Abstract:
The invention relates to regulators of UCP3 gene transcription, including novel UCP3 transcriptional promoters. UCP3 promoters are used in diagnosis and pharmaceutical development. In particular, transfected cells comprising UCP3 transcriptional regulators operably linked to a reporter are used in high-throughput pharmaceutical screens.

Description:
This is a divisional application under 35 U.S.C. 120 of U.S. Ser. No. 08/948,277, filed Oct. 9, 1997, now U.S. Pat. No. 5,849,581, which is incorporated herein by reference. 
    
    
     INTRODUCTION 
     1. Field of the Invention 
     The field of this invention is the transcriptional promoter of the UCP3 gene and its use in drug screening. 
     2. Background 
     A mitochondrial protein called uncoupling protein (UCP1) is thought to play an important role in the body&#39;s regulation of energy utilization. Such regulation provides wide spread physiological controls including body weight, appetite, glucose metabolism, temperature, immune responses, etc. Mechanistically, UCP1 is thought to create a pathway that allows dissipation of the proton electrochemical gradient across the inner mitochondrial membrane in brown adipose tissue, without coupling to any other energy consuming process (for review, see Nicholis &amp; Locke (1984) Physiol Rev 64, 1-64). Unfortunately, the role of UCP1 in physiologies such as body weight regulation in large adult mammals such as people, cattle, pigs, etc. is likely to be limited, since there is little brown adipose tissue in such animals. 
     UCP2 is a second, related uncoupling protein that is much more widely expressed in large adult mammals (see, e.g. Fleury et al. (1997) Nature Genetics 15, 269-272 and Tartaglia et al. (1996) WO96/05861). Consistent with a role in the regulation of energy utilization generally, and in diabetes and obesity in particular, the UCP2 gene is upregulated in response to fat feeding and maps to regions of the human and mouse genomes linked to hyperinsulinaemia and obesity. Accordingly, upregulators of this gene hold great therapeutic promise for these diseases. To provide regulators of UCP2 gene expression, we cloned the endogenous promoter of the human UCP2 gene and identified various deletion mutants having transcriptional regulatory activity (U.S. Ser. No. 08/846,012, filed Apr. 25, 1997). 
     UCP3 is a third, related uncoupling protein also widely expressed in large adult mammals. Accordingly, upregulators of this gene hold great therapeutic promise for diseases such as hyperinsulinaemia and obesity. To provide regulators of UCP3 gene expression, we have cloned the endogenous promoter of natural UCP3 genes and identified various deletion mutants having transcriptional regulatory activity. 
     SUMMARY OF THE INVENTION 
     The invention provides methods and compositions relating to the UCP3 gene transcriptional promoter. The compositions include recombinant regulators of gene expression comprising the UCP3 promoter of at least one of SEQ ID NOS:1 and 2, or a deletion mutant thereof at least 50 bp in length having cis transcriptional regulatory activity. Exemplary such deletion mutants comprise at least one of SEQ ID NO:1, bases 411-460, bases 461-510, bases 401-563, bases 319-326, bases 98-104, bases 49-56, bases 49-104 and bases 547-554. In preferred embodiments, the regulators comprise at least one of a GC/SP1, GH-TRE and PR/GR binding site. In further embodiments, the regulators comprise a 5&#39; untranslated UCP3 gene exon. Frequently, the regulators may further comprise a UCP3 or non-UCP3 core promoter operatively joined to said mutant. 
     The invention also provides hybridization probes and replication/amplification primers having a hitherto novel UCP3 specific sequence contained in SEQ ID NO:1 or 2 (including its complement and analogs and complements thereof having the corresponding sequence, e.g . in RNA) and sufficient to effect specific hybridization thereto (i.e. specifically hybridize with the corresponding SEQ ID NO:1 or 2 in the presence of genomic DNA). Such primers or probes are at least 12, preferably at least 24, more preferably at least 36 bases in length. 
     The invention also provides cells and vectors comprising the disclosed UCP3 regulators, including cells comprising such regulators operably linked to non-UCP3 genes. Such cells find used in the disclosed methods for identifying agents which regulate the activity of a UCP3 promoter. In an exemplary such method, the cells are contacted with a candidate agent, under conditions wherein, but for the presence of said agent, the gene exhibits a first expression; detecting the presence of a second expression of the gene, wherein a difference between said first and said second expression indicates said agent regulates the activity of a UCP3 gene promoter. 
     The invention also provides other assays for transcriptional regulators including transcription complex formation assays. An exemplary such assay involves combining a DNA comprising a disclosed regulator with a transcription factor and a candidate agent, under conditions wherein, but for the presence of said agent, the regulator and transcription factor form a first association; detecting the presence of a second association of the regulator and transcription factor, wherein a difference between the first and second associations indicates the agent modulates the association of a UCP3 promoter and transcription factor. The subject nucleic acid regulators also find a variety of other applications, including uses in diagnosis. In particular, hybridization probes and PCR primers derived from the disclosed promoters are used to identify genetic mutations in samples comprising a UCP3 gene. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG. 1. Diagram of mUCP3 promoter constructs driving expression of luciferase enzymatic activity in CaPO 4  transfected HeLa cells. Cells are harvested 18 hrs post transfection and assayed for luciferase. 
