Abstract:
The invention relates to a method for efficient RNA silencing of target genes in eucaryotic cells, particularly plant cells. Consequently, the method can be used to reduce the phenotypic expression of an endogenous gene in a plant cell. Furthermore, the method can be applied in a high throughput screening for mutant phenotypes as a result of RNA silencing of any endogene.

Description:
CROSS-REFERENCE TO RELATED APPLICATION  
       [0001]    This application is a continuation of PCT International Patent Application No. PCT/EP/02/11188, filed on Oct. 2, 2002, designating the United States of America, and published, in English, as PCT International Publication No. WO 03/031632 A1 on Apr. 17, 2003, the contents of the entirety of which is incorporated by this reference. 
     
    
     
       TECHNICAL FIELD  
         [0002]    The invention relates generally to biotechnology, and more particularly to a method for efficient RNA silencing in eucaryotic cells, particularly plant cells. Consequently, the method can be used to reduce the phenotypic expression of an endogenous gene in a plant cell. Furthermore, the method can be applied in a high throughput screening for RNA silencing.  
         BACKGROUND  
         [0003]    “RNA silencing” is a type of gene regulation based on sequence-specific targeting and degradation of RNA. The term encompasses related pathways found in a broad range of eukaryotic organisms, including fungi, plants, and animals.  
           [0004]    In plants, RNA silencing serves as an antiviral defense and many plant viruses encode suppressors of silencing. Also, it becomes clear that elements of the RNA silencing system are essential for gene regulation in development. The emerging view is that RNA silencing is part of a sophisticated network of interconnected pathways for cellular defense, transposon surveillance, and regulation of development. Based on the sequence specific RNA degradation, RNA silencing has become a powerful tool to manipulate gene expression experimentally. RNA silencing was first discovered in transgenic plants, where it was termed co-suppression or posttranscriptional gene silencing (PTGS). Sequence-specific RNA degradation processes related to PTGS have also been found in ciliates, fungi, and a variety of animals from  Caenorhabditis elegans  to mice (RNA interference).  
           [0005]    A key feature uniting the RNA silencing pathways in different organisms is the importance of double-stranded RNA (dsRNA) as a trigger or an intermediate. The dsRNA is cleaved into small interfering RNAs (21 to 25 nucleotides) of both polarities, and these are thought to act as guides to direct the RNA degradation machinery to the target RNAs. An intriguing aspect of RNA silencing in plants is that it can be triggered locally and then spread via a mobile silencing signal. In plants, RNA silencing is correlated with methylation of homologous transgene DNA in the nucleus. Other types of epigenetic modifications may be associated with silencing in other organisms.  
           [0006]    It is known from the art that transgenes encoding ds or self-complementary (hairpin) RNAs of endogenous gene sequences are highly effective at directing the cell&#39;s degradation mechanism against endogenous (ss) mRNAs, thus giving targeted gene suppression. This discovery has enabled the transgenic enhancement of a plant&#39;s defense mechanism against viruses that it is unable to combat unaided. It has also shed light on how antisense and co-suppression might operate: by the inadvertent integration of two copies of the transgenes in an inverted repeat orientation, such that read-through transcription from one gene into the adjacent copy produces RNA with self-complementary sequences.  
           [0007]    RNA silencing is induced in plants by transgenes designed to produce either sense or antisense transcripts. Furthermore, transgenes engineered to produce self-complementary transcripts (dsRNAs) are potent and consistent inducers of RNA silencing. Finally, replication of plant viruses, many of which produce dsRNA replication intermediates, causes a type of RNA silencing called Virus Induced Gene Silencing (VIGS). Whether VIGS, and the different types of transgene-induced RNA silencing in plants result from similar or distinct mechanisms is still a matter of debate. However, recent genetic evidence raises the possibility that the RNA silencing pathway is branched and that the branches converge in the production of dsRNA.  
         SUMMARY OF THE INVENTION  
         [0008]    Until recently, RNA silencing was viewed primarily as a thorn in the side of plant molecular geneticists, limiting expression of transgenes and interfering with a number of applications that require consistent, high-level transgene expression. With our present understanding of the process, however, it is clear that RNA silencing could have enormous potential for engineering control of gene expression, as well as for the use as a tool in functional genomics. It could be experimentally induced and targeted to a single specific gene or even to a family of related genes. Likewise, ds RNA-induced TGS may have similar potential to control gene expression. Although methods for RNA silencing have been described in the art (e.g., WO99/53050, WO99/32619, WO99/61632, and WO98/53083), a need exists to develop alternative and more efficient tools for RNA silencing.  
