Abstract:
This invention provides a procedure for authentication of plant and animal materials used as traditional Chinese medicine is described. This method amplifies and detects the discrete and species-specific RFLP patterns in the region of rDNA. The present invention offers a reliable and definite way to identify morphologically similar Chinese medicine, using a minute amount of biological samples. Its application in the authentication of American and Oriental ginsengs is illustrated in detail.

Description:
FIELD OF THE INVENTION 
     This invention is directed to the authentication of Chinese medicinal materials based upon RFLP patterns of the PCR-amplified rDNA. 
     BACKGROUND OF THE INVENTION 
     Traditional Chinese medicine refers to the medicinal materials and clinical application of such materials in the framework of the theoretical and empirical parameters cicumscribed by the Chinese people in the last 2-3 millennia. This medical system and many of the medicinal materials has spread to and adopted by other Oriental countries such as Japan and Korea and evolved into Oriental medicine in those countries. As a result, traditional Chinese medicine should not be limited to only the herbs and other natural products used in Chinese medicine, but also to Oriental medicine. Traditional Chinese medicine currently in mainland China also covers the practice and medicinal materials used by Tibetan, Mongolian and other ethnic minorities. 
     The herbs and other natural products (animals and minerals) used in Chinese medicine have been recorded in a) classical herbals, e.g. Bencao Gangmu () and Bencao Gangmu Shiyi () which recorded American ginseng (the two together contain about 2,500 items; b) pharmacopoeia, e.g. Pharmacopoeia of the People&#39;s Republic of China () which contains some 600 items; and c) treaties, e.g. Encyclopedia of Chinese Materia Medica (Zhongyao Dacidian ), which contain 5,767 items. 
     Traditionally the authentication of Chinese herbs relied upon morphological and histological inspection. In many cases, such as in the authentication of different ginseng species, and in the authentication of Acorus species, this method is unreliable. An effective program of authentication of Chinese herbs is essential and central issue in the healthy development of the herbal industry. It provides a necessary protection for consumers, minimises unfair business competition and prevents the health hazard of many adulterants. 
     In plant nuclear genome, genes for ribosomal RNA (rDNA) are normally clustered in an array of multiple tandemly repeated copies of the cistron of 18S-ITS1-5.8S-ITS2-28S (Hillis, D. M and Dixon, M. T., 1991, The Quarterly Review of Biology, 66: 411-453). The sequence separates the 18S and 5.8S rRNA genes is designated as ITS1 (Internal Transcribed Spacer 1) and the sequence between 5.8S and 28S is designated as ITS2. The coding regions of the three rDNA genes are highly conserved, whereas the sequence homology within the ITS1 and ITS2 regions are lower across the plant kingdom. Furthermore within a given individual organism or species, the rDNA sequence is usually very similar due to the homogenization of the sequence by gene conversion and crossing over. This invention takes advantage of these features of plant rDNA, and use PCR to amplify the DNA of ITS1-5.8S-ITS2 regions with the conserved DNA sequences flanking to the regions as primers, and explores the DNA polymorphism in different plant species within the ITS1-5.8S-ITS2 region as a means of authentication. 
     The roots of Panax quinquefolius (American Ginseng) and P. ginseng (Oriental ginseng) are important herbal medicinal materials widely applied in the Orient as tonic, prophylactic and anti-aging agents. In recent years the American ginseng, cultivated mainly in Wisconsin, USA, and British Colombia and Ontaria, Canada, enjoys increasingly popularity as a health food in Western countries. The ginseng trade is a big industry, in 1993 Hong Kong imports more than HK$1,500 million worth of American and Oriental ginsengs. The retail price of cultivated American ginseng is usually much more expensive than that of cultivated Oriental ginseng produced in China, and that prompts wild-spread practice of disguising Orient ginseng as American ginseng by dishonest merchants. Tremendous financial incentive is also responsible for the imitation or adulteration of ginsengs with some herbal products including several poisonous plants that bear morphological similarity with ginsengs. The two ginsengs also have different medical values and potency. 
     Both American and Oriental ginsengs, together with several important Chinese medicines including sanchi (P. notoginseng), belong to the genus of Panax in the family of Araliaceae. American ginseng and Oriental ginseng have similar morphological appearance. Furthermore many commercial ginseng products exist in the forms of powder or shredded slice, rendering their authentication by morphological and histological methods difficult and unpractical. In recent years, techniques have been developed to authenticate ginseng samples by examination of their ginsenoside profiles (Lang, Z., Lou, W S. and But, P P H, 1993, J. Clin. Pharm. Sci., 2:133-143). However, the application of chemical analysis may be limited as the amount of ginsenosides are significantly affected by many environmental factors such as the storage condition, the freshness of the products and the different post-harvest processing. In addition, the chemical method demands large quantity of materials for proper analysis. 
