Abstract:
An erythropoietin-inducible, erythroid-specific DNA construct is disclosed. The DNA construct comprises a promoter sequence from a sheep juvenile beta globin gene, an erythroid-specific enhancer sequence from the locus control region of the human beta globin gene and a nucleotide coding sequence of interest.

Description:
TECHNICAL FIELD 
     The present invention generally relates to the treatment of osteopenias such as osteoporosis, and non-human transgenic mammals expressing therapeutic transgenes for the same. 
     BACKGROUND OF THE INVENTION 
     Osteopenias are characterized by decreased mass density and calcification of bone. These diseases are generally attributable to inadequate osteoid synthesis thus causing an imbalance in the bone deposition to resorption ratio. Osteopenias include Paget&#39;s disease and primary and secondary osteoporosis. Various treatment protocols have been attempted the objects of which are to stimulate bone formation. 
     Growth hormone (GH) has been found to stimulate bone formation through stimulation of insulin-like growth factor-1 (IGF-1) production primarily by the liver and secondarily by osteoblasts. Osteoblasts are responsible for synthesis of bone matrix and are known to express osteoblasts are known to express GH receptors. IGF-1 further stimulates osteoblast proliferation. The biological half-lives of IGFs are increased when associated with IGF binding proteins that are also locally produced in the bone. It has been suggested that GH stimulates bone formation through a localized mechanism that increases IGF and IGF binding protein production which in turn stimulates osteoblast proliferation. 
     Hormonal treatments such as direct infusion of human growth hormone (hGH) have been attempted to stimulate bone deposition. They have not been successful to date. Therapies involving direct GH injections can produce adverse systemic effects. These include sterility or at least reduced fertility and adverse lactogenic effects. Because GH injections induce systemic stimulation of IGFs, which bind insulin receptors when present in excess, severe hypoglycemia may also result. 
     Attempts have been made to stimulate osteoblasts through localized production of GH in the bone so as to avoid adverse systemic effects through injection of GH. One approach achieved expression of a GH transgene in osteoblasts which stimulated increased growth in the long bones of transgenic mice. (Baker, A. R. et al. (1992) Mol. Cell. Biol 12: 5541-47). However, this approach would be difficult to adapt for treatment of osteopenias. After birth, osteoblast isolation from a human patient is highly impractical, thus eliminating use of transfected osteoblasts in any gene therapy. Further, no specific inducible regulatory sequences were included in the transgenic model to provide higher levels of expression, if desired in a given therapy. 
     Prior to the development of the present invention, no erythroid-specific gene therapy existed for localized and controlled stimulation of GH production to enhance bone deposition using a gene construct which could be up-regulatable based on therapeutic need and capable of being applied to more readily available and collectable tissues, such as bone marrow stem cells. 
     Erythroid-specific gene expression in transgenic non-human mammals has been previously reported in the art, principally using human beta globin genes. For example, WO 9(1/05041 published Apr. 18, 1991 discloses expression of human beta globin gene in erythroid tissue of transgenic non-human mammals having such expression enhanced through use of selected DNase hypersensitive sites (HS) from the locus control region (LCR) of the human beta globin gene. Palmiter, et al. Science 222, 809 (1983) discloses transgenic mice containing the human growth hormone gene fused to a metallothionein promoter sequence. These references neither suggest nor disclose expression of an erythroid-specific gene capable of being up-regulatable in response to therapeutic needs. 
     A juvenile beta globin gene (β c ), possessed by artiodactyls (goats and sheep of the haplotype A), exhibits a unique developmental expression pattern. Most mammals have embryonic beta globin genes active early in development. These embryonic genes are then down-regulated in favor of beta globin genes encoding for adult forms of beta globin. Sheep of the A haplotype have a gene switching pattern comprising &#34;embryonic-fetal-juvenile+adult-adult.&#34; When mature sheep become anemic or exposed to high altitude, which causes erythropoietin (EPO) levels to rise, the switching pattern can be reversed and β c  up-regulated with adult beta globin being down regulated. The juvenile beta globin gene can also be reinduced artificially in healthy adult sheep through administration of EPO. 
     No gene construct known in the prior art is up-regulatable, erythroid-specific and ligatable to hGH gene for gene therapeutic treatment of osteopenias. 
     SUMMARY OF THE INVENTION 
     According to the present invention, a method for the treatment of osteopenias has been developed using a transgene comprising erythroid-specific and up-regulatable β c  gene regulatory elements to which hGH gene is ligated. The transgene of the present invention includes, preferably, only the β c  promoter with an erythroid-specific enhancer cloned upstream, preferably including at least 5&#39;HS2 from the human beta globin LCR. 
     Transgenic non-human mammals carrying the transgene of the present invention have been produced that exhibit enhanced but controlled bone deposition in the range of 10%-30% greater than normal. Additional bone deposition may be inducible by up-regulating the transgene through induction of anemia by phenylhydrazine administration or by EPO injections. 
     In the therapeutic methods of the present invention, a patient&#39;s bone marrow tissue, preferably erythroid stem cells, are collected. Next, the stem cells are transfected with the HS2+β c  hGH transgene and retransfused to repopulate the marrow. Bone marrow stem cells are preferred as they home naturally back to the marrow when reinfused into the bloodstream. 
     The method of the present invention avoids for the most part the systemic side effects accompanied by injections of recombinant GH by exerting primarily a local effect on bone formation and deposition. 
     Other advantages and aspects of the present invention will become apparent upon reading the following description of the drawings and detailed description of the invention. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The file of this patent contains at least one black and white photograph. Copies of this patent with black and white photographs will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. 
     FIG. 1A is a diagrammatic representation of the HS2+β c  hGH transgene of the present invention; 
     FIG. 1B depicts the nucleotide sequence, SEQ ID No:1, of the HS2+β c  hGH transgene as cloned; 
     FIG. 2 discloses RNase protection analysis of the TG420 transgenic mouse line showing phenylhydrazine and EPO induction of the transcription of the hGH transgene; 
     FIGS. 3A-3C are graphic representations disclosing growth rates of the TG420, TG450 and TG435 transgenic lines compared to nontransgenic littermates wherein &#34;M&#34; means male, &#34;F&#34; means female and &#34;TG&#34; means transgenic; 
     FIG. 4 discloses in situ hybridization of fetal mouse sagittal sections to detect erythroid-specific expression of the transgene of the present invention and endogenous mouse alpha globin in transgenic and nontransgenic control mouse fetuses; 
     FIG. 5A is a radiographic image comparing representative caudal vertebrae of a male mouse from the TG420 line and an age and sex matched nontransgenic control; 
     FIG. 5B is a graph comparing cortical bone densities between male mice from the TG420 line and nontransgenic male controls; 
     FIG. 6A is stained semi-thin histological sections of proximal tibiae having a nontransgenic control depicted at the top and a male from the TG450 line at the bottom; and, 
     FIG. 6B is a graph comparing average cortical bone thickness between older mice from the TG450 line and nontransgenic age-matched controls. 
    
    
     DETAILED DESCRIPTION 
     While this invention is susceptible of embodiment in many different forms, there is shown in the drawings and will herein be described in detail a preferred embodiment of the invention with the understanding that the present disclosure is to be considered as an exemplification of the principles of the invention and is not intended to limit the broad aspect of the invention to the embodiments illustrated. 
     Synthesis of HS2+β c  hGH Transgene 
     As disclosed in FIG. 1A, the transgene of the present invention contains three parts: 5&#39;HS2 from the human beta globin LCR, sheep β c  promoter, and hGH gene. The transgene of the present invention embodies the nucleotide sequence, SEQ ID No:1, disclosed in FIG. 1B. The transgene was originally constructed in vector pBS. 
     The DNA fragment containing the sheep β c  gene is a 3.2 kb Hind III fragment obtained from a plasmid clone available from J. Lingrel, University of Cincinnati; GenBank accession No. X 14728. The sequence for sheep β c  is disclosed in Garner and Lingrel. (1989) J. Mol. Evol. 28:175-184 which is incorporated herein by reference in its entirety. This fragment contains the entire β c  gene with 827 bp of 5&#39; flanking sequence and 791 bp of 3&#39; flanking sequence. The β c  promoter is contained in an 819 bp HindIII-PvuII fragment. 
     The β c  promoter and a small amount of pUC18 vector flanking sequence was purified as a 908 bp DNA fragment. ApaI linkers were blunt-end ligated to this and the fragment was digested with ApaI. 
     As disclosed in FIG. 1A, the linkered β c  promoter fragment was ligated to the unique ApaI site in the proximal promoter of the hGH gene. The hGH gene, carried in plasmid pghGH2.6-3, was supplied by H. Goodman, Mass. General Hospital. (See also GenBank accession nos. J00148 and K00612) The sequence of hGH gene is disclosed in DeNoto et al. (1981) Nucl. Acids Res. 9:3719-3730 which is also incorporated herein by reference in its entirety. 
     The total length of the promoter used in the transgene construct of the present invention is 842 bp. Of this the β c  promoter contributed 819 bp to the promoter with the balance of the promoter sequence derived from hGH gene itself and the ApaI linker. The cap site and all downstream sequences were derived entirely from the hGH gene. 
     A 1.9 kb HindIII fragment containing the 5&#39;HS2 element of the human beta globin LCR was inserted into the HindIII site immediately upstream of the β c  promoter to complete the construct. The 5&#39;HS2 HindIII fragment is defined as nucleotides 1527 through 3451 in GenBank file Humhbb-part 3. 
     Production of Transgenic Mice Containing HS2+β c  hGH Transgene 
     The transgene of the present invention was purified from vector sequence before microinjection into mouse embryos. No vector sequence was microinjected. After the DNA was purified free of vector sequences and concentrated using Elutip columns (Schleicher and Schuell, Inc.) the purified and concentrated DNA was microinjected into male or female pronuclei of fertilized one-cell mouse eggs using techniques known in the art. DNA is microinjected at a concentration of 2 ng/μl in 10 mM Tris (pH 7.5), 0.1 mM EDTA. 
     The injected eggs were then implanted into pseudopregnant female mice following the procedures disclosed in Hogan, et al. (1986 Cold Spring Harbor Lab.) Manipulating the Mouse Embryo, A Laboratory Manual. The eggs in the foster females were allowed to develop to term, usually 20-21 days. 
     Mice were obtained from The Jackson Laboratory. Embryos for microinjection were produced by breeding B6SJL/F 1  J mice. Genomic DNA was purified from tail tissue and transgenic individuals were determined by slot blot hybridization. Transgene integrity was confirmed by genomic Southern blot hybridization. 
     Analysis of hGH Transgenic Mice 
     Six HS2+β c  hGH transgenic lines were produced by breeding founder mice (TG407, TG409, TG416, TG420, TG435, TG450). The TG420 TG435 and TG450 lines were the primary focus of analysis. Expression of the hGH transgene was analyzed in blood by the quantitative RNase protection assay at key developmental time points. 
     RNA was prepared for RNase protection assay by collecting blood samples from embryos and fetuses by first dissecting individuals from the uterus in their amniotic sacs, rinsing in cold phosphate-buffered saline (PBS; 0.8% NaCl, 2.7 mM KCl, 1.5 mM KH 2  PO 4 , 20 mM NA 2  HPO 2 ) containing 10 units/ml of heparin, and then bleeding each individual from the amnion into fresh cold PBS-heparin. Blood was collected from very young mice into cold PBS-heparin following decapitation. Older mice were bled from the tail vein. 
     FIG. 2 discloses the results of RNase protection assay using five 5-6 month old mice referred to in FIG. 2 as A through E. Endogenous alpha globin provided the internal standard in each sample. In FIG. 2 the ratio of hGH to alpha globin in the baseline sample is compared to the ratio of hGH to alpha globin in the treated sample to indicate fold induction of the hGH gene. 
     As disclosed in FIG. 2, the TG420 line was able to up-regulate expression of the transgene in response to phenylhydrazine-induced anemia and EPO. The TG420 line increased expression of hGH mRNA four to eight-fold with phenylhydrazine and three to twenty-fold with EPO induction. The TG435 line induced expression two to 14-fold with these treatments. This suggests that the 819 bp β c  promoter fragment that was used contains an &#34;EPO-response element.&#34; It presently is unknown in the art what exact DNA sequence comprises an &#34;EPO response element&#34; or how EPO acts through it to induce expression of a gene. 
     The most dramatic physiological characteristics of the HS2+β c  hGH transgenic mice is that the mice in three of the lines TG420, TG450, and TG435 grow 5% to 40% larger than normal. Representative plots of transgenic and nontransgenic littermate growth rates are shown in FIGS. 3A through 3C. Although the mice produce their highest levels of hGH mRNA as embryos and fetuses, their enhanced growth does not become apparent until approximately 3 weeks of age. This is most likely due to the absence of GH receptors in the liver preventing full expression of growth hormone&#39;s systematic effects via IGF production (Maes et al., 1983, Endocrinology 113: 1325-1332). The same growth trend was reported for mt-GH (metallothionein-growth hormone) transgenic mice (Hammer et al., 1985, Cold Spring Harbor Symp. Ouant. Biol. 50: 379-387). The mice of three other transgenic lines (TG407, TG409 and TG416) express the β c  hGH transgene at lower levels and do not grow large. Quantitation of their serum hGH levels and comparison to the measurements already made reveal the threshold amount of systemic hGH that produces a growth response. 
     Radioimmunoassay (RIA) was used to measure hGH levels in the serum and trapped within the red blood cells (rbcs) of the mice. Efforts focused on the two lines showing the most obvious enhanced growth (TG450 and TG420). In these lines hGH levels are approximately 5-10 times greater inside the rbcs than in the serum of TG450 mice and 50-100 times greater in rbcs than serum for TG420 mice (volumetric comparisons), indicating that most of the hGH is probably never released systemically. Although it is difficult to compare directly between RIAs run in different labs using different tracers, it appears that serum levels of hGH are low in our mouse lines when compared to the mt-GH transgenic mice (Palmiter et al., 1982, Nature 300: 611-615). For example, some representative serum hGH values from TG420 mice were 270 ng/ml at 23 days, 110 ng/ml at 13 weeks, and 59 ng/ml at 5 months. This compares to values of 280, 310, 3.2×10 4 , 5.7×10 4 , 1.1×10 5  ng/ml in Palmiter&#39;s mice (ages not specified). 
     To evaluate tissue specificity of transgene expression in these mice, in situ hybridization studies were conducted. Ten micron sagittal sections of TG and control mouse fetuses were mounted onto nitrocellulose filters and probed with  35  S-labeled antisense hGH or a globin probes. The hGH mRNA specific probe was hybridized to sagittal sections 18.5 gestational day (nearly full-term) mouse fetuses representing the three lines that demonstrated enhanced growth. Adjacent sections were probed for mouse a globin mRNA to identify erythroid tissues and pooled blood. Representative control and transgenic sections are shown in FIG. 4. Fetal liver was hybridized by both a globin and hGH probes indicating expression of both genes in the tissue. Liver is the major erythropoietic organ at this age. Pooled blood produces an a globin signal in the hearts of both TG and nonTG fetuses. By the same token, high hGH mRNA, also present in the blood, is detected in the heart of the TG fetus. The large arrow in FIG. 4 indicates the hybridization signal in liver and the small arrow indicates the heart. Transgenic fetuses from the TG420, TG435 and TG450 lines produced the same results. 
     Transgenic mice from the TG420 and TG450 lines had spines that from simple observation were unquestionably longer than their littermate controls. To confirm this observation, the bones of both anesthetized live mice and eviscerated carcasses were measured and visualized radiographically by mammography according to the method disclosed in Schneider et al., (1994) J. Bone Min. Res. 9:581-591 which is incorporated herein in its entirety. The development of the vertebrae and knees were monitored from birth through maturity in these two lines and the TG435 line. 
     FIG. 5A is a radiographic image of representative caudal vertebrae and FIG. 5B is a graph of relative densities of these vertebrae over time. TG420 and TG450 transgenics have greater bone density than nontransgenics starting around three weeks. The relative difference in density increases with age, despite the extremely how levels of hGH typically measured in the serum of mature transgenics. 
     For more detailed evaluation of differences due to transgene expression, histological specimens were collected and prepared following the methods disclosed in Schneider, et al.,(1988) J. Bone Min. Res. 3:225-232 which is incorporated in its entirety herein by reference. Cortical bone thickness was measured in some of the oldest members of the mouse colony. Semi-thin sections were prepared from transgenic and age and sex-matched control mice. 
     A representative comparison of cortical bone between an age and sex-matched control mouse is shown in FIG. 6A at the top and a 13 month male from the TG450 line is shown in FIG. 6A bottom. As disclosed in FIG. 6B, at 13 and 18 months cortical bone was significantly thicker in the TG450 line. 
     Gene Therapy Using HS2+β c  hGH Transgene 
     The transgene of the present invention has produced dramatic bone development in certain transgenic mice, increasing the thickness and density of the bone. The enhanced bone deposition continues into advanced maturity in the mice, without increasing hGH mRNA in the red blood cells and serum hormone levels in older animals. In many instances, serum hGH levels were within the normal ranges for endogenous mouse growth hormone. 
     Application of this same delivery mechanism for hGH to humans at risk or suffering from osteoporosis and other osteopenias may improve their bone deposition to resorption ratio. Currently, the most effective treatments for osteoporosis are based on prevention and early warning for those who are especially at risk. The efficacies of current prescriptions such as estrogen, calcium, vitamin supplements, and weight-bearing exercise are sharply reduced when initiated late in life or after menopause. 
     Gene therapies using the transgene of the present invention would utilize the steps of; collecting a patient&#39;s erythroid stem cells from their bone marrow or circulation, the collected stem cells are transfected with an hGH gene construct such as the transgene of the present invention, and the transfected stem cells are retransfused into the patient. They home naturally to the bone marrow compartment in all its locations throughout the body to repopulate the marrow. Individuals identified to be at risk prenatally, e.g. osteogenesis imperfecta patients, could be treated with stem cells isolated from cord blood as well. 
     Similar gene therapies may be employed in elderly patients who have already experienced fractures or are at serious risk. Likewise, children or young adults with osteopenias or those predisposed to the development of osteopenias could also be treated with transfected erythroid stem cells. The osteopenias include primary osteoporosis such as involutional (postmenopausal and senile) and juvenile osteoporosis and secondary osteoporosis such as those included in Table I below: 
     
                       TABLE I______________________________________Secondary Osteoporosis (Osteopenias)______________________________________A.      Endocrine       1.    Andrenal cortex    Cushing&#39;s disease       Addison&#39;s disease  2. Gonadal disorders   Hypogonadism  3. Pituitary  Acromegaly Hypopituitarism  4. PancreasDiabetes mellitus  5. Thyroid    Hyperthyroidism   Hypothyroidism  6. Parathyroid   HyperparathyroidismB.      Marrow replacement and expansion       1.    Myeloma  2. Leukemia  3. Metastatic disease  4. Gauchr&#39;s disease  5. Amemias (sickle cell, thalassemia)C.      Drugs and substances       1.    Corticosteroids  2. Heparin  3. Anticonvulsants  4. Immunosuppressants  5. AlcoholD.      Chronic disease       1.    Chronic renal disease  2. Hepatic insufficiency  3. GI malasorption  4. Chronic inflammatory polyarthropathies  5. Chronic debility/immobilizationE.      Deficiency states       1.    Vitamin D  2. Vitamin C (scurvy)  3. Calcium  4. MalnutritionF.      Inborn errors of metabolism       1.    Osteogenesis imperfecta  2. Homocystinuria______________________________________ 
    
     Therapies using the transgene of the present invention could limit the risk of overuse or unnecessarily high systemic levels of GH. 
     Deposits 
     Plasmid containing the HS2+β c  hGH transgene have been deposited with the American Type Culture Collection on Oct. 13, 1994 and given ATCC Accession No. 75908. 
     While the specific embodiments have been illustrated and described, numerous modifications come to mind without significantly departing from the spirit of the invention and the scope of protection is only limited by the scope of the accompanying Claims. 
     
         __________________________________________________________________________#             SEQUENCE LISTING   - -  - - (1) GENERAL INFORMATION:   - -    (iii) NUMBER OF SEQUENCES: 1   - -  - - (2) INFORMATION FOR SEQ ID NO:1:   - -      (i) SEQUENCE CHARACTERISTICS:       (A) LENGTH: 4456 bp       (B) TYPE: nucleic acid       (C) STRANDEDNESS: single       (D) TOPOLOGY: linear   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:   - - AAGCTTCAGT TTTTCCTTAG TTCCTGTTAC ATTTCTGTGT GTCTCCATTA GT -#GACCTCCC     60   - - ATAGTCCAAG CATGAGCAGT TCTGGCCAGG CCCCTGTCGG GGTCAGTGCC CC -#ACCCCCGC    120   - - CTTCTGGTTC TGTGTAACCT TCTAAGCAAA CCTTCTGGCT CAAGCACAGC AA -#TGCTGAGT    180   - - CATGATGAGT CATGCTGAGG CTTAGGGTGT GTGCCCAGAT GTTCTCAGCC TA -#GAGTGATG    240   - - ACTCCTATCT GGGTCCCCAG CAGGATGCTT ACAGGGCAGA TGGCAAAAAA AA -#GGAGAAGC    300   - - TGACCACCTG ACTAAAACTC CACCTCAAAC GGCATCATAA AGAAAATGGA TG -#CCTGAGAC    360   - - AGAATGTGAC ATATTCTAGA ATATATTATT TCCTGAATAT ATATATATAT AT -#ATATACAC    420   - - ATATACGTAT ATATATATAT ATATATATAT TTGTTGTTAT CAATTGCCAT AG -#AATGATTA    480   - - GTTATTGTGA ATCAAATATT TATCTTGCAG GTGGCCTCTA TACCTAGAAG CG -#GCAGAATC    540   - - AGGCTTTATT AATACATGTG TATAGATTTT TAGGATCTAT ACACATGTAT TA -#ATATGAAA    600   - - CAAGGATATG GAAGAGGAAG GCATGAAAAC AGGAAAAGAA AACAAACCTT GT -#TTGCCATT    660   - - TTAAGGCACC CCTGGACAGC TAGGTGGCAA AAGGCCTGTG CTGTTAGAGG AC -#ACATGCTC    720   - - ACATACGGGG TCAGATCTGA CTTGGGGTGC TACTGGGAAG CTCTCATCTT AA -#GGATACAT    780   - - CTCAGGCCAG TCTTGGTGCA TTAGGAAGAT GTAGGCAACT CTGATCCTGA GA -#GGAAAGAA    840   - - ACATTCCTCC AGGAGAGCTA AAAGGGTTCA CCTGTGTGGG TAACTGTGAA GG -#ACTACAAG    900   - - AGGATGAAAA ACAATGACAG ACAGACATAA TGCTTGTGGG AGAAAAAACA GG -#AGGTCAAG    960   - - GGGATAGAGA AGGCTTCCAG AAGAATGGCT TTGAAGCTGG CTTCTGTAGG AG -#TTCACAGT   1020   - - GGCAAAGATG TTTCAGAAAT GTGACATGAC TTAAGGAACT ATACAAAAAG GA -#ACAAATTT   1080   - - AAGGAGAGGC AGATAAATTA GTTCAACAGA CATGCAAGGA ATTTTCAGAT GA -#ATGTTATG   1140   - - TCTCCACTGA GCTTCTTGAG GTTAGCAGCT GTGAGGGTTT TGCAGGCCCA GG -#ACCCATTA   1200   - - CAGGACCTCA CGTATACTTG ACACTGTTTT TTGTATTCAT TTGTGAATGA AT -#GACCTCTT   1260   - - GTCAGTCTAC TCGGTTTCGC TGTGAATGAA TGATGTCTTG TCAGCCTACT TG -#GTTTCGCT   1320   - - AAGAGCACAG AGAGAAGATT TAGTGATGCT ATGTAAAAAC TTCCTTTTTG GT -#TCAAGTGT   1380   - - ATGTTTGTGA TAGAAATGAA GACAGGCTAC ATGATGCATA TCTAACATAA AC -#ACAAACAT   1440   - - TAAGAAAGGA AATCAACCTG AAGAGTATTT ATACAGATAA CAAAATACAG AG -#AGTGAGTT   1500   - - AAATGTGTAA TAACTGTGGC ACAGGCTGGA ATATGAGCCA TTTAAATCAC AA -#ATTAATTA   1560   - - GAAAAAAAAC AGTGGGGAAA AAATTCCATG GATGGGTCTA GAAAGACTAG CA -#TTGTTTTA   1620   - - GGTTGAGTGG CAGTGTTTAA AGGGTGATAT CAGACTAAAC TTGAAATATG TG -#GCTAAATA   1680   - - ACTAGAATAC TCTTTATTTT TTCGTATCAT GAATAGCAGA TATAGCTTGA TG -#GCCCCATG   1740   - - CTTGGTTTAA CATCCTTGCT GTTCCTGACA TGAAATCCTT AATTTTTGAC AA -#AGGGGCTA   1800   - - TTCATTTTCA TTTTATATTG GGCCTAGAAA TTATGTAGAT GGTCCTGAGG AA -#AAGTTTAT   1860   - - AGCTTGTCTA TTTCTCTCTC TAACATAGTT GTCAGCACAA TGCCTAGGCT AT -#AGGAAGTA   1920   - - CTCAAAGCTT CTCTGTTCTT CCAGACACTT TCACAGGTCA GTCCATGAGG AA -#ACAAATTA   1980   - - TATGCCTTGA TTTAGTCTTT GAACACATTG CCTCTCTCAG GGTTACTGAA AC -#TTTGGGGT   2040   - - TATGATTGAA TTTTTACTTA TTTGTATTTC TTACTACATG GAGAGGTCTC TA -#CGTTTTCA   2100   - - CATATGGATT AATGGAGTAA GTATTTCACA GGATCTCTCA ATCCACACTT AT -#TCTATTTT   2160   - - TCTATCATTA TTCATTTGGT CGTAAATTAA ATTAAGAAAA AATTGAATGA GT -#AGATAAGT   2220   - - AAATGAATAT TTGTTTTCAT ACCAGAATGA CTTAATCCCA AACAGGCGAA CA -#AAAGAGAT   2280   - - GCATATTTAG AACAGAGGCA GAAGTTTTAT CCATGGTGTC CTTGTAATTC TT -#TTGCATAT   2340   - - TCTGAAGGCA CAGGAGGTGA TCCATCCACA TAGTCTTAAG TTGAATCATA GT -#GTACAAAT   2400   - - CCTTTCCACT TTCTGGAGCC CAGATTCTTA ATTTGTGTTA TAAAAAAATT GA -#GGAAGTGT   2460   - - TTTCCAAGAG ATTACCTCGT TGAGATTCTA AAATCTCTAC AAGCAAACTT GC -#TAAGGAAG   2520   - - ATGATTTTAG TAGCAATGTG TATTGCTGGA ATGACTGAGA CCTTGAGATG CC -#CAGAAAGA   2580   - - GGGCTGACGG TCTCAAGTCA GTACCAGGAA GACCAAGGAG AAGTATGACT AT -#CATCATTC   2640   - - AAGCCTCACC CTGTGGAACC ACAACTTGGA ATGAGCCAAT CTGCTCACAG AA -#GCAGGGAG   2700   - - GGCAGGAGGC AGGGCTGGGC ATAAAAGGAA GAGCCGGGCC AGGGGGCCCA CA -#AGAGACCG   2760   - - GCTCAAGGAT CCCAAGGCCC AACTCCCCGA ACCACTCAGG GTCCTGTGGA CG -#CTCACCTA   2820   - - GCTGCAATGG CTACAGGTAA GCGCCCCTAA AATCCCTTTG GGCACAATGT GT -#CCTGAGGG   2880   - - GAGAGGCAGC GACCTGTAGA TGGGACGGGG GCACTAACCC TCAGGTTTGG GG -#CTTCTGAA   2940   - - TGAGTATCGC CATGTAAGCC CAGTATGGCC AATCTCAGAA AGCTCCTGGT CC -#CTGGAGGG   3000   - - ATGGAGAGAG AAAAACAAAC AGCTCCTGGA GCAGGGAGAG TGCTGGCCTC TT -#GCTCTCCG   3060   - - GCTCCCTCTG TTGCCCTCTG GTTTCTCCCC AGGCTCCCGG ACGTCCCTGC TC -#CTGGCTTT   3120   - - TGGCCTGCTC TGCCTGCCCT GGCTTCAAGA GGGCAGTGCC TTCCCAACCA TT -#CCCTTATC   3180   - - CAGGCTTTTT GACAACGCTA GTCTCCGCGC CCATCGTCTG CACCAGCTGG CC -#TTTGACAC   3240   - - CTACCAGGAG TTTGTAAGCT CTTGGGGAAT GGGTGCGCAT CAGGGGTGGC AG -#GAAGGGGT   3300   - - GACTTTCCCC CGCTGGGAAA TAAGAGGAGG AGACTAAGGA GCTCAGGGTT TT -#TCCCGAAG   3360   - - CGAAAATGCA GGCAGATGAG CACACGCTGA GTGAGGTTCC CAGAAAAGTA AC -#AATGGGAG   3420   - - CTGGTCTCCA GCGTAGACCT TGGTGGGCGG TCCTTCTCCT AGGAAGAAGC CT -#ATATCCCA   3480   - - AAGGAACAGA AGTATTCATT CCTGCAGAAC CCCCAGACCT CCCTCTGTTT CT -#CAGAGTCT   3540   - - ATTCCGACAC CCTCCAACAG GGAGGAAACA CAACAGAAAT CCGTGAGTGG AT -#GCCTTGAC   3600   - - CCCAGGCGGG GATGGGGGAG ACCTGTAGTC AGAGCCCCCG GGCAGCACAG GC -#CAATGCCC   3660   - - GTCCTTCCCC TGCAGAACCT AGAGCTGCTC CGCATCTCCC TGCTGCTCAT CC -#AGTCGTGG   3720   - - CTGGAGCCCG TGCAGTTCCT CAGGAGTGTC TTCGCCAACA GCCTGGTGTA CG -#GCGCCTCT   3780   - - GACAGCAACG TCTATGACCT CCTAAAGGAC CTAGAGGAAG GCATCCAAAC GC -#TGATGGGG   3840   - - GTGGGGGTGG CGCTAGGGGT CCCCAATCTT GGAGCCCCAC TGACTTTGAG AG -#CTGTGTTA   3900   - - GAGAAACACT GCTGCCCTCT TTTTAGCAGT CCAGGCCCTG ACCCAAGAGA AC -#TCACCTTA   3960   - - TTCTTCATTT CCCCTCGTGA ATCCTCTAGC CTTTCTCTAC ACCCTGAAGG GG -#AGGGAGGA   4020   - - AAATGAATGA ATGAGAAAGG GAGGGAGCAG TACCCAAGCG CTTGGCCTCT CC -#TTCTCTTC   4080   - - CTTCACTTTG CAGAGGCTGG AAGATGGCAG CCCCCGGACT GGGCAGATCT TC -#AAGCAGAC   4140   - - CTACAGCAAG TTCGACACAA ACTCACACAA CGATGACGCA CTACTCAAGA AC -#TACGGGCT   4200   - - GCTCTACTGC TTCAGGAAGG ACATGGACAA GGTCGAGACA TTCCTGCGCA TC -#GTGCAGTG   4260   - - CCGCTCTGTG GAGGGCAGCT GTGGCTTCTA GCTGCCCGGG TGGCATCCCT GT -#GACCCCTC   4320   - - CCCAGTGCCT CTCCTGGCCT TGGAAGTTGC CACTCCAGTG CCCACCAGCC TT -#GTCCTAAT   4380   - - AAAATTAAGT TGCATCATTT TGTCTGACTA GGTGTCCTCT ATAATATTAT GG -#GGTGGAGG   4440   - - GGGGTGGTTT GGAGCA             - #                  - #- #  4456__________________________________________________________________________