           [0009]    In the present invention, we have developed a highly efficient method for RNA silencing that can also be used as a tool for high throughput silencing. The method uses a host that carries already a silenced locus and a second recombinant gene comprising a region that is homologous with the silenced locus. Although it is known that the recombinant gene will be silenced, we have surprisingly found that also target genes, which have no significant homology with the silenced locus but have homology with the recombinant gene, are efficiently silenced.  
           [0010]    The present invention deals with an efficient method for RNA silencing in a eucaryotic host. The method makes use of a host that already comprises a silenced locus. Such a silenced locus can for example be generated by methods known in the art. For example the publication of De Buck and Depicker, 2001 and other publications, and also PCT patent publications WO99/53050, WO99/32619, WO99/61632, and WO98/53083 describe methods to obtain RNA silencing and for generating a silenced recombinant locus. The ‘target gene’ is here defined as the gene of interest for silencing or to down-regulate its expression. An important aspect of this invention is that the target gene has no significant homology with the silenced locus. No significant homology means that either the overall homology is less than 40, 35, 30, 25% or even less or that no contiguous stretch of at least 23 identical nucleotides are present (Thomas et al., 2001). Homology is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of homology to various insertions, deletions, substitutions, and other modifications. Silencing of the target gene in the present invention occurs via an intermediate step and hence our method is designated as domino silencing (FIG. 1). In the intermediate step a recombinant gene construct is introduced by transformation into the host comprising the silenced locus. The recombinant gene construct has a region of homology with the silenced locus already present. The region of homology is preferably more than 60, 70, 80, 90, 95 or even more than 99% homologous. The homologous region between the silenced locus and the recombinant gene can be found in the 5′ untranslated or 3′ untranslated region of the recombinant gene construct. Furthermore, the recombinant gene construct has a region of minimal 23 nucleotides (Thomas et al., 2001), but preferably longer, that are identical with the target gene, or has a region of overall homology of more than 60, 70, 80, 90, 95 or even more than 99%. A recombinant gene is defined herein as a construct which does not naturally occur in nature. A non-limiting example of a recombinant gene construct is a construct wherein the coding region of a gene is operably linked to a 5′ untranslated region and/or to a 3′ untranslated region of one or more other genes, alternatively the 5′ or 3′ untranslated region is an artificial sequence.  
           [0011]    Thus, in one embodiment the invention provides a method for obtaining efficient RNA silencing of a target gene comprising the introduction of a recombinant gene into a host that comprises a silenced locus and an unsilenced target gene whereby the recombinant gene comprises a region that is homologous with the silenced locus and whereby the target gene has homology with the recombinant gene but has no significant homology with the silenced locus.  
           [0012]    In another embodiment, the method is used wherein the host is a plant or plant cell.  
           [0013]    In another embodiment, the method of the invention can be used for high throughput gene silencing. Indeed, a recombinant gene library can be made wherein for example every gene or coding region thereof is combined with (operably linked with) a region of homology with the silenced gene that resides in the silenced locus and the recombinant gene library can be transformed to an eukaryotic host or individual (specific) genes derived from the recombinant gene library can be transformed into an eukaryotic host wherein silencing of specific genes is wanted.  
           [0014]    In yet another embodiment, the invention provides a plant or plant cell that comprises a silenced locus and wherein a silenced target gene is obtained through the introduction of a recombinant gene according to the current method of the invention.  
           [0015]    In yet another embodiment, the RNA silencing of the target gene is obtained in more than 80, 85, 90 or 95% of the transgenic organisms.  
           [0016]    In yet another embodiment, the RNA silencing of the target gene occurs at an efficiency of more than 80, 85, 90 or 95% as compared to the level of the unsilenced expression of the target gene.  
       