     SUMMARY OF THE INVENTION 
     This invention is based upon the DNA polymorphism in the ITS1-5.8S-ITS2 region of rDNA. Accordingly genomic DNA was isolated and the ITS regions of rDNA was selectively amplified using pairs of primers that correspond to the consensus DNA sequence within the rDNA. The resultant PCR products were then subject to the fragmentation by selected restriction endonuclease to generate, after electrophoresis, discrete and species-specific RFLP patterns. Application of this invention to authenticate American ginseng from Oriental ginseng and several common adulterants are detailed as examples. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1. ITS1-5.8S-ITS2 DNA sequence of P. quinquefolius (SEQ ID NO.2). 
     FIG. 2. (A). ITS1-5.8S-ITS2 DNA sequence of P. ginseng (SEQ ID NO.2). (B) G1 DNA is the same as the sequnece of P. ginseng; (SEQ ID NO.3) the IC DNA is the sequence from a Russian cultivar of P. ginseng. The four variables between the two are underlined (SEQ ID NO.4). 
     FIG. 3. ITS1-5.8S-ITS2 DNA sequence of P. japonicus (SEQ ID NO.5). 
     FIG. 4. ITS1-5.8S-ITS2 DNA sequence of P. notoginseng (SEQ ID NO.6). 
     FIG. 5. ITS1-5.8S-ITS2 DNA sequence of P. trifolium (SEQ ID NO.7). 5&#39; AGCCATCCTCGCTGCCCGCCACAC 3&#39; (SEQ ID NO.8) 3&#39; ACTCGCCGTTACTAGGGGAA 5&#39; (SEQ ID NO.9) 
     FIG. 6. The primers, 18d and 28cc, used to amplify ITS1-5.8S-ITS2 regions of the plant rDNA genes. 
     FIG. 7. The Hinf1 RFLP patterns of the ITS1-5.8S-ITS2 region for P. quinquefolius (American Ginseng), P. ginseng (Oriental ginseng) and their adulterants. Lane M, DNA size marker; lane 1, P. quinquefolius (American Ginseng); lane 2, P. ginseng (Oriental ginseng); lane 3, adulterant M. jalapa; lane 4, adulterant P. acinosa. Lane 5 to 9 represent the RFLP patterns of the mixed samples of American Ginseng and Oriental ginseng in different ratio. Lane 5, American Ginseng and Oriental ginseng in the ratio of 9:1; Lane 6, in the ratio of 7:3; Lane 7, in the ratio of 1:1; Lane 8, in the ratio of 3:7; and Lane 9, in the ration of 1:9. Two fragments of 0.1 kb and 0.06 kb present in American ginseng but are absent from Oriental ginsen, while a fragment of 0.17 kb present in Oriental ginseng but absent from American ginseng. M. jalapa contains two characteristic fragments of 0.4 kb and 0.3 kb in size. The plant DNA were extracted using CTAB (cetyl triethylammonium bromide) method as described in Experimental Details and their rDNA ITS regions were amplified by PCR using the primers specified in FIG. 6. The resultant PCR products were subject to restriction of Hinf1, fractionated on SDS-PAGE and silver stained. 
     FIG. 8. The Taq1 RFLP patterns of the ITS1-5.8S-ITS2 region for P. quinquefolius (American Ginseng), P. ginseng (Oriental ginseng) and their adulterants. Lane M, DNA size marker; lane 1, P. quinquefolius (American Ginseng); lane 2, P. ginseng (Oriental ginseng); lane 3, adulterant M. jalapa; lane 4, adulterant P. acinosa. Lane 5 to 9 represent the RFLP patterns of the mixed samples of American Ginseng and Oriental ginseng in different ratio. Lane 5, American Ginseng and Oriental ginseng in the ratio of 9:1; Lane 6, in the ratio of 7:3; Lane 7, in the ratio of 1:1; Lane 8, in the ratio of 3:7; and Lane 9, in the ration of 1:9. A 0.18 kb fragment is present in American ginseng but absent from Oriental ginseng; while a 0.27 kb fragment is present in Oriental ginseng but absent from American ginseng. Both adulternats contains characteristic fragments of 0.28 kb and 0.4 kb in size. The plant DNA were extracted using CTAB method and their rDNA ITS regions were amplified by PCR using the primers specified in FIG. 6. The resultant PCR products were subject to restriction of Taq1, fractionated on SDS-PAGE and silver stained. 