     FIG. 2. Diagram of hUCP3 promoter constructs driving expression of luciferase enzymatic activity in CaPO 4  transfected HeLa cells. Cells are harvested 18 hrs post transfection and assayed for luciferase. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The subject nucleic acids are of synthetic/non-natural sequences and/or are isolated, i.e. unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, preferably at least about 5% by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome. Nucleic acids comprising the nucleotide sequence of SEQ ID NO:1 or 2, or fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by a sequence other than that which it is joined to on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, which is at a terminus or is immediately flanked by a sequence other than that which it is joined to on a natural chromosome. While the nucleic acids are usually RNA or DNA, it is sometimes advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability, etc. 
     The subject nucleic acids find a wide variety of applications including use as hybridization probes, PCR primers, therapeutic nucleic acids, etc.; use in detecting the presence of UCP3 genes and gene transcripts, in detecting or amplifying nucleic acids encoding additional UCP3 homologs and structural analogs, in gene therapy applications and in a variety of screening assays. 
     In diagnosis, UCP3-promoter specific hybridization probes find use in identifying wild-type and mutant UCP3 alleles in clinical and laboratory samples. Mutant alleles are used to generate allele-specific oligonucleotide (ASO) probes for high-throughput clinical diagnoses. In therapy, therapeutic UCP3 nucleic acids are used to modulate cellular expression or intracellular concentration or availability of active UCP3. For example, UCP3 nucleic acids are used to modulate cellular expression or intracellular concentration or availability of active UCP3 protein. UCP3 inhibitory nucleic acids are typically antisense: single-stranded sequences comprising complements of the disclosed natural UCP3 transcript sequences, particularly the untranslated exon 1. Antisense modulation of the expression of a given UCP3 protein may employ antisense nucleic acids operably linked to gene regulatory sequences. Cells are transfected with a vector comprising a UCP3 sequence with a promoter sequence oriented such that transcription of the gene yields an antisense transcript capable of binding to endogenous UCP3 encoding mRNA. Alternatively, single-stranded antisense nucleic acids that bind to genomic DNA or mRNA encoding UCP3 protein may be administered to the target cell, in or temporarily isolated from a host, at a concentration that results in a substantial reduction in expression of the targeted protein. An enhancement in UCP3 expression is effected by introducing into the targeted cell type UCP3 nucleic acids which increase the functional expression of the corresponding gene products. Such nucleic acids may be UCP3 expression vectors, vectors which upregulate the functional expression of an endogenous allele, or replacement vectors for targeted correction of mutant alleles. Techniques for introducing the nucleic acids into viable cells are known in the art and include retroviral-based transfection, viral coat protein-liposome mediated transfection, etc. 
     The invention provides efficient methods of identifying pharmacological agents or lead compounds for agents active at the level of UCP3 gene transcription. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. A wide variety of assays for transcriptional regulators are provided including cell-based transcription assays, promoter-protein binding assays, etc. For example, the disclosed luciferase reporter constructs are used to transfect cells such as HeLa cells for cell-based transcription assays. Specifically, HeLa cells are plated onto microtiter plates and used to screen libraries of candidate agents for lead compounds which modulate the transcriptional regulation of the UCP3 gene promoter, as monitored by luciferase expression. An exemplary promoter-protein binding assay is described below. The following examples, exemplary promoter deletion mutants and screening assays are offered by way of illustration and not by way of limitation. 
     EXAMPLES 
     Transfection of cultured HeLa cells: 
     Transient transfections were carried out using cultured HeLa cells by calcium phosphate precipitation. 5 ug of promoter-luciferase plasmid DNA were co-transfected with either 1 ug of pMSV expression vector or 1 ug of pMSV-TR expression vector. Samples were co-precipitated with 2 ug of salmon sperm DNA and 0.2 ug of a β-galactosidase internal control expression vector, then applied atop adherent HeLa cells in 6 well tissue culture plates. After 16 hr cells were washed in phosphate buffered saline and refed with fresh DMEM/F12 culture medium supplemented with 10% fetal bovine serum. After an additional 24 hr cells were harvested, lysed and assayed for luciferase and β-galactosidase enzymatic activity according to manufacturer&#39;s recommendations (Promega). 
     Isolation of human and mouse UCP3 genomic clones. 
     Genomic clones containing the promoter region, the first exon and the remaining 5&#39; untranslated region of the human and mouse UCP3 gene were obtained by hybridization screening of bacteriophage 1 libraries using PCR amplified probes derived from hUCP3 and mUCP3 encoding sequences. The clones were further confirmed by rehybridization using PCR probes derived from 5&#39; untranslated region sequence, which were obtained from RACE PCR amplification. Genomic clones were subcloned into pBluescript KSII (Stratagene), and then sequenced using an Applied Biosystems DNA sequencer. The promoter sequences were subjected to BLAST search on the NCBI server; no homologies to any known sequence were found. Indentically conserved oligonucleotides (see alignment, Table 1) are used in primers and probes for UCP3 genes. 