    
    
     BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS  
       [0017]    [0017]FIG. 1: Schematic outline of homology between a silenced locus X, a recombinant gene Y and a target gene Z.  
         [0018]    [0018]FIG. 2: Schematic outline of the T-DNA constructs that are present in silenced locus X 1 , recombinant gene Y 1  and target gene Z 1  (T-DNAs of pGVCHS287, pGUSchsS and pXD610 respectively) and of the transcript homology between X 1 , Y 1  and Z 1 .  
         [0019]    LB and RB: left and right T-DNA border respectively; Pnos: nopaline synthase promoter; hpt: hygromycin phosphotransferase coding sequence; 3′nos: 3′untranslated region of the nopaline synthase gene; P35S; Cauliflower mosaic virus 35S promoter; nptII c.s., neomycin phosphotransferase II coding sequence; 3′chs: 3′untranslated region of the chalcone synthase gene of Anthirrinum majus; +1: transcription start; A n : poly A-tail; gus c.s.: β-glucuronidase coding sequence; Pss: promoter of the small subunit of rubisco; bar: phosphinotricine transferase coding sequence; 3′g7: 3′untranslated region of the  Agrobacterium octopine  T-DNA gene 7; 3′ocs: 3′untranslated region of octopine synthase gene.  
         [0020]    [0020]FIG. 3: Schematic outline of the T-DNA construct present in silenced locus X 1  and of the transiently introduced T-DNAs Y 2  (T-DNAs of pGVCHS287 and pPs35SCAT1S3chs, respectively) and of the transcript homology between X 1 , Y 2  and Z 2  (the catalase1 endogene). Abbreviations as in FIG. 2 
         [0021]    [0021]FIG. 4: Schematic outline of the T-DNA constructs present in silenced locus X 2  and of the transiently introduced T-DNAs Y 2  (T-DNAs of pGUSchsS+pGUSchsAS, and pPs35SCAT1S3chs, respectively) and of the transcript homology between X 2 , Y 2  and Z 2  (the catalase1 endogene). Abbreviations as in FIG. 2 
         [0022]    [0022]FIG. 5: pPs35SCAT1S3chs 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0023]    A post-transcriptionally silenced inverted repeat transgene locus can trigger silencing of a reporter gene producing non-homologous transcripts.  
         [0024]    We studied the interaction between three transgene loci X 1 , Y 1  and Z 1  (FIG. 2. For a detailed description of all loci and constructs, see materials and methods) to address the question whether or not a stepwise homology between loci can lead to silencing.  
         [0025]    It has been demonstrated previously that the post-transcriptionally silenced nptII genes in locus XI are capable to in trans silence transiently expressed genes with partial transcript homology to their nptII transcripts (Van Houdt et al., 2000 b). We subsequently found that also a stably expressed β-glucuronidase (gus) gene (in locus Y 1 ), with partial transcript homology to the nptII transcripts of the silencing inducing locus X 1 , becomes efficiently silenced in trans (FIG. 2: X 1  and Y 1  and table 1: X 1 Y 1  compared to Y 1 ). On the contrary, the nptII genes of locus X 1  are not able to trigger silencing of the gus genes in locus Z 1  which is expected as the genes of both loci produce transcripts without significant homology (FIG. 2). The homology between the two transcripts of X 1  and Y 1  is mainly situated in the 3′untranslated region (250 nucleotides), but also the 5′untranslated sequences show a small region of homology (29 nucleotides). These results demonstrate that the in trans silencing effects are not triggered by promoter homology. When Y 1  and Z 1  loci are combined in so called Y 1 Z 1  hybrids both types of gus genes, having transcript homology in the gus coding sequence of 1809 nucleotides, remain highly expressed as reflected in the normal gus activity showing that the RNA silencing mechanism does not become activated (Table 1: Y 1 Z 1  compared to Y 1  and Z 1 ). Surprisingly, upon creation of a stepwise homology between X 1  and Z 1  by introducing locus Y 1 , the new observation described here is that also the gus expression in locus Z 1  is reduced in X 1 Y 1 Z 1  plants (Table 1: X 1 Y 1 Z 1  compared to Y 1 Z 1 ). Thus, creating a stepwise homology between a silenced locus and a target gene by introducing a recombinant gene is sufficient to trigger silencing of the target.  
