.
It is another object of the invention to provide an enzyme-linked immunosorbent assay method for determining the presence of APC mutations.
It is still another object of the invention to provide an enzyme-linked immunosorbent assay method for determining the presence of APC mutations in a body sample by comparing the amount of binding of two antibodies which bind to APC in different portions of the protein.
It is another object of the invention to provide a method for determining the presence of an APC mutation by looking for loss of reactivity to a single antibody to APC protein.
It is an object of the invention to provide solid supports for use in performing enzyme-linked immunosorbent assays.
It is another object of the invention to provide hybridoma cells which produce antibodies useful for assaying APC proteins.
These and other objects of the invention are provided by one or more of the embodiments which are described below. In one embodiment of the invention, an antibody preparation is provided in which the antibodies are specifically immunoreactive with an epitope contained within the amino terminal 1103 amino acids of APC.
In another embodiment of the invention, an antibody preparation is provided in which the antibodies are specifically immunoreactive with an epitope contained within the carboxy terminal 306 amino acids of APC.
In still another embodiment of the invention a method for determining the presence of mutations in a human APC gene is provided. The method comprises the following steps: extracting proteins from a human body sample; separating said extracted proteins on a polyacrylamide gel; blotting said separated proteins onto a filter; contacting said filter with antibodies which are specifically immunoreactive with APC protein to bind said antibodies to proteins blotted on said filter; detecting the location of said antibodies which bind to said proteins; determining the size of the proteins which are bound to said antibodies, wherein a size of less than about 300 kDa indicates the presence of an APC mutation in the tissue sample.
In still another embodiment of the invention, a method for determining the presence of APC mutations in a human is provided. The method comprises: contacting a first sample of a tissue or body fluid with a first antibody which is specifically immunoreactive with an epitope contained within the amino terminal 1103 amino acids of APC; determining the amount of binding of the first antibody to said first tissue sample or body fluid; contacting a second sample of the tissue or body fluid with a second antibody which is specifically immunoreactive with an epitope contained within the carboxy terminal 306 amino acids of APC; determining the amount of binding of the second antibody to said second tissue or body fluid sample; comparing the determined amount of binding of said first antibody to the determined amount of binding of said second antibody, wherein finding a tissue or body fluid which binds substantially more of the first antibody than the second antibody indicates the presence of an APC mutation in the human.
In yet another embodiment of the present invention an enzyme-linked immunosorbent assay (ELISA) is provided for determining the presence of APC mutations. The method comprises the steps of: coating a solid support with a first antibody which is specifically immunoreactive with a first epitope, said epitope being contained within the amino terminal 1103 amino acids of APC or the carboxy terminal 306 amino acids of APC; contacting the coated solid support with an aliquot of test sample comprising a body fluid or lysate of a tissue to bind components of said aliquot to said solid support by means of said first antibody; contacting the solid support-bound components of said test sample with a second antibody which is specifically immunoreactive with a second epitope on APC, to bind said second antibody to said solid support by means of said components and said first antibody; determining the amount of second antibody which is bound to said solid support. Alternatively, the test sample comprising a body fluid or tissue lysate can be coated directly onto a solid support without use of a first antibody.
In another embodiment of the invention a method for determining the presence of an APC mutation in a human is provided. The method comprises the steps of: contacting a first tissue sample with an antibody which is specifically immunoreactive with an epitope contained within the carboxy terminal 306 amino acids of APC (as shown in SEQ ID NO:2); and determining the amount of binding of said antibody to said first tissue sample, a tissue which fails to bind the antibody indicating an APC mutation in the human.
In yet another embodiment of the invention a method for detecting truncated forms of APC is provided. The method comprises the steps of: lysing a test sample of cells from a human; fractionating said cells into a soluble and a particulate fraction; contacting said soluble fraction with an antibody which is specifically immunoreactive with an epitope contained within the amino terminal 1103 amino acids of APC (as shown in SEQ ID NO:2); detecting any of said antibody which bound to said soluble fraction, the presence of antibody bound to the soluble fraction indicating a truncated form of APC in said cells.
In still another embodiment of the invention hybridoma cells are provided which secrete an antibody which is specifically immunoreactive with an epitope contained within the amino terminal 1103 amino acids of APC or with an epitope contained within the carboxy terminal 306 amino acids of APC.
These and other embodiments of the invention provide the art with less expensive and less labor intensive means for detecting mutations in APC than are provided by genetic techniques currently available.

BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A, 1B, and 1C show the detection of APC protein by Western blot analysis. Total protein lysates (100 .mu.g) were assayed by Western blot with either polyclonal Anti-NAPC (FIG. 1A), polyclonal Anti-MAPC (FIG. 1B), or monoclonal FE9 (FIG. 1C). Protein lysates were made from the colorectal carcinoma line SW480 containing a stop codon in APC at 1338 (lanes 1) and four different FAP lymphoblastoid cell lines with known truncating APC mutations at codons 1061, 1175, 1211, or 1309 respectively (lanes 2-5). Full length protein is indicated as "FL" while mutant truncated proteins are indicated as "MT".
FIG. 2 displays the characterization of APC protein in tumor cell lines by Western blot analysis. Total protein lysates (100 .mu.g) were assayed by Western blot with monoclonal FE9. Examples from one colon cancer (C1), three colon adenomas (C2 to C4), four prostatic cancers (lanes P1 to P4), and four pancreatic cancers (lanes A1 to A4) cell lines are shown. Full length protein is indicated as "FL" and mutant truncated protein is indicated as "MT". The truncated proteins migrate as 100, 130, 100, and 135 kDa polypeptides (left to right) as compared to standard molecular weight markers.
FIGS. 3A and 3B illustrate the subcellular fractionation of normal and mutant APC protein. Cell lines were fractionated and each fraction was analyzed by Western blot using polyclonal Anti-APC antibodies. FIG. 3A shows the results obtained with SW480 cells, and FIG. 3B shows the results obtained with a lymphoblastoid cell line derived from a patient heterozygous for a truncating mutation at codon 1309 of APC. For both panels, equivalent proportions of the total protein lysate (lane 1), the 100,000.times.g insoluble "membrane fraction" (lane 2), the "nuclear fraction" (lane 3) and the "cytoplasmic fraction" (lane 4) were loaded. Full length protein is indicated by "FL" while mutant truncated protein is indicated by "MT".
FIGS. 4A, 4B, 4C, 4D, 4E and 4F photographically capture immunohistochemistry of normal colonic mucosa using affinity purified polyclonal anti-NAPC antibodies. The pattern of staining with polyclonal anti-NAPC antibody (FIG. 4A, low power and FIG. 4B, medium power) is not seen with a three-fold higher concentration of normal rabbit immunoglobulin (FIG. 4D, low power and FIG. 4E, medium power). This signal was effectively competed with GST-NAPC (FIG. 4F, medium power) but was not competed with a three-fold higher concentration of GST-MDM2 (FIG. 4C, medium power).

DETAILED DESCRIPTION OF THE INVENTION
We have found that the presence of many mutations in the APC gene can be identified in human tissues using immunological methods. Studies utilizing DNA-based analyses of leukocytes of FAP patients have shown that greater than 90% of the mutations are predicted to result in truncations of the APC protein. (Miyoshi et al., Proc. Natl. Acad. Sci., 89:4452, 1992.) Other studies of primary, sporadic, colorectal tumors indicate that at least 60% of colorectal tumors contain truncating APC mutations (Miyoshi, et al., Human Molecular Genetics, 1:229-233 (1992); Powell, et al., Nature, 17:235-237 (1992)). These mutations include point mutations which generate stop codons, and deletions and insertions which generate frameshifts which create stop codons immediately downstream from the mutations. In accordance with the present invention, an antibody specifically immunoreactive with an epitope contained within the amino terminal 1103 amino acids of APC detects truncated APC proteins and can be used to detect the majority of both inherited and somatic mutations in the APC gene.
Fusion proteins can and have been made which comprise a portion of the APC protein fused to the carboxyl end of another protein, or portion thereof. These fusion proteins are obtained by expressing gene fusions of the protein coding regions. Any other protein can be used to fuse with APC. Usually the other protein is selected for some desirable property. For example maltose binding protein (MBP) can be used, as it renders the fusion protein easily purified by means of an amylose column. The bacterial gene trpE can also be used advantageously, as it is inducible by means of tryptophan starvation.
Portions of the APC protein can be selected for fusion to other proteins, as desired. Typically a segment of the large APC protein of about 100-500 amino acid residues is chosen. In a preferred embodiment of the invention an amino terminal segment and optionally a carboxy terminal segment are chosen. For example, in a particular embodiment of the invention a fusion protein is used which comprises APC amino acids 1-220. In another embodiment a peptide which comprises APC amino acids 2537-2843 (the carboxy terminal 306 amino acids) is used. In still another embodiment of the invention a peptide which comprises amino acids 221 to 1103 of APC is used.
The fusion proteins can be used to immunize animals in order to obtain antibodies specifically immunoreactive with epitopes contained within the chosen fragment. Antisera of the immunized animals can be collected and optionally affinity purified. It is desirable to use as an affinity reagent either the fusion protein which was used to immunize the animals, or preferably a fusion protein having the same portion of APC protein, but having a different amino terminal protein. Affinity purification of polyclonal antibodies is well known in the art, and can be accomplished using routine methods.
Fusion proteins are not the only immunogens which can be used to raise the antibodies of the present invention. Synthetic, recombinant, or naturally produced peptides containing amino acid sequences of the APC protein can be used alone, or linked to other proteins, such as keyhole limpet hemocyanin (KLH), to generate a sufficient immune response. Other means of administering an immunogen to bolster the immune response may be used, as are known in the art.
Spleen cells of the immunized animals may also be fused to myeloma cells, according to the method of Kohler and Milstein, to generate hybridoma cells which secrete monoclonal antibodies. The hybridoma cells can be screened for production of appropriate antibodies by use of the original immunogen, or another form of an immunogen which contains the same, or an overlapping portion of the APC protein. Cloning and subcloning of the hybridoma cells is accomplished, as is known in the art, to achieve a cell line which stably produces antibodies which are specifically immunoreactive with epitopes on APC protein. Specific immunoreactivity means that the antibodies do not bind to other human proteins.
Particular monoclonal antibodies which have been found to be useful in the practice of the present invention are listed in Tables I and II. Not all of the antibodies which will immunoprecipitate APC will bind to APC on Western blot. This may be due to conformational epitopes which are destroyed under conditions of SDS-polyacrylamide gel electrophoresis. Other antibodies which can neither immunoprecipitate nor detect APC on Western blots can detect APC by immunohistocheinistry, e.g., on cryostat sections.
In one method according to the present invention APC mutations are detected by running proteins extracted from a test sample on an SDS-PAGE. Samples for testing according to the present invention are any tissue, whether suspected of being neoplastic or not. Also useful are body fluids, such as urine, blood, serum, plasma, fecal material, saliva, etc. Either cells or shed antigen, can be detected in such body fluids. Lysates of cells are also useful. The proteins are blotted onto filters and the blotted proteins are then contacted with one of the antibodies of the present invention (Western blotting). Antibodies which bind to blotted proteins are detected by an appropriate staining technique. The size of the proteins which have bound proteins is determined. If the protein which binds to antibody has a size less than about 300 kDa, this is an indication that there is an APC mutation in the tissue sample which is a "truncation mutation", typically a nonsense, frameshifting deletion, or frameshifting insertion mutation. In order to detect the maximum number of such truncation mutations, it is desirable to use an antibody which specifically immunoreacts with an epitope in the amino terminal portion of the protein. Use of a carboxy terminal epitope-binding antibody could result in the failure to detect truncation proteins which are actually present in the tissue.
Although small truncation proteins may be adequately detected using standard Western blotting techniques, larger truncation proteins and full length APC protein may fail to be sufficiently blotted from an SDS-PAGE due to their large size. Therefore it is desirable to use techniques which lead to enhanced transfer from gel matrix to filter. These may include, for example, the use of 3% low melting point agarose to form the gel matrix.
According to the present invention, patients can be tested to determine whether they have inherited a germ-line mutation of APC, or to determine somatic mutations. In the former case, a suitable and convenient tissue sample comprises peripheral blood mononuclear cells or leukocytes. If a germ-line mutation is present, it can be detected in any tissue of the body. In the latter case (somatic mutations), the test tissue sample should comprise tumor tissue or preneoplastic tissue. A control sample comprising non-tumor tissue, preferably of adjacent noncancerous tissue, can also be tested.
In an alternative method for detecting APC mutations according to the present invention, immunohistochemistry or immunofluorescence is employed. A tissue sample or cell sample is prepared by any standard technique known in the art. Peripheral blood mononuclear cells can be fixed according to known techniques for immunochemical staining. Tissue samples can be frozen and embedded in a mounting compound, for example O.C.T. (Miles Laboratories). Thin sections can then be cut, incubated with an antibody specific for APC, stained, and observed. According to one method, an antibody which is immunoreactive with an epitope contained within the amino terminal portion of APC is used to stain a first sample of a tissue. A second antibody which is specifically immunoreactive with an epitope contained within the carboxy terminal portion of APC is used to stain a second sample of the same tissue. The amount of binding of each antibody is determined. If substantially more of the amino terminal antibody binds to the tissue sample than does the carboxy terminal antibody, an APC truncation mutation is indicated. In one embodiment of the invention antibodies are used which are labelled with a detectable tag, such as an immunofluorescent compound or radioactive molecule. Such a detectable tag can also be attached to an additional antibody which is immunoreactive with the amino terminal or carboxy terminal anti-APC antibodies. In another embodiment of the invention an antibody is labelled by linkage to an enzyme which upon contact with an appropriate enzyme substrate, generates a colored reaction product. In still another embodiment of the invention detectable tags are linked to the antibody via an avidin:biotin ligand binding pair, as is generally known in the art. Typically, the antibody is linked to biotin and the detectable tag, e.g., alkaline phosphatase, is conjugated to avidin. According to one aspect of the invention the amino terminal and carboxy terminal antibodies are incubated with the same tissue sample or cell sample. In that case the antibodies are labelled with different detectable tags.
Enzyme-linked immunosorbent assays (ELISA) are also contemplated within the scope of the present invention. Such assays employ solid supports, such as microtiter plates or dipsticks, which are coated with a first antibody which is specifically immunoreactive with an epitope which is contained within the amino terminal portion of APC. A test sample is contacted with the coated solid support under conditions where antigen-antibody complexes form and are stable to allow components of the test sample to bind to the antibody which has been coated onto the support, if such components are present in the test sample. Appropriate washing of the solid support is used, as is known in the art, to remove components of the test sample which are not specifically bound to the antibodies. A second antibody is added which is specifically immunoreactive with a second epitope on APC. Thus the components of the test sample which bound to the first (amino terminal) antibody, are now sandwiched between two antibodies. Second antibody which has not specifically bound to the support-bound components of the test sample are washed away, under conditions in which antigen-antibody complexes which have formed are stable. The amount of second antibody which is bound to the solid support (by means of the first antibody and the components of the test sample) is determined, typically by adding a chromogenic substrate which generates a colored product upon contact with an enzyme which is linked to the second antibody. Alternative formats for running such ELISA assays are contemplated and include binding the test sample directly to the solid support without using a first antibody.
According to another aspect of the present invention, a second aliquot of the test sample may be run through a parallel enzyme-linked immunosorbent assay, as described above. However, in this parallel ELISA different antibodies are coated onto the solid support. In this version, a carboxy terminal antibody is used as the first antibody. Thus when the results of the first and second parallel ELISAs are compared, a substantially smaller amount of binding occurring in the second ELISA than in the first indicates an APC mutation in the tissue from which the sample was derived. Samples can be, for example, lysates of blood cells or of solid tumors. Other assays which can be performed using the antibodies of the present invention include immunoprecipitations, and protein dot blots. The antibodies used in the practice of this invention may be either polyclonal or monoclonal or combinations thereof.
In another aspect of the invention, tissue samples can be analyzed, for example by immunohistochemistry, for the ability to bind to an antibody which is specific for an epitope in the carboxy terminal 306 amino acids of APC. If the tissue sample fails to bind the antibody, then truncation and/or deletion mutations of APC are indicated. If desired, a control sample of an adjacent, normal tissue may be analyzed using the same antibody. The ability of the control sample to bind the antibody when the test sample failed to do so indicates a somatic mutation of APC.
According to still another aspect of the invention, immunological detection is combined with subcellular fractionation to yield information about a human's APC alleles. It has been found that full-length APC protein is found in a particulate fraction, whereas the soluble or cytoplasmic fraction contains only truncated forms. If full-length APC and truncated APC are present in the same cell, the truncated form is also detected in the particulate fraction, presumably due to oligomerization of the APC proteins. Thus if only particulate APC is detected, one infers that the human has two wild-type alleles. If both forms of APC are detected, than one infers that the human has one mutant and one wild-type allele. If only cytosolic APC is observed, then one infers that the human has two mutant alleles of APC. One mutant allele indicates a predisposition to sporadic or familial colon cancer. Two mutant alleles indicate a worse prognosis.
Subcellular fractionation, according to the present invention can be accomplished by any means which separates typically cytoplasmic cellular components from typically membrane-associated cellular components. In one method of accomplishing the fractionation, cells are first lysed (either mechanically or osmotically, for example) and then nuclei and unbroken cells are removed by a slow centrifugation, typically at about 500.times.g. The supernatant can then be centrifuged at about 100,000.times.g for 1 hour to pellet the particulate or membrane-associated fraction. The remaining supernatant is considered to be the soluble or cytoplasmic fraction. As stated above, full-length APC has been found in the particulate fraction, whereas truncated APC is found in the soluble fraction. If full-length APC is present in the cell along with truncated APC, then truncated APC also can be found in the particulate fraction.
After subcellular fractionation has been accomplished, the proteins can be analyzed immunologically, by any means taught herein. Typically the proteins of the individual fractions are separated electrophorectically, and immunoblotted. Alternatively, the fractions can be assayed in an ELISA format using both an antibody which binds to the amino terminal portion of APC and optionally an antibody which binds to the carboxy terminal portion of APC.
Antibody-secreting hybridomas according to the present invention have been deposited at the American Type Culture Collection (ATCC), 12301 Parklawn Avenue, Rockville, Md. 20852-1996 on Mar. 11, 1993. These include the antibodies known as FE9, AC4, CC1, CF11, DB1, IA1, IE1, and HG2 which have been given the accession numbers HB 11294, HB 11292, HB 11296, HB 11288, HB 11295, HB 11297, HB 11298, and HB 11293, respectively. Other hybridomas making antibodies which bind to the same epitopes can be made, e.g., using anti-idiotype technology. Antibodies can be tested for binding to the same epitope as the deposited antibodies by examining the ability to completely inhibit the binding of the deposited antibody to its cognate antigen. It has been found that FE9 binds to an epitope comprising amino acids 16-29 of APC. AC4 binds to an epitope comprising amino acids 109-170 of APC. Isotype switching variants of these antibodies can also be made, as is known in the art. See e.g., Spira et al., (1985) "The Generation of Better Monoclonal Antibodies Through Somatic Mutations", Hybridoma Technology in the Biosciences and Medicine (ed. Springer), pp. 77-78, Plenum Press, N.Y.
Solid supports according to the present invention may be any plastic, paper, or glass to which antibodies will adhere. Most typically solid supports comprise a plastic microtiter plate. Precoated paper sheets and sticks are also contemplated, as well as microspheres, as carriers of the antibodies of the present invention.
The antibodies of the present invention can also be used as imaging agents. They can be labeled, as is known in the art, and administered to a patient to assess tumor load in the colon, or to detect metastases.
The antibodies of the present invention are useful for determining the presence of germ-line or somatic mutations which indicate a predisposition to colon cancer. APC mutations may also be involved in the development of gastric, esophageal, pancreatic and small cell lung cancers. The presence of germ-line APC mutations is associated with FAP and portends a high probability that the patient will develop colon carcinoma. Somatic APC mutations have been found to be associated with the early development of colonic tumors.
EXAMPLES
Example 1
This example demonstrates the construction of bacterial fusion proteins useful for immunizing and screening to obtain antibodies specific for portions of the APC protein.
Bacterial fusion proteins containing the amino terminal portion of the APC protein were constructed from partial APC CDNA clones as follows. A fragment of the APC gene containing nucleotides -15 to 734 was engineered to have an EcoRI site 10 nucleotides 5' of the initiating methionine by PCR amplification with the following primers, 5'-CAAGGGAATTCAAGGATG-3' (SEQ ID NO:3), and 5'TGCTTCTGTTGCTTGAGGC-3' (SEQ ID NO:4); (underlined bases were altered to create the EcoRI site). After digestion with EcoRI, a fragment extending from the engineered EcoRI site to the endogenous EcoRI site at nucleotide 660 was cloned into the EcoRI site of pATH3 (Koerner, T. J., et al., Methods Enzymol. 194:477-490 (1991)) and pGEX-3X (Pharmacia) resulting in a trpE fusion (pATH-NAPC) or a glutathione-S-transferase (GST) fusion (pGEX-NAPC).
A second non-overlapping set of fusion proteins was constructed from an APC EcoRi fragment extending from the endogenous EcoRi site at nucleotide 660 to an exogenous EcoRI site at nucleotide 3309 introduced during cDNA cloning. This fragment was cloned into pATH3, pMAL-C2 (New England Biolabs), and pGEX-2T (Pharmacia) resulting in a trpe fusion (pATH-MAPC), a maltose binding protein (MBP) fusion (MBP-MAPC), or a GST fusion (pGEX-MAPC). The third set of fusion proteins contain the 306 extreme carboxy terminal amino acids of APC cloned into pATH3 and pMAL-C2 expression vectors to give fusion proteins trpE-XC and MBP-XC, respectively. The full length fusion protein (trpE-XC) has a molecular weight of 75 kDa.
The pATH encoded trpE fusion proteins were produced in temperature sensitive E. coli CAG456 (Baker, T. A., et al., Proc. Natl. Acad. Sci. USA 81: 6779-6783 (1984)). Stationary phase E. coli were diluted 1:5 and induced for 12 hr by tryptophan starvation. The cells were harvested by centrifugation and resuspended in 1/60 volume TEN (50 mM Tris HCl (pH 8), 1 mM EDTA, 50 mM NaCl). Lysozyme was added to 20 mg/ml and the culture was incubated on ice for 15 minutes. After addition of NP-40 to a final concentration of 0.25%, the sample was sonicated, the insoluble fusion proteins were pelleted (12,000.times.g for 5 minutes at 4.degree. C.), washed with TEN, and frozen at -80.degree. C.
The pGEX encoded glutathione-S-transferase fusion proteins were produced in E. coli JM101 cells. Cells were grown at 37.degree. C. to an absorbance (A.sub.600) of 0.5 at 550 nm and induced by the addition of isopropyl-.beta.-D-thiogalactoside (0.5 mM). After a 4 hour induction, the cells were harvested by centrifugation, resuspended in 1/30 volume MTPBS (150 mM sodium chloride, 16 mM monobasic sodium phosphate and 4 mM dibasic sodium phosphate, pH 7.3) and sonicated. The insoluble fusion proteins were pelleted, washed once with 0.03% SDS and frozen at -80.degree. C.
Soluble forms of the pMAL-C2 encoded maltose binding protein fusion proteins MBP-XC and MBP-MAPC expressed in E. coli cells were produced and purified as follows: Cells were grown at 37.degree. C. to an absorbance (A.sup.600) of 0.5. Fusion protein expression was then induced by addition of isopropyl-.beta.-D-thiogalactoside to 0.5 mM. After a 4 hour incubation, the cells were harvested by cenyrifugation, resuspended in 1/10 volume of STE (150 mM NaCl, 10 mM Tris-Cl, pH 7.4, and 1 mM EDTA) and passed twice through a 20K French Pressure cell (SLM Aminco, SLM Instruments, Inc. Urbana, Ill.) at 15,000 PSI to break open cells. Insoluble material was removed by centrifugation at 16,000.times.g for 10 minutes and the remanining soluble material was applied to an amylose column. Soluble MBP-XC and MBP-MAPC were eluted from the column with amylose. Peak fractions were identified by SDS/PAGE and pooled.
Example 2
This example demonstrates the production of polyclonal antibodies specifically immunoreactive with amino or carboxy terminal segments of APC protein.
The insoluble bacterial pellets containing the trpE bacterial fusion proteins were purified by SDS-PAGE. Gel slices containing approximately 300 .mu.g of fusion protein were homogenized in Freund's complete adjuvant (Sigma) for the primary injection and Freund's incomplete adjuvant (Sigma) for booster injections. Two New Zealand white rabbits were injected with each protein preparation. Rabbits were injected every 2 weeks and bled 10 days after each booster injection. The antibodies were affinity purified by binding to the glutathione-S-transferase insoluble bacterial fusion proteins. The bacterial pellets containing 200 .mu.g of fusion protein were thawed, washed with TEN and pelleted. One ml of antisera was added to the pellet and incubated on ice for one hour. The antibody-insoluble protein complexes were then pelleted and washed with TBS. The antibodies were released with 500 .mu.l of 0.2 M glycine, pH 2.3 on ice for five minutes. The remaining insoluble proteins were pelleted. The antibody solution was neutralized with 70 .mu.l of 1M Tris pH 9.5. Bovine serum albumin and sodium azide were added to a final concentration of 5 mg/ml and 0.05%, respectively.
Example 3
This example demonstrates the production of monoclonal antibodies specifically immunoreactive with amino or carboxy terminal segments of APC.
Female CB6/F1 mice were immunized by intraperitoneal injection on three successive occasions with 20 .mu.g of trpE-NAPC or trpE-XC fusion protein which had been expressed in E. coli and purified by electroelution following SDS-PAGE. Hybridomas were produced as described (McKenzie, S. J., et al., Oncogene 4: 543-548 (1989)), except fused cells were plated at a density of 1.times.10.sup.6 /ml. Mouse test bleeds and hybridomas were screened for anti-APC reactivity by an antigen capture ELISA using purified GST-NAPC (Smith, D. B., et al., Gene 67:31-40 (1988)) or soluble MBP-XC, purified as described above, coated on plates at a concentration of 500 ng/ml or 3 .mu.g/ml, respectively. Purified GST-MDM2, expressed and purified as described (Oliner, J. D., et al., Nature 358:80-83 (1992)) and purified MBP-MAPC were was used as the negative control antigens. Antigen/antibody complexes were detected by incubation with horseradish peroxidase conjugated goat anti-mouse IgG heavy and light chain (Kirkegaard and Perry Labs, Inc.) followed by development with tetramethylbenzidine as described (McKenzie, S. J., et al., Oncogene 4:543-548 (1989)). All positive hybridomas were subcloned twice by limiting dilution. Monoclonal antibodies directed against the amino terminal portion of APC were initially tested for specific recognition of APC by Western blot using GST-NAPC and trpE-NAPC, to show anti-APC reactivity, while GST-MDM2 and trpE-MAPC were used as negative controls. The monoclonal antibodies directed against the carboxy terminal portion of APC were initially tested for specific recognition of APC by ELISA using soluble fusion protein MBP-XC to show anti-APC reactivity and MBP-MAPC as negative control.
Ten hybridomas were isolated which reacted specifically with an epitope contained within amino acids 1-220 of APC. Six hybridomas were isolated which specifically reacted with an epitope contained within the carboxy terminal 306 amino acid residues of APC. These are noted in Tables I and II which also show their isotypes.
TABLE I______________________________________Summary of APC-NH MonoclonalsAntibody Isotype Western Blot Immunoprecipitation______________________________________AC4-2F.sup.1 IgG.sub.1 +BD2-2E IgG.sub.2a +BD12-2I IgG.sub.2b + +BE7-2J IgG.sub.1 +CC1-2H IgG.sub.2b + +CF11-1G IgG.sub.2b + +EF4-1H IgG.sub.1 + +FC5-1C IgG.sub.1 +FE9-1B.sup.2 IgG.sub.1 + +HA94-2J IgG.sub.1 +______________________________________ .sup.1 = AC4 is able to detect APC by immunohistochemistry on cryostat sections. .sup.2 = FE9 is also able to detect APC by Western blot in cell lines listed in Smith et al.
TABLE II______________________________________Summary of APC-XC MonoclonalsAntibody Isotype Western Blot Immunoprecipitation______________________________________DB1-2C IgG.sub.1 + +HG2-1C.sup.3 IgG.sub.1 + +IA1-1D IgG.sub.1 + +IB6-1L IgG.sub.1IE1-2J IgG.sub.1 + +IE8-2G IgG.sub.1 + +______________________________________ .sup.3 = HG21C is able to detect APC by immunohistochemistry on cryostat sections.
Example 4
This example demonstrates the characterization of the ten monoclonal antibodies specific for APC amino terminus by Western blot and immunoprecipitation.
For the western blots, 1 .mu.g each of GST-NAPC, GST-MDM-2, trpE-NAPC and trpE-MAPC, were separated by SDS-PAGE and transferred to nitrocellulose. The blots were probed with 10 ml of undiluted hybridoma supernatant.
By western blot, all of the antibodies are able to recognize full length, 48 kDa, GST-NAPC and full length trpE-NAPC. None recognize GST-hMDM2 or trpE-MAPC. They all, however, detect a number of lower molecular weight bands in the GST-NAPC lane. These bands are most likely caused by proteolytic degradation of full length GST-NAPC.
For immunoprecipitation, SW 480 cells were metabolically labeled with .sup.35 S-methionine. The lysate was then precipitated using 12 .mu.g, in hybridoma supernatant, of each antibody. Five of the antibodies, BD12, CC1, CF11, EF4, and FE9 are able to detect the 147 kDa APC protein in SW 480 lysates by immunoprecipitation. See, e.g., FIG. 1C, lane 1. A summary of the characterization is presented in Table 1.
Example 5
This example demonstrates the detection of APC protein in seven lymphoblastoid and a colorectal cell line by monoclonal and polyclonal antibodies in Western blots.
Polyclonal and monoclonal antibodies were used for Western blot analysis of protein lysates from the colorectal cancer cell line SW480 and from seven EBV-immortalized lymphoblastoid lines generated from FAP patients. The SW480 cell line contains only one allele of the APC gene, and that allele has a nonsense mutation at codon 1338 resulting in a predicted protein size of 147 kDa (Nishisho, I., et al., Science 253:665-669 (1991)). All seven FAP cell lines contain a normal allele as well as a known truncating mutation of the APC gene (Miyoshi, Y. et al., Proc. Natl. Acad. Sci USA 89:4452-4456 (1992)).
The normal APC gene is predicted to encode a protein of 312 kDa while the mutations result in proteins with predicted relative sizes ranging from 27 kDa to 147 kDa. Analysis of protein lysates by standard Western Blot techniques following SDS-PAGE failed to detect full length APC protein. To facilitate detection of large proteins, protein lysates were subjected to electrophoresis through a denaturing agarose gel system and protein transfer was accomplished by capillary action. This transfer system coupled with enhanced chemiluminescence allowed detection of full length APC protein. More specifically, electrophoresis was performed in a 3% low melting point agarose gel formed in 0.1% SDS in Tris-borate buffer (89 mM Tris base, 89 mM boric acid, 2 mM EDTA), Trisglycine buffer (25 mM Tris base, 191 mM glycine, 0.05% SDS) was used as the running buffer. Cell protein lysates were prepared by boiling for five min in SDS-PAGE loading buffer (63 mM Tris-HCl, pH 6.8, 10% glycerol, 5% 2-mercaptoethanol, 2% SDS, 0.025% bromophenol blue). Protein concentrations were determined following Amido Black staining as previously described (Sheffield, J. N., et al., Analytical Biochemistry 166:49-54 (1987)) and 100 .mu.g of total protein was loaded in each lane. The proteins were transferred by capillary action to a polyvinyldifluoride membrane (Immobilon, Millipore) in Tris buffered saline (0.1M Tris-HCl, pH 7.5, 0.9% sodium chloride) (TBS) with 0.04% SDS overnight. The filters were briefly rinsed with TBS and blocked with 10% nonfat dried milk, 10% goat serum, 0.1% Tween 20 in TBS for 1 hr. After incubation with 1-2 .mu.g/ml of primary antibody in 5% nonfat dried milk, 0.1% Tween 20 in TBS for two hr, the filters were washed for 30 min in 0.1% Tween 20 in TBS with three changes. Horseradish peroxidase conjugated goat anti-rabbit or goat anti-mouse antibodies (0.05 .mu.g/ml in 5% nonfat dried milk, 0.5% Tween 20 in TBS) were incubated with the filter for 45 min. The filters were then washed six times, 20 min. each, with 0.1% Tween 20 in TBS. Peroxidase activity was detected using Amersham's enhanced chemiluminescence detection kit following the manufacturer's protocol.
Polyclonal anti-NAPC, anti-MAPC and monoclonal FE9 antibodies all detected an approximately 300 kDa protein in the FAP cell lines which was absent in the SW480 cell line (examples in FIGS. 1A, 1B and 1C). Furthermore, truncated proteins corresponding to the expected sizes were detected in SW480 and four of the FAP lines (FIG. 1). In one case, freshly drawn blood was obtained from an FAP patient and peripheral mononuclear cells were isolated. Peripheral mononuclear cells were isolated from EDTA anticoagulated blood using an Histopaque 1077 step gradient (Sigma) following the manufacturer's protocol. Both mutant (117 kDa) and full length APC protein were detectable providing evidence that this analysis could be done directly on blood cells. In three of the FAP lymphoblastoid samples, truncated proteins were not detected, even though full length APC proteins were easily detectable (data not shown). These included lines with nonsense mutations at codons 232, 301 and 625. Three independent cell lysates from each of these cell lines were analyzed with anti-NAPC or FE9 using a variety of different gel electrophoresis conditions that should have allowed detection of proteins in the size range predicted by the mutations. These results suggest that certain forms of truncated APC are expressed at very low levels relative to full length, likely due to instability of the mutant transcript or protein.
Similar results were obtained with monoclonals FE9, CF11 and EF4. A fourth monoclonal CC1 failed to detect endogenous APC on Western blots even though CC1 was able to immunoprecipitate metabolically labelled endogenous APC (data not shown).
Example 6
This example demonstrates the use of antibodies specific for APC to detect altered APC protein in human tumor cell lines.
We determined the status of APC protein in human tumors. In order to avoid problems with contaminating normal tissue, we examined APC protein in human cell lines derived from colonic, breast, prostatic, cervical, lung and pancreatic tumors. A total of 32 colorectal tumor cell lines were studied including 6 derived from sporadic adenomas, 22 from sporadic carcinomas, 3 from FAP adenomas, and one from an FAP carcinoma. Western blot analysis revealed that 24 (75%) of these lines contained truncated APC protein (examples in FIG. 2). Cell lines were scored positive for truncated APC protein only if the novel band reacted with Anti-NAPC, Anti-MAPC, and FE9 antibodies. Interestingly, greater than 80% (27 of 32) of the colon lines were totally devoid of full length APC protein.
In contrast, seven breast, six prostatic, nine cervical, nine pancreatic and nine lung carcinoma cell lines each contained only full-length APC protein by Western blot analysis (examples in FIG. 2). The results suggest that APC mutations might not play a role in the development of these tumors, although it is possible that missense APC mutations and/or very early truncating mutations occur in these tumor types. These results also indicate that the APC protein is expressed at detectable levels in many different tissue types, specifically, lymphoid, lung, breast, prostate, pancreas and cervix.
Example 7
This example demonstrates that APC is localized to the cytoplasm.
Several experiments were undertaken to localize APC at the subcellular level. When cell fractionation using SW480 cells was performed, the truncated protein was found exclusively in the cytosolic fraction (FIG. 3A, lane 4). The procedure was performed as previously described (Sprott, et al., Analytical Biochemistry, 166:49-54 (1987)) with the following modifications. Cells were harvested by centrifugation at 200.times.g and washed three times with Dulbecco's phosphate buffered saline containing 0.5 mM calcium chloride. The pellet was resuspended in lysis buffer (5 mM Tris HCl, pH 7.8, 2 mM MgCl.sub.2, 1 mM PMSF, 0.5 .mu.g/ml leupeptin (Sigma), 10 .mu.g/ml trypsin inhibitor (Sigma)). The sample was then centrifuged at 500.times.g to pellet nuclei and unlysed cells. The pellet was washed once with lysis buffer and saved as the "nuclear fraction". The supernatant was centrifuged at 100,000.times.g for 1 hr. The resulting pellet, was designated as the "membrane fraction", and the supernatant was designated the "cytosolic fraction". All three fractions were solubilized directly in SDS-PAGE loading buffer prior to Western blot analysis. These fractions were verified by finding that p53 and DCC were predominantly found in the "nuclear" and membrane" fractions, respectively.
When an FAP lymphoblastoid line containing full length and truncated APC protein was fractionated, the results were unexpected. The full-length APC protein was found in the 100,000.times.g insoluble "membrane fraction" while the truncated form was present in the 100,000.times.g fraction and in the cytosolic fraction (FIG. 3B, lane 2 and 4). APC fractionation was unaffected by Triton X-100 treatment but deoxycholate, a weak ionic detergent, completely solubilized full length and truncated APC (data not shown). This suggested that APC was not a membrane protein, but rather that the full length APC protein was complexed in an insoluble aggregate. This pattern of fractionation was reproduced with a second FAP lymphoblastoid line containing a different truncated APC protein.
To further define the subcellular localization of APC, immunohistochemical studies were performed on frozen normal colonic mucosa sections using affinity purified polyclonal antibodies. Frozen normal colonic mucosa samples were embedded in O.C.T. (Miles Laboratories, Inc.) and cut into 12 .mu.m sections. The sections were immediately fixed in 0.3% hydrogen peroxide in absolute methanol at room temperature for 30 min. The tissues were washed three times in Dulbecco's phosphate buffered saline (PBS). The APC antigen was unmasked using the Antigen Retrieval System (Biogenex Laboratories) following the manufacturer's protocol. The tissues were briefly washed three times with H.sub.2 O and three times with PBS, blocked with goat serum for 30 min, and then incubated with affinity purified Anti-NAPC (1.5 .mu.g/ml) or Anti-MAPC antibodies (1.5 .mu.g/ml) or normal rabbit immunoglobulins (4.5 .mu.g/ml) diluted in goat serum for 2 hr. The tissues were washed with PBS three times for 5 min each. Biotinylated goat anti-rabbit antibody (Vector Laboratories) was diluted 1:200 in goat serum and exposed to the tissues for 30 min. The tissues were washed in PBS three times for five min. each. Immunoperoxidase staining was performed using the Vectastain Elite ABC System (Vector Laboratories) following the manufacturer's protocol. DAPI staining was performed as a nuclear counterstain by overlaying the immunoperoxidase stained tissue with 0.1 mg/ml DAPI and viewing under U.V. excitation. Competition studies were performed using soluble GST fusion proteins purified as previously described (Smith D. B., et al. Gene 67: 3-40, (1988)). GST-NAPC and GST-MDM2 fusion protein were included in the primary antibody incubations at a final concentration of 3 .mu.g/ml and 9 .mu.g/ml, respectively.
Cytoplasmic staining concentrated at the basolateral portion of crypt epithelial cells was seen with Anti-NAPC (FIGS. 4A and 4B), but not with a three fold higher concentration of normal rabbit IgG (FIGS. 4D and 4E). When counterstained with DAPI, the darkest APC specific staining was shown to be basolateral to the nuclei. The staining of epithelial cells displays a marked increase from the base of the crypt to the luminal surface. The identical pattern was observed with polyclonal Anti-MAPC antibodies. To confirm that this antibody binding was specific for APC, competitions were performed with soluble bacterial GST-NAPC fusion protein or a control unrelated fusion protein (GST-MDM2). The signal was fully competed by the NAPC fusion protein (FIG. 4F), but was unaffected by three-fold higher concentration of the MDM2 fusion protein (FIG. 4C). The signal seen with Anti-MAPC antibodies could not be competed away by soluble bacterial NAPC fusion protein, as expected. The same pattern of staining was observed in FAP colonic mucosa from two individuals with either a codon 625 or 1309 truncating mutation (data now shown).
__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 4- (2) INFORMATION FOR SEQ ID NO:1:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 9606 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: NO- (iv) ANTI-SENSE: NO- (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapi - #ens- (viii) POSITION IN GENOME: (B) MAP POSITION: 5q21- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 34..8562- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:- GGACTCGGAA ATGAGGTCCA AGGGTAGCCA AGG ATG GCT GCA GC - #T TCA TAT GAT 54# Met Ala Ala Ala Ser Tyr Asp#5 1- CAG TTG TTA AAG CAA GTT GAG GCA CTG AAG AT - #G GAG AAC TCA AAT CTT 102Gln Leu Leu Lys Gln Val Glu Ala Leu Lys Me - #t Glu Asn Ser Asn Leu# 20- CGA CAA GAG CTA GAA GAT AAT TCC AAT CAT CT - #T ACA AAA CTG GAA ACT 150Arg Gln Glu Leu Glu Asp Asn Ser Asn His Le - #u Thr Lys Leu Glu Thr# 35- GAG GCA TCT AAT ATG AAG GAA GTA CTT AAA CA - #A CTA CAA GGA AGT ATT 198Glu Ala Ser Asn Met Lys Glu Val Leu Lys Gl - #n Leu Gln Gly Ser Ile# 55- GAA GAT GAA GCT ATG GCT TCT TCT GGA CAG AT - #T GAT TTA TTA GAG CGT 246Glu Asp Glu Ala Met Ala Ser Ser Gly Gln Il - #e Asp Leu Leu Glu Arg# 70- CTT AAA GAG CTT AAC TTA GAT AGC AGT AAT TT - #C CCT GGA GTA AAA CTG 294Leu Lys Glu Leu Asn Leu Asp Ser Ser Asn Ph - #e Pro Gly Val Lys Leu# 85- CGG TCA AAA ATG TCC CTC CGT TCT TAT GGA AG - #C CGG GAA GGA TCT GTA 342Arg Ser Lys Met Ser Leu Arg Ser Tyr Gly Se - #r Arg Glu Gly Ser Val# 100- TCA AGC CGT TCT GGA GAG TGC AGT CCT GTT CC - #T ATG GGT TCA TTT CCA 390Ser Ser Arg Ser Gly Glu Cys Ser Pro Val Pr - #o Met Gly Ser Phe Pro# 115- AGA AGA GGG TTT GTA AAT GGA AGC AGA GAA AG - #T ACT GGA TAT TTA GAA 438Arg Arg Gly Phe Val Asn Gly Ser Arg Glu Se - #r Thr Gly Tyr Leu Glu120 1 - #25 1 - #30 1 -#35- GAA CTT GAG AAA GAG AGG TCA TTG CTT CTT GC - #T GAT CTT GAC AAA GAA 486Glu Leu Glu Lys Glu Arg Ser Leu Leu Leu Al - #a Asp Leu Asp Lys Glu# 150- GAA AAG GAA AAA GAC TGG TAT TAC GCT CAA CT - #T CAG AAT CTC ACT AAA 534Glu Lys Glu Lys Asp Trp Tyr Tyr Ala Gln Le - #u Gln Asn Leu Thr Lys# 165- AGA ATA GAT AGT CTT CCT TTA ACT GAA AAT TT - #T TCC TTA CAA ACA GAT 582Arg Ile Asp Ser Leu Pro Leu Thr Glu Asn Ph - #e Ser Leu Gln Thr Asp# 180- TTG ACC AGA AGG CAA TTG GAA TAT GAA GCA AG - #G CAA ATC AGA GTT GCG 630Leu Thr Arg Arg Gln Leu Glu Tyr Glu Ala Ar - #g Gln Ile Arg Val Ala# 195- ATG GAA GAA CAA CTA GGT ACC TGC CAG GAT AT - #G GAA AAA CGA GCA CAG 678Met Glu Glu Gln Leu Gly Thr Cys Gln Asp Me - #t Glu Lys Arg Ala Gln200 2 - #05 2 - #10 2 -#15- CGA AGA ATA GCC AGA ATT CAG CAA ATC GAA AA - #G GAC ATA CTT CGT ATA 726Arg Arg Ile Ala Arg Ile Gln Gln Ile Glu Ly - #s Asp Ile Leu Arg Ile# 230- CGA CAG CTT TTA CAG TCC CAA GCA ACA GAA GC - #A GAG AGG TCA TCT CAG 774Arg Gln Leu Leu Gln Ser Gln Ala Thr Glu Al - #a Glu Arg Ser Ser Gln# 245- AAC AAG CAT GAA ACC GGC TCA CAT GAT GCT GA - #G CGG CAG AAT GAA GGT 822Asn Lys His Glu Thr Gly Ser His Asp Ala Gl - #u Arg Gln Asn Glu Gly# 260- CAA GGA GTG GGA GAA ATC AAC ATG GCA ACT TC - #T GGT AAT GGT CAG GGT 870Gln Gly Val Gly Glu Ile Asn Met Ala Thr Se - #r Gly Asn Gly Gln Gly# 275- TCA ACT ACA CGA ATG GAC CAT GAA ACA GCC AG - #T GTT TTG AGT TCT AGT 918Ser Thr Thr Arg Met Asp His Glu Thr Ala Se - #r Val Leu Ser Ser Ser280 2 - #85 2 - #90 2 -#95- AGC ACA CAC TCT GCA CCT CGA AGG CTG ACA AG - #T CAT CTG GGA ACC AAG 966Ser Thr His Ser Ala Pro Arg Arg Leu Thr Se - #r His Leu Gly Thr Lys# 310- GTG GAA ATG GTG TAT TCA TTG TTG TCA ATG CT - #T GGT ACT CAT GAT AAG1014Val Glu Met Val Tyr Ser Leu Leu Ser Met Le - #u Gly Thr His Asp Lys# 325- GAT GAT ATG TCG CGA ACT TTG CTA GCT ATG TC - #T AGC TCC CAA GAC AGC1062Asp Asp Met Ser Arg Thr Leu Leu Ala Met Se - #r Ser Ser Gln Asp Ser# 340- TGT ATA TCC ATG CGA CAG TCT GGA TGT CTT CC - #T CTC CTC ATC CAG CTT1110Cys Ile Ser Met Arg Gln Ser Gly Cys Leu Pr - #o Leu Leu Ile Gln Leu# 355- TTA CAT GGC AAT GAC AAA GAC TCT GTA TTG TT - #G GGA AAT TCC CGG GGC1158Leu His Gly Asn Asp Lys Asp Ser Val Leu Le - #u Gly Asn Ser Arg Gly360 3 - #65 3 - #70 3 -#75- AGT AAA GAG GCT CGG GCC AGG GCC AGT GCA GC - #A CTC CAC AAC ATC ATT1206Ser Lys Glu Ala Arg Ala Arg Ala Ser Ala Al - #a Leu His Asn Ile Ile# 390- CAC TCA CAG CCT GAT GAC AAG AGA GGC AGG CG - #T GAA ATC CGA GTC CTT1254His Ser Gln Pro Asp Asp Lys Arg Gly Arg Ar - #g Glu Ile Arg Val Leu# 405- CAT CTT TTG GAA CAG ATA CGC GCT TAC TGT GA - #A ACC TGT TGG GAG TGG1302His Leu Leu Glu Gln Ile Arg Ala Tyr Cys Gl - #u Thr Cys Trp Glu Trp# 420- CAG GAA GCT CAT GAA CCA GGC ATG GAC CAG GA - #C AAA AAT CCA ATG CCA1350Gln Glu Ala His Glu Pro Gly Met Asp Gln As - #p Lys Asn Pro Met Pro# 435- GCT CCT GTT GAA CAT CAG ATC TGT CCT GCT GT - #G TGT GTT CTA ATG AAA1398Ala Pro Val Glu His Gln Ile Cys Pro Ala Va - #l Cys Val Leu Met Lys440 4 - #45 4 - #50 4 -#55- CTT TCA TTT GAT GAA GAG CAT AGA CAT GCA AT - #G AAT GAA CTA GGG GGA1446Leu Ser Phe Asp Glu Glu His Arg His Ala Me - #t Asn Glu Leu Gly Gly# 470- CTA CAG GCC ATT GCA GAA TTA TTG CAA GTG GA - #C TGT GAA ATG TAT GGG1494Leu Gln Ala Ile Ala Glu Leu Leu Gln Val As - #p Cys Glu Met Tyr Gly# 485- CTT ACT AAT GAC CAC TAC AGT ATT ACA CTA AG - #A CGA TAT GCT GGA ATG1542Leu Thr Asn Asp His Tyr Ser Ile Thr Leu Ar - #g Arg Tyr Ala Gly Met# 500- GCT TTG ACA AAC TTG ACT TTT GGA GAT GTA GC - #C AAC AAG GCT ACG CTA1590Ala Leu Thr Asn Leu Thr Phe Gly Asp Val Al - #a Asn Lys Ala Thr Leu# 515- TGC TCT ATG AAA GGC TGC ATG AGA GCA CTT GT - #G GCC CAA CTA AAA TCT1638Cys Ser Met Lys Gly Cys Met Arg Ala Leu Va - #l Ala Gln Leu Lys Ser520 5 - #25 5 - #30 5 -#35- GAA AGT GAA GAC TTA CAG CAG GTT ATT GCA AG - #T GTT TTG AGG AAT TTG1686Glu Ser Glu Asp Leu Gln Gln Val Ile Ala Se - #r Val Leu Arg Asn Leu# 550- TCT TGG CGA GCA GAT GTA AAT AGT AAA AAG AC - #G TTG CGA GAA GTT GGA1734Ser Trp Arg Ala Asp Val Asn Ser Lys Lys Th - #r Leu Arg Glu Val Gly# 565- AGT GTG AAA GCA TTG ATG GAA TGT GCT TTA GA - #A GTT AAA AAG GAA TCA1782Ser Val Lys Ala Leu Met Glu Cys Ala Leu Gl - #u Val Lys Lys Glu Ser# 580- ACC CTC AAA AGC GTA TTG AGT GCC TTA TGG AA - #T TTG TCA GCA CAT TGC1830Thr Leu Lys Ser Val Leu Ser Ala Leu Trp As - #n Leu Ser Ala His Cys# 595- ACT GAG AAT AAA GCT GAT ATA TGT GCT GTA GA - #T GGT GCA CTT GCA TTT1878Thr Glu Asn Lys Ala Asp Ile Cys Ala Val As - #p Gly Ala Leu Ala Phe600 6 - #05 6 - #10 6 -#15- TTG GTT GGC ACT CTT ACT TAC CGG AGC CAG AC - #A AAC ACT TTA GCC ATT1926Leu Val Gly Thr Leu Thr Tyr Arg Ser Gln Th - #r Asn Thr Leu Ala Ile# 630- ATT GAA AGT GGA GGT GGG ATA TTA CGG AAT GT - #G TCC AGC TTG ATA GCT1974Ile Glu Ser Gly Gly Gly Ile Leu Arg Asn Va - #l Ser Ser Leu Ile Ala# 645- ACA AAT GAG GAC CAC AGG CAA ATC CTA AGA GA - #G AAC AAC TGT CTA CAA2022Thr Asn Glu Asp His Arg Gln Ile Leu Arg Gl - #u Asn Asn Cys Leu Gln# 660- ACT TTA TTA CAA CAC TTA AAA TCT CAT AGT TT - #G ACA ATA GTC AGT AAT2070Thr Leu Leu Gln His Leu Lys Ser His Ser Le - #u Thr Ile Val Ser Asn# 675- GCA TGT GGA ACT TTG TGG AAT CTC TCA GCA AG - #A AAT CCT AAA GAC CAG2118Ala Cys Gly Thr Leu Trp Asn Leu Ser Ala Ar - #g Asn Pro Lys Asp Gln680 6 - #85 6 - #90 6 -#95- GAA GCA TTA TGG GAC ATG GGG GCA GTT AGC AT - #G CTC AAG AAC CTC ATT2166Glu Ala Leu Trp Asp Met Gly Ala Val Ser Me - #t Leu Lys Asn Leu Ile# 710- CAT TCA AAG CAC AAA ATG ATT GCT ATG GGA AG - #T GCT GCA GCT TTA AGG2214His Ser Lys His Lys Met Ile Ala Met Gly Se - #r Ala Ala Ala Leu Arg# 725- AAT CTC ATG GCA AAT AGG CCT GCG AAG TAC AA - #G GAT GCC AAT ATT ATG2262Asn Leu Met Ala Asn Arg Pro Ala Lys Tyr Ly - #s Asp Ala Asn Ile Met# 740- TCT CCT GGC TCA AGC TTG CCA TCT CTT CAT GT - #T AGG AAA CAA AAA GCC2310Ser Pro Gly Ser Ser Leu Pro Ser Leu His Va - #l Arg Lys Gln Lys Ala# 755- CTA GAA GCA GAA TTA GAT GCT CAG CAC TTA TC - #A GAA ACT TTT GAC AAT2358Leu Glu Ala Glu Leu Asp Ala Gln His Leu Se - #r Glu Thr Phe Asp Asn760 7 - #65 7 - #70 7 -#75- ATA GAC AAT TTA AGT CCC AAG GCA TCT CAT CG - #T AGT AAG CAG AGA CAC2406Ile Asp Asn Leu Ser Pro Lys Ala Ser His Ar - #g Ser Lys Gln Arg His# 790- AAG CAA AGT CTC TAT GGT GAT TAT GTT TTT GA - #C ACC AAT CGA CAT GAT2454Lys Gln Ser Leu Tyr Gly Asp Tyr Val Phe As - #p Thr Asn Arg His Asp# 805- GAT AAT AGG TCA GAC AAT TTT AAT ACT GGC AA - #C ATG ACT GTC CTT TCA2502Asp Asn Arg Ser Asp Asn Phe Asn Thr Gly As - #n Met Thr Val Leu Ser# 820- CCA TAT TTG AAT ACT ACA GTG TTA CCC AGC TC - #C TCT TCA TCA AGA GGA2550Pro Tyr Leu Asn Thr Thr Val Leu Pro Ser Se - #r Ser Ser Ser Arg Gly# 835- AGC TTA GAT AGT TCT CGT TCT GAA AAA GAT AG - #A AGT TTG GAG AGA GAA2598Ser Leu Asp Ser Ser Arg Ser Glu Lys Asp Ar - #g Ser Leu Glu Arg Glu840 8 - #45 8 - #50 8 -#55- CGC GGA ATT GGT CTA GGC AAC TAC CAT CCA GC - #A ACA GAA AAT CCA GGA2646Arg Gly Ile Gly Leu Gly Asn Tyr His Pro Al - #a Thr Glu Asn Pro Gly# 870- ACT TCT TCA AAG CGA GGT TTG CAG ATC TCC AC - #C ACT GCA GCC CAG ATT2694Thr Ser Ser Lys Arg Gly Leu Gln Ile Ser Th - #r Thr Ala Ala Gln Ile# 885- GCC AAA GTC ATG GAA GAA GTG TCA GCC ATT CA - #T ACC TCT CAG GAA GAC2742Ala Lys Val Met Glu Glu Val Ser Ala Ile Hi - #s Thr Ser Gln Glu Asp# 900- AGA AGT TCT GGG TCT ACC ACT GAA TTA CAT TG - #T GTG ACA GAT GAG AGA2790Arg Ser Ser Gly Ser Thr Thr Glu Leu His Cy - #s Val Thr Asp Glu Arg# 915- AAT GCA CTT AGA AGA AGC TCT GCT GCC CAT AC - #A CAT TCA AAC ACT TAC2838Asn Ala Leu Arg Arg Ser Ser Ala Ala His Th - #r His Ser Asn Thr Tyr920 9 - #25 9 - #30 9 -#35- AAT TTC ACT AAG TCG GAA AAT TCA AAT AGG AC - #A TGT TCT ATG CCT TAT2886Asn Phe Thr Lys Ser Glu Asn Ser Asn Arg Th - #r Cys Ser Met Pro Tyr# 950- GCC AAA TTA GAA TAC AAG AGA TCT TCA AAT GA - #T AGT TTA AAT AGT GTC2934Ala Lys Leu Glu Tyr Lys Arg Ser Ser Asn As - #p Ser Leu Asn Ser Val# 965- AGT AGT AAT GAT GGT TAT GGT AAA AGA GGT CA - #A ATG AAA CCC TCG ATT2982Ser Ser Asn Asp Gly Tyr Gly Lys Arg Gly Gl - #n Met Lys Pro Ser Ile# 980- GAA TCC TAT TCT GAA GAT GAT GAA AGT AAG TT - #T TGC AGT TAT GGT CAA3030Glu Ser Tyr Ser Glu Asp Asp Glu Ser Lys Ph - #e Cys Ser Tyr Gly Gln# 995- TAC CCA GCC GAC CTA GCC CAT AAA ATA CAT AG - #T GCA AAT CAT ATG GAT3078Tyr Pro Ala Asp Leu Ala His Lys Ile His Se - #r Ala Asn His Met Asp# 10151005 - # 1010- GAT AAT GAT GGA GAA CTA GAT ACA CCA ATA AA - #T TAT AGT CTT AAA TAT3126Asp Asn Asp Gly Glu Leu Asp Thr Pro Ile As - #n Tyr Ser Leu Lys Tyr# 10305- TCA GAT GAG CAG TTG AAC TCT GGA AGG CAA AG - #T CCT TCA CAG AAT GAA3174Ser Asp Glu Gln Leu Asn Ser Gly Arg Gln Se - #r Pro Ser Gln Asn Glu# 10450- AGA TGG GCA AGA CCC AAA CAC ATA ATA GAA GA - #T GAA ATA AAA CAA AGT3222Arg Trp Ala Arg Pro Lys His Ile Ile Glu As - #p Glu Ile Lys Gln Ser# 10605- GAG CAA AGA CAA TCA AGG AAT CAA AGT ACA AC - #T TAT CCT GTT TAT ACT3270Glu Gln Arg Gln Ser Arg Asn Gln Ser Thr Th - #r Tyr Pro Val Tyr Thr# 10750- GAG AGC ACT GAT GAT AAA CAC CTC AAG TTC CA - #A CCA CAT TTT GGA CAG3318Glu Ser Thr Asp Asp Lys His Leu Lys Phe Gl - #n Pro His Phe Gly Gln# 10951085 - # 1090- CAG GAA TGT GTT TCT CCA TAC AGG TCA CGG GG - #A GCC AAT GGT TCA GAA3366Gln Glu Cys Val Ser Pro Tyr Arg Ser Arg Gl - #y Ala Asn Gly Ser Glu# 11105- ACA AAT CGA GTG GGT TCT AAT CAT GGA ATT AA - #T CAA AAT GTA AGC CAG3414Thr Asn Arg Val Gly Ser Asn His Gly Ile As - #n Gln Asn Val Ser Gln# 11250- TCT TTG TGT CAA GAA GAT GAC TAT GAA GAT GA - #T AAG CCT ACC AAT TAT3462Ser Leu Cys Gln Glu Asp Asp Tyr Glu Asp As - #p Lys Pro Thr Asn Tyr# 11405- AGT GAA CGT TAC TCT GAA GAA GAA CAG CAT GA - #A GAA GAA GAG AGA CCA3510Ser Glu Arg Tyr Ser Glu Glu Glu Gln His Gl - #u Glu Glu Glu Arg Pro# 11550- ACA AAT TAT AGC ATA AAA TAT AAT GAA GAG AA - #A CGT CAT GTG GAT CAG3558Thr Asn Tyr Ser Ile Lys Tyr Asn Glu Glu Ly - #s Arg His Val Asp Gln# 11751165 - # 1170- CCT ATT GAT TAT AGT TTA AAA TAT GCC ACA GA - #T ATT CCT TCA TCA CAG3606Pro Ile Asp Tyr Ser Leu Lys Tyr Ala Thr As - #p Ile Pro Ser Ser Gln# 11905- AAA CAG TCA TTT TCA TTC TCA AAG AGT TCA TC - #T GGA CAA AGC AGT AAA3654Lys Gln Ser Phe Ser Phe Ser Lys Ser Ser Se - #r Gly Gln Ser Ser Lys# 12050- ACC GAA CAT ATG TCT TCA AGC AGT GAG AAT AC - #G TCC ACA CCT TCA TCT3702Thr Glu His Met Ser Ser Ser Ser Glu Asn Th - #r Ser Thr Pro Ser Ser# 12205- AAT GCC AAG AGG CAG AAT CAG CTC CAT CCA AG - #T TCT GCA CAG AGT AGA3750Asn Ala Lys Arg Gln Asn Gln Leu His Pro Se - #r Ser Ala Gln Ser Arg# 12350- AGT GGT CAG CCT CAA AAG GCT GCC ACT TGC AA - #A GTT TCT TCT ATT AAC3798Ser Gly Gln Pro Gln Lys Ala Ala Thr Cys Ly - #s Val Ser Ser Ile Asn# 12551245 - # 1250- CAA GAA ACA ATA CAG ACT TAT TGT GTA GAA GA - #T ACT CCA ATA TGT TTT3846Gln Glu Thr Ile Gln Thr Tyr Cys Val Glu As - #p Thr Pro Ile Cys Phe# 12705- TCA AGA TGT AGT TCA TTA TCA TCT TTG TCA TC - #A GCT GAA GAT GAA ATA3894Ser Arg Cys Ser Ser Leu Ser Ser Leu Ser Se - #r Ala Glu Asp Glu Ile# 12850- GGA TGT AAT CAG ACG ACA CAG GAA GCA GAT TC - #T GCT AAT ACC CTG CAA3942Gly Cys Asn Gln Thr Thr Gln Glu Ala Asp Se - #r Ala Asn Thr Leu Gln# 13005- ATA GCA GAA ATA AAA GGA AAG ATT GGA ACT AG - #G TCA GCT GAA GAT CCT3990Ile Ala Glu Ile Lys Gly Lys Ile Gly Thr Ar - #g Ser Ala Glu Asp Pro# 13150- GTG AGC GAA GTT CCA GCA GTG TCA CAG CAC CC - #T AGA ACC AAA TCC AGC4038Val Ser Glu Val Pro Ala Val Ser Gln His Pr - #o Arg Thr Lys Ser Ser# 13351325 - # 1330- AGA CTG CAG GGT TCT AGT TTA TCT TCA GAA TC - #A GCC AGG CAC AAA GCT4086Arg Leu Gln Gly Ser Ser Leu Ser Ser Glu Se - #r Ala Arg His Lys Ala# 13505- GTT GAA TTT CCT TCA GGA GCG AAA TCT CCC TC - #C AAA AGT GGT GCT CAG4134Val Glu Phe Pro Ser Gly Ala Lys Ser Pro Se - #r Lys Ser Gly Ala Gln# 13650- ACA CCC AAA AGT CCA CCT GAA CAC TAT GTT CA - #G GAG ACC CCA CTC ATG4182Thr Pro Lys Ser Pro Pro Glu His Tyr Val Gl - #n Glu Thr Pro Leu Met# 13805- TTT AGC AGA TGT ACT TCT GTC AGT TCA CTT GA - #T AGT TTT GAG AGT CGT4230Phe Ser Arg Cys Thr Ser Val Ser Ser Leu As - #p Ser Phe Glu Ser Arg# 13950- TCG ATT GCC AGC TCC GTT CAG AGT GAA CCA TG - #C AGT GGA ATG GTA AGT4278Ser Ile Ala Ser Ser Val Gln Ser Glu Pro Cy - #s Ser Gly Met Val Ser# 14151405 - # 1410- GGC ATT ATA AGC CCC AGT GAT CTT CCA GAT AG - #C CCT GGA CAA ACC ATG4326Gly Ile Ile Ser Pro Ser Asp Leu Pro Asp Se - #r Pro Gly Gln Thr Met# 14305- CCA CCA AGC AGA AGT AAA ACA CCT CCA CCA CC - #T CCT CAA ACA GCT CAA4374Pro Pro Ser Arg Ser Lys Thr Pro Pro Pro Pr - #o Pro Gln Thr Ala Gln# 14450- ACC AAG CGA GAA GTA CCT AAA AAT AAA GCA CC - #T ACT GCT GAA AAG AGA4422Thr Lys Arg Glu Val Pro Lys Asn Lys Ala Pr - #o Thr Ala Glu Lys Arg# 14605- GAG AGT GGA CCT AAG CAA GCT GCA GTA AAT GC - #T GCA GTT CAG AGG GTC4470Glu Ser Gly Pro Lys Gln Ala Ala Val Asn Al - #a Ala Val Gln Arg Val# 14750- CAG GTT CTT CCA GAT GCT GAT ACT TTA TTA CA - #T TTT GCC ACA GAA AGT4518Gln Val Leu Pro Asp Ala Asp Thr Leu Leu Hi - #s Phe Ala Thr Glu Ser# 14951485 - # 1490- ACT CCA GAT GGA TTT TCT TGT TCA TCC AGC CT - #G AGT GCT CTG AGC CTC4566Thr Pro Asp Gly Phe Ser Cys Ser Ser Ser Le - #u Ser Ala Leu Ser Leu# 15105- GAT GAG CCA TTT ATA CAG AAA GAT GTG GAA TT - #A AGA ATA ATG CCT CCA4614Asp Glu Pro Phe Ile Gln Lys Asp Val Glu Le - #u Arg Ile Met Pro Pro# 15250- GTT CAG GAA AAT GAC AAT GGG AAT GAA ACA GA - #A TCA GAG CAG CCT AAA4662Val Gln Glu Asn Asp Asn Gly Asn Glu Thr Gl - #u Ser Glu Gln Pro Lys# 15405- GAA TCA AAT GAA AAC CAA GAG AAA GAG GCA GA - #A AAA ACT ATT GAT TCT4710Glu Ser Asn Glu Asn Gln Glu Lys Glu Ala Gl - #u Lys Thr Ile Asp Ser# 15550- GAA AAG GAC CTA TTA GAT GAT TCA GAT GAT GA - #T GAT ATT GAA ATA CTA4758Glu Lys Asp Leu Leu Asp Asp Ser Asp Asp As - #p Asp Ile Glu Ile Leu# 15751565 - # 1570- GAA GAA TGT ATT ATT TCT GCC ATG CCA ACA AA - #G TCA TCA CGT AAA GGC4806Glu Glu Cys Ile Ile Ser Ala Met Pro Thr Ly - #s Ser Ser Arg Lys Gly# 15905- AAA AAG CCA GCC CAG ACT GCT TCA AAA TTA CC - #T CCA CCT GTG GCA AGG4854Lys Lys Pro Ala Gln Thr Ala Ser Lys Leu Pr - #o Pro Pro Val Ala Arg# 16050- AAA CCA AGT CAG CTG CCT GTG TAC AAA CTT CT - #A CCA TCA CAA AAC AGG4902Lys Pro Ser Gln Leu Pro Val Tyr Lys Leu Le - #u Pro Ser Gln Asn Arg# 16205- TTG CAA CCC CAA AAG CAT GTT AGT TTT ACA CC - #G GGG GAT GAT ATG CCA4950Leu Gln Pro Gln Lys His Val Ser Phe Thr Pr - #o Gly Asp Asp Met Pro# 16350- CGG GTG TAT TGT GTT GAA GGG ACA CCT ATA AA - #C TTT TCC ACA GCT ACA4998Arg Val Tyr Cys Val Glu Gly Thr Pro Ile As - #n Phe Ser Thr Ala Thr# 16551645 - # 1650- TCT CTA AGT GAT CTA ACA ATC GAA TCC CCT CC - #A AAT GAG TTA GCT GCT5046Ser Leu Ser Asp Leu Thr Ile Glu Ser Pro Pr - #o Asn Glu Leu Ala Ala# 16705- GGA GAA GGA GTT AGA GGA GGA GCA CAG TCA GG - #T GAA TTT GAA AAA CGA5094Gly Glu Gly Val Arg Gly Gly Ala Gln Ser Gl - #y Glu Phe Glu Lys Arg# 16850- GAT ACC ATT CCT ACA GAA GGC AGA AGT ACA GA - #T GAG GCT CAA GGA GGA5142Asp Thr Ile Pro Thr Glu Gly Arg Ser Thr As - #p Glu Ala Gln Gly Gly# 17005- AAA ACC TCA TCT GTA ACC ATA CCT GAA TTG GA - #T GAC AAT AAA GCA GAG5190Lys Thr Ser Ser Val Thr Ile Pro Glu Leu As - #p Asp Asn Lys Ala Glu# 17150- GAA GGT GAT ATT CTT GCA GAA TGC ATT AAT TC - #T GCT ATG CCC AAA GGG5238Glu Gly Asp Ile Leu Ala Glu Cys Ile Asn Se - #r Ala Met Pro Lys Gly# 17351725 - # 1730- AAA AGT CAC AAG CCT TTC CGT GTG AAA AAG AT - #A ATG GAC CAG GTC CAG5286Lys Ser His Lys Pro Phe Arg Val Lys Lys Il - #e Met Asp Gln Val Gln# 17505- CAA GCA TCT GCG TCG TCT TCT GCA CCC AAC AA - #A AAT CAG TTA GAT GGT5334Gln Ala Ser Ala Ser Ser Ser Ala Pro Asn Ly - #s Asn Gln Leu Asp Gly# 17650- AAG AAA AAG AAA CCA ACT TCA CCA GTA AAA CC - #T ATA CCA CAA AAT ACT5382Lys Lys Lys Lys Pro Thr Ser Pro Val Lys Pr - #o Ile Pro Gln Asn Thr# 17805- GAA TAT AGG ACA CGT GTA AGA AAA AAT GCA GA - #C TCA AAA AAT AAT TTA5430Glu Tyr Arg Thr Arg Val Arg Lys Asn Ala As - #p Ser Lys Asn Asn Leu# 17950- AAT GCT GAG AGA GTT TTC TCA GAC AAC AAA GA - #T TCA