     The DNA sequence of the first untranslated exon and upstream DNA of the human and mouse UCP3 genes are shown in SEQ ID NOS:1 and 2, respectively. A number of transcription factor binding sites, splice sites and transcriptional start sites for the human and mouse genes are are shown in Tables I and II, respectively. 
     
                       TABLE I______________________________________Human UCP3 gene transcriptional start, splice and factor binding sites.              SEQ ID NO: 1,               SITE nucleotides______________________________________c-Myc          1132-1138  IBP-1 1355-1360  C/EBP 1006-1013  NF-IL6 266-274  GH-CSE2 843-849  HNF5 566-572  AP-1 1944-1950  START SITE 1461, 1399-1548  HiNF-A 1115-1121  AP-2 961-968  HC3 269-274  GCF 396-403  GH-CSE1 853-859  GR  602-1607  AP-2 1525-1532  INTRON I 1549-2000______________________________________ 
    
     
                       TABLE II______________________________________Mouse UCP3 gene transcriptional start,  splice and factor binding sites.               SEQ ID NO: 2,  SITE nucleotides______________________________________c-Myc           4716-4722  gamma IRE 4851-4859  PR 4861-4869  C/EBP 4287-4295  SRF 3915-3925  NF-IL6 3204-3214  HiNF-A 2968-2976  AP-1 2410-2418  Insulin-Responsive 1152-1159  AP-2 293-301  ApoE-B2 5381-5393  EXON 4935-5080  MyoD 4675-4681  NF-kB 4701-4712  NFIL6 4405-4414  MyoD 3929-3935  AP-2 3706-3714  p53 3062-3072  b-α-tabuli 2801-2810  GH-CSE1 1974-1982  CREB 791-799  GcF 4996-5003  START 4935-4948  INTRON 5081-5436______________________________________ 
    
     Deletion mutant construction and activity analysis 
     The promoter activity of the 5&#39; flanking region of human UCP3 gene and a variety of deletion mutants thereof are conveniently screened in a transient transfection assay using mammalian cell lines. An exemplary assay is the HeLa-cell based luciferase reporter assay of FIGS. 1 and 2. Selected promoter deletions are amplified by PCR using targeting primers. Amplification primer pairs for exemplified deletions are as follows: 
     
         __________________________________________________________________________Tagged/untagged       Nucleotide  endonuclease siteSequence__________________________________________________________________________  att-Mlu1 ATTACGCGT   - att-Hind III ATTAAGCTT   - att-EcoR1 ATTGAATTC   - Mlu1 (core)CGCG   -H1:   att-Mlu1- (SEQ ID NO:1, nucleotides 1-20)  att-Hind III (reverse compliment of SEQ ID NO:1, nucleotides 1981-2000)   - H2att-Mlu1- (SEQ ID NO:1, nucleotides 1-20) (reverse compliment of SEQ ID NO:1, nucleotides 1529-1548)   - H3att-Mlu1- (SEQ ID NO:1, nucleotides 200-219) (reverse compliment of SEQ ID NO:1, nucleotides 1529-1548) (SEQ ID NO:1, nucleotides 1091-1110) (reverse compliment of SEQ ID NO:1, nucleotides 1529-1548)   - H5att-Mlu1 (SEQ ID NO:1, nucleotides 1286-1306) (reverse compliment of SEQ ID NO:1, nucleotides 1529-1548) (SEQ ID NO:1, nucleotides 1462-1482) (reverse compliment of SEQ ID NO:1, nucleotides 1529-1548) (SEQ ID NO:1, nucleotides 1-20) (reverse compliment of SEQ ID NO:1, nucleotides 1068-1090) (SEQ ID NO:1, nucleotides 1286-1306) (reverse compliment of SEQ ID NO:1, nucleotides 1441-1461) (SEQ ID NO:2, nucleotides 1-25)  att-Hind III - (reverse compliment of SEQ ID NO:2, nucleotides 5411-5436 (SEQ ID NO:2, nucleotides 1-25) att-Hind III - (reverse compliment of SEQ ID NO:2, nucleotides 5054-5080) (SEQ ID NO:2, nucleotides 3751-3778) att-Hind III - (reverse compliment of SEQ ID NO:2, nucleotides 5054-5080) (SEQ ID NO:2, nucleotides 3940-3967) att-Hind III - (reverse compliment of SEQ ID NO:2, nucleotides 5054-5080) (SEQ ID NO:2, nucleotides 4581-4612) att-Hind III - (reverse compliment of SEQ ID NO:2, nucleotides 5054-5080) (SEQ ID NO:2, nucleotides 4840-4867) att-Hind III - (reverse compliment of SEQ ID NO:2, nucleotides 5054-5080) (SEQ ID NO:2, nucleotides 4930-4958) att-Hind III - (reverse compliment of SEQ ID NO:2, nucleotides 5054-5080)__________________________________________________________________________ 
    