         [0026]    Silencing inducing transgene loci can trigger silencing of a non-homologous endogene.  
         [0027]    We further assessed the universality and the usefulness in high throughput functional gene analyses of silencing elicited by a stepwise homology in trans, called domino silencing. Therefore, we evaluated whether the expression of the tobacco endogenous catalase1 (cat1) genes is reduced in plants carrying a silencing locus (X locus) showing no significant homology with the catalase endogene by introducing a recombinant gene (Y construct). As silencing locus we used either X 1  or X 2  (FIG. 2: locus X 1 , FIG. 3: locus X 2 ), in either case containing the 3′ chalcone synthase sequences of Anthirrinum majus (3′chs). As transmitter for silencing we constructed a recombinant gene composed of the catalase1 coding sequence and the 3′ chs region under control of the 35S promoter (P35S) (residing on T-DNA pPs35SCAT1S3chs, FIGS. 2 and 3: T-DNA in Y 2 ). The recombinant cat1 3′chs genes (Y2) were introduced in tobacco leaves bearing locus X 1  (or X 2 ) via Agrobacterium injection. As a negative control, we introduced a recombinant gene in which the cat1 coding sequence is replaced by the gus coding sequence (pGUSchsS, T-DNA construct as in locus Y 1  FIG. 1). In this case, no stepwise homology is created between the silencing inducing locus and the target catalase endogenes. As a positive control, the recombinant construct Y 2  was also introduced in transgenic tobacco with silenced catalase1 genes by the presence of a catalase1 antisense construct (Cat1AS in Champnongpol et al., 1996). Sixteen days after Agrobacterium injection, the catalase activity was determined in protein extracts of injected leaf tissue and compared with the activity in non-injected wild type (SR1) leaf tissue (Table 2). The results indicate that domino silencing is also applicable to endogenes since the catalase activity is clearly reduced in 6 out of 7 samples, while it remains high in the negative controls. In conclusion, not only an inverted repeat-bearing silencing-inducing transgene locus, but also a silencing-inducing locus in which the two residing chimeric genes give rise to transcripts with complementarity in the 3′UTR (3′chs)(FIG. 3: X 2 ), is able to trigger domino silencing reducing endogenous catalase expression.  
                                                           TABLE 1                           Results of a GUS-activity determination in protein extracts of leaf       tissue harvested from tobacco plants containing different       combinations of the loci X 1 , Y 1  and Z 1         (FIG. 2). The mean values of a number of plants (n) are given                GUS-act. 1  4 weeks 2         GUS-act. Mature 2             Genotype   U GUS/mg TSP   N   U GUS/mg TSP   n                    X 1     &lt; 3     1   &lt;   1       Y 1         368 ± 165 4     9   n.d.   —       Z 1     126 ± 30    10   48 ± 8    5       X 1 Y 1     2 ± 1   4   4 ± 2   4       X 1 Z 1     139 ± 35    9   46 ± 14   5       Y 1 Z 1     477 ± 101   10   231 ± 106   6       X 1 Y 1 Z 1   5  → Y 1 Z 1  →   195 ± 104   16   315 ± 46   8       X 1 Y 1 Z 1     4 ± 3   22   12 ± 4    9                                                                  
 