     FIG. 9. The Sau3A1 RFLP patterns of the ITS1-5.8S-ITS2 region for P. quinquefolius (American Ginseng), P. ginseng (Oriental ginseng) and their adulterants. Lane M, DNA size marker; lane 1, P. quinquefolius (American Ginseng); lane 2, P. ginseng (Oriental ginseng); lane 3, adulterant M. jalapa; lane 4, adulterant P. acinosa. Lane 5 to 9 represent the RFLP patterns of the mixed samples of American Ginseng and Oriental ginseng in different ratio. Lane 5, American Ginseng and Oriental ginseng in the ratio of 9:1; Lane 6, in the ratio of 7:3; Lane 7, in the ratio of 1:1; Lane 8, in the ratio of 3:7; and Lane 9, in the ration of 1:9. When Compared to American ginseng, Oriental ginseng contains three additional DNA fragments at the size of 0.7 kb, 0.35 kb and 0.17 kb. The 0.35 kb fragment is absent from the two adulterants. On the other hand, in comparison of ginsengs, M. jalapa contains three additional fragment of 0.3 kb, 0.07 kb and 0.05 kb in size, and P. acinosa contains an additional fragment of 0.05 kb in size. The plant DNA were extracted using CTAB method and their rDNA ITS regions were amplified by PCR using the primers specified in FIG. 6. The resultant PCR products were subject to restriction of Sau3A1, fractionated on SDS-PAGE and silver stained. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     This invention provides an isolated nucleic acid molecule having the ITS1-5.8S-ITS2 sequence. This invention also provides isolated nucleic acid molecules having the ITS1-5.8S-ITS2 sequence from plant cultivers. It is expected even within the same species, there will be a minor variation between them. Such variation may be up to 1% or less. It is intent of this invention to cover this variation and it is easily appreciated by a person of ordinary skill in the art that the claimed invention work the same with this variation. 
     This invention provides an isolated nucleic acid molecule having the ITS1-5.8S-ITS2 sequence of P. quinquefolius. In an embodiment, this sequence is as set forth in FIG. 1. 
     In an embodiment, the ITS1-5.8S-ITS2 sequence is the DNA sequence between the oligonucleotide primers 18d and 28cc. 
     This invention provides an isolated nucleic acid molecule having the TS1-5.8S-ITS2 sequence of P. ginseng. In an embodiment, the sequence is as set forth in FIG. 2A or B. 
     This invention provides an isolated nucleic acid molecule having the ITS1-5.8S-ITS2 sequence of P. japonicus. In a perferred embodiment, the sequence is as set forth in FIG. 3. 
     This invention provides an isolated nucleic acid molecule having the ITS1-5.8S-ITS2 sequence of P. notoginseng. In a perferred embodiment, the sequence is as set forth in FIG. 4. 
     This invention provides an isolated nucleic acid molecule having the ITS1-5.8S-ITS2 sequence of P. trifolium. In an embodiment, the sequence is as set forth in FIG. 5. 
     This invention also provides a method for authenticating the identity of herbs comprising the following steps: (a) extracting rDNA from a herb sample with known identity determined by traditional means; (b) amplifying the ITS1-5.8S-ITS2 region of the extracted rDNA using oligonucleotide primers that are conserved across plant kingdom and that flank to the ITS1-5.8S-ITS2 region by polymerase chain reaction; (c) digesting the amplified ITS1-5.8S-ITS2 region with appropriate restriction endonucleases to generate restriction fragments; and (d) separating the restriction fragments resulted from step (c) to generate profiles and comparing these profiles with the known profiles from an authenticated sample with the same identity, wherein similar profiles confirms the identity of the herbal sample. 
     This invention further provides a method for identifying a herbal material comprising the following steps: (a) extracting rDNA from the herbal material; (b) amplifying the ITS1-5.8S-ITS2 region of the extracted rDNA using oligonucleotide primers that are conserved across plant kingdom and that flank to the ITS1-5.8S-ITS2 region by polymerase chain reaction; (c) digesting the amplified ITS1-5.8S-ITS2 region with appropriate restriction endonucleases to generate restriction fragments; and (d) separating the restriction fragments resulted from step (c) to generate a profile of the herbal material and comparing this profile with known profiles from different herbs, wherein the showing of similar profile with a known herb identifies the herbal material. 