     The deletions may be recombined in any desired variation. For example internal deletions are readily prepared by amplifying both 5&#39; and 3&#39; deletions followed by ligation. Alternatively, a UCP3 promoter deletion may be fused with non-UCP3 promoter element(s) to form heterohybrid promoters. Internal deletions and heterohybrid constructs are exemplified as follows: 
     
         M8·1a&amp;1b pair; 2a&amp;b pair (SEQ ID NO:2, nucleotides 1-25) (reverse compliment of SEQ ID NO:2, nucleotides 3727-3751) (SEQ ID NO:2, nucleotides 4840-4870) (reverse compliment of SEQ ID NO:2, nucleotides 5054-5080) (SEQ ID NO:2, nucleotides 3249-3274) (reverse compliment of SEQ ID NO:2, nucleotides 3727-3751) (SEQ ID NO:2, nucleotides 4840-4870) (reverse compliment of SEQ ID NO:2, nucleotides 5054-5080)   - M10  att-Mlu1- (SEQ ID NO:2, nucleotides 1-25)  att-EcoR1- (reverse compliment of SEQ ID NO:2, nucleotides3727-3751) (SEQ ID NO:2, nucleotides 3940-3967)  att-EcoR1- (reverse compliment of SEQ ID NO:2, nucleotides4910-4935)   - M12  1a. att-Mlu1- (SEQ ID NO:2, nucleotides 3940-3967) (reverse compliment of SEQ ID NO:2, nucleotides 4823-4842) (SEQ ID NO:2, nucleotides 4863-4887) (reverse compliment of SEQ ID NO:2, nucleotides 4910-4935)   - M13 &amp;M14 - Oligo anealing no PCR (SEQ ID NO:2, nucleotides 4843-4862) (reverse compliment of SEQ ID NO:2, nucleotides 4843-4862) 
    
     The PCR fragments are restriction enzyme digested by MluI and HindIII, and then subcloned into MluI and HindIII sites of pGL-2B or pGL-2P (Promega). Transient transfections are carried out using cultured HeLa cells by calcium phosphate precitation method. After 40 hours, cells are harvested, lysed and assayed for luciferase activity. Exemplary mutants are shown to a range of transcriptional activity (FIGS. 1, 2). 
     Protocol for AP-2 - UCP3 gene promoter binding assay. 
     A. Reagents: 
     Neutralite Avidin: 20 μg/ml in PBS. 
     Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hr, RT. 
     Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 0.25 mM EDTA, 1% glycerol, 0.5% NP-40, 50 mM BME, 1 mg/ml BSA, cocktail of protease inhibitors. 
       33  P AP-2 10× stock: 10 -6  -10 -8  M &#34;cold&#34; AP-2 supplemented with 200,000-250,000 cpm of labeled AP-2 (Beckman counter). Place in the 4° C. microfridge during screening. 
     Protease inhibitor cocktail (1000×): 10 mg Trypsin Inhibitor (BMB # 109894), 10 mg Aprotinin (BMB # 236624), 25 mg Benzamidine (Sigma # B-6506), 25 mg Leupeptin (BMB # 1017128), 10 mg APMSF (BMB # 917575), and 2 mM NaVo 3  (Sigma # S-6508) in 10 ml of PBS. 
     Oligonucleotide stock: (specific biotinylated). Biotinylated oligo at 17 pmole/μl, UCP3 gene promoter containing AP-2 site: (BIOTIN)- (SEQ ID NO:1, bases 950-970). 
     B. Preparation of assay plates: 
     Coat with 120 μl of stock N-Avidin per well overnight at 4° C. 
     Wash 2× with 200 μl PBS. 
     Block with 150 μl of blocking buffer. 
     Wash 2× with 200 μl PBS. 
     C. Assay: 
     Add 40 μl assay buffer/well. 
     Add 10 μl compound or extract. 
     Add 10 μl  33  P-AP-2 (20,000-25,000 cpm/0.1-10 pmoles/well=10 -9  -10 -7  M final concentration). 
     Shake at 25C for 15 min. 
     Incubate additional 45 min. at 25C. 
     Add 40 μl oligo mixture (1.0 pmoles/40 ul in assay buffer with 1 ng of ss-DNA) 
     Incubate 1 hr at RT. 
     Stop the reaction by washing 4× with 200 μl PBS. 
     Add 150 μl scintillation cocktail. 
     Count in Topcount. 
     D. Controls for all assays (located on each plate): 
     a. Non-specific binding (no oligo added) 
     b. Specific soluble oligo at 80% inhibition. 
     All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. 