         [0028]    [0028]                                               TABLE 2                           Results of a catalase-activity determination in protein       extracts of leaf tissue harvested from Agrobacterium       injected tobacco leaves.            Genotype injected   Construct introduced via   catalase activity 16 days       Plant   Agrobacterium injection   after injection (60 μg TSP)                    WT (SR1)   -(non-injected)   −0.2116 2     100% 3          X 1     PGUSchsS   −0.2556   121%        X 1     Y 2     −0.0589   27%       X 1   4     Y 2     −0.0698   33%       X 2     PGUSchsS   −0.1782   84%       X 2     Y 2     −0.0641   30%       X 2     Y 2     −0.0987   47%       X 2   4     Y 2     −0.0914   43%       X 2   4     Y 2     −0.1996   94%       X 2   4     Y 2     −0.0627   30%       Cat1AS   Y 2     −0.0439   21%                                                    
       EXAMPLES  
     Materials and Methods  
       [0029]    Plasmid Construction  
         [0030]    pPs35SCAT1S3chs: The T-DNA of this plasmid is schematically shown in FIG. 3: Y 2  and the nucleotide sequence is depicted in SEQ ID NO:1 of the accompanying and incorporated herein SEQUENCE LISTING.  
         [0031]    Description of the Transgene Loci and Production of Hybrid Plants  
         [0032]    Locus X 1  harbors an inverted repeat about the right T-DNA border of construct pGVCHS287, carrying a neomycin phosphotransferase II (nptII) gene under the control of the Cauliflower mosaic virus 35S promoter (P35S) and the 3′signalling sequences of the Anthirrinum majus chalcone synthase gene (3′chs). The nptII genes are post-transcriptionally silenced and can trigger in trans silencing and methylation of homologous target genes (Van Houdt et al., 2000 a and b and FIG. 2).  
         [0033]    Locus Y 1  contains a single copy of the pGUSchsS T-DNA, containing a gus gene under the control of P35S and 3′chs (in transformant GUSchsS29) and shows normal levels of gus expression (FIG. 2).  
         [0034]    Locus Z 1  contains more than one copy of the pXD610 T-DNA, harboring the gus gene under control of P35S and the 3′untranslated region (UTR) of the nopaline synthase gene (3′nos), (in plant LXD610-2) and shows normal gus expression (De Loose et al., 1995 and FIG. 2).  
         [0035]    Locus X 2  contains a single copy of both the pGUSchsS and pGUSchsAS T-DNA (in transformant GUSchsS+GUSchsAS11) and triggers silencing in cis of the gus genes, but also in trans of (partially) homologous genes (FIG. 4).  
         [0036]    X 1  and Z 1  hemizygous plants were obtained as hybrid progeny of the crossing of tobacco plants homozygous for locus X 1  (=Holo1; Van Houdt et al., 2000 a and b) and homozygous for locus Z 1  (=LXD610-2/9 De Loose et al., 1995) to wild type SR1 respectively. Y 1  hemizygous plants were obtained by crossing the hemizygous primary tobacco transformant GUSchsS29 to SR1 and selecting for the presence of locus Y 1  in the hybrid progeny. X 1 Y 1  and Y 1 Z 1  hemizygous plants are the hybrid progeny plants of the cross between Holo1 and GUSchsS29 and between GUSchsS29 and LXD610-2/9 respectively that are selected for the presence of Y 1 . X 1 Z 1  hemizygous plants are the hybrid progeny of the cross between Holo1 and LXD610-2/9. X 1 Y 1 Z 1  hemizygous plants were obtained by crossing X 1 Y 1  hemizygous plants to LXD610-2/9; as we only selected for the presence of Y 1  in the hybrid progeny both Y 1 Z 1  and X 1 Y 1 Z 1  hemizygous plants were obtained.  
         [0037]    Preparation of Agrobacteria and Injection  
         [0038]    The Agrobacteria C58C1Rif R  (pGV2260) (pGUSchsS)Cb R ,PPT R  or C58C1Rif R (pMP90)(pPs35SCAT1S3chs)Gm R ,PPT R  were mainly grown as described by Kapila et al., 1997 except that the Agrobacteria were resuspended in MMA to a final OD 600  of 1. Greenhouse grown plants of 10 to 15 cm in height were used. Half of the third top leaf was injected via the lower surface using a 5 ml syringe while the leaf remained attached to the plant. The plants were kept in the greenhouse and 16 days after injection three to four discs of 11 mm in diameter were excised from the injected tissue for the preparation of a fresh protein extract to determine the catalase activity.  
         [0039]    Enzymatic Assays  
         [0040]    Preparation of the protein extracts and GUS-activity measurements were done as previously described (Van Houdt et al., 2000 b). Preparation of the protein extracts for catalase-activity measurement and the spectrophotometric catalase-activity determination was done according to Champnongpol et al., 1996.  
       REFERENCES  
       [0041]    Van Houdt, H., Kovarik, A., Van Montagu, M., and Depicker, A. (2000 a). Cross-talk between posttranscriptionally silenced neomycin phosphotransferase II transgenes.  FEBS Lett.  467, 41-46.  
         [0042]    Van Houdt, H., Kovarik, A., Van Montagu, M., and Depicker, A. (2000 b) Both sense and antisense RNAs are targets for the sense transgene-induced posttranscriptional silencing mechanism.  Mol. Gen. Genet.  263, 995-1002.  
         [0043]    De Loose, M., Danthinne, X., Van Bockstaele, E., Van Montagu, M. and Depicker, A., (1995) Different 5′leader sequences modulate β-glucuronidase accumulation levels in transgenic  Nicotiana tobacum  plants.  Euphytica  85, 209-216.  
         [0044]    Kapila, J., De Rycke, R., Van Montagu, M. and Angenon, G. (1997) An Agrobacterium-mediated transient gene expression system for intact leaves.  Plant Science  122, 101-108.  
         [0045]    Champnongpol, S., Willekens, H., Langebartels, C., Van Montagu, M., Inzé, D., and Van Camp, W. (1996) Transgenic tobacco with a reduced catalase activity develops necrotic lesions and induces pathogenesis-related expression under high light.  Plant J.  10(3), 491-503.  
         [0046]    Thomas, C. L., Jones, L., Baulcombe, D. C. and Maule, A. J. (2001) Size constraints for targetting post-transcriptional gene silencing and for RNA-directed methylation in  Nicotiana benthamiana  using potato virus X vector.  Plant J.  25(4), 417-425.  
         [0047]    De Buck, S. and Depicker, A. (2001) Disruption of their palindromic arrangement leads to selective loss of DNA methylation in inversely repeated gus transgenes in Arabidopsis.  Mol. Gen. Genom.  265, 1060-1068.  
     