     This invention provides an isolated nucleic acid molecule having the ITS1-5.8S-ITS2 sequence. In an embodiment, the sequence is from an animal. 
     This invention also provide a method for authenticating the identity of an animal traditional Chinese medicine comprising the following steps: (a) extracting rDNA from an animal traditional Chinese medicine sample with known identity determined by traditional means; (b) amplifying the ITS1-5.8S-ITS2 region of the extracted rDNA using oligonucleotide primers that are conserved across animal kingdom and that flank to the ITS1-5.8S-ITS2 region by polymerase chain reaction; (c) digesting the amplified ITS1-5.8S-ITS2 region with appropriate restriction endonucleases to generate restriction fragments; and (d) separating the restriction fragments resulted from step (c) to generate profiles and comparing these profiles with the known profiles from an authenticated sample with the same identity, wherein similar profiles confirms the identity of the animal traditional Chinese medicine. 
     Finally, this invention provides a method for identifying an animal traditional Chinese medicine comprising the following steps: (a) extracting rDNA from the Chinese medicine; (b) amplifying the ITS1-5.8S-ITS2 region of the extracted rDNA using oligonucleotide primers that are conserved across animal kingdom and that flank to the ITS1-5.8S-ITS2 region by polymerase chain reaction; (c) digesting the amplified ITS1-5.8S-ITS2 region with appropriate restriction endonucleases to generate restriction fragments; and (d) separating the restriction fragments resulted from step (c) to generate a profile of the herbal material and comparing this profile with known profiles from different known animal Chinese medicine sample, wherein the showing of similar profile with a known animal sample identifies the animal Chinese medicine. 
     In order to facilitate an understanding of the following examples, certain frequently occurring methods and/or terms are best described in Sambrook, et al. (Sambrook, et al. (1989)). 
     This invention will be better understood by reference to the Experimental Details section which follows, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter. 
     EXPERIMENTAL DETAILS 
     Extraction of plant DNA. Dried roots of P. quinquefolius were from Canada, P. ginseng from China; M. jalapa L and P. acinosa Roxb from Hong Kong. The dried samples were rinsed with 70% ethanol and then distilled water to remove surface contaminants. The samples were then ground into fine powder in liquid nitrogen by a mortar and pestle. Powders of P. quinquefolius and P. ginseng were mixed in different proportion of 9:1, 7:3, 1:1, 3:7 and 1:9 in the mixed sampling assay. Ground sample powder was added into 12 vol. of 1×CTAB extraction buffer  50 mM Tris-HCl, pH 8.0, 0.7M NaCl, 10 mM EDTA, 1% cetyl triethylammonium bromide (CTAB), 20 mM 2-mercaptoethanol! and incubated for 30 min at 56° C. with occasional shaking. The CTAB extraction buffer was pre-warmed to 56° C. The mixture was then cooled down to room temperature and extracted with an equal volume of chloroform/isoamyl alcohol (24:1). After centrifugation at 13,000×g for 10 min., the aqueous phase was extracted with 0.1 vol. of 10% CTAB solution. It was then extracted again with an equal volume of chloroform/isoamyl alcohol (24:1). The aqueous phase was collected, and added with an equal volume of 1×CTAB precipitation buffer  50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% CTAB!. After standing at room temperature for 1 hour, the solution was centrifuged at 13,000×g for 15 min. The resultant pellet was resuspended in 400 ul 1M NaCl, added with 800 ul of cooled absolute ethanol and stored at -20 C. overnight. The suspension was centrifuged at 13,000×g for 10 min. and the pellet was washed with 70% ethanol twice. It was then dried and resuspended in 50 ul TE buffer  10 mM Tris-HCl, pH 8.0, 1 mM EDTA!. Further purification by CsCl gradient ultracentrifugation is optional. 
     Amplification of rDNA. The plant rDNA was amplified using a pair of primers 18d and 28cc (Hillis, D. M and Dixon, M. T., 1991, The Quarterly Review of Biology, 66: 411-453), which correspond to the conserved regions of plant 18S and 28S respectively. 
     18d: CACAC CGCCC GTCGC TCCTA CCGA (SEQ ID NO.10) 
     28cc: ACTCG CCGTT ACTAG GGGAA (SEQ ID NO.11) 
     The reaction was performed in a 50 ul mixture containing 1 ng plant DNA, 1×Taq buffer  10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.001% gelatin!, 0.2 mM dNTPs, 1 uM of each primer and 1 unit of Taq polymerase. Reaction mixtures were overlaid with mineral oil and reaction was carried out in a Thermolyne thermocycler. Initial template denaturation was programmed at 94 C., 5 min. It was then subjected to 35 cycles of 94 C. for 1 min.; 60 C. for 1 min.; 72 C. for 2 min., and with a final extension of 72 C. for 10 min. After the reaction, the products were resolved by a 1.4% TBE agarose gel. 