     
         __________________________________________________________________________#             SEQUENCE LISTING   - -  - - (1) GENERAL INFORMATION:   - -    (iii) NUMBER OF SEQUENCES: 2   - -  - - (2) INFORMATION FOR SEQ ID NO:1:   - -      (i) SEQUENCE CHARACTERISTICS:       (A) LENGTH: 2000 base - #pairs       (B) TYPE: nucleic acid       (C) STRANDEDNESS: double       (D) TOPOLOGY: linear   - -     (ii) MOLECULE TYPE: DNA (genomic)   - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:1:   - - AAGCTTTGCA CTTGAACATC CATGCTTCTG ACCACCTGCC CTGTGACGCT GG -#CTCTGTGC     60   - - CCCAGTCCAG AAAAGACTTC TGCCTACTCC TCCTCTGCCC TACCCAGTTA AC -#TCCCTTTC    120   - - CTTCCCTCCC TTCTGCTTCT CACTCCTCCC CTCCCTTCTC TTCTTCTTCT CC -#CCTTCCCC    180   - - CATCACCTGG GGCCCGATTC AGCTGTGCCC AGCCCTTACT CTGAGTGCCC AC -#AGATGGAG    240   - - CCTCCAGTAG CTTCTGTGGG GCACCCTTCC ACCAGGTCCC AGCTCCCTTG GC -#TCCAGCAG    300   - - TGTCCATGCT AAAGCCTCCA AGTGTCATGT TGGAGAGAAT GGTGTTCACA GT -#AGATAAGC    360   - - CCAAAATGCC TTACAGTTTA CAGGCTGGAG TCAGGCCCCG CCACGTTGCT GG -#CTACATGA    420   - - CTTCCCTGAG ATTCCATTTC CTCCTCAGTA AAATAAGTGG TAAGATTTTA GG -#ATCCCCAG    480   - - CACTAAAAAG AAACGAAATA CTGATACAGG CTCCAACATG GATGAATTTT GA -#AAGCATTA    540   - - CTATACTAAG TGAAAGAAGC CAGTCACAAA CAAGCACATA TTGGATGATT CC -#ATTTCTAG    600   - - GAAGTGTTCA GAACAGGCAA ATTTATAGAG ACAGAAAGTA GATTGATTAG TG -#GTTGCCTG    660   - - AGGCTGGGGA GCGGGGGAAG GGAGGTGACT ACCAATGTGT ATGGAGTTTT TC -#CAGGGTGA    720   - - GAGGGTGATG AAAATGTTCT AAAATAGATT GTGTTGATGG TTGTGCCACT CA -#GAATATAC    780   - - TAAAAACCAT TTGAATTGTG CACTTGAAAC AGATGAATTG TACGGTATGT GA -#ATTCTATA    840   - - TCAATAAATC TGTAATTTAA AAAAAAAAAA TTAGGTCGGG TGCAGTGGCT CA -#CACCTATA    900   - - ATCCCAGCAC TTTGCCAGAC TGAGGCAGGA GGATCACTTA AGCCCAGGAG TT -#CAAGACCA    960   - - GCCTGGGGAA CACAGCAAGA CCTCGTCTCT ACTAAAAAAT TTTAAATTAC AA -#AAAAAAAA   1020   - - AGTAAAAAAA ATAGAATCCT AATAGTACCT ATCTCATAGG ATTGTGGAAA AT -#AGTAGTAA   1080   - - TGTATGTAAA ATATTTAGCA CATAGTAGGC ACAAAGAAAT GACATTATTA TT -#AAGAGACC   1140   - - TGGGAGAGCT GTGCCCAGCC TATCGTGGGA GGCCTTGACC TTTGGACTCA AA -#AGTGGCAG   1200   - - CAGGTCCACC CCCCCATACA CCCTTGTCAC CAAGGAAGCG TCCACAGCTT AA -#AGGAGCTA   1260   - - TATTAAAGCA CCCCAAGTCA AGAGGACTGA ACCAGATCTG GAACTCACTC AC -#CTCCCCTC   1320   - - TCACCTCACT GCCCTCACCA GCCAGCCTCT TGTCAAGTGA TCAGGCTGTC AA -#CCAACTTC   1380   - - TCTAGGATAA GGTTTCAGGT CAGCCCGTGT GTATAAGACC AGTGCCAAGC CA -#GAAGCAGC   1440   - - AGAGACAACA GTGAATGACA AGGAGGGGCC ATCCAATCCC TGCTGCCACC TC -#CTGGGATG   1500   - - GAGCCCTAGG GAGCCCCTGT GCTGCCCCTG CCGTGGCAGG ACTCACAGGT AA -#GACCGGTT   1560   - - TCTCCTCCCT CATCCCTTCC CCTCTCCCTC TCCCTTCTCC TTGTTCTCCC TT -#TCATTGGA   1620   - - GGCTTTCAGA GAGCAGCCCC GAGCAGTCAG GGCTCACTAG CTGCAGCTTG TC -#AGACCTGA   1680   - - TAGAGATTCA GTCCAGCCGC CACCTTATGA AAAGGGAGCT GTGGCCTTGA TG -#AGGGTACT   1740   - - GTGGCAGGGC TGGGGCTTGA ACCCAACACC CGTGTCACTC ACTCAAGACT CA -#CACCCCCT   1800   - - TTGCCTTGCT GGCTGCCTCT GGTGGGATTT TGCAAATCCC CATAGACAGG AA -#GTGGCTTT   1860   - - CTTCTTTGCC TGCCCCAGAA TCTCTGCGAT TCCTCCAGAG CATAAATCCC TC -#TCTTTCCA   1920   - - TGAGGACCCT GGGGCCCTCT TCCTGAGTAG GGATGACAGG GGCACTTCTG AC -#CTGAGGCG   1980   - - TGGTCCAGGT CATTTGCTGG            - #                  - #  200 - #0  - -  - - (2) INFORMATION FOR SEQ ID NO:2:  - -      (i) SEQUENCE CHARACTERISTICS:      (A) LENGTH: 5436 base - #pairs      (B) TYPE: nucleic acid      (C) STRANDEDNESS: double      (D) TOPOLOGY: linear  - -     (ii) MOLECULE TYPE: DNA (genomic)  - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:2:  - - ACTAGTGACC CAAAAGATCT GTCTATAGCT ACAGCCAGAG CACCGAATGG GA -#CAAGTATC     60   - - CTCTAAACAC CAAAGATCTC AGGATCTTGA GATGAGGCAC ACAGAAGCTT AA -#AAGTCTCT    120   - - CAGAGAACCC AAAGGGTATA TCCACAGTAG ACCACTACCA TTGGTTCTAG TG -#GAATGAAT    180   - - GAGGCATTTA CTGCAGAGGC AGTCTCCTTC CAACCTGATC TAAACCAGAT CT -#TCATGACC    240   - - CAGTCCTACC CATTCACTGG TCTCATAGCA TCCGAGCCCT CGTCTTGACC TG -#GCCTGCCC    300   - - GGGGTTGTGT ATTGAAGCTC TAGTTCATGC CCTCAGAAGT GCCTGGTCTG GA -#GCAGAGGG    360   - - CAATATGGAC ACTGGCCTCT TGGCCCTCCC CTGGCCTGAG GTGTTACTTT AG -#AAGGAAGA    420   - - ACTGTGAACC AAAAACAACA CCCTTCCTGT TAGCCTAGAC CTCCCCAGAA CA -#CAGAGCTG    480   - - TACCTCCAGG CTCTGCCAAG CATCCTAGCT TGACACTGCT TATTCTTGGC CA -#CCAGGAAG    540   - - GCTTGCCTAA GGGCCTGCTC GGCTTGCCCA CCTCCCCCTC AGTGCTTAGA GA -#TCTGGACT    600   - - GACAGGGATA GTCCTGACTA TTTGCCCAGC CCCCTTGGCC ATGGTTAGGA AA -#CTAGCACC    660   - - CAAGCAGCAG AGAGGACACA AGCTCATTCC CCTTACCCCA TTCTTTGGGT AG -#GTCACTGT    720   - - GTCTTGCCTG TTTTACTCTG AGCTGTGAAG CCAGGATCTG CTGTCATTTC TG -#CCTCCTGT    780   - - TGACGTGTAT TGTTCTATAA AAAGGAATAA TGGGATTTCT AAGTGTGCTC AT -#CCTTGACC    840   - - CTCTCTAGCC AGGCCTGACC TTTCTTGTTA GCAGGCTCAC GGGGTACAAG CT -#GGAGAGGT    900   - - GGAACAGTGT ATTAGGGTCC GTCAGCATGG GAACAAAAAA GATCTACCGG CC -#TGTGGGGA    960   - - CAGCCTCCGT TTCTTTCTGC ACTGGCTCTT CCTGCCTGGC CTCCATCTCT TT -#CCTAGGGG   1020   - - CCTCATTCTC AGCCCACCAG GCAACTTTGC TATGTAGATC TGAGCCCTTC AG -#CACAGGAA   1080   - - CCTGACAAGA TACCAAAAAG GGGAAAGTGG CTGAGCACAC GGCTTTGTCT AT -#GGTAGCTG   1140   - - CTCAAGTGGG ATTTCCACCT GCCTCAGCTC TGCCACTCAG AGCTGCACGA CC -#TTGAACTA   1200   - - ACTAAGTGCA CCTGACCTTG TGATGTCATG GCCAGGGTTC CACAAGACAG GG -#TGTATGAG   1260   - - GTATTTGCAA ACCAGGGACC TAGAGAAAAT GTTAGCCCAA GGACCAGACT CG -#CCTCTGCA   1320   - - ATGCTGTATC CTCAGGACTA ATCTAGATGA CTTCCTTCTC TCCAGGACAA GA -#GTGAAGCC   1380   - - CACCAGGGGG AGCCCTTGCC CAGAGGCCTT GTCGCCCCAG ACCTTACTCC AT -#CCCTCCCA   1440   - - ACCTTCCTGA GGAAGAGACT CGCAGGATTG CACGGATATT TTCTTCCCAG TA -#TTCCAAAA   1500   - - AGACTGAGGA AACCTGAGGG ACGTGGCTAC CAGCCAGCCT CTCAGCTCTG TA -#AAGCTTGC   1560   - - AAGAGACAAC AGGCCTATTT CCCTCAGCAT CCTCAGGCCA CTAGCATCAC CC -#TATTCCAT   1620   - - GGGTATCTTG AGACCTAATC AGCTAAGACC AGAGGAGTGT CCCAAACCAC CT -#AGCAGCTG   1680   - - CCTACCAGGA CCTCAGCTTC CTTCTGTAAT GAGGTGACTG CTGGAAGTGA GA -#CTCAAGCA   1740   - - CCCAAGTGTC CCACCCATCT TCTGACTGTG GGGCCTAAGG AGGCCTCTCT GA -#GCAGAGAA   1800   - - ACCAGTGTCC TTGACACAGC CTACTGCTGA CCAGAGCCCA CCTTCTGCTT AA -#ATAGGACA   1860   - - GGCTATTTTT GCCATAAATA TTGGAAGAAA CATGGAATAA GTATGTACAT AG -#CTGAGAAA   1920   - - ATTCCAGAGC CCTGTCCAGC TTGTTTGTTT GGGAATGAGG TATTTTATTG AT -#ATAAATTA   1980   - - TTTTTATGAT GTATTAATCA ATAGAACAGG GGAATTGCCT CCTTTAAAAC TG -#TATTCGGT   2040   - - TGTCTGAAAT TTAACCATGT TTTTAACATA ATGTTTGTTT CTACTCAGCT CT -#GAAATTCA   2100   - - TTGCTCTTGC CTTTGTTTTT AATAAAAGTC TGGCATTTGT ATTTGTGTAC AA -#GTGTTTTT   2160   - - TGGGTTGGTC ACTGGGAAAA GGTGTTGCAG AAAGAAGGAT ACAGCCATCA CT -#CCACCACC   2220   - - TGCAACATGA GTCGCCATGA GACTTGCTAG TTCTTCCAAC TGTCAGATTG AC -#CCATGTTA   2280   - - GAGGGATGCT AGTGTTCCAC TTCTGGTGGT TTGGGCTTTT GCTTGCTTGC TT -#GGTTTGTC   2340   - - TTTTCCTCTG ATGGCCCTTT AAATCTTGCT CAGCACAGTG AGCGTCCAAC TA -#AGATCTCG   2400   - - TCCCCGGTGT GACTCACGAG GGCACTTCTG TCAGAACACA GCCACTTGGG CA -#GCTACCAT   2460   - - AGACAAAGTC CTCTGAGGAT AATCTGAACG GAATAGATGT CCAGTGCGTC TG -#GTAGTGAG   2520   - - GCGGAAAGGA GCTACCTGAG GGCTCTGCTT GTCACCCTGT GTGTCATTTC TC -#TAAATCCT   2580   - - CACAACTCTA TGATGCCCCT CCCACAATTA CCCTCAACAC CATGGAAGTC GG -#ATGCATTG   2640   - - CTTCTGTATC GGTACAAAGT GCATCAGGAC CGAAGACATA CCCCAGAGAT AA -#GGACTCAG   2700   - - ACCAAAGCAG GATACAGGCA GTAAACGCCC TGAATTCGGG CGGTCTCAGG CT -#GCTAAATC   2760   - - CAGGGAGGGA TTTCTGGAAG AAACCCGTAC TGGGGTAAAG CCCAGAGAAT GG -#TATAGACC   2820   - - AGGTCATGGA CAGCTGCAAA GAGAACCAGA GAATGTTCTG GACCATATCA GT -#GTCCAGCC   2880   - - TGGCTGAAAC TGTGGCTCTG GCCTCATCTG GAAGGCACAG ACACGTAGAC TC -#TGGCTTCG   2940   - - TATGGTCGCT GTGAAGATCA AATGGGACAT TTCTGAAAAT GCTTCGCTGG CA -#TTTGGCAC   3000   - - ATAATGGAAG CTTGAGAAAT GTCAGCCCTT CCCACTCTTC CTAAGCACCG GG -#GTGAATTA   3060   - - GAGGCAAGCT CACTGGCTAC CCAGAACGCA GAGCACTCCA GCCTCCCATA AG -#GGCCATGA   3120   - - ACCTTCAGCC AAACCTGCCC CTCAGATCTC TGTTCCTTGG AGGTCACCTT GC -#AAACCTGC   3180   - - CCAGTCCTTA GCCAGGGGTT CCCCTTGCCC CAGGCCTTGC CTTCGGCCAG AA -#CAGAGCTT   3240   - - CCAGTACCTG GAGTTCCACG TCCCTCGGGG TAAGAAACTT CTAGGTTCCA AG -#CCTAAGGG   3300   - - GTAAAGCCTA GGGTGAAGAA GATCACTGCC AAATCCTGAT CCTGTAAATA AC -#CTGAAGGA   3360   - - GTCAAGTGAG AACAGGGCCA CAGGAGGGCA CGGGCTGCAG ACAAGGTGAA GG -#TCTGAGAC   3420   - - ACAGACGACA TGCCCAATTT GTTATTTACC AGTCTCTCCC AGTGATAGCT CT -#GAGGCAAC   3480   - - TTGACTTGCC AAGTTGTACA ACACATTTGT CACCACAACC AGAATGAACC CT -#GACTTCCT   3540   - - TATGCATGCC CTCCACAGAT GCCTGGAAAG TATTTCTGGT GACCCAGAGA CT -#GTAGCAGC   3600   - - CAACCTTAAA ACTCCCAGCA GAGGTTTGAC TCTAGCTAGG CCTGAGCTCT CC -#TCCCTCTG   3660   - - GGTTTCTCCA GAACCTGCTG CCTCTAGTTT GACTTCTACT TGTAGCCCCA GC -#CCCCACAT   3720   - - GGTTCCCACA GTCCTGCCCA TGTCCAGATT AATAGTTCCA GACTGAGCTC AA -#AGCAGTGA   3780   - - GCAGGTCCTG GTCCAAGAAA CAGGATAGAC AGTCTAGATG TCACAGCACC CA -#CCCTGCCA   3840   - - TGGCGCTGCG CTCAAGTTCC AAAATGTCCT CTACCTTCTC TGTCCTACTT AT -#CTCCTCTC   3900   - - CCCTCTCCTT TTAGTTTCCC TTCTATGCCA CCTGGCTCCC AACTTCAGCT GT -#GCCCACCC   3960   - - AGTCTTTAAC CTCAGCGCCT GCAGATGGAC CAGCCTACAG TGGTTGCTGC AA -#GGGATTTT   4020   - - GCTCACCCTG CCCCATCTCC GGTCCCAGCA TGTCCATGTC ATGAGGAATC AG -#GATTGCAG   4080   - - CTGATAGACC CAAAGTGTGG TATAGGTTAC AGGCAAGTCA GGCCTGCAGT GA -#CCTAGCTG   4140   - - TCTGTCCTTG ACAGTCAGTT TTCTGTGTAA AGTAAGCATT GACACATGAG GG -#CTCTACAC   4200   - - AGTACGTCAG TGTCTACCGG TACAGTGCAG TGTGTTAACA CCACTGTGAA AG -#AAACCAGC   4260   - - CACAAGAGGC TATACATTCT ATATAATCCT ACTCAAACAA AGCATCTAGA AC -#AAGCAAAT   4320   - - TAAGAAGCAG AATAAAGACG AGTGGTTGCC TGAAGCTGGG GGAAGGGAAA GG -#GGGTGACT   4380   - - TGTTCTGTTT TTGTCTTTGA GCCATGATGA AAGTGTTCTG AACTAGCATG TA -#GTAGTTGA   4440   - - TCAACCAGAC TGTACTAAAC ACTATTGTGT GCTCTATGTG GGCAAATCAC AT -#GCTGTGTA   4500   - - CAAAACATCT GTTGTCCTTT GTTTTTAAAT TTAGGATCCT GCTTCCTAGA GA -#TGTGGGAA   4560   - - ATAGAAGCGC TGTGCCTGAA ATATCAAGCA TATCTTGGCA CCAAGATGTC CT -#CTACCTTC   4620   - - TCTGCCCCGT CTTCTCCTCT CCCCTCTTGA GAATGTCAGG CCTCTAAGAA GT -#GACACCTG   4680   - - TAACCATTGT ATAGGATCCT GGAGAGCCCC TGTCCTAAGA GACCTTGTCC TT -#TGGGCTCT   4740   - - CAAAGGTGAC AAATGCTGTC ACACACCTCC TGGCCACCAA GGTAGCTCTC CT -#CTTGAAAG   4800   - - CTCAAAGGAG CCACATTAAA GAGCCCCAGG TCACGGAAGC TAAACCAGAT CT -#GGAACTCA   4860   - - CTGGTCCCCT CCCCGCAGCC TGCCTCTTGT CAAGTGATCA GACTGTCAAC TA -#GCTTCTCA   4920   - - GAATTAGGTT TCAGGTCAGC TGGTGCACAG GGCCAGTGCC GAGCCAGGGA CA -#GCAGAGAC   4980   - - AACAGTGAAT GGTGAGGCCC GGCCGTCAGA TCCTGCTGCT ACCTAATGGA GT -#GGAGCCTT   5040   - - AGGGTGGCCC TGCACTACCC AACCTTGGCT AGACGCACAG GTAAGACCCC AT -#ACTCTGCT   5100   - - CTCCTCTCCC TTTTTCCCTT CCATGGATGC TCACAGCCAG GAGCTTGCTG GG -#ATCACTCA   5160   - - GCACTGCGTG AGAGACCGAG AGTGAGCCGG TCTAGCTCCC ACCTAGTAAA GA -#TGAAGGAA   5220   - - CTGCAGGCCT GGGGAGGGCC TTGACTTCCA CATCTATGTG ACTCCTCACA AC -#TCCCGTGT   5280   - - TTTGCTGACT CCTCTGCTGG GATCTTACAA ATGCCAAATG AAAAGTGTCC CT -#CTCCTTTG   5340   - - GCCCAGGATC CCCACGGAGC ACAAAGCCCT CTCCAGCAAG GATCCTGGGG CC -#CTTCCTGG   5400   - - GTAAAAATAA TGAGGGCACT CTGGCCTGAA GCCTGG      - #- #     5436__________________________________________________________________________