       
       
         1 
         
           
             1  
           
           
             1  
             10635  
             DNA  
             Artificial Sequence  
             
               pPs35SCAT1S3chs  
             
           
            1 

agattcgaag ctcggtcccg tgggtgttct gtcgtctcgt tgtacaacga aatccattcc     60 

cattccgcgc tcaagatggc ttcccctcgg cagttcatca gggctaaatc aatctagccg    120 

acttgtccgg tgaaatgggc tgcactccaa cagaaacaat caaacaaaca tacacagcga    180 

cttattcaca cgcgacaaat tacaacggta tatatcctgc cagtactcgg ccgtcgaata    240 

acttcgtata atgtatgcta tacgaagtta tgaattcgcg ctctatcata gatgtcgcta    300 

taaacctatt cagcacaata tattgttttc attttaatat tgtacatata agtagtaggg    360 

tacaatcagt aaattgaacg gagaatatta ttcataaaaa tacgatagta acgggtgata    420 

tattcattag aatgaaccga aaccggcggt aaggatctga gctacacatg ctcaggtttt    480 

ttacaacgtg cacaacagaa ttgaaagcaa atatcatgcg atcataggcg tctcgcatat    540 

ctcattaaag cagctggaag atttgatgga tcctcatcag atctcggtga cgggcaggac    600 

cggacggggc ggtaccggca ggctgaagtc cagctgccag aaacccacgt catgccagtt    660 

cccgtgcttg aagccggccg cccgcagcat gccgcggggg gcatatccga gcgcctcgtg    720 

catgcgcacg ctcgggtcgt tgggcagccc gatgacagcg accacgctct tgaagccctg    780 

tgcctccagg gacttcagca ggtgggtgta gagcgtggag cccagtcccg tccgctggtg    840 

gcggggggag acgtacacgg tcgactcggc cgtccagtcg taggcgttgc gtgccttcca    900 

ggggcccgcg taggcgatgc cggcgacctc gccgtccacc tcggcgacga gccagggata    960 

gcgctcccgc agacggacga ggtcgtccgt ccactcctgc ggttcctgcg gctcggtacg   1020 

gaagttgacc gtgcttgtct cgatgtagtg gttgacgatg gtgcagaccg ccggcatgtc   1080 

cgcctcggtg gcacggcgga tgtcggccgg gcgtcgttct gggctcatgg tagatctgtt   1140 

taaacgttaa cggattgaga gtgaatatga gactctaatt ggataccgag gggaatttat   1200 

ggaacgtcag tggagcattt ttgacaagaa atatttgcta gctgatagtg accttaggcg   1260 

acttttgaac gcgcaataat ggtttctgac gtatgtgctt agctcattaa actccagaaa   1320 

cccgcggctg agtggctcct tcaatcgttg cggttctgtc agttccaaac gtaaaacggc   1380 

ttgtcccgcg tcatcggcgg gggtcataac gtgactccct taattctccg ctcatgatca   1440 

agctacctca gcaggatccg gcgcgccatg gtcgataaga aaaggcaatt tgtagatgtt   1500 

aattcataac atctcctcca tgacttaaaa aacttgcaaa agatttatat agaaatactt   1560 

aaatattttg actaaaaaaa aaaaaaaaaa aacacacaca taaaccaaca aataacataa   1620 

attattttta tatagccttt atttcaatga tcacaacgaa acaatacaag tacaaagcgt   1680 

tacaagagag aaatcgccaa tatagctcac atgcagcaca catcacaata ataggtaacc   1740 

atgtccactt ttttattacg gaaataagaa aataacccaa cccccgtacc cgggttcata   1800 

tgcttggtct cacattaagc ctagaagcta gcttttgacc cagagatttg tcagcctgag   1860 

accagtatga gatccaaatg ctgcggatct cataagtgat acgaggatca gacaaggtct   1920 

ccacccaccg acgaataaag cgttcttgcc tgtctggtgt gaatgagcgg tacctttctc   1980 

ctggttgctt gaaattgttc tctttctgaa tgacacactt ctcgcgtttg ccagtgcaca   2040 

ttgtagaagg aataggatac ttctcagcat ggcgaacagg atcatacctt gaagggaagt   2100 

agtcgatctc ctcatccctg tgcataaaat tcatggagcc atcgtagtga ttgttgtgat   2160 

gagcgcattt tggagcatta gcaggtagtt gcaaatagtt tggtccaagt cgatacctct   2220 

gggtatcaga gtaggagaaa atacgagttt gaagcatctt atcatctgag taataaaccc   2280 

ctggaacaac aatagaaggg cagaaagcta gctgctcatt ctcattagag aagttatcaa   2340 

tgttcttgtt cagaactaat cttcccaccg gctgcaaagg caagatatcc tctggccaag   2400 

tttttgtcac atcaagtgga tcaaaatcaa atctgtcttc atgatctgga tccatagtcc   2460 

ccgggcagtg ggcgatttga tttaaatctc tagaatagta aattgtaatg ttgtttgttg   2520 

tttgttttgt tgtggtaatt gttgtaaaaa tacggatcgt cctgcagtcc tctccaaatg   2580 

aaatgaactt ccttatatag aggaagggtc ttgcgaagga tagtgggatt gtgcgtcatc   2640 

ccttacgtca gtggagatat cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc   2700 

ttctttttcc acgatgctcc tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga   2760 

ggcatcttga acgatagcct ttcctttatc gcaatgatgg catttgtagg tgccaccttc   2820 

cttttctact gtccttttga tgaagtgaca gatagctggg caatggaatc cgaggaggtt   2880 

tcccgatatt accctttgtt gaaaagtctc aatagccctt tggtcttctg agactgtatc   2940 

tttgatattc ttggagtaga cgagagtgtc gtgctccacc atgttgacga agattttctt   3000 

cttgtcattg agtcgtaaaa gactctgtat gaactgttcg ccagtcttca cggcgagttc   3060 

tgttagatcc tcgatctgaa tttttgactc catggccttt gattcagtag gaactacttt   3120 

cttagagact ccaatctcta ttacttgcct tggtttatga agcaagcctt gaatcgtcca   3180 

tactggaata gtacttctga tcttgagaaa tatatctttc tctgtgttct tgatgcagtt   3240 

agtcctgaat cttttgactg catctttaac cttcttggga aggtatttga tctcctggag   3300 

attattactc gggtagatcg tcttgatgag acctgccgcg taggcctctc taaccatctg   3360 

tgggtcagca ttctttctga aattgaagag gctaatcttc tcattatcgg tggtgaacat   3420 

ggtatcgtca ccttctccgt cgaactttct tcctagatcg tagagataga gaaagtcgtc   3480 

catggtgatc tccggggcaa aggagatctc tagagtcgag atttaaatcc taaatcctgc   3540 

aggaagctta ccggtataac ttcgtatagc atacattata cgaagttatc catggagcca   3600 

tttacaattg aatatatcct gccgccgctg ccgctttgca cccggtggag cttgcatgtt   3660 

ggtttctacg cagaactgag ccggttaggc agataatttc cattgagaac tgagccatgt   3720 

gcaccttccc cccaacacgg tgagcgacgg ggcaacggag tgatccacat gggactttta   3780 

aacatcatcc gtcggatggc gttgcgagag aagcagtcga tccgtgagat cagccgacgc   3840 

accgggcagg cgcgcaacac gatcgcaaag tatttgaacg caggtacaat cgagccgacg   3900 

ttcacggtac cggaacgacc aagcaagcta gcttagtaaa gccctcgcta gattttaatg   3960 

cggatgttgc gattacttcg ccaactattg cgataacaag aaaaagccag cctttcatga   4020 

tatatctccc aatttgtgta gggcttatta tgcacgctta aaaataataa aagcagactt   4080 

gacctgatag tttggctgtg agcaattatg tgcttagtgc atctaacgct tgagttaagc   4140 

cgcgccgcga agcggcgtcg gcttgaacga attgