Abstract:
The present invention includes a DNA clone from the myotonic muscular dystrophy gene, a cosmid probe to the myotonic dystrophy site, as well as methods of detecting myotonic muscular dystrophy using RFLP. The method involves the steps of digesting DNA from an individual to be tested with a restriction endonuclease and detecting the restriction fragment length polymorphism with hybridization to probes within the myotonic muscular locus and southern blot analysis. Alternatively, the myotonic muscular dystrophy gene can be measured by determining the amount of mRNA or measuring the amount of protein with an antibody. Further, the myotonic muscular dystrophy gene defect can be detected using either fluorescence in situ hybridization or pulsed field gel electrophoresis using the probes described herein.

Description:
This application is a continuation of application Ser. No. 08/019,940, filed Feb. 19, 1993, abandoned. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to the field of molecular diagnosis of myotonic muscular dystrophy. 
     BACKGROUND 
     The myotonic muscular dystrophy (DM) disease is the most common adult muscular dystrophy in man with a prevalence of 1 in 10,000. The disorder is inherited in an autosomal dominant manner with variable expression of symptoms from individual to individual within a given family. Furthermore, the phenomenon of anticipation (increasing disease severity over generations) is well documented for DM. This is particularly evident when an affected mother transmits the gene for the disease to her offspring. These offspring have a high incidence of mental retardation and profound infantile myotonia. Adult patients with DM manifest a pleiotropic set of symptoms including myotonia, cardiac arrhythmias, cataracts, frontal baldness, hypogonadism, and other endocrine dysfunctions. There is no evidence that myotonic muscular dystrophy may be caused by defects in more than one gene. 
     A myotonic muscular dystrophy gene has been mapped to human chromosome position 19q13.3. Both a genetic and physical map of the region was developed by a group of investigators acting as a voluntary consortium under sponsorship of the Muscular Dystrophy Association. The genetic linkage studies identified two RFLP alleles, D10 and X75, which are polymerase chain reaction (PCR)-based dinucleotide polymorphisms and are tightly linked to DM. 
     Two disorders, Kennedy disease and fragile X syndrome, are associated with triplet nucleotide amplification. The triplet is CAG in the Kennedy disease gene and CGG in the fragile X syndrome gene. Repeat lengths in Kennedy disease have been reported as 40-57 units, whereas the normal range is 11-31 repeats. In the case of fragile X syndrome, the CGG repeat sequence becomes unstable once greater than 52 units long and is predisposed to amplification during female meiosis. The molecular basis of the Sherman paradox has now been explained for fragile X syndrome. The generation-to-generation progressive amplification of the CGG triplet repeat in fragile X syndrome correlates with increasing disease severity and lack of expression of the FMR-1 gene. 
     The present application provides a new method of sequence scanning for triplet repeats which are GC-rich and thus suspect for genetic instability by amplification/deletion/translocation. This method successfully identified a putative protein kinase gene in patients with DM. This application also provides rapid and simple methods for accurate means of DM diagnosis. The gene, myotonin protein kinase, was discovered by molecular cloning, predicted to be a protein kinase on the basis of sequence motif homology, characterized with regard to its sequence and alternative spliced forms, and found to be altered in expression in tissues of patients with DM. 
     SUMMARY OF THE INVENTION 
     An object of the present invention is a method for cloning human disease genes with GC-rich oligonucleotides screening method. 
     An object of the present invention is a method for diagnosing myotonic muscular dystrophy. 
     A further object of the present invention is a provision of a sequence of the DM gene. 
     Another object of the present invention is a method of detecting the myotonic muscular dystrophy desease by measuring GCT repeats. 
     An additional object of the present invention is a method of detecting the myotonic muscular dystrophy disease by measuring the mRNA or protein from the DM gene. 
     A further object of the present invention is a vector for expression of myotonic protein kinase. 
     Another object of the present invention is the provision of antibodies to myotonic protein kinase. 
     An additional object of the present invention is a diagnostic test for myotonic muscular dystrophy. 
     Thus in accomplishing the foregoing objects there is provided in accordance with one aspect of the present invention as a composition of matter, a 3.2 kb cDNA clone containing the DM gene. A further aspect is a 11613 bp genomic DNA sequence (SEQ ID NO. 10) containing the DM gene. 
     A further embodiment of the present invention is a method of detecting DM comprising the steps of digesting DNA from an individual to be tested with a restriction endonuclease and detecting the restriction fragment length polymorphism by hybridization to probes within the DM locus and southern blot analysis. In a preferred embodiment of the present invention, the probe is pMDY1 and the restriction endonucleases are selected from the group consisting of Nco1, Ban1, and Taq1. 
     Alternate embodiments of the present invention include detecting DM by measuring the expression of the DM gene either as the amount of mRNA expressed or as the amount of DM protein produced. Another embodiment of the present invention includes a method of detecting DM comprising the steps of detecting variation in the (CTG)n repeat at the 3&#39; end of the DM gene by measuring the length of the repeat, wherein n for normal ranges between 5 to 33 and n for DM is greater than 35. A variety of methods are available to detect the dosage measurements of the repeat. These procedures can be selected from the group consisting of visual examination, densitometry measurement, quantitative radioactivity, and quantitative fluorescence as well as pulsed field gel electrophoresis and fluorescence in situ hybridization. 
     Other and further objects, features and advantages will be apparent and eventually more readily understood from a reading of the following specification and by reference to the accompanying drawings forming a part thereof, wherein examples of the present preferred embodiments of the invention are given for the purpose of the disclosure. 
    
    
     DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a sequence of the GCT triplet repeat (upper case) and its flanking regions (lower case). The locations of PCR primers are shown by solid lines with arrows. The complete 1.4 kb sequence (PMDY1) is in SEQ. ID. No. 1. 
     FIG. 2 shows Southern analysis of leukocyte DNA with probes containing the GCT repeat from pMDY1 and demonstrates that sequence expansion is the molecular alteration associated with myotonic dystrophy. 
     FIG. 3 shows Southern analysis of leukocyte DNA with probes containing the GCT repeat from pMDY1 and demonstrates that sequence expansion is the molecular alteration associated with myotonic dystrophy. 
     FIG. 4 shows the polymorphic nature of the GCT locus in normal human genomic DNAs. Amplification of genomic DNA was carried out as described in Example 3 and analyzed on a denaturing DNA sequencing gel. 
     FIG. 5 shows the sequence based GCT alleles determined by PCR in control families and myotonic dystrophy families. Control families exhibit Mendelian inheritance of alleles. In myotonic dystrophy families, all affected individuals show only one allele, that from the normal parent. 
     FIG. 6 shows prenatal diagnosis of myotonic dystrophy using PCR analysis of the GCT repeat locus from MDY1. The control family is on the left and the filled symbols represent affected individuals. The size standard in the left lane is a 123 bp ladder. 
     FIG. 7 shows the triplet repeat sequences. 
     FIG. 8 shows the gene structure of Mt-PK and various isoforms of Mt-PK mRNA. Filled and patterned boxes indicate the locations of exons. The exons that coincide with the prediction by GRAIL as described in Example 5 are shown with asterisks beneath. All the exons predicted by GRAIL with excellent scores coincide with real exon positions. All the mRNA isoforms were obtained as described in Example 8. In the isoforms II-VIII, only the exons involved in the changes are shown. Isoforms II, III and IV are alternatively spliced variants of form I. Restriction enzymes: A-XbaI, B-BamHI, E-EcoRI, H-HindIII, X-XhoI. 
     FIG. 9 shows synthetic peptide locations (shown as a line under the amino acid sequence positions) and consensus regions of Mt-PK. The hatched box indicates the truncated protein expressed in E. coli. The corresponding number of antibody to synthetic peptide is: SEQ. ID. NO. 7-9828, SEQ. ID. NO. 8-254, SEQ. ID. NO. 9-10257. 
     FIG. 10 shows the plasmid map for the construct which express myotonic protein kinase in bacteria. 
     FIG. 11 shows western blot analysis of human and mouse tissues with the myotonic protein kinase antibody. The rodent Mt-PK isoforms run at lower apparent molecular weight (52,000) than the human isoforms. 
     FIG. 12 shows quantitation of mRNA levels for normal and DM adult patients by RTPCR (reverse transcriptase PCR). Each dot represents the average of nine determinations which were carried out as described in Example 11 for each RNA sample. The numbers shown here are the ratio of the Mt-PK RTPCR product to the internal control, human transferrin receptor, RTPCR product. 
     FIG. 13 Southern blot for PCR products from genomic DNA and total RNA. The mutant allele GCT repeat sizes for these samples are: TM-120, MR-68(the smallest one), LS-160, KH-205, CH-800. 
     FIG. 14 Evaluation of Mt-PK protein expression in adult muscle. Skeletal muscle biopsies from normal individuals and DM patients were processed for western blots as described in Example 10. Exactly 50 μg total protein was loaded per lane. The numbers below the lanes show the amount of Mt-PK detected in the samples by RIA as described in Example 13. The reported values are the mean from triplicate determinations. The values were expressed as ng/50 μg tissue protein. 
     FIG. 15 shows the correlation between Mt-PK protein levels and the disease severity of adult DM. 
     The drawings and figures are not necessarily to scale and certain features mentioned may be exaggerated in scale as shown in schematic form in the interest of clarity and conciseness. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     It will be readily apparent to one skilled in the art that variations, substitutions and modifications may be made to the invention disclosed herein without departing from the scope and the spirit of the invention. 
     Each sample to be tested herein for the DM mutation site is derived from genomic DNA, mRNA or protein. The source of the genomic DNA to be tested can be any medical specimen which contains DNA. Some examples of medical specimens include blood, semen, vaginal swabs, buccal mouthwash, tissue, hair, skin, amniotic fluid and mixture of body fluids. 
     As used herein the term &#34;polymerase chain reaction&#34; or &#34;PCR&#34; refers to the PCR procedure described in the patents to Mullis, et al., U.S. Pat. Nos. 4,683,195 and 4,683,202. The procedure basically involves: (1) treating extracted DNA to form single-stranded complementary strands; (2) adding a pair of oligonucleotide primers, wherein one primer of the pair is substantially complementary to part of the sequence in the sense strand and the other primer of each pair is substantially complementary to a different part of the same sequence in the complementary antisense strand; (3) annealing the paired primers to the complementary sequence; (4) simultaneously extending the annealed primers from a 3&#39; terminus of each primer to synthesize an extension product complementary to the strands annealed to each primer wherein said extension products after separation from the complement serve as templates for the synthesis of an extension product for the other primer of each pair; (5)separating said extension products from said templates to produce single-stranded molecules; and (6) amplifying said single-stranded molecules by repeating at least once said annealing, extending and separating steps. 
     As used herein fluorescence in situ hybridization or &#34;FISH&#34; refers to the procedure described in Wotta, et al., Am. J. of Human Genetics, 46, 95-106 (1988) and Kievits, et al., Cytogenet. Cell Genet., 53134-136 (1990). The procedure basically involves the steps of preparing interphase or metaphase spreads from cells of peripheral blood lymphocytes and hybridizing labeled probes to the interphase or metaphase spreads. Using probes with mixed labels allows visualization of space, order and distance between hybridization sites. After hybridization the labels are examined to determine the order and distance between the hybridization sites. 
     As used herein, the term &#34;pulsed field gel electrophoresis&#34; or &#34;PFGE&#34; refers to a procedure described by Schwartz, et al., Cold Springs Harbor Symposium, Quantitative Biology, 47:189-195 (1982). The procedure basically comprises running a standard electrophoresis gel (agarose, polyacrylamide or other gel known to those skilled in the art) under pulsing conditions. One skilled in the art recognizes that the strength of the field as well the direction of the field is pulsed and rotated in order to separate megabase DNA molecules. Current commercial systems are computer controlled and select the strength, direction and time of pulse depending on the molecular weight of DNA to be separated. 
     The following examples are offered by way of illustration and are not intended to limit the invention in any manner. In the examples all percentages are by weight for solids and by volume for liquids and all temperatures are in degrees celsius unless otherwise noted. 
     EXAMPLE 1 
     Isolation and Identification of DM Locus 
     Yeast artificial chromosomes (YACs) isolated from the St. Louis library which span the DM locus were used. YAC clones 231G8 and 483E7 were subcloned into cosmids and human clones were identified by the presence of common repeat sequences. 
     YACs 231G8 and 483E7 DNA were partially digested by Sau3A and cloned into cosmid vector &#34;Super Cos&#34; (Stratagene). Human clones were identified by their hybridization with radiolabeled total human DNA, selected and arrayed on a gridded plate. Duplicate filter lifts were screened for clonal specific triplet repeats by their hybridization to a mixture of 4 radiolabeled oligonucleotides. Two positive cosmids (MDY1 and MDY2) were identified on the grid. These 2 cosmids were then found to contain sequences in common including BamHI fragments of 1.4 and 1.35 kb. The 1.4 kb BamHI fragment was then identified to contain the triplet repeat sequence. 
     Using hybridization techniques, a mixture of 4 oligonucleotides consisting of tandemly repeated GC-rich trinucleotides (CAC, GCT, TCC, TCG) identified 2 out of 300 cosmids (cosmids MDY1 and MDY2). This set of 4 triplet repeats (each 21 nucleotides in length) include 24 of 60 possible triplet repeats with emphasis on the GC-rich ones. The CGG repeat was examined separately. The 2 positive cosmids were found to be overlapping with each other. A 1.4 kb BamHI fragment which specifically hybridized to the GCT repeat was identified and subcloned into pBluescript (pMDY1). The sequence of pMDY1 was determined by means of the dideoxynucleotide termination method and an ABI 373 automated fluorescent DNA sequencer (FIG. 1). Sequence of the pMDY1 was determined by using a combination of dideoxynucleotide termination reaction and the Taq DyeDeoxy™ terminator cycle sequencing reaction (Applied Biosystems). The sequencing reactions were analyzed on an automated DNA sequencer (ABI 373). As predicted by the oligonucleotide hybridization, a region containing 11 repeats of the GCT triplet was identified. This triplet is known to be highly polymorphic and unstable in the androgen receptor gene. Thus, a mixed oligonucleotide probe has successfully identified a short (1.4 kb) candidate sequence for genetic instability from 2 YACs at the DM locus. 
     EXAMPLE 2 
     Genetic Instability of DM Locus 
     To test genetic instability at the DM locus by studying families with congenital DM born to affected DM mothers were studied. Evidence of genetic instability at the DM locus is illustrated in FIGS. 2-3. NcoI digestion and Southern Blot analysis was performed on samples from families in which a congenitally affected child has been born using the cloned 1.4 kb BamHI fragment from cosmid MDY1 as a probe (Families 1585, 1127, and 1800) or the mixed probe (1.4 kb and 1.35 kb BamHI fragments) (Family 1860). Sequence enlargement in each congenitally affected child was demonstrated. Sequence enlargement to a lesser extent was also detected in the affected mother from Families 1127 and 1800 and in an increasing pattern in the affected grandmother from Family 1860. Family 1860 in FIG. 3 shows an example where a three-generation transmission of DM exhibits progressive enlargement (8.8 kb to 12.7 kb) of an NcoI fragment. 
     After digestion with the 3 restriction enzymes indicated, probes comprised of the 1.4 kb and the 1.35 kb BamHI fragments from cosmid MDY1 clearly identified enlarged DNA fragments from the congenitally affected child born to the affected mother from Family 953. The enlarged sequence was detected in neither parent and, by examination of the BamHI data, is at least 6 kb larger than sequence detected in the parents. Other restriction endonucleases including BanI and TaqI also identified fragment enlargements (FIG. 2). 
     Further it was found that 9 of 9 congenital DM patients and 14 of 16 adult DM patients had fragment enlargements. An exception is shown in FIG. 3 (Family 1585). There were no fragment enlargements or reductions among 31 controls examined. Since each congenital DM patient had unique enlarged restriction fragments which cannot be attributed to the parents, it is concluded that this DNA sequence expansion is the mutational basis of DM. In each of these families non-parentage was excluded by the linkage study. 
     EXAMPLE 3 
     PCR Analysis 
     100 ng of genomic DNAs were mixed with 3 pmole of each primer (SEQ. ID. Nos. 2 and 3 ) in a total volume of 15 μl containing 10 mM Tris-HCl pH8.3, 50 mM KCL, 1.5 mM MgCl 2 , 200 μM of each of the 4 dNTPs, 4 μCi of  32  p-dCTP and 0.75 units of AmpliTaq DNA polymerase. The reactions were heated to 95° C. for 10 min. and followed by 25 cycles of denaturation (95° C., 1 min), DNA reannealing (54° C., 1 min), and elongation (72° C., 2 min). The radioactive PCR products were combined with 95% formamide loading dye and then heated to 95° .C. for 2 min before electrophoresis through a 6% denaturing DNA sequencing gel. Allele sizes were determined by their migration relative to an M13 sequencing ladder. For analysis by 3% agarose gel electrophoresis, 200 ng of genomic DNA was amplified in a final volume of 100 μl using the same buffer, 250 μM of the 4 dNTPs and 0.5 units of AmpliTaq DNA polymerase. The reactions were heated to 95° C. for 5 min and then subjected to 32 cycles of 94° C. for 1 min 57° C. for 1 min, and 72° C. for 3 min. 