AAG AAA CAG AAT5478Asn Ala Glu Arg Val Phe Ser Asp Asn Lys As - #p Ser Lys Lys Gln Asn# 18151805 - # 1810- TTG AAA AAT AAT TCC AAG GAC TTC AAT GAT AA - #G CTC CCA AAT AAT GAA5526Leu Lys Asn Asn Ser Lys Asp Phe Asn Asp Ly - #s Leu Pro Asn Asn Glu# 18305- GAT AGA GTC AGA GGA AGT TTT GCT TTT GAT TC - #A CCT CAT CAT TAC ACG5574Asp Arg Val Arg Gly Ser Phe Ala Phe Asp Se - #r Pro His His Tyr Thr# 18450- CCT ATT GAA GGA ACT CCT TAC TGT TTT TCA CG - #A AAT GAT TCT TTG AGT5622Pro Ile Glu Gly Thr Pro Tyr Cys Phe Ser Ar - #g Asn Asp Ser Leu Ser# 18605- TCT CTA GAT TTT GAT GAT GAT GAT GTT GAC CT - #T TCC AGG GAA AAG GCT5670Ser Leu Asp Phe Asp Asp Asp Asp Val Asp Le - #u Ser Arg Glu Lys Ala# 18750- GAA TTA AGA AAG GCA AAA GAA AAT AAG GAA TC - #A GAG GCT AAA GTT ACC5718Glu Leu Arg Lys Ala Lys Glu Asn Lys Glu Se - #r Glu Ala Lys Val Thr# 18951885 - # 1890- AGC CAC ACA GAA CTA ACC TCC AAC CAA CAA TC - #A GCT AAT AAG ACA CAA5766Ser His Thr Glu Leu Thr Ser Asn Gln Gln Se - #r Ala Asn Lys Thr Gln# 19105- GCT ATT GCA AAG CAG CCA ATA AAT CGA GGT CA - #G CCT AAA CCC ATA CTT5814Ala Ile Ala Lys Gln Pro Ile Asn Arg Gly Gl - #n Pro Lys Pro Ile Leu# 19250- CAG AAA CAA TCC ACT TTT CCC CAG TCA TCC AA - #A GAC ATA CCA GAC AGA5862Gln Lys Gln Ser Thr Phe Pro Gln Ser Ser Ly - #s Asp Ile Pro Asp Arg# 19405- GGG GCA GCA ACT GAT GAA AAG TTA CAG AAT TT - #T GCT ATT GAA AAT ACT5910Gly Ala Ala Thr Asp Glu Lys Leu Gln Asn Ph - #e Ala Ile Glu Asn Thr# 19550- CCA GTT TGC TTT TCT CAT AAT TCC TCT CTG AG - #T TCT CTC AGT GAC ATT5958Pro Val Cys Phe Ser His Asn Ser Ser Leu Se - #r Ser Leu Ser Asp Ile# 19751965 - # 1970- GAC CAA GAA AAC AAC AAT AAA GAA AAT GAA CC - #T ATC AAA GAG ACT GAG6006Asp Gln Glu Asn Asn Asn Lys Glu Asn Glu Pr - #o Ile Lys Glu Thr Glu# 19905- CCC CCT GAC TCA CAG GGA GAA CCA AGT AAA CC - #T CAA GCA TCA GGC TAT6054Pro Pro Asp Ser Gln Gly Glu Pro Ser Lys Pr - #o Gln Ala Ser Gly Tyr# 20050- GCT CCT AAA TCA TTT CAT GTT GAA GAT ACC CC - #A GTT TGT TTC TCA AGA6102Ala Pro Lys Ser Phe His Val Glu Asp Thr Pr - #o Val Cys Phe Ser Arg# 20205- AAC AGT TCT CTC AGT TCT CTT AGT ATT GAC TC - #T GAA GAT GAC CTG TTG6150Asn Ser Ser Leu Ser Ser Leu Ser Ile Asp Se - #r Glu Asp Asp Leu Leu# 20350- CAG GAA TGT ATA AGC TCC GCA ATG CCA AAA AA - #G AAA AAG CCT TCA AGA6198Gln Glu Cys Ile Ser Ser Ala Met Pro Lys Ly - #s Lys Lys Pro Ser Arg# 20552045 - # 2050- CTC AAG GGT GAT AAT GAA AAA CAT AGT CCC AG - #A AAT ATG GGT GGC ATA6246Leu Lys Gly Asp Asn Glu Lys His Ser Pro Ar - #g Asn Met Gly Gly Ile# 20705- TTA GGT GAA GAT CTG ACA CTT GAT TTG AAA GA - #T ATA CAG AGA CCA GAT6294Leu Gly Glu Asp Leu Thr Leu Asp Leu Lys As - #p Ile Gln Arg Pro Asp# 20850- TCA GAA CAT GGT CTA TCC CCT GAT TCA GAA AA - #T TTT GAT TGG AAA GCT6342Ser Glu His Gly Leu Ser Pro Asp Ser Glu As - #n Phe Asp Trp Lys Ala# 21005- ATT CAG GAA GGT GCA AAT TCC ATA GTA AGT AG - #T TTA CAT CAA GCT GCT6390Ile Gln Glu Gly Ala Asn Ser Ile Val Ser Se - #r Leu His Gln Ala Ala# 21150- GCT GCT GCA TGT TTA TCT AGA CAA GCT TCG TC - #T GAT TCA GAT TCC ATC6438Ala Ala Ala Cys Leu Ser Arg Gln Ala Ser Se - #r Asp Ser Asp Ser Ile# 21352125 - # 2130- CTT TCC CTG AAA TCA GGA ATC TCT CTG GGA TC - #A CCA TTT CAT CTT ACA6486Leu Ser Leu Lys Ser Gly Ile Ser Leu Gly Se - #r Pro Phe His Leu Thr# 21505- CCT GAT CAA GAA GAA AAA CCC TTT ACA AGT AA - #T AAA GGC CCA CGA ATT6534Pro Asp Gln Glu Glu Lys Pro Phe Thr Ser As - #n Lys Gly Pro Arg Ile# 21650- CTA AAA CCA GGG GAG AAA AGT ACA TTG GAA AC - #T AAA AAG ATA GAA TCT6582Leu Lys Pro Gly Glu Lys Ser Thr Leu Glu Th - #r Lys Lys Ile Glu Ser# 21805- GAA AGT AAA GGA ATC AAA GGA GGA AAA AAA GT - #T TAT AAA AGT TTG ATT6630Glu Ser Lys Gly Ile Lys Gly Gly Lys Lys Va - #l Tyr Lys Ser Leu Ile# 21950- ACT GGA AAA GTT CGA TCT AAT TCA GAA ATT TC - #A GGC CAA ATG AAA CAG6678Thr Gly Lys Val Arg Ser Asn Ser Glu Ile Se - #r Gly Gln Met Lys Gln# 22152205 - # 2210- CCC CTT CAA GCA AAC ATG CCT TCA ATC TCT CG - #A GGC AGG ACA ATG ATT6726Pro Leu Gln Ala Asn Met Pro Ser Ile Ser Ar - #g Gly Arg Thr Met Ile# 22305- CAT ATT CCA GGA GTT CGA AAT AGC TCC TCA AG - #T ACA AGT CCT GTT TCT6774His Ile Pro Gly Val Arg Asn Ser Ser Ser Se - #r Thr Ser Pro Val Ser# 22450- AAA AAA GGC CCA CCC CTT AAG ACT CCA GCC TC - #C AAA AGC CCT AGT GAA6822Lys Lys Gly Pro Pro Leu Lys Thr Pro Ala Se - #r Lys Ser Pro Ser Glu# 22605- GGT CAA ACA GCC ACC ACT TCT CCT AGA GGA GC - #C AAG CCA TCT GTG AAA6870Gly Gln Thr Ala Thr Thr Ser Pro Arg Gly Al - #a Lys Pro Ser Val Lys# 22750- TCA GAA TTA AGC CCT GTT GCC AGG CAG ACA TC - #C CAA ATA GGT GGG TCA6918Ser Glu Leu Ser Pro Val Ala Arg Gln Thr Se - #r Gln Ile Gly Gly Ser# 22952285 - # 2290- AGT AAA GCA CCT TCT AGA TCA GGA TCT AGA GA - #T TCG ACC CCT TCA AGA6966Ser Lys Ala Pro Ser Arg Ser Gly Ser Arg As - #p Ser Thr Pro Ser Arg# 23105- CCT GCC CAG CAA CCA TTA AGT AGA CCT ATA CA - #G TCT CCT GGC CGA AAC7014Pro Ala Gln Gln Pro Leu Ser Arg Pro Ile Gl - #n Ser Pro Gly Arg Asn# 23250- TCA ATT TCC CCT GGT AGA AAT GGA ATA AGT CC - #T CCT AAC AAA TTA TCT7062Ser Ile Ser Pro Gly Arg Asn Gly Ile Ser Pr - #o Pro Asn Lys Leu Ser# 23405- CAA CTT CCA AGG ACA TCA TCC CCT AGT ACT GC - #T TCA ACT AAG TCC TCA7110Gln Leu Pro Arg Thr Ser Ser Pro Ser Thr Al - #a Ser Thr Lys Ser Ser# 23550- GGT TCT GGA AAA ATG TCA TAT ACA TCT CCA GG - #T AGA CAG ATG AGC CAA7158Gly Ser Gly Lys Met Ser Tyr Thr Ser Pro Gl - #y Arg Gln Met Ser Gln# 23752365 - # 2370- CAG AAC CTT ACC AAA CAA ACA GGT TTA TCC AA - #G AAT GCC AGT AGT ATT7206Gln Asn Leu Thr Lys Gln Thr Gly Leu Ser Ly - #s Asn Ala Ser Ser Ile# 23905- CCA AGA AGT GAG TCT GCC TCC AAA GGA CTA AA - #T CAG ATG AAT AAT GGT7254Pro Arg Ser Glu Ser Ala Ser Lys Gly Leu As - #n Gln Met Asn Asn Gly# 24050- AAT GGA GCC AAT AAA AAG GTA GAA CTT TCT AG - #A ATG TCT TCA ACT AAA7302Asn Gly Ala Asn Lys Lys Val Glu Leu Ser Ar - #g Met Ser Ser Thr Lys# 24205- TCA AGT GGA AGT GAA TCT GAT AGA TCA GAA AG - #A CCT GTA TTA GTA CGC7350Ser Ser Gly Ser Glu Ser Asp Arg Ser Glu Ar - #g Pro Val Leu Val Arg# 24350- CAG TCA ACT TTC ATC AAA GAA GCT CCA AGC CC - #A ACC TTA AGA AGA AAA7398Gln Ser Thr Phe Ile Lys Glu Ala Pro Ser Pr - #o Thr Leu Arg Arg Lys# 24552445 - # 2450- TTG GAG GAA TCT GCT TCA TTT GAA TCT CTT TC - #T CCA TCA TCT AGA CCA7446Leu Glu Glu Ser Ala Ser Phe Glu Ser Leu Se - #r Pro Ser Ser Arg Pro# 24705- GCT TCT CCC ACT AGG TCC CAG GCA CAA ACT CC - #A GTT TTA AGT CCT TCC7494Ala Ser Pro Thr Arg Ser Gln Ala Gln Thr Pr - #o Val Leu Ser Pro Ser# 24850- CTT CCT GAT ATG TCT CTA TCC ACA CAT TCG TC - #T GTT CAG GCT GGT GGA7542Leu Pro Asp Met Ser Leu Ser Thr His Ser Se - #r Val Gln Ala Gly Gly# 25005- TGG CGA AAA CTC CCA CCT AAT CTC AGT CCC AC - #T ATA GAG TAT AAT GAT7590Trp Arg Lys Leu Pro Pro Asn Leu Ser Pro Th - #r Ile Glu Tyr Asn Asp# 25150- GGA AGA CCA GCA AAG CGC CAT GAT ATT GCA CG - #G TCT CAT TCT GAA AGT7638Gly Arg Pro Ala Lys Arg His Asp Ile Ala Ar - #g Ser His Ser Glu Ser# 25352525 - # 2530- CCT TCT AGA CTT CCA ATC AAT AGG TCA GGA AC - #C TGG AAA CGT GAG CAC7686Pro Ser Arg Leu Pro Ile Asn Arg Ser Gly Th - #r Trp Lys Arg Glu His# 25505- AGC AAA CAT TCA TCA TCC CTT CCT CGA GTA AG - #C ACT TGG AGA AGA ACT7734Ser Lys His Ser Ser Ser Leu Pro Arg Val Se - #r Thr Trp Arg Arg Thr# 25650- GGA AGT TCA TCT TCA ATT CTT TCT GCT TCA TC - #A GAA TCC AGT GAA AAA7782Gly Ser Ser Ser Ser Ile Leu Ser Ala Ser Se - #r Glu Ser Ser Glu Lys# 25805- GCA AAA AGT GAG GAT GAA AAA CAT GTG AAC TC - #T ATT TCA GGA ACC AAA7830Ala Lys Ser Glu Asp Glu Lys His Val Asn Se - #r Ile Ser Gly Thr Lys# 25950- CAA AGT AAA GAA AAC CAA GTA TCC GCA AAA GG - #A ACA TGG AGA AAA ATA7878Gln Ser Lys Glu Asn Gln Val Ser Ala Lys Gl - #y Thr Trp Arg Lys Ile# 26152605 - # 2610- AAA GAA AAT GAA TTT TCT CCC ACA AAT AGT AC - #T TCT CAG ACC GTT TCC7926Lys Glu Asn Glu Phe Ser Pro Thr Asn Ser Th - #r Ser Gln Thr Val Ser# 26305- TCA GGT GCT ACA AAT GGT GCT GAA TCA AAG AC - #T CTA ATT TAT CAA ATG7974Ser Gly Ala Thr Asn Gly Ala Glu Ser Lys Th - #r Leu Ile Tyr Gln Met# 26450- GCA CCT GCT GTT TCT AAA ACA GAG GAT GTT TG - #G GTG AGA ATT GAG GAC8022Ala Pro Ala Val Ser Lys Thr Glu Asp Val Tr - #p Val Arg Ile Glu Asp# 26605- TGT CCC ATT AAC AAT CCT AGA TCT GGA AGA TC - #T CCC ACA GGT AAT ACT8070Cys Pro Ile Asn Asn Pro Arg Ser Gly Arg Se - #r Pro Thr Gly Asn Thr# 26750- CCC CCG GTG ATT GAC AGT GTT TCA GAA AAG GC - #A AAT CCA AAC ATT AAA8118Pro Pro Val Ile Asp Ser Val Ser Glu Lys Al - #a Asn Pro Asn Ile Lys# 26952685 - # 2690- GAT TCA AAA GAT AAT CAG GCA AAA CAA AAT GT - #G GGT AAT GGC AGT GTT8166Asp Ser Lys Asp Asn Gln Ala Lys Gln Asn Va - #l Gly Asn Gly Ser Val# 27105- CCC ATG CGT ACC GTG GGT TTG GAA AAT CGC CT - #G ACC TCC TTT ATT CAG8214Pro Met Arg Thr Val Gly Leu Glu Asn Arg Le - #u Thr Ser Phe Ile Gln# 27250- GTG GAT GCC CCT GAC CAA AAA GGA ACT GAG AT - #A AAA CCA GGA CAA AAT8262Val Asp Ala Pro Asp Gln Lys Gly Thr Glu Il - #e Lys Pro Gly Gln Asn# 27405- AAT CCT GTC CCT GTA TCA GAG ACT AAT GAA AG - #T CCT ATA GTG GAA CGT8310Asn Pro Val Pro Val Ser Glu Thr Asn Glu Se - #r Pro Ile Val Glu Arg# 27550- ACC CCA TTC AGT TCT AGC AGC TCA AGC AAA CA - #C AGT TCA CCT AGT GGG8358Thr Pro Phe Ser Ser Ser Ser Ser Ser Lys Hi - #s Ser Ser Pro Ser Gly# 27752765 - # 2770- ACT GTT GCT GCC AGA GTG ACT CCT TTT AAT TA - #C AAC CCA AGC CCT AGG8406Thr Val Ala Ala Arg Val Thr Pro Phe Asn Ty - #r Asn Pro Ser Pro Arg# 27905- AAA AGC AGC GCA GAT AGC ACT TCA GCT CGG CC - #A TCT CAG ATC CCA ACT8454Lys Ser Ser Ala Asp Ser Thr Ser Ala Arg Pr - #o Ser Gln Ile Pro Thr# 28050- CCA GTG AAT AAC AAC ACA AAG AAG CGA GAT TC - #C AAA ACT GAC AGC ACA8502Pro Val Asn Asn Asn Thr Lys Lys Arg Asp Se - #r Lys Thr Asp Ser Thr# 28205- GAA TCC AGT GGA ACC CAA AGT CCT AAG CGC CA - #T TCT GGG TCT TAC CTT8550Glu Ser Ser Gly Thr Gln Ser Pro Lys Arg Hi - #s Ser Gly Ser Tyr Leu# 28350- GTG ACA TCT GTT TAAAAGAGAG GAAGAATGAA ACTAAGAAAA TT - #CTATGTTA8602Val Thr Ser Val2840- ATTACAACTG CTATATAGAC ATTTTGTTTC AAATGAAACT TTAAAAGACT GA - #AAAATTTT8662- GTAAATAGGT TTGATTCTTG TTAGAGGGTT TTTGTTCTGG AAGCCATATT TG - #ATAGTATA8722- CTTTGTCTTC ACTGGTCTTA TTTTGGGAGG CACTCTTGAT GGTTAGGAAA AA - #ATAGAAAG8782- CCAAGTATGT TTGTACAGTA TGTTTTACAT GTATTTAAAG TAGCATCCCA TC - #CCAACTTC8842- CTTAATTATT GCTTGTCTAA AATAATGAAC ACTACAGATA GGAAATATGA TA - #TATTGCTG8902- TTATCAATCA TTTCTAGATT ATAAACTGAC TAAACTTACA TCAGGGGAAA AT - #TGGTATTT8962- ATGCAAAAAA AAAATGTTTT TGTCCTTGTG AGTCCATCTA ACATCATAAT TA - #ATCATGTG9022- GCTGTGAAAT TCACAGTAAT ATGGTTCCCG ATGAACAAGT TTACCCAGCC TG - #CTTTGCTT9082- ACTGCATGAA TGAAACTGAT GGTTCAATTT CAGAAGTAAT GATTAACAGT TA - #TGTGGTCA9142- CATGATGTGC ATAGAGATAG CTACAGTGTA ATAATTTACA CTATTTTGTG CT - #CCAAACAA9202- AACAAAAATC TGTGTAACTG TAAAACATTG AATGAAACTA TTTTACCTGA AC - #TAGATTTT9262- ATCTGAAAGT AGGTAGAATT TTTGCTATGC TGTAATTTGT TGTATATTCT GG - #TATTTGAG9322- GTGAGATGGC TGCTCTTTAT TAATGAGACA TGAATTGTGT CTCAACAGAA AC - #TAAATGAA9382- CATTTCAGAA TAAATTATTG CTGTATGTAA ACTGTTACTG AAATTGGTAT TT - #GTTTGAAG9442- GGTTTGTTTC ACATTTGTAT TAATTAATTG TTTAAAATGC CTCTTTTAAA AG - #CTTATATA9502- AATTTTTTCT TCAGCTTCTA TGCATTAAGA GTAAAATTCC TCTTACTGTA AT - #AAAAACAT9562# 960 - #6ACCATTCC ATGCGTTGGC ACTT- (2) INFORMATION FOR SEQ ID NO:2:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 2843 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:- Met Ala Ala Ala Ser Tyr Asp Gln Leu Leu Ly - #s Gln Val Glu Ala Leu# 15- Lys Met Glu Asn Ser Asn Leu Arg Gln Glu Le - #u Glu Asp Asn Ser Asn# 30- His Leu Thr Lys Leu Glu Thr Glu Ala Ser As - #n Met Lys Glu Val Leu# 45- Lys Gln Leu Gln Gly Ser Ile Glu Asp Glu Al - #a Met Ala Ser Ser Gly# 60- Gln Ile Asp Leu Leu Glu Arg Leu Lys Glu Le - #u Asn Leu Asp Ser Ser# 80- Asn Phe Pro Gly Val Lys Leu Arg Ser Lys Me - #t Ser Leu Arg Ser Tyr# 95- Gly Ser Arg Glu Gly Ser Val Ser Ser Arg Se - #r Gly Glu Cys Ser Pro# 110- Val Pro Met Gly Ser Phe Pro Arg Arg Gly Ph - #e Val Asn Gly Ser Arg# 125- Glu Ser Thr Gly Tyr Leu Glu Glu Leu Glu Ly - #s Glu Arg Ser Leu Leu# 140- Leu Ala Asp Leu Asp Lys Glu Glu Lys Glu Ly - #s Asp Trp Tyr Tyr Ala145 1 - #50 1 - #55 1 -#60- Gln Leu Gln Asn Leu Thr Lys Arg Ile Asp Se - #r Leu Pro Leu Thr Glu# 175- Asn Phe Ser Leu Gln Thr Asp Leu Thr Arg Ar - #g Gln Leu Glu Tyr Glu# 190- Ala Arg Gln Ile Arg Val Ala Met Glu Glu Gl - #n Leu Gly Thr Cys Gln# 205- Asp Met Glu Lys Arg Ala Gln Arg Arg Ile Al - #a Arg Ile Gln Gln Ile# 220- Glu Lys Asp Ile Leu Arg Ile Arg Gln Leu Le - #u Gln Ser Gln Ala Thr225 2 - #30 2 - #35 2 -#40- Glu Ala Glu Arg Ser Ser Gln Asn Lys His Gl - #u Thr Gly Ser His Asp# 255- Ala Glu Arg Gln Asn Glu Gly Gln Gly Val Gl - #y Glu Ile Asn Met Ala# 270- Thr Ser Gly Asn Gly Gln Gly Ser Thr Thr Ar - #g Met Asp His Glu Thr# 285- Ala Ser Val Leu Ser Ser Ser Ser Thr His Se - #r Ala Pro Arg Arg Leu# 300- Thr Ser His Leu Gly Thr Lys Val Glu Met Va - #l Tyr Ser Leu Leu Ser305 3 - #10 3 - #15 3 -#20- Met Leu Gly Thr His Asp Lys Asp Asp Met Se - #r Arg Thr Leu Leu Ala# 335- Met Ser Ser Ser Gln Asp Ser Cys Ile Ser Me - #t Arg Gln Ser Gly Cys# 350- Leu Pro Leu Leu Ile Gln Leu Leu His Gly As - #n Asp Lys Asp Ser Val# 365- Leu Leu Gly Asn Ser Arg Gly Ser Lys Glu Al - #a Arg Ala Arg Ala Ser# 380- Ala Ala Leu His Asn Ile Ile His Ser Gln Pr - #o Asp Asp Lys Arg Gly385 3 - #90 3 - #95 4 -#00- Arg Arg Glu Ile Arg Val Leu His Leu Leu Gl - #u Gln Ile Arg Ala Tyr# 415- Cys Glu Thr Cys Trp Glu Trp Gln Glu Ala Hi - #s Glu Pro Gly Met Asp# 430- Gln Asp Lys Asn Pro Met Pro Ala Pro Val Gl - #u His Gln Ile Cys Pro# 445- Ala Val Cys Val Leu Met Lys Leu Ser Phe As - #p Glu Glu His Arg His# 460- Ala Met Asn Glu Leu Gly Gly Leu Gln Ala Il - #e Ala Glu Leu Leu Gln465 4 - #70 4 - #75 4 -#80- Val Asp Cys Glu Met Tyr Gly Leu Thr Asn As - #p His Tyr Ser Ile Thr# 495- Leu Arg Arg Tyr Ala Gly Met Ala Leu Thr As - #n Leu Thr Phe Gly Asp# 510- Val Ala Asn Lys Ala Thr Leu Cys Ser Met Ly - #s Gly Cys Met Arg Ala# 525- Leu Val Ala Gln Leu Lys Ser Glu Ser Glu As - #p Leu Gln Gln Val Ile# 540- Ala Ser Val Leu Arg Asn Leu Ser Trp Arg Al - #a Asp Val Asn Ser Lys545 5 - #50 5 - #55 5 -#60- Lys Thr Leu Arg Glu Val Gly Ser Val Lys Al - #a Leu Met Glu Cys Ala# 575- Leu Glu Val Lys Lys Glu Ser Thr Leu Lys Se - #r Val Leu Ser Ala Leu# 590- Trp Asn Leu Ser Ala His Cys Thr Glu Asn Ly - #s Ala Asp Ile Cys Ala# 605- Val Asp Gly Ala Leu Ala Phe Leu Val Gly Th - #r Leu Thr Tyr Arg Ser# 620- Gln Thr Asn Thr Leu Ala Ile Ile Glu Ser Gl - #y Gly Gly Ile Leu Arg625 6 - #30 6 - #35 6 -#40- Asn Val Ser Ser Leu Ile Ala Thr Asn Glu As - #p His Arg Gln Ile Leu# 655- Arg Glu Asn Asn Cys Leu Gln Thr Leu Leu Gl - #n His Leu Lys Ser His# 670- Ser Leu Thr Ile Val Ser Asn Ala Cys Gly Th - #r Leu Trp Asn Leu Ser# 685- Ala Arg Asn Pro Lys Asp Gln Glu Ala Leu Tr - #p Asp Met Gly Ala Val# 700- Ser Met Leu Lys Asn Leu Ile His Ser Lys Hi - #s Lys Met Ile Ala Met705 7 - #10 7 - #15 7 -#20- Gly Ser Ala Ala Ala Leu Arg Asn Leu Met Al - #a Asn Arg Pro Ala Lys# 735- Tyr Lys Asp Ala Asn Ile Met Ser Pro Gly Se - #r Ser Leu Pro Ser Leu# 750- His Val Arg Lys Gln Lys Ala Leu Glu Ala Gl - #u Leu Asp Ala Gln His# 765- Leu Ser Glu Thr Phe Asp Asn Ile Asp Asn Le - #u Ser Pro Lys Ala Ser# 780- His Arg Ser Lys Gln Arg His Lys Gln Ser Le - #u Tyr Gly Asp Tyr Val785 7 - #90 7 - #95 8 -#00- Phe Asp Thr Asn Arg His Asp Asp Asn Arg Se - #r Asp Asn Phe Asn Thr# 815- Gly Asn Met Thr Val Leu Ser Pro Tyr Leu As - #n Thr Thr Val Leu Pro# 830- Ser Ser Ser Ser Ser Arg Gly Ser Leu Asp Se - #r Ser Arg Ser Glu Lys# 845- Asp Arg Ser Leu Glu Arg Glu Arg Gly Ile Gl - #y Leu Gly Asn Tyr His# 860- Pro Ala Thr Glu Asn Pro Gly Thr Ser Ser Ly - #s Arg Gly Leu Gln Ile865 8 - #70 8 - #75 8 -#80- Ser Thr Thr Ala Ala Gln Ile Ala Lys Val Me - #t Glu Glu Val Ser Ala# 895- Ile His Thr Ser Gln Glu Asp Arg Ser Ser Gl - #y Ser Thr Thr Glu Leu# 910- His Cys Val Thr Asp Glu Arg Asn Ala Leu Ar - #g Arg Ser Ser Ala Ala# 925- His Thr His Ser Asn Thr Tyr Asn Phe Thr Ly - #s Ser Glu Asn Ser Asn# 940- Arg Thr Cys Ser Met Pro Tyr Ala Lys Leu Gl - #u Tyr Lys Arg Ser Ser945 9 - #50 9 - #55 9 -#60- Asn Asp Ser Leu Asn Ser Val Ser Ser Asn As - #p Gly Tyr Gly Lys Arg# 975- Gly Gln Met Lys Pro Ser Ile Glu Ser Tyr Se - #r Glu Asp Asp Glu Ser# 990- Lys Phe Cys Ser Tyr Gly Gln Tyr Pro Ala As - #p Leu Ala His Lys Ile# 10050- His Ser Ala Asn His Met Asp Asp Asn Asp Gl - #y Glu Leu Asp Thr Pro# 10205- Ile Asn Tyr Ser Leu Lys Tyr Ser Asp Glu Gl - #n Leu Asn Ser Gly Arg# 10401030 - # 1035- Gln Ser Pro Ser Gln Asn Glu Arg Trp Ala Ar - #g Pro Lys His Ile Ile# 10550- Glu Asp Glu Ile Lys Gln Ser Glu Gln Arg Gl - #n Ser Arg Asn Gln Ser# 10705- Thr Thr Tyr Pro Val Tyr Thr Glu Ser Thr As - #p Asp Lys His Leu Lys# 10850- Phe Gln Pro His Phe Gly Gln Gln Glu Cys Va - #l Ser Pro Tyr Arg Ser# 11005- Arg Gly Ala Asn Gly Ser Glu Thr Asn Arg Va - #l Gly Ser Asn His Gly# 11201110 - # 1115- Ile Asn Gln Asn Val Ser Gln Ser Leu Cys Gl - #n Glu Asp Asp Tyr Glu# 11350- Asp Asp Lys Pro Thr Asn Tyr Ser Glu Arg Ty - #r Ser Glu Glu Glu Gln# 11505- His Glu Glu Glu Glu Arg Pro Thr Asn Tyr Se - #r Ile Lys Tyr Asn Glu# 11650- Glu Lys Arg His Val Asp Gln Pro Ile Asp Ty - #r Ser Leu Lys Tyr Ala# 11805- Thr Asp Ile Pro Ser Ser Gln Lys Gln Ser Ph - #e Ser Phe Ser Lys Ser# 12001190 - # 1195- Ser Ser Gly Gln Ser Ser Lys Thr Glu His Me - #t Ser Ser Ser Ser Glu# 12150- Asn Thr Ser Thr Pro Ser Ser Asn Ala Lys Ar - #g Gln Asn Gln Leu His# 12305- Pro Ser Ser Ala Gln Ser Arg Ser Gly Gln Pr - #o Gln Lys Ala Ala Thr# 12450- Cys Lys Val Ser Ser Ile Asn Gln Glu Thr Il - #e Gln Thr Tyr Cys Val# 12605- Glu Asp Thr Pro Ile Cys Phe Ser Arg Cys Se - #r Ser Leu Ser Ser Leu# 12801270 - # 1275- Ser Ser Ala Glu Asp Glu Ile Gly Cys Asn Gl - #n Thr Thr Gln Glu Ala# 12950- Asp Ser Ala Asn Thr Leu Gln Ile Ala Glu Il - #e Lys Gly Lys Ile Gly# 13105- Thr Arg Ser Ala Glu Asp Pro Val Ser Glu Va - #l Pro Ala Val Ser Gln# 13250- His Pro Arg Thr Lys Ser Ser Arg Leu Gln Gl - #y Ser Ser Leu Ser Ser# 13405- Glu Ser Ala Arg His Lys Ala Val Glu Phe Pr - #o Ser Gly Ala Lys Ser# 13601350 - # 1355- Pro Ser Lys Ser Gly Ala Gln Thr Pro Lys Se - #r Pro Pro Glu His Tyr# 13750- Val Gln Glu Thr Pro Leu Met Phe Ser Arg Cy - #s Thr Ser Val Ser Ser# 13905- Leu Asp Ser Phe Glu Ser Arg Ser Ile Ala Se - #r Ser Val Gln Ser Glu# 14050- Pro Cys Ser Gly Met Val Ser Gly Ile Ile Se - #r Pro Ser Asp Leu Pro# 14205- Asp Ser Pro Gly Gln Thr Met Pro Pro Ser Ar - #g Ser Lys Thr Pro Pro# 14401430 - # 1435- Pro Pro Pro Gln Thr Ala Gln Thr Lys Arg Gl - #u Val Pro Lys Asn Lys# 14550- Ala Pro Thr Ala Glu Lys Arg Glu Ser Gly Pr - #o Lys Gln Ala Ala Val# 14705- Asn Ala Ala Val Gln Arg Val Gln Val Leu Pr - #o Asp Ala Asp Thr Leu# 14850- Leu His Phe Ala Thr Glu Ser Thr Pro Asp Gl - #y Phe Ser Cys Ser Ser# 15005- Ser Leu Ser Ala Leu Ser Leu Asp Glu Pro Ph - #e Ile Gln Lys Asp Val# 15201510 - # 1515- Glu Leu Arg Ile Met Pro Pro Val Gln Glu As - #n Asp Asn Gly Asn Glu# 15350- Thr Glu Ser Glu Gln Pro Lys Glu Ser Asn Gl - #u Asn Gln Glu Lys Glu# 15505- Ala Glu Lys Thr Ile Asp Ser Glu Lys Asp Le - #u Leu Asp Asp Ser Asp# 15650- Asp Asp Asp Ile Glu Ile Leu Glu Glu Cys Il - #e Ile Ser Ala Met Pro# 15805- Thr Lys Ser Ser Arg Lys Gly Lys Lys Pro Al - #a Gln Thr Ala Ser Lys# 16001590 - # 1595- Leu Pro Pro Pro Val Ala Arg Lys Pro Ser Gl - #n Leu Pro Val Tyr Lys# 16150- Leu Leu Pro Ser Gln Asn Arg Leu Gln Pro Gl - #n Lys His Val Ser Phe# 16305- Thr Pro Gly Asp Asp Met Pro Arg Val Tyr Cy - #s Val Glu Gly Thr Pro# 16450- Ile Asn Phe Ser Thr Ala Thr Ser Leu Ser As - #p Leu Thr Ile Glu Ser# 16605- Pro Pro Asn Glu Leu Ala Ala Gly Glu Gly Va - #l Arg Gly Gly Ala Gln# 16801670 - # 1675- Ser Gly Glu Phe Glu Lys Arg Asp Thr Ile Pr - #o Thr Glu Gly Arg Ser# 16950- Thr Asp Glu Ala Gln Gly Gly Lys Thr Ser Se - #r Val Thr Ile Pro Glu# 17105- Leu Asp Asp Asn Lys Ala Glu Glu Gly Asp Il - #e Leu Ala Glu Cys Ile# 17250- Asn Ser Ala Met Pro Lys Gly Lys Ser His Ly - #s Pro Phe Arg Val Lys# 17405- Lys Ile Met Asp Gln Val Gln Gln Ala Ser Al - #a Ser Ser Ser Ala Pro# 17601750 - # 1755- Asn Lys Asn Gln Leu Asp Gly Lys Lys Lys Ly - #s Pro Thr Ser Pro Val# 17750- Lys Pro Ile Pro Gln Asn Thr Glu Tyr Arg Th - #r Arg Val Arg Lys Asn# 17905- Ala Asp Ser Lys Asn Asn Leu Asn Ala Glu Ar - #g Val Phe Ser Asp Asn# 18050- Lys Asp Ser Lys Lys Gln Asn Leu Lys Asn As - #n Ser Lys Asp Phe Asn# 18205- Asp Lys Leu Pro Asn Asn Glu Asp Arg Val Ar - #g Gly Ser Phe Ala Phe# 18401830 - # 1835- Asp Ser Pro His His Tyr Thr Pro Ile Glu Gl - #y Thr Pro Tyr Cys Phe# 18550- Ser Arg Asn Asp Ser Leu Ser Ser Leu Asp Ph - #e Asp Asp Asp Asp Val# 18705- Asp Leu Ser Arg Glu Lys Ala Glu Leu Arg Ly - #s Ala Lys Glu Asn Lys# 18850- Glu Ser Glu Ala Lys Val Thr Ser His Thr Gl - #u Leu Thr Ser Asn Gln# 19005- Gln Ser Ala Asn Lys Thr Gln Ala Ile Ala Ly - #s Gln Pro Ile Asn Arg# 19201910 - # 1915- Gly Gln Pro Lys Pro Ile Leu Gln Lys Gln Se - #r Thr Phe Pro Gln Ser# 19350- Ser Lys Asp Ile Pro Asp Arg Gly Ala Ala Th - #r Asp Glu Lys Leu Gln# 19505- Asn Phe Ala Ile Glu Asn Thr Pro Val Cys Ph - #e Ser His Asn Ser Ser# 19650- Leu Ser Ser Leu Ser Asp Ile Asp Gln Glu As - #n Asn Asn Lys Glu Asn# 19805- Glu Pro Ile Lys Glu Thr Glu Pro Pro Asp Se - #r Gln Gly Glu Pro Ser# 20001990 - # 1995- Lys Pro Gln Ala Ser Gly Tyr Ala Pro Lys Se - #r Phe His Val Glu Asp# 20150- Thr Pro Val Cys Phe Ser Arg Asn Ser Ser Le - #u Ser Ser Leu Ser Ile# 20305- Asp Ser Glu Asp Asp Leu Leu Gln Glu Cys Il - #e Ser Ser Ala Met Pro# 20450- Lys Lys Lys Lys Pro Ser Arg Leu Lys Gly As - #p Asn Glu Lys His Ser# 20605- Pro Arg Asn Met Gly Gly Ile Leu Gly Glu As - #p Leu Thr Leu Asp Leu# 20802070 - # 2075- Lys Asp Ile Gln Arg Pro Asp Ser Glu His Gl - #y Leu Ser Pro Asp Ser# 20950- Glu Asn Phe Asp Trp Lys Ala Ile Gln Glu Gl - #y Ala Asn Ser Ile Val# 21105- Ser Ser Leu His Gln Ala Ala Ala Ala Ala Cy - #s Leu Ser Arg Gln Ala# 21250- Ser Ser Asp Ser Asp Ser Ile Leu Ser Leu Ly - #s Ser Gly Ile Ser Leu# 21405- Gly Ser Pro Phe His Leu Thr Pro Asp Gln Gl - #u Glu Lys Pro Phe Thr# 21602150 - # 2155- Ser Asn Lys Gly Pro Arg Ile Leu Lys Pro Gl - #y Glu Lys Ser Thr Leu# 21750- Glu Thr Lys Lys Ile Glu Ser Glu Ser Lys Gl - #y Ile Lys Gly Gly Lys# 21905- Lys Val Tyr Lys Ser Leu Ile Thr Gly Lys Va - #l Arg Ser Asn Ser Glu# 22050- Ile Ser Gly Gln Met Lys Gln Pro Leu Gln Al - #a Asn Met Pro Ser Ile# 22205- Ser Arg Gly Arg Thr Met Ile His Ile Pro Gl - #y Val Arg Asn Ser Ser# 22402230 - # 2235- Ser Ser Thr Ser Pro Val Ser Lys Lys Gly Pr - #o Pro Leu Lys Thr Pro# 22550- Ala Ser Lys Ser Pro Ser Glu Gly Gln Thr Al - #a Thr Thr Ser Pro Arg# 22705- Gly Ala Lys Pro Ser Val Lys Ser Glu Leu Se - #r Pro Val Ala Arg Gln# 22850- Thr Ser Gln Ile Gly Gly Ser Ser Lys Ala Pr - #o Ser Arg Ser Gly Ser# 23005- Arg Asp Ser Thr Pro Ser Arg Pro Ala Gln Gl - #n Pro Leu Ser Arg Pro# 23202310 - # 2315- Ile Gln Ser Pro Gly Arg Asn Ser Ile Ser Pr - #o Gly Arg Asn Gly Ile# 23350- Ser Pro Pro Asn Lys Leu Ser Gln Leu Pro Ar - #g Thr Ser Ser Pro Ser# 23505- Thr Ala Ser Thr Lys Ser Ser Gly Ser Gly Ly - #s Met Ser Tyr Thr Ser# 23650- Pro Gly Arg Gln Met Ser Gln Gln Asn Leu Th - #r Lys Gln Thr Gly Leu# 23805- Ser Lys Asn Ala Ser Ser Ile Pro Arg Ser Gl - #u Ser Ala Ser Lys Gly# 24002390 - # 2395- Leu Asn Gln Met Asn Asn Gly Asn Gly Ala As - #n Lys Lys Val Glu Leu# 24150- Ser Arg Met Ser Ser Thr Lys Ser Ser Gly Se - #r Glu Ser Asp Arg Ser# 24305- Glu Arg Pro Val Leu Val Arg Gln Ser Thr Ph - #e Ile Lys Glu Ala Pro# 24450- Ser Pro Thr Leu Arg Arg Lys Leu Glu Glu Se - #r Ala Ser Phe Glu Ser# 24605- Leu Ser Pro Ser Ser Arg Pro Ala Ser Pro Th - #r Arg Ser Gln Ala Gln# 24802470 - # 2475- Thr Pro Val Leu Ser Pro Ser Leu Pro Asp Me - #t Ser Leu Ser Thr His# 24950- Ser Ser Val Gln Ala Gly Gly Trp Arg Lys Le - #u Pro Pro Asn Leu Ser# 25105- Pro Thr Ile Glu Tyr Asn Asp Gly Arg Pro Al - #a Lys Arg His Asp Ile# 25250- Ala Arg Ser His Ser Glu Ser Pro Ser Arg Le - #u Pro Ile Asn Arg Ser# 25405- Gly Thr Trp Lys Arg Glu His Ser Lys His Se - #r Ser Ser Leu Pro Arg# 25602550 - # 2555- Val Ser Thr Trp Arg Arg Thr Gly Ser Ser Se - #r Ser Ile Leu Ser Ala# 25750- Ser Ser Glu Ser Ser Glu Lys Ala Lys Ser Gl - #u Asp Glu Lys His Val# 25905- Asn Ser Ile Ser Gly Thr Lys Gln Ser Lys Gl - #u Asn Gln Val Ser Ala# 26050- Lys Gly Thr Trp Arg Lys Ile Lys Glu Asn Gl - #u Phe Ser Pro Thr Asn# 26205- Ser Thr Ser Gln Thr Val Ser Ser Gly Ala Th - #r Asn Gly Ala Glu Ser# 26402630 - # 2635- Lys Thr Leu Ile Tyr Gln Met Ala Pro Ala Va - #l Ser Lys Thr Glu Asp# 26550- Val Trp Val Arg Ile Glu Asp Cys Pro Ile As - #n Asn Pro Arg Ser Gly# 26705- Arg Ser Pro Thr Gly Asn Thr Pro Pro Val Il - #e Asp Ser Val Ser Glu# 26850- Lys Ala Asn Pro Asn Ile Lys Asp Ser Lys As - #p Asn Gln Ala Lys Gln# 27005- Asn Val Gly Asn Gly Ser Val Pro Met Arg Th - #r Val Gly Leu Glu Asn# 27202710 - # 2715- Arg Leu Thr Ser Phe Ile Gln Val Asp Ala Pr - #o Asp Gln Lys Gly Thr# 27350- Glu Ile Lys Pro Gly Gln Asn Asn Pro Val Pr - #o Val Ser Glu Thr Asn# 27505- Glu Ser Pro Ile Val Glu Arg Thr Pro Phe Se - #r Ser Ser Ser Ser Ser# 27650- Lys His Ser Ser Pro Ser Gly Thr Val Ala Al - #a Arg Val Thr Pro Phe# 27805- Asn Tyr Asn Pro Ser Pro Arg Lys Ser Ser Al - #a Asp Ser Thr Ser Ala# 28002790 - # 2795- Arg Pro Ser Gln Ile Pro Thr Pro Val Asn As - #n Asn Thr Lys Lys Arg# 28150- Asp Ser Lys Thr Asp Ser Thr Glu Ser Ser Gl - #y Thr Gln Ser Pro Lys# 28305- Arg His Ser Gly Ser Tyr Leu Val Thr Ser Va - #l# 2840- (2) INFORMATION FOR SEQ ID NO:3:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 18 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (genomic)- (iii) HYPOTHETICAL: NO- (iv) ANTI-SENSE: NO- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:# 18 TG- (2) INFORMATION FOR SEQ ID NO:4:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 19 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (genomic)- (iii) HYPOTHETICAL: NO- (iv) ANTI-SENSE: NO- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:# 19 GGC__________________________________________________________________________

Antibodies and assay for detecting mutant APC proteins

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Date Issued

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Foreign Referenced Citations (1)

Non-Patent Literature Citations (23)

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Entry
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