     Sequencing ITS1-5.8S-ITS2. The ITS1-5.8S-ITS2 region of plant rDNA was sequenced in both strands using a set of primers on the conserved regions of the flanking 18S and 28S rDNA. The sequences of the primers used were as follows (Hillis, D. M and Dixon, M. T., 1991, The Quarterly Review of Biology, 66: 411-453): 
     18d: CACAC CGCCC GTCGC TCCTA CCGA (SEQ ID NO.12) 
     5.8c: TTGCG TTCAA AGACT CGATG (SEQ ID NO.13) 
     5.8d: AACCA TCGAG TCTTT GAACG CA (SEQ ID NO.14) 
     28cc: ACTCG CCGTT ACTAG GGGAA (SEQ ID NO.15) 
     SequiThermTM Cycle Sequencing Kit (Epicentre, Madison, Wis.) was used to direct-sequence the PCR-amplified rDNA containing ITS-5.8S-ITS2. The sequencing procedure was done according to the manufacturers&#39;s instruction. The products were resolved onto a standard 8% polyacrylamide sequencing gel. 
     Determination of Restriction Fragment Length Polymorphisms in the ITS1-5.8S-ITS2 region. Plant rDNA amplified using primers 18d and 28cc was purified using Geneclean kit (Bio101, Inc.) and digested with selected restriction endonucleases TaqI, Sau3AI or HinfI. 1.5 ug rDNA was used for each digestion in a volume of 50 ul. For TaqI. a buffer of 100 mM NaCL, 10 mM Tris-HCl, 10 mM MgCl2, 10 mM 2-mercaptoethanol, pH 8.4, supplemented with 100 ug/ml bovine serum albumin was used and the digestion was carried out at 65 C. for 4 hours. For Sau3AI, a buffer of 100 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, pH 7.3, supplemented with 100 ug/ml bovine serum albumin was used. For HinfI a buffer of 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9 was used. The reaction for both Sau3AI and HinfI was incubated at 37 C. for 4 hours. The products were then purified by phenol: chloroform: isoamyl alcohol (25:24:1) and dissolved in 20 ul distilled water. They were resolved in a 5% PAGE and detected with Bio-Rad silver stain kit (Bio-Rad, Ltd). 
     ADVANTAGES OF THE INVENTION 
     The method of authentication of ginsengs described above are expected to be suitable for authentication of other herbal traditional Chinese medicine as well, considering the existence of the conserved DNA sequences flanking to the ITS1-5.8S-ITS2 region in plant kingdom and the variation in the ITS1 and ITS2 regions among different plant species. In comparison to the existing procedures of authentication of traditional Chinese medicine, this invention provides the following advantages: 
     a. the authentication results are reliable and reproducible, and are not affected by the physical forms and age of the plant samples; 
     b. it is a method of high sensitivity: microgram sample is sufficient; 
     c. more than one distinctive profiles with different enzymatic digestion can be produced and that makes the interpretation of results straightforward; 
     d. the contamination of other biological materials can be detected. 
     REFERENCE 
     Hillis, D. M and Dixon, M. T. (1991) Ribosomal DNA: molecular evolution and phylogenic infrrence. Quar. Rev. Biol., 66: 411-453. 
     Lang, Z., Lou, W S. and But, P P H. (1993) High performance liquid chromatographical analysis of ginsenosides in Panax ginseng and P. notoginseng. J. Clin. Pharm. Sci., 2:133-143. 
     Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual. 
     
         __________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 15(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 874 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:ACTCGCCGTTACTAGGGGAATCCTTGTAAGTTTCTTTTCCTCCGCTTATTGATATGCTTA60AACTCAGCGGGTAGTCCCGCTGACCTGGGGTCGCGGTCGGAGCGCACGTCGAGGAGCGCA120ACAGGGTCATGAGAGCTTTTGCTGGCGACGGGTCACCGCACGACATGAGAAGAGGGCTTT180TTACAACCACCACTTGTCGTGACGTCCATCGCCAAGGACTCGCATTTGGGCCAACCGCGC240GGTGAGACACGGGAGGCCATTATCCGCCCCTCCGCCTCGACTCCCGCAAAGGAGTGATGG300GTTGGGGGGCGACGCGATGCGTGAACGCCCAGGCAGACGTGCCCTCGGCCTAATGGCTTC360GGGCGCAACTTGCGTTCAAAGACTCGATGGTTCACGGGATTCTGTAATTCACACCAAGTA420TCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGT480TTGTGTTTTAGAAAGACGCTTCCGCCGCCCGCAAACGGGGGGGACGCGTGCAGTTCAGTT540TGATTTCCTTGGCGCATTCCGCGCCGGGGGGTCGTTGTTCGGACGAGAGCCACCCAAGGG600TGGTCCCCGACCATGGGTTTGCAACTTGGGGAGCTTGCGCACCCCTCGTCCCTCACCCGG660TATTGTAACGTGTTCGCGGGTCGTTCTGCTATGCAGGTTTCGACAATGATCCTTCCGCAG720GTTCACCTACGGAAACCTTGTTACGACTTCTCCTTCCTCTAAATGATAAGGTTCAGTGGA780CTTCTTTCGACGTCGCGGGCAGCGAACCGCCCACGTCGCCGCAATCCGAACACTTCACCG840GACCATTCAATCGGTAGGAGCGACGGGCGGTGTG874(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 875 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:ACTCGCCGTTACTAGGGGAATCCTTGTAAGTTTCTTTTCCTCCGCTTATTGATATGCTTA60AACTCAGCGGGTAGTCCCGCTGACCTGGGGTCGCGGTCGGAGCGCACGTCGAGGACGGCG120CAACAGGGTCATGAGAGCTTTTGCTGGCGACGGGTCACCGCACGACATGAGAAGAGGGCT180TTTTACAACCACCACTTGTCGTGACGTCCATCGCCAAGGACTCGCATTTGGGCCAACCGC240GCGGTGAGACACGGGAGGCCATTATCCGCCCCTCCGCCTCAACTCCCGCAAGGGAGTGAT300GGGTTGGGGGGCGACGCGATGCGTGACGCCCAGGCAGACGTGCCCTCGGCCTAATGGCTT360CGGGCGCAACTTGCGTTCAAAGACTCGATGGTTCACGGGATTCTGCAATTCACACCAAGT420ATCGCATTTCGCTACGTTCTTCATCGATGCGAGACGCGAGATATCCGTTGTCGAGAGTCG480TTTGTGTTTTAGAAAGACGCTTCCGCCGCCCGCAAACGGGGGGGACGCGTGCAGTTCAGT540TTGATTTCCTTGGCGCATTCCGCGCCGGGGGGTCGTTGTTCGGACGAGATCCACCCAAGG600GTGGTCCCCGACCATGGGTTTGCAACTTGGGGAGCTTGCGCACCCCTCGTCCCTCACCCG660GTATTGTAACGTGTTCGCGGGTCGTTCTGCTATGCAGGTTTCGACAATGATCCTTCCGCA720GGTTCACCTACGGAAACCTTGTTACGACTTCTCCTTCCTCTAAATGATAAGGTTCAGTGG780ACTTCTTTCGACGTCGCGGGCAGCGAACCGCCCACGTCGCCGCAATCCGAACACTTCACC840GGACCATTCAATCGGTAGGAGCGACGGGCGGTGTG875(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 875 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:ACTCGCCGTTACTAGGGGAATCCTTGTAAGTTTCTTTTCCTCCGCTTATTGATATGCTTA60AACTCAGCGGGTAGTCCCGCTGACCTGGGGTCGCGGTCGGAGCGCACGTCGAGGACGGCG120CAACAGGGTCATGAGAGCTTTTGCTGGCGACGGGTCACCGCACGACATGAGAAGAGGGCT180TTTTACAACCACCACTTGTCGTGACGTCCATCGCCAAGGACTCGCATTTGGGCCAACCGC240GCGGTGAGACACGGGAGGCCATTATCCGCCCCTCCGCCTCAACTCCCGCAAGGGAGTGAT300GGGTTGGGGGGCGACGCGATGCGTGACGCCCAGGCAGACGTGCCCTCGGCCTAATGGCTT360CGGGCGCAACTTGCGTTCAAAGACTCGATGGTTCACGGGATTCTGCAATTCACACCAAGT420ATCGCATTTCGCTACGTTCTTCATCGATGCGAGACGCGAGATATCCGTTGTCGAGAGTCG480TTTGTGTTTTAGAAAGACGCTTCCGCCGCCCGCAAACGGGGGGGACGCGTGCAGTTCAGT540TTGATTTCCTTGGCGCATTCCGCGCCGGGGGGTCGTTGTTCGGACGAGATCCACCCAAGG600GTGGTCCCCGACCATGGGTTTGCAACTTGGGGAGCTTGCGCACCCCTCGTCCCTCACCCG660GTATTGTAACGTGTTCGCGGGTCGTTCTGCTATGCAGGTTTCGACAATGATCCTTCCGCA720GGTTCACCTACGGAAACCTTGTTACGACTTCTCCTTCCTCTAAATGATAAGGTTCAGTGG780ACTTCTTTCGACGTCGCGGGCAGCGAACCGCCCACGTCGCCGCAATCCGAACACTTCACC840GGACCATTCAATCGGTAGGAGCGACGGGCGGTGTG875(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 870 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:ACTCGCCGTTACTAGGGGAATCCTTGTAAGTTTCTTTTCCTCCGCTTATTGATATGCTTA60AACTCAGCGGGTAGTCCGGCCTGACCTGGGGTCGCGGTCGGAGCGCACGTCGAGGACGGC120GCAACAGGGTCATGAGAGCTTTTGCTGGCGACGGGTCACCGCACGACATGAGAAGAGGGC180TTTTTACAACCACCACTTGTCGTGACGTCCATCGCCAAGGACTCGCATTTGGGCCAACCG240CGCGGTGAGACACGGGAGGCCATTATCCGCCCCTCCGCCTCAACTCCCGCAAGGGAGTGA300TGGGTTGGGGGGCGACGCGATGCGTGACGCCCAGGCAGACGTGCCCTCGGCCTAATGGCT360TCGGGCGCAACTTGCGTTCAAAGACTCGATGGTTCACGGGATTCTGCAATTCACACCAAG420TATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTC480GTTTGTGTTTTAGAAAGACGCTTCCGCCGCCCGCAAACGGGGGGGACGCGTGCAGTTCAG540TTTGATTTCCTTGGCGCATTCCGCGCCGGGGGGTCGTTGTTCGGACGAGATCCACCCAAG600GGTGGTCCCCGACCATGGGTTTGCAACTTGGGGAGCTTGCGCACCCCTCGTCCCTCACCC660GGTATTGTAACGTGTTCGCGGGTCGTTCTGCTATGCAGGTTTCGACAATGATCCTTCCGC720AGGTTCACCTACGGAAACCTTGTTACGACTTCTCCTTCCTCTAAATGATAAGGTTCAGTG780GACTTCTTTCGACGTCGCGAGCAGCGAACCGCCCACGTCGCCGCAATCCGAACACTTCAC840CGGACCATTCAATCGGTAGGAGCGACGGGG870(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 875 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:ACTCGCCGTTACTAGGGGAATCCTTGTAAGTTTCTTTTCCTCCGCTTATTGATATGCTTA60AACTCAGCGGGTAGTCCCGCCTGACCTGGGGTCGCGGTCGGAGCGCACGTCGAGGACGGC120GCAACAGGGTCATGAGAGCTTTTGTTGGCGAAGGGTCACCGCACGACATGAGAAGAGGGC180TTTTTACAACCACCACTTGTCGTGACGTCCATCGCCAAGGACTCGCATTTGGGCCAACCG240CACGGTGAGACACGGGAGGCCAATATCCGCCCCTCCGCCTCGACTCCCGCAAGGGAGTGA300TGGGTTGGGGGGCGACGCGATGCGTGAACGCCCAGGCAGACGTGCCCTCGGCCTAATGGC360TTAGGGCGCAACTTGCGTTCAAAGACTCGATGGTTCACGGGATTCTGCAATTCACACCAA420GTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGT480CGTTTGTGTTTTAGAAAGACGCTTCCGCCGCCCGCAAATGGGGGGGACGCGTGCAGTTCA540GTTTGATTTCCTTGGCACATTCCGCGCCGGGGGGTCGTTGTTCGGACGAGATCCACCAAG600GGTGTCCCCGACCATGGGTTTGCAACTTGGGGAGCTTGCGCACGCCTCGTCCCTCACCCG660GTATTGTAACGTGTTCACGGGTCGTTCTGCTATGCAGGTTTCGACAATGATCCTTCCGCA720GGTTCACCTACGGAAACCTTGTTACGACTTCTCCTTCCTCTAAATGATAAGGTTCAGTGG780ACTTCTTTCGACGTCGCGGGCAGCGAACCGCCCACGTCGCCGCAATCCGAACACTTCACC840GGACCATTCAATCGGTAGGAGCGACGGGCGGTGTG875(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 876 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:ACTCGCCGTTACTAGGGGAATCCTTGTAAGTTTCTTTTCCTCCGCTTATTGATATGCTTA60AACTCAGCGGGTAGTCCCGCTGACCTGGGGTCGCGGTCGGAGCGCACGTCGAGGACGGCG120CAACAGGGTCATGAGAGCTTTTGCTGGCGACGGGTCACCGCACGACATGAGAAGAGGGCT180TTTTACAACCACCACTTGTCGTGACGTCCATCGCCAAGGACTCGCATTTGGGCCAACCGC240GCGGTGAGACACGGGAGGCCATTATCCGCCCCTCCGCCTCGACTCCCGCAAAGGAGTGAT300GGGTTGGGGGGCGACGCGATGCGTGAACGCCCAGGCAGACGTGCCCTCGGCCTAATGGCT360TCGGGCGCAACTTGCGTTCAAAGACTCGATGATTCACGGGATTCTGCAATTCACACCAAG420TATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTC480GTTTGTGTTTTAGAAAGACGCTTCCGCCGCCCGCAAACGGGGGGGACGCGTGCAGTTCAG540TTTGATTTCCTTGGCGCATTCCGCGCCGGGGGGTCGTTGTTCGGACGAGAGCCACCCAAG600GGTGGTCCCCGACCATGGGTTTGCAACTTGGGGAGCTTGCGCACCCCTCGTCCCTCACCC660GGTATTGTAACGTGTTCGCGGGTCGTTCTGCTATGCAGGTTTCGACAATGATCCTTCCGC720AGGTTCACCTACGGAAACCTTGTTACGACTTCTCCTTCCTCTAAATGATAAGGTTCAGTG780GACTTCTTTCGACGTCGCAGGCAGCGAACCGCCCACGTCGCCGCAATCCGAACACTTCAC840CGGACCATTCAATCGGTAGGAGCGACGGGCGGTGTG876(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 875 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:ACTCGCCGTTACTAGGGGAATCCTTGTAAGTTTCTTTTCCTCCGCTTATTGATATGCTTA60AACTCAGCGGGTAGTCCCGCCTGACCTGGGGTCGCGGTCGGAGCGCGCGTCGGGGACGGC120GCAACAGGGTCGTGAGAGCCTTTGCCGGCGACGGGTCACCGCACGACTTGAGAAGAGGGC180TTTTTACAACCACCACTTGTCGTGACGTCCGTCGCCGAGGACTCGCATTTGGGCCAACCG240CGCGGTTAGACACGGGAGGCCAATATCCGCCCCTCCGCCTCGACTCCCGTAAGGGAGTGA300TGGGTTGGGGGGCGACGCGATGCGTGACGCCCAGGCAGACGTGCCCTCGGCCTAATGGCT360TAGGGCGCAACTTGCGTTCAAAGACTCGATGGTTCACGGGATTCTGCAATTCACACCAAG420TATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTC480GTTTGTGTTTTAGAAAGACGCTTCCGCCGCCCGCAAACGGGGGGGACGCGTGCAGTTCAG540TTTGATTTCCTTGGCGCATTCCGCGCCGGGGGGTCGTTGTTCGGACGGGGAGCACCCGGG600GGCGGCCCCCGACCATGGGTTCGGAACTTGGGGGGCTTGCGCACCCTTCGTCCCTCACCC660GGTGTTGAAACGTGTTCGCGGGTCGTTCTGCTGTGCAGGTTTCGACAATGATCCTTCCGC720AGGTTCACCTACGGAAACCTTGTTACGACTTCTCCTTCCTCTAAATGATAAGGTTCAGTG780GACTTCTTTCGACGTCGCGGCAGCGAACCGCCCACGTCGCCGCAATCCGAACACTTCACC840GGACCATTCAATCGGTAGGAGCGACGGGCGGTGTG875(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:AGCCATCCTCGCTGCCCGCCACAC24(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:ACTCGCCGTTACTAGGGGAA20(2) INFORMATION FOR SEQ ID NO:10:(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:10:CACACCGCCCGTCGCTCCTACCGA24(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:ACTCGCCGTTACTAGGGGAA20(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:CACACCGCCCGTCGCTCCTACCGA24(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:TTGCGTTCAAAGACTCGATG20(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:AACCATCGAGTCTTTGAACGCA22(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:ACTCGCCGTTACTAGGGGAA20__________________________________________________________________________