     EXAMPLE 4 
     DNA Sequence Characterization 
     In an effort to delineate the sequence involved in the DNA expansion, the GCT repeat size variation was examined using PCR amplification, followed by agarose and polyacrylamide gel electrophoresis. Using synthetic oligonucleotides which immediately flank the GCT repeat (FIG. 1), analysis of the radioactive amplification products indicated that the region is highly polymorphic (FIG. 4). The most common allele is 5 repeats with extremes of 5 to 30 from 40 normal individuals analyzed. The heterozygote frequency is 85%. This length polymorphism can also be observed by agarose gel analysis but with less detailed resolution. Examination of this sequence polymorphism in 3 DM and 4 control families is shown in FIG. 5. Unaffected individuals have the expected frequency of pairs of alleles, while DM patients have only one, the allele of the unaffected parent. Mendelian inheritance of alleles is observed in the control families. Thus, in these family studies, the DM GCT allele (as measured by PCR) is not detectable. Southern analysis indicates that each affected individual has a large expanded fragment. A repeat sequence of longer than 3 kb is beyond current ability to amplify using PCR. The simplest interpretation of these data is that the GCT repeat has meiotic instability at the DM locus and is responsible for the mutation in DM. Examination by PCR of the regions immediately flanking the GCT repeats indicated in FIG. 1 shows them to be non-polymorphic and unaltered in DM families. 
     EXAMPLE 5 
     Since both the FMR-1 (Fragile X) and androgen receptor (Kennedy) mRNA contain triplet repeats, the pMDY1 sequence was examined by the computer program Grail (Gene Recognition and Analysis Internet Link). GRAIL Computer searches are available to general users by the Oak Ridge National Laboratory File server at GRAIL@ornl.gov. This program revealed an &#34;excellent&#34; exon identification score possibly biased by the inclusion of triplet repeat sequences. The transcript was directly searched using brain and skeletal muscle mRNA copied by reverse transcriptase (cDNA). This study identified amplified products of the expected size supporting the computer prediction. Furthermore, the pMDY1 probe successfully identified brain cDNA clones whose GCT repeat alleles differed. These data collectively indicate the repeat sequence is in a gene. 
     EXAMPLE 6 
     The utility of DNA-based detection of DM mutations is illustrated by prenatal diagnosis. In this pregnancy, the fetus was predicted to have a greater than 97% risk to be affected on the basis of linkage analysis. The result from the family is shown in FIG. 6. Amplification of the polymorphic region using DNA extracted from leukocytes or cultured chorionic villi cells and the oligonucleotide primers shown in FIG. 1 followed by electrophoresis through a 3% agarose gel identified a Mendelian pattern of inheritance of the informative paternal PCR amplified GCT alleles in a control family (left). All individuals affected with myotonic dystrophy (filled symbols) appeared to be hemizygous (or homozygous) for the GCT locus. A maternal allele was not detected in the congenitally affected child. The fetus, which had been judged to be at greater than 97% risk to be affected by linkage analysis, inherited the opposite paternal allele and was confirmed to be affected by the absence of an amplifiable maternal allele. Thus, direct detection of the DM mutation by PCR was in agreement with the linkage prediction. This procedure which is an additional DNA-based method for diagnosis of DM utilizes direct mutation detection. It provides greater ease and accuracy. 
     EXAMPLE 7 
     Detecting Triplet Repeat Mutations 
     The synthetic oligonucleotides of GC rich triplet character as shown in FIG. 7 were used in a scanning strategy to identify unstable genetic sequences. Oligonueleotides were labelled with gamma- 32  P-ATP at the 5&#39; end and used as probes to screen libraries which can be cDNA, cosmid, lamda, and plasmid genomic libraries. The scanning procedure detected a highly polymorphic GCT repeat at the DM locus. This repeat was characterized. It identified an unstable and expanding sequence found in DM patients. In FIG. 7 is shown the GC rich triplets useful in scanning for autosomal disease. 
     EXAMPLE 8 
     Gene Structure and Isoforms of Mt-PK Gene 
     The genomic sequence of MT-Pk was determined from M13 subclones of a cosmid clone found to contain the structural gene on the basis of homology to the MT-Pk cDNA and GCT repeat. A graphic representation of the gene is given in FIG. 8. Grail computer analysis of this sequence identified putative exons which are illustrated in FIG. 8 for sequences whose score was &#34;excellent&#34;. Additional exons were identified from the cDNA clones and from that of alternative splice forms determined by sequence of reverse transcriptase PCR (RTPCR). The RTPCR sequences were obtained from human adult muscle, brain and heart tissues while the cDNA clone was obtained from brain tissue. The alternative splice forms are graphically represented in FIG. 8. In each case the RT cDNA sequence was determined by automated DNA sequencing from plasmid subclone of the PCR product. Each isoform was amplified from muscle or heart mRNA using primers from exons flanking the intron sequence to avoid nuclear DNA amplification products. Eight alternative splice forms which differ primarily in the 5&#39; and 3&#39; regions of the mRNA of MT-Pk have been identified. In all cases, protein kinases sequence homology (located in exons 2-6) is preserved and not affected by the alternative splice events. Several interesting splice forms would suggest deleted (Forms V, VI, VII, and VIII) or alternate coding sequence (Form VIII) for the MT-Pk. Form VIII lacks both exons 12 and 13. This generates a termination codon immediately after the splice acceptor which removes a predicted carboxyl-terminal transmembrane domain of the protein. 
     EXAMPLE 9 
     Development of Antibodies for Mt-PK Protein 
     In an effort to understand better the Mt-PK protein isoforms, antibodies were developed to both synthetic peptides and purified Mt-PK protein expressed in E. coli. Antibodies were developed against synthetic peptide immunogens (SEQ. ID. NOS. 7,8 and 9) using selected amino acid sequences of Mt-PK as designated on FIG. 9. Three anti-peptide antisera (9828, 10257, 254) were generated that recognize a 55,000 molecular weight protein in muscle, the expected size of the Mt-PK. In addition to antipeptide antibodies, we developed a specific and high affinity antibody (10033) using as antigen a truncated Mt-PK protein produced with the prokaryotic expression vector pRSET (Invitrogen Co.). This construct (as shown in FIG. 10) incorporated the isoform VIII 3&#39; termination codon and was devoid of the Mt-PK putative membrane spanning domain. In addition, the metal binding domain of the pRSET vector was fused in-frame at the amino terminus of Mt-PK with the AUG of bp 842. This recombinant vector provided a chimeric peptide that was subsequently purified by nickel affinity chromatography. Following purification of the expressed protein on a nickel column, N-terminal amino acid sequencing was carried out to verify the identity of the purified product. The sequence obtained corresponded to the nickel binding epitope, followed by four residues of the Mt-PK sequence initiating with methionine encoded at bp 842 (M-K-Q-T), confirming the authenticity of the chimeric Mt-PK protein. The fusion protein was then used to validate the specificity of antibodies raised against peptides and the prokaryotically expressed antigen. 
     EXAMPLE 10 
     Western Blot Analysis 
     We have used these antibodies (10033 and 254) to detect proteins by western analysis in selected tissues (FIG. 11). These studies indicate a high level of Mt-PK protein expression in both human and rodent muscle, heart and to a lesser extent in brain. Human and mouse tissues were disrupted in isotonic buffer containing protease inhibitors and spun at 48,000 xg for 30 min. The supernatant was assayed for protein content using a modified Lowry (Micro BCA, Pierce Chem Co.) and 50 μg were loaded per lane onto denaturing 12-23% acrylamide gradient gels. The proteins were transferred to nitrocellulose and probed with antisera. 
     EXAMPLE 11 
     Quantitation of MT-PK mRNA 
     In order to determine if the CTG repeat expansion altered the level of the mRNA in tissues, we developed a quantitative RTPCR assay. Total RNA was extracted from various tissues by RNAzol. 1 ug of total RNA was used for the reverse transcription reaction using Superscript RT (BRL) following the manufacturer&#39;s instructions. The reverse transcription reaction was heat terminated and diluted 2.5X with H 2  O. 2 ul of the RT product were then used for the PCR reaction which used two sets of primers; one set for the Mt-PK gene (SEQ. ID. Nos. 12 and 13) and one set for the human transferrin receptor gene (an internal control) (SEQ. ID. Nos. 4 and 5). The PCR products were analyzed on a 2% agarose gel and scanned by a Gene Scanner (ABI). mRNA of transferrin receptor (TFR), a stably-expressed gene, was used as an internal quantitation standard. The ratio of Mt-PK/TFR for different individuals and clinical conditions is given in FIG. 12. In adults with myotonic dystrophy the mRNA levels were found to be consistently low. Using primer pairs which permit a distinction between the two different alleles (i.e. primers flanking the CTG repeats) it was found that the mRNA level of the mutant allele from the adult DM expressed at lower levels--i.e. lack of coequal expression of wild type and mutant mRNA. 
     EXAMPLE 12 
     PCR-Southern Analysis of mRNA Level 
     Genomic DNA and total RNA were isolated from lymphoblastoid cell lines. RNA was treated with DNaseI before RTPCR reaction. PCR primers flanking the CTG repeat were used and PCR products were loaded onto 2% agarose gel. Oligonucleotide of 21 residues in length was used as probe for the Southern analysis. We were able to detect the mRNA level of normal alleles, reduced levels from permutation alleles (up to 205 repeats), and at extremely low levels from mutant allele with 800 repeats (See FIG. 13). Genomic DNA with the same number of repeats were used as the control for evaluating PCR efficiency. Thus, in adult DM, the level of mRNA identified by the RTPCR was mostly to fully accounted for by the normal Mt-PK alleles. These observations contrast with our coequal detection of two normal alleles in the control individuals. 
     EXAMPLE 13 
     Quantitation of Mt-PK Protein by Radioimmunoassay (RIA) 
     The level of Mt-PK protein has been determined in normal and diseased adult muscle by two methods, western blot and radioimmunoassay (RIA). Tissue extracts (1 to 10 ug) were incubated with  125  I-labelled Mt-PK fusion protein (7 uCi/ug protein) and anti Mt-PK antisera (10033, at a final dilution of 1/1000), for 2 hours at room temperature. The immune complex was precipitated with protein A agarose for 45 minutes. The precipitate was washed three times with buffer containing Tris-HCl (pH8.3), 1% NP-40, 3% BSA, and 0.5M NaCl. The relative level of Mt-PK in adult muscle is shown in FIG. 14. A total 20 DM patients and 7 normal individuals were examined. Decreased Mt-PK expression was found in 18 out of 20 adult patients, and the amount of the decrease was proportional to the severity of disease as shown in FIG. 15. Thus, by two independent methods the Mt-PK gene expression has been shown to be decreased in adult DM muscle compared to normal adult muscle. 
     
         __________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 13(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1383 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:GGATCCACCTTCCCATGTAAGACCCCTCTCTTTCCCCTGCCTCAGACCTGCTGCCCATTC60TGCAGATCCCCTCCCTGGCTCCTGGTCTCCCCGTCCAGATATAGGGCTCACCCTACGTCT120TTGCGACTTTAGAGGGCAGAAGCCCTTTATTCAGCCCCAGATCTCCCTCCGTTCAGGCCT180CACCAGATTCCCTCCGGGATCTCCCTAGATAACCTCCCCAACCTCGATTCCGCTCGCTGT240CTCTCGCCCCACCGCTGAGGGCTGGGCTGGGCTCCGATCGGGTCACCTGTCCCTTCTCTC300TCCAGCTAGATGGCCCCCCGGCCGTGGCTGTGGGCCAGTGCCCGCTGGTGGGGCCAGGCC360CCATGCACCGCCGCCACCTGCTGCTCCCTGCCAGGGTACGTCCGGCTGCCCACGCCCCCC420TCCGCCGTCGCGCCCCGCGCTCCACCCGCCCCGTGCCACCCGCTTAGCTGCGCATTTGCG480GGGCTGGGCCCACGGTAGGAGGGCGGATCTTCGGGCAGACAATCAACACAGGCCGCTAGG540AAGCAGCCAATGACGAGTTCGGACGGGATTCGAGGCGTGCGAGTGGACTAACAACAGCTG600TAGGCTGTTGGGGCGGGGGCGGGGCGCAGGGAAGAGTGCGGGCCCACCTATGGGCGTAGG660CGGGGCGAGTCCCAGGAGCCAATCAGAGGCCCATGCCGGGTGTTGACCTCGCCCTCTCCC720CGCAGGTCCCTAGGCCTGGCCTATCGGAGGCGCTTTCCCTGCTCCTGTTCGCCGTTGTTC780TGTCTCGTGCCGCCGCCCTGGGCTGCATTGGGTTGGTGGCCCACGCCGGCCAACTCACCG840CAGTCTGGCGCCCGCCCAGGAGCCGCCCGCGCTCCCTGAACCCTAGAACTGTCTTCGACT900CCGGGGCCCCGTTGGAAGACTGAGTGCCCGGGGCACGGCACAGAAGCCGCGCCCACCGCC960TGCCAGTTCACAACCGCTCCGAGCGTGGGTCTCCGCCCAGCTCCAGTCCTGTGACCGGGC1020CCGCCCCCTAGCGGCCGGGGAGGGAGGGGCCGGGTCCGCGGCCGGCGAACGGGGCTCGAA1080GGGTCCTTGTAGCCGGGAATGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGGGGGGA1140TCACAGACCATTTCTTTCTTTCGGCCAGGCTGAGGCCCTGACGTGGATGGGCAAACTGCA1200GGCCTGGGAAGGCAGCAAGCCGGGCCGTCCGTGTTCCATCCTCCACGCACCCCCACCTAT1260CGTTGGTTCGCAAAGTGCAAAGCTTTCTTGTGCATGACGCCCTGCTCTGGGGAGCGTCTG1320GCGCGATCTCTGCCTGCTTACTCGGGAAATTTGCTTTTGCCAAACCCGCTTTTTCGGGGA1380TCC1383(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:GCTCGAAGGGTCCTTGTAGCCGGG24(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:CTGGCCGAAAGAAAGAAATGGTC23(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:CAGCTCCCTGAATAGTCCAAGTAG24(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:GAATTGAACCTGGACTATGAGAGG24(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 555 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:MetLysGlnThrGlyGlnValTyrAlaMetLysIleMetAsnLysTrp151015AspMetLeuLysArgGlyGluValSerCysPheArgGluGluArgAsp202530ValLeuValAsnGlyAspArgArgTrpIleThrGlnLeuHisPheAla354045PheGlnAspGluAsnTyrLeuTyrLeuValMetGluTyrTyrValGly505560GlyAspLeuLeuThrLeuLeuSerLysPheGlyGluArgIleProAla65707580GluMetAlaArgPheTyrLeuAlaGluIleValMetAlaIleAspSer859095ValHisArgLeuGlyTyrValHisArgAspIleLysProAspAsnIle100105110LeuLeuAspArgCysGlyHisIleArgLeuAlaAspPheGlySerCys115120125LeuLysLeuArgAlaAspGlyThrValArgSerLeuValAlaValGly130135140ThrProAspTyrLeuSerProGluIleLeuGlnAlaValGlyGlyGly145150155160ProGlyThrGlySerTyrGlyProGluCysAspTrpTrpAlaLeuGly165170175ValPheAlaTyrGluMetPheTyrGlyGlnThrProPheTyrAlaAsp180185190SerThrAlaGluThrTyrGlyLysIleValHisTyrLysGluHisLeu195200205SerLeuProLeuValAspGluGlyValProGluGluAlaArgAspPhe210215220IleGlnArgLeuLeuCysProProGluThrArgLeuGlyArgGlyGly225230235240AlaGlyAspPheArgThrHisProPhePhePheGlyLeuAspTrpAsp245250255GlyLeuArgAspSerValProProPheThrProAspPheGluGlyAla260265270ThrAspThrCysAsnPheAspLeuValGluAspGlyLeuThrAlaMet275280285ValSerGlyGlyGlyGluThrLeuSerAspIleArgGluGlyAlaPro290295300LeuGlyValHisLeuProPheValGlyTyrSerTyrSerCysMetAla305310315320LeuArgAspSerGluValProGlyProThrProMetGluLeuGluAla325330335GluGlnLeuLeuGluProHisValGlnAlaProSerLeuGluProSer340345350ValSerProGlnAspGluThrAlaGluValAlaValProAlaAlaVal355360365ProAlaAlaGluAlaGluAlaGluValThrLeuArgGluLeuGlnGlu370375380AlaLeuGluGluGluValLeuThrArgGlnSerLeuSerArgGluMet385390395400GluAlaIleArgThrAspAsnGlnAsnPheAlaSerGlnLeuArgGlu405410415AlaGluAlaArgAsnArgAspLeuGluAlaHisValArgGlnLeuGln420425430GluArgMetGluLeuLeuGlnAlaGluGlyAlaThrAlaValThrGly435440445ValProSerProArgAlaThrAspProProSerHisLeuAspGlyPro450455460ProAlaValAlaValGlyGlnCysProLeuValGlyProGlyProMet465470475480HisArgArgHisLeuLeuLeuProAlaArgValProArgProGlyLeu485490495SerGluAlaLeuSerLeuLeuLeuPheAlaValValLeuSerArgAla500505510AlaAlaLeuGlyCysIleGlyLeuValAlaHisAlaGlyGlnLeuThr515520525AlaValTrpArgProProArgSerArgProArgSerLeuAsnProArg530535540ThrValPheAspSerGlyAlaProLeuGluAsp545550555(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:AsnGlyAspArgArgTrpIleThrGln15(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 12 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:LeuValGluAspGlyLeuThrAlaMetValSerGly1510(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:PheAspSerGlyAlaProLeuGluAsp15(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 11613 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:CCATGGCCTCTCTGCACCCCGCCTCAGGGTCAGGGTCAGGGTCATGCTGGGAGCTCCCTC60TCCTAGGACCCTCCCCCCAAAAGTGGGCTCTATGGCCCTCTCCCCTGGTTTCCTGTGGCC120TGGGGCAAGCCAGGAGGGCCAGCATGGGGCAGCTGCCAGGGGCGCAGCCGACAGGCAGGT180GTTCGGCGCCAGCCTCTCCAGCTGCCCCAACAGGTGCCCAGGCGCTGGGAGGGCGGTGAC240TCACGCGGGCCCTGTGGGAGAACCAGCTTTGCAGACAGGCGCCACCAGTGCCCCCTCCTC300TGCGATCCAGGAGGGACAACTTTGGGTTCTTCTGGGTGTGTCTCCTTCTTTTGTAGGTTC360TGCACCCACCCCCACCCCCAGCCCCAAAGTCTCGGTTCCTATGAGCCGTGTGGGTCAGCC420ACCATTCCCGCCACCCCGGGTCCCTGCGTCCTTTAGTTCTCCTGGCCCAGGGCCTCCAAC480CTTCCAGCTGTCCCACAAAACCCCTTCTTGCAAGGGCTTTCCAGGGCCTGGGGCCAGGGC540TGGAAGGAGGATGCTTCCGCTTCTGCCAGCTGCCTTGTCTGCCCACCTCCTCCCCAAGCC600CAGGACTCGGGCTCACTGGTCACTGGTTTCTTTCATTCCCAGCACCCTGCTCCTCTGGCC660CTCATATGTCTGGCCCTCAGTGACTGGTGTTTGGTTTTTGGCCTGTGTGTAACAAACTGT720GTGTGACACTTGTTTCCTGTTTCTCCGCCTTCCCCTGCTTCCTCTTGTGTCCATCTCTTT780CTGACCCAGGCCTGGTTCCTTTCCCTCCTCCTCCCATTTCACAGATGGGAAGGTGGCGGC840CAAGAAGGGCCAGGCCATTCAGCCTCTGGAAAAACCTTCTCCCAACCTCCCACAGCCCCT900AATGACTCTCCTGGCCTCCCTTTAGTAGAGGATGAAGTTGGGTTGGCAGGGTAAACTGAG960ACCGGGTGGGGTAGGGGTCTGGCGCTCCCGGGAGGAGCACTCCTTTTGTGGCCCGAGCTG1020CATCTCGCGGCCCCTCCCCTGCAAGGCCTGGGGCGGGGGAGGGGGCCAGGGTTCCTGCTG1080CCTTAAAAGGGCTCAATGTCTTGGCTCTCTCCTCCCTCCCCCGTCCTCAGCCCTGGCTGG1140TTCGTCCCTGCTGGCCCACTCTCCCGGAACCCCCCGGAACCCCTCTCTTTCCTCCAGAAC1200CCACTGTCTCCTCTCCTTCCCTCCCCTCCCATACCCATCCCTCTCTCCATCCTGCCTCCA1260CTTCTTCCACCCCCGGGAGTCCAGGCCTCCCTGTCCCCACAGTCCCTGAGCCACAAGCCT1320CCACCCCAGCTGGTCCCCCACCCAGGCTGCCCAGTTTAACATTCCTAGTCATAGGACCTT1380GACTTCTGAGAGGCCTGATTGTCATCTGTAAATAAGGGGTAGGACTAAAGCACTCCTCCT1440GGAGGACTGAGAGATGGGCTGGACCGGAGCACTTGAGTCTGGGATATGTGACCATGCTAC1500CTTTGTCTCCCTGTCCTGTTCCTTCCCCCAGCCCCAAATCCAGGGTTTTCCAAAGTGTGG1560TTCAAGAACCACCTGCATCTGAATCTAGAGGTACTGGATACAACCCCACGTCTGGGCCGT1620TACCCAGGACATTCTACATGAGAACGTGGGGGTGGGGCCCTGGCTGCACCTGAACTGTCA1680CCTGGAGTCAGGGTGGAAGGTGGAAGAACTGGGTCTTATTTCCTTCTCCCCTTGTTCTTT1740AGGGTCTGTCCTTCTGCAGACTCCGTTACCCCACCCTAACCATCCTGCACACCCTTGGAG1800CCCTCTGGGCCAATGCCCTGTCCCGCAAAGGGCTTCTCAGGCATCTCACCTCTATGGGAG1860GGCATTTTTGGCCCCCAGAACCTTACACGGTGTTTATGTGGGGAAGCCCCTGGGAAGCAG1920ACAGTCCTAGGGTGAAGCTGAGAGGCAGAGAGAAGGGGAGACAGACAGAGGGTGGGGCTT1980TCCCCCTTGTCTCCAGTGCCCTTTCTGGTGACCCTCGGTTCTTTTCCCCCACCACCCCCC2040CAGCGGAGCCCATCGTGGTGAGGCTTAAGGAGGTCCGACTGCAGAGGGACGACTTCGAGA2100TTCTGAAGGTGATCGGACGCGGGGCGTTCAGCGAGGTAAGCCGAACCGGGCGGGAGCCTG2160ACTTGACTCGTGGTGGGCGGGGCATAGGGGTTGGGGCGGGGCCTTAGAAATTGATGAATG2220ACCGAGCCTTAGAACCTAGGGCTGGGCTGGAGGCGGGGCTTGGGACCAATGGGCGTGGTG2280TGGCAGGTGGGGCGGGGCCACGGCTGGGTGCAGAAGCGGGTGGAGTTGGGTCTGGGCGAG2340CCCTTTTGTTTTCCCGCCGTCTCCACTCTGTCTCACTATCTCGACCTCAGGTAGCGGTAG2400TGAAGATGAAGCAGACGGGCCAGGTGTATGCCATGAAGATCATGAACAAGTGGGACATGC2460TGAAGAGGGGCGAGGTGAGGGGCTGGGCGGACGTGGGGGGCTTTGAGGATCCGCGCCCCG2520TCTCCGGCTGCAGCTCCTCCGGGTGCCCTGCAGGTGTCGTGCTTCCGTGAGGAGAGGGAC2580GTGTTGGTGAATGGGGACCGGCGGTGGATCACGCAGCTGCACTTCGCCTTCCAGGATGAG2640AACTACCTGGTGAGCTCCGGGCCGGGGGGACTAGGAAGAGGGACAAGAGCCCGTGCTGTC2700ACTGGACGAGGAGGTGGGGAGAGGAAGCTCTAGGATTGGGGGTGCTGCCCGGAAACGTCT2760GTGGGAAAGTCTGTGTGCGGTAAGAGGGTGTGTCAGGTGGATGAGGGGCCTTCCCTATCT2820GAGACGGGGATGGTGTCCTTCACTGCCCGTTTCTGGGGTGATCTGGGGGACTCTTATAAA2880GATGTCTCTGTTGCGGGGGGTCTCTTACCTGGAATGGGATAGGTCTTCAGGAATTCTAAC2940GGGGCCACTGCCTAGGGAAGGAGTGTCTGGGACCTATTCTCTGGGTGTTGGGTGGCCTCT3000GGGTTCTCTTTCCCAGAACATCTCAGGGGGAGTGAATCTGCCCAGTGACATCCCAGGAAA3060GTTTTTTTGTTTGTGTTTTTTTTTGAGGGGCGGGGGCGGGGGCCGCAGGTGGTCTCTGAT3120TTGGCCCGGCAGATCTCTATGGTTATCTCTGGGCTGGGGCTGCAGGTCTCTGCCCAAGGA3180TGGGGTGTCTCTGGGAGGGGTTGTCCCAGCCATCCGTGATGGATCAGGGCCTCAGGGGAC3240TACCAACCACCCATGACGAACCCCTTCTCAGTACCTGGTCATGGAGTATTACGTGGGCGG3300GGACCTGCTGACACTGCTGAGCAAGTTTGGGGAGCGGATTCCGGCCGAGATGGCGCGCTT3360CTACCTGGCGGAGATTGTCATGGCCATAGACTCGGTGCACCGGCTTGGCTACGTGCACAG3420GTGGGCGCAGCATGGCCGAGGGGATAGCAAGCTTGTTCCCTGGCCGGGTTCTTGGAAGGT3480CAGAGCCCAGAGAGGCCAGGGCCTGGAGAGGGACCTTCTTGGTTGGGGCCCACCGGGGGG3540TGCCTGGGAGTAGGGGTCAGAACTGTAGAAGCCCTACAGGGGCGGAACCCGAGGAAGTGG3600GGTCCCAGGTGGCACTGCCCGGAGGGGCGGAGCCTGGTGGGACCACAGAAGGGAGGTTCA3660TTTATCCCACCCTTCTCTTTTCCTCCGTGCAGGGACATCAAACCCGACAACATCCTGCTG3720GACCGCTGTGGCCACATCCGCCTGGCCGACTTCGGCTCTTGCCTCAAGCTGCGGGCAGAT3780GGAACGGTGAGCCAGTGCCCTGGCCACAGAGCAACTGGGGCTGCTGATGAGGGATGGAAG3840GCACAGAGTGTGGGAGCGGGACTGGATTTGGAGGGGAAAAGAGGTGGTGTGACCCAGGCT3900TAAGTGTGCATCTGTGTGGCGGAGTATTAGACCAGGCAGAGGGAGGGGCTAAGCATTTGG3960GGAGTGGTTGGAAGGAGGGCCCAGAGCTGGTGGGCCCAGAGGGGTGGGCCCAAGCCTCGC4020TCTGCTCCTTTTGGTCCAGGTGCGGTCGCTGGTGGCTGTGGGCACCCCAGACTACCTGTC4080CCCCGAGATCCTGCAGGCTGTGGGCGGTGGGCCTGGGACAGGCAGCTACGGGCCCGAGTG4140TGACTGGTGGGCGCTGGGTGTATTCGCCTATGAAATGTTCTATGGGCAGACGCCCTTCTA4200CGCGGATTCCACGGCGGAGACCTATGGCAAGATCGTCCACTACAAGGTGAGCACGGCCGC4260AGGGAGACCTGGCCTCTCCCGGTAGGCGCTCCCAGCTATCGCCTCCTCTCCCTCTGAGCA4320GGAGCACCTCTCTCTGCCGCTGGTGGACGAAGGGGTCCCTGAGGAGGCTCGAGACTTCAT4380TCAGCGGTTGCTGTGTCCCCCGGAGACACGGCTGGGCCGGGGTGGAGCAGGCGACTTCCG4440GACACATCCCTTCTTCTTTGGCCTCGACTGGGATGGTCTCCGGGACAGCGTGCCCCCCTT4500TACACCGGATTTCGAAGGTGCCACCGACACATGCAACTTCGACTTGGTGGAGGACGGGCT4560CACTGCCATGGTGAGCGGGGGCGGGGTAGGTACCTGTGGCCCCTGCTCGGCTGCGGGAAC4620CTCCCCATGCTCCCTCCATAAAGTTGGAGTAAGGACAGTGCCTACCTTCTGGGGTCCTGA4680ATCACTCATTCCCCAGAGCACCTGCTCTGTGCCCATCTACTACTGAGGACCCAGCAGTGA4740CCTAGACTTACAGTCCAGTGGGGGAACACAGAGCAGTCTTCAGACAGTAAGGCCCCAGAG4800TGATCAGGGCTGAGACAATGGAGTGCAGGGGGTGGGGGACTCCTGACTCAGCAAGGAAGG4860TCCTGGAGGGCTTTCTGGAGTGGGGAGCTATCTGAGCTGAGACTTGGAGGGATGAGAAGC4920AGGAGAGGACTCCTCCTCCCTTAGGCCGTCTCTCTTCACCGTGTAACAAGCTGTCATGGC4980ATGCTTGCTCGGCTCTGGGTGCCCTTTTGCTGAACAATACTGGGGATCCAGCACGGACCA5040GATGAGCTCTGGTCCCTGCCCTCATCCAGTTGCAGTCTAGAGAATTAGAGAATTATGGAG5100AGTGTGGCAGGTGCCCTGAAGGGAAGCAACAGGATACAAGAAAAAATGATGGGGCCAGGC5160ACGGTGCTCACGCCTGTAACCCCAGCAATTTGGCAGGCCGAAGTGGGTGGATTGCTTGAG5220CCCAGGAGTTCGAGACCAGCCTGGGCAATGTGGTGAGACCCCCGTCTCTACAAAAATGTT5280TTAAAAATTGGTTGGGCGTGGTGGCGCATGCCTGTATACTCAGCTACTAGGGTGGCCGAC5340GTGGGCTTGAGCCCAGGAGGTCAAGGCTGCAGTGAGCTGTGATTGTGCCACTGCACTCCA5400GCCTGGGCAACGGAGAGAGACTCTGTCTCAAAAATAAGATAAACTGAAATTAAAAAATAG5460GCTGGGCTGGCCGGGCGTGGTGGCTCACGCCTGTAATCTCAGCACTTTGGGAGGCCGAGG5520CGGGTGGATCACGAGGTCAGAAGATGGAGACCAGCCTGGCCAGCGTGGCGAAACCCCGTC5580TCTACCAAAAATATAAAAAATTAGCCAGGCGTGGTAGAGGGCGCCTGTAATCTCAGCTAC5640TCAGGACGCTGAGGCAGGAGAATCGCCTGAACCTGGGAGGCGGAGGTTGCAGTGAGCTGA5700GATTGCACCACTGCACTCCAGCCTGGGTAACAGAGCGAGACTCCGTATCAAAGAAAAAGA5760AAAAAGAAAAAATGCTGGAGGGGCCACTTTAGATAAGCCCTGAGTTGGGGCTGGTTTGGG5820GGGAACATGTAAGCCAAGATCAAAAAGCAGTGAGGGGCCCGCCCTGACGACTGCTGCTCA5880CATCTGTGTGTCTTGCGCAGGAGACACTGTCGGACATTCGGGAAGGTGCGCCGCTAGGGG5940TCCACCTGCCTTTTGTGGGCTACTCCTACTCCTGCATGGCCCTCAGGTAAGCACTGCCCT6000GGACGGCCTCCAGGGGCCACGAGGCTGCTTGAGCTTCCTGGGTCCTGCTCCTTGGCAGCC6060AATGGAGTTGCAGGATCAGTCTTGGAACCTTACTGTTTTGGGCCCAAAGACTCCTAAGAG6120GCCAGAGTTGGAGGACCTTAAATTTTCAGATCTATGTACTTCAAAATGTTAGATTGAATT6180TTAAAACCTCAGAGTCACAGACTGGGCTTCCCAGAATCTTGTAACCATTAACTTTTACGT6240CTGTAGTACACAGAGCCACAGGACTTCAGAACTTGGAAAATATGAAGTTTAGACTTTTAC6300AATCAGTTGTAAAAGAATGCAAATTCTTTGAATCAGCCATATAACAATAAGGCCATTTAA6360AAGTATTAATTTAGGCGGGCCGCGGTGGCTCACGCCTGTAATCCTAGCACTTTGGGAGGC6420CAAGGCAGGTGGATCATGAGGTCAGGAGATCGAGACCATCCTGGCTAACACGGTGAAACC6480CCGTCTCTACTAAAAATACAAAAAAATTAGCCGGGCATGGTGGCGGGCGCTTGCGGTCCC6540AGCTACTTGGGAGGCGAGGCAGGAGAATGGCATGAACCCGGGAGGCGGAGCTTGCAGTGA6600GCCGAGATCATGCCACTGCACTCCAGCCTGGGCGACAGAGCAAGACTCCGTCTCAAAAAA6660AAAAAAAAAAAAAGTATTTATTTAGGCCGGGTGTGGTGGCTCACGCCTGTAATTCCAGTG6720CTTTGGGAGGATGAGGTGGGTGGATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGACCA6780ACGTGGAGAAACCTCATCTCTACTAAAAAACAAAATTAGCCAGGCATGGTGGCATATACC6840TGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCAGAACCCAGGAGGGGGAGGTT6900GTGGTTAGCTGAGATCGTGCCATTGCATTCCAGCCTGGGCAACAAGAGTGAAACTTCATC6960TCAAAAAAAAAAAAAAAAAAGTACTAATTTACAGGCTGGGCATGGTGGCTCACGCTTGGA7020ATCCCAGCACTTTGGGAGGCTGAAGTGGACGGATTGCTTCAGCCCAGGAGTTCAAGACCA7080GCCTGAGCAACATAATGAGACCCTGTCTCTACAAAAAATTGAAAAAATCGTGCCAGGCAT7140GGTGGTCTGTGCCTGCAGTCCTAGCTACTCAGGAGTCTGAAGTAGGAGAATCACTTGAGC7200CTGGAGTTTGAGGCTTCAGTGAGCCATGATAGATTCCAGCCTAGGCAACAAAGTGAGACC7260TGGTCTCAACAAAAGTATTAATTACACAAATAATGCATTGCTTATCACAAGTAAATTAGA7320AAATACAGATAAGGAAAAGGAAGTTGATATCTCGTGAGCTCACCAGATGGCAGTGGTCCC7380TGGCTCACACGTGTACTGACACATGTTTAAATAGTGGAGAACAGGTGTTTTTTTGGTTTG7440TTTTTTTCCCCTTCCTCATGCTACTTTGTCTAAGAGAACAGTTGGTTTTCTAGTCAGCTT7500TTATTACTGGACAACATTACACATACTATACCTTATCATTAATGAACTCCAGCTTGATTC7560TGAACCGCTGCGGGGCCTGAACGGTGGGTCAGGATTGAACCCATCCTCTATTAGAACCCA7620GGCGCATGTCCAGGATAGCTAGGTCCTGAGCCGTGTTCCCACAGGAGGGACTGCTGGGTT7680GGAGGGGACAGCCACTTCATACCCCAGGGAGGAGCTGTCCCCTTCCCACAGCTGAGTGGG7740GTGTGCTGACCTCAAGTTGCCATCTTGGGGTCCCATGCCCAGTCTTAGGACCACATCTGT7800GGAGGTGGCCAGAGCCAAGCAGTCTCCCCATCAGGTCGGCCTCCCTGTCCTGAGGCCCTG7860AGAAGAGGGGTCTGCAGCGGTCACATGTCAAGGGAGGAGATGAGCTGACCCTAGAACATG7920GGGGTCTGGACCCCAAGTCCCTGCAGAAGGTTTAGAAAGAGCAGCTCCCAGGGGCCCAAG7980GCCAGGAGAGGGGCAGGGCTTTTCCTAAGCAGAGGAGGGGCTATTGGCCTACCTGGGACT8040CTGTTCTCTTCGCTCTGCTGCTCCCCTTCCTCAAATCAGGAGGTCTTGGAAGCAGCTGCC8100CCTACCCACAGGCCAGAAGTTCTGGTTCTCCACCAGATAATCAGCATTCTGTCTCCCTCC8160CCACTCCCTCCTCCTCTCCCCAGGGACAGTGAGGTCCCAGGCCCCACACCCATGGAAGTG8220GAGGCCGAGCAGCTGCTTGAGCCACACGTGCAAGCGCCCAGCCTGGAGCCCTCGGTGTCC8280CCACAGGATGAAACAGTAAGTTGGTGGAGGGGAGGGGGTCCGTCAGGGACAATTGGGAGA8340GAAAAGGTGAGGGCTTCCCGGGTGGCGTGCACTGTAGAGCCCTCTAGGGACTTCCTGAAC8400AGAAGCAGACAGAAACCACGGAGAGACGAGGTTACTTCAGACATGGGACGGTCTCTGTAG8460TTACAGTGGGGCATTAAGTAAGGGTGTGTGTGTTGCTGGGGATCTGAGAAGTCGATCTTT8520GAGCTGAGCGCTGGTGAAGGAGAAACAAGCCATGGAAGGAAAGGTGCCAAGTGGTCAGGC8580GAGAGCCTCCAGGGCAAAGGCCTTGGGCAGGTGGGAATCCTGATTTGTTCCTGAAAGGTA8640GTTTGGCTGAATCATTCCTGAGAAGGCTGGAGAGGCCAGCAGGAAACAAAACCCAGCAAG8700GCCTTTTGTCGTGAGGGCATTAGGGAGCTGGAGGGATTTTGAGCAGCAGAGGGACATAGG8760TTGTGTTAGTGTTTGAGCACCAGCCCTCTGGTCCCTGTGTAGATTTAGAGGACCAGACTC8820AGGGATGGGGCTGAGGGAGGTAGGGAAGGGAGGGGGCTTGGATCATTGCAGGAGCTATGG8880GGATTCCAGAAATGTTGAGGGGACGGAGGAGTAGGGGATAAACAAGGATTCCTAGCCTGG8940AACCAGTGCCCAAGTCCTGAGTCTTCCAGGAGCCACAGGCAGCCTTAAGCCTGGTCCCCA9000TACACAGGCTGAAGTGGCAGTTCCAGCGGCTGTCCCTGCGGCAGAGGCTGAGGCCGAGGT9060GACGCTGCGGGAGCTCCAGGAAGCCCTGGAGGAGGAGGTGCTCACCCGGCAGAGCCTGAG9120CCGGGAGATGGAGGCCATCCGCACGGACAACCAGAACTTCGCCAGGTCGGGATCGGGGCC9180GGGGCCGGGGCCGGGATGCGGGCCGGTGGCAACCCTTGGCAGCCCCTCTCGTCCGGCCCG9240GACGGACTCACCGTCCTTACCTCCCCACAGTCAACTACGCGAGGCAGAGGCTCGGAACCG9300GGACCTAGAGGCACACGTCCGGCAGTTGCAGGAGCGGATGGAGTTGCTGCAGGCAGAGGG9360AGCCACAGGTGAGTCCCTCATGTGTCCCCTTCCCCGGAGGACCGGGAGGAGGTGGGCCGT9420CTGCTCCGCGGGGCGTGTATAGACACCTGGAGGAGGGAAGGGACCCACGCTGGGGCACGC9480CGCGCCACCGCCCTCCTTCGCCCCTCCACGCGCCCTATGCCTCTTTCTTCTCCTTCCAGC9540TGTCACGGGGGTCCCCAGTCCCCGGGCCACGGATCCACCTTCCCATGTAAGACCCCTCTC9600TTTCCCCTGCCTCAGACCTGCTGCCCATTCTGCAGATCCCCTCCCTGGCTCCTGGTCTCC9660CCGTCCAGATATAGGGCTCACCCTACGTCTTTGCGACTTTAGAGGGCAGAAGCCCTTTAT9720TCAGCCCCAGATCTCCCTCCGTTCAGGCCTCACCAGATTCCCTCCGGGATCTCCCTAGAT9780AACCTCCCCAACCTCGATTCCGCTCGCTGTCTCTCGCCCCACCGCTGAGGGCTGGGCTGG9840GCTCCGATCGGGTCACCTGTCCCTTCTCTCTCCAGCTAGATGGCCCCCCGGCCGTGGCTG9900TGGGCCAGTGCCCGCTGGTGGGGCCAGGCCCCATGCACCGCCGCCACCTGCTGCTCCCTG9960CCAGGGTACGTCCGGCTGCCCACGCCCCCCTCCGCCGTCGCGCCCCGCGCTCCACCCGCC10020CCGTGCCACCCGCTTAGCTGCGCATTTGCGGGGCTGGGCCCACGGCAGGAGGGCGGATCT10080TCGGGCAGCCAATCAACACAGGCCGCTAGGAAGCAGCCAATGACGAGTTCGGACGGGATT10140CGAGGCGTGCGAGTGGACTAACAACAGCTGTAGGCTGTTGGGGCGGGGGCGGGGCGCAGG10200GAAGAGTGCGGGCCCACCTATGGGCGTAGGCGGGGCGAGTCCCAGGAGCCAATCAGAGGC10260CCATGCCGGGTGTTGACCTCGCCCTCTCCCCGCAGGTCCCTAGGCCTGGCCTATCGGAGG10320CGCTTTCCCTGCTCCTGTTCGCCGTTGTTCTGTCTCGTGCCGCCGCCCTGGGCTGCATTG10380GGTTGGTGGCCCACGCCGGCCAACTCACCGCAGTCTGGCGCCGCCCAGGAGCCGCCCGCG10440CTCCCTGAACCCTAGAACTGTCTTCGACTCCGGGGCCCCGTTGGAAGACTGAGTGCCCGG10500GGCACGGCACAGAAGCCGCGCCCACCGCCTGCCAGTTCACAACCGCTCCGAGCGTGGGTC10560TCCGCCCAGCTCCAGTCCTGTGATCCGGGCCCGCCCCCTAGCGGCCGGGGAGGGAGGGGC10620CGGGTCCGCGGCCGGCGAACGGGGCTCGAAGGGTCCTTGTAGCCGGGAATGCTGCTGCTG10680CTGCTGCTGCTGCTGCTGCTGCTGGGGGGATCACAGACCATTTCTTTCTTTCGGCCAGGC10740TGAGGCCCTGACGTGGATGGGCAAACTGCAGGCCTGGGAAGGCAGCAAGCCGGGCCGTCC10800GTGTTCCATCCTCCACGCACCCCCACCTATCGTTGGTTCGCAAAGTGCAAAGCTTTCTTG10860TGCATGACGCCCTGCTCTGGGGAGCGTCTGGCGCGATCTCTGCCTGCTTACTCGGGAAAT10920TTGCTTTTGCCAAACCCGCTTTTTCGGGGATCCCGCGCCCCCCTCCTCACTTGCGCTGCT10980CTCGGAGCCCCAGCCGGCTCCGCCCGCTTCGGCGGTTTGGATATTTATTGACCTCGTCCT11040CCGACTCGCTGACAGGCTACAGGACCCCCAACAACCCCAATCCACGTTTTGGATGCACTG11100AGACCCCGACATTCCTCGGTATTTATTGTCTGTCCCCACCTAGGACCCCCACCCCCGACC11160CTCGCGAATAAAAGGCCCTCCATCTGCCCAAAGCTCTGGACTCCACAGTGTCCGCGGTTT11220GCGTTGTGGGCCGGAGCTCCGCAGCGGGCCAATCCGGAGGCGTGTGGAGGCGGCCGAAGG11280TCTGGGAGGAGCTAGCGGGATGCGAAGCGGCCGAATCAGGGTTGGGGGAGGAAAAGCCAC11340GGGGCGGGGCTTTGGCGTCCGGCCAATAGGAGGGCGAGCGGGCCACCCGGAGGCACCGCC11400CCCGCCCAGCTGTGGCCCAGCTGTGCCACCGAGCGTCGAGAAGAGGGGGCTGGGCTGGCA11460GCGCGCGCGGCCATCCTCCTTCCACTGCGCCTGCGCACGCCACGCGCATCCGCTCCTGGG11520ACGCAAGCTCGAGAAAAGTTGCTGCAAACTTTCTAGCCCGTTCCCCGCCCCTCCTCCCGG11580CCAGACCCGCCCCCCCTGCGGAGCCGGGAATTC11613(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 3182 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:GCCACAAGCCTCCACCCCAGCTGGTCCCCCACCCAGGCTGCCCAGTTTAACATTCCTAGT60CATAGGACCTTGACTTCTGAGAGGCCTGATTGTCATCTGTAAATAAGGGGTAGGACTAAA120GCACTCCTCCTGGAGGACTGAGAGATGGGCTGGACCGGAGCACTTGAGTCTGGGATATGT180GACCATGCTACCTTTGTCTCCCTGTCCTGTTCCTTCCCCCAGCCCCAAATCCAGGGTTTT240CCAAAGTGTGGTTCAAGAACCACCTGCATCTGAATCTAGAGGTACTGGATACAACCCCAC300GTCTGGGCCGTTACCCAGGACATTCTACATGAGAACGTGGGGGTGGGGCCCTGGCTGCAC360CTTGAACTGTCACCTGGAGTCAGGGTGGAAGGTGGAAGAACTGGGTCTTATTTCCTTCTC420CCCTTGTTCTTTAGGGTCTGTCCTTCTGCAGACTCCGTTACCCCACCCTAACCATCCTGC480ACACCCTTGGAGCCCTCTGGGCCAATGCCCTGTCCCGCAAAGGGCTTCTCAGGCATCTCA540CCTCTATGGGAGGGCATTTTTGGCCCCCAGAACCTTACACGGTGTTTATGTGGGGAAGCC600CCTGGGAAGCAGACAGTCCTAGGGTGAAGCTGAGAGGCAGAGAGAAGGGGAGACAGACAG660AGGGTGGGGCTTTCCCCCTTGTCTCCAGTGCCCTTTCTGGTGACCCTCGGTTCTTTTCCC720CCACCACCCCCCCAGCGGAGCCCATCGTGGTGAGGCTTAAGGAGGTCCGACTGCAGAGGG780ACGACTTCGAGATTCTGAAGGTGATCGGACGCGGGGCGTTCAGCGAGGTAGCGGTAGTGA840AGATGAAGCAGACGGGCCAGGTGTATGCCATGAAGATCATGAACAAGTGGGACATGCTGA900AGAGGGGCGAGGTGTCGTGCTTCCGTGAGGAGAGGGACGTGTTGGTGAATGGGGACCGGC960GGTGGATCACGCAGCTGCACTTCGCCTTCCAGGATGAGAACTACCTGTACCTGGTCATGG1020AGTATTACGTGGGCGGGGACCTGCTGACACTGCTGAGCAAGTTTGGGGAGCGGATTCCGG1080CCGAGATGGCGCGCTTCTACCTGGCGGAGATTGTCATGGCCATAGACTCGGTGCACCGGC1140TTGGCTACGTGCACAGGGACATCAAACCCGACAACATCCTGCTGGACCGCTGTGGCCACA1200TCCGCCTGGCCGACTTCGGCTCTTGCCTCAAGCTGCGGGCAGATGGAACGGTGCGGTCGC1260TGGTGGCTGTGGGCACCCCAGACTACCTGTCCCCCGAGATCCTGCAGGCTGTGGGCGGTG1320GGCCTGGGACAGGCAGCTACGGGCCCGAGTGTGACTGGTGGGCGCTGGGTGTATTCGCCT1380ATGAAATGTTCTATGGGCAGACGCCCTTCTACGCGGATTCCACGGCGGAGACCTATGGCA1440AGATCGTCCACTACAAGGAGCACCTCTCTCTGCCGCTGGTGGACGAAGGGGTCCCTGAGG1500AGGCTCGAGACTTCATTCAGCGGTTGCTGTGTCCCCCGGAGACACGGCTGGGCCGGGGTG1560GAGCAGGCGACTTCCGGACACATCCCTTCTTCTTTGGCCTCGACTGGGATGGTCTCCGGG1620ACAGCGTGCCCCCCTTTACACCGGATTTCGAAGGTGCCACCGACACATGCAACTTCGACT1680TGGTGGAGGACGGGCTCACTGCCATGGTGAGCGGGGGCGGGGAGACACTGTCGGACATTC1740GGGAAGGTGCGCCGCTAGGGGTCCACCTGCCTTTTGTGGGCTACTCCTACTCCTGCATGG1800CCCTCAGGGACAGTGAGGTCCCAGGCCCCACACCCATGGAACTGGAGGCCGAGCAGCTGC1860TTGAGCCACACGTGCAAGCGCCCAGCCTGGAGCCCTCGGTGTCCCCACAGGATGAAACAG1920CTGAAGTGGCAGTTCCAGCGGCTGTCCCTGCGGCAGAGGCTGAGGCCGAGGTGACGCTGC1980GGGAGCTCCAGGAAGCCCTGGAGGAGGAGGTGCTCACCCGGCAGAGCCTGAGCCGGGAGA2040TGGAGGCCATCCGCACGGACAACCAGAACTTCGCCAGTCAACTACGCGAGGCAGAGGCTC2100GGAACCGGGACCTAGAGGCACACGTCCGGCAGTTGCAGGAGCGGATGGAGTTGCTGCAGG2160CAGAGGGAGCCACAGCTGTCACGGGGGTCCCCAGTCCCCGGGCCACGGATCCACCTTCCC2220ATCTAGATGGCCCCCCGGCCGTGGCTGTGGGCCAGTGCCCGCTGGTGGGGCCAGGCCCCA2280TGCACCGCCGCCACCTGCTGCTCCCTGCCAGGGTCCCTAGGCCTGGCCTATCGGAGGCGC2340TTTCCCTGCTCCTGTTCGCCGTTGTTCTGTCTCGTGCCGCCGCCCTGGGCTGCATTGGGT2400TGGTGGCCCACGCCGGCCAACTCACCGCAGTCTGGCGCCCGCCCAGGAGCCGCCCGCGCT2460CCCTGAACCCTAGAACTGTCTTCGACTCCGGGGCCCCGTTGGAAGACTGAGTGCCCGGGG2520CACGGCACAGAAGCCGCGCCCACCGCCTGCCAGTTCACAACCGCTCCGAGCGTGGGTCTC2580CGCCCAGCTCCAGTCCTGTGACCGGGCCCGCCCCCTAGCGGCCGGGGAGGGAGGGGCCGG2640GTCCGCGGCCGGCGAACGGGGCTCGAAGGGTCCTTGTAGCCGGGAATGCTGCTGCTGCTG2700CTGGGGGGATCACAGACCATTTCTTTCTTTCGGCCAGGCTGAGGCCCTGACGTGGATGGG2760CAAACTGCAGGCCTGGGAAGGCAGCAAGCCGGGCCGTCCGTGTTCCATCCTCCACGCACC2820CCCACCTATCGTTGGTTCGCAAAGTGCAAAGCTTTCTTGTGCATGACGCCCTGCTCTGGG2880GAGCGTCTGGCGCGATCTCTGCCTGCTTACTCGGGAAATTTGCTTTTGCCAAACCCGCTT2940TTTCGGGGATCCCGCGCCCCCCTCCTACTTGCGCTGCTCTCGGAGCCCCAGCCGCTCCGC3000CCGCTTCGGCGGTTTGGATATTTATTGACCTCGTCCTCCGACTCGCTGACAGGCTACAGG3060ACCCCCAACAACCCCAATCCACGTTTTGGATGCACTGAGACCCCGACATTCCTCGGTATT3120TATTGTCTGTCCCCACCTAGGACCCCCACCCCCGACCCTCGCGAATAAAAGGCCCTCCAT3180CG3182(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:CACCTCTCTCTGCCGCTGGTGGAC24(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:CCTAGCGGCGCACCTTCCCGAATG24__________________________________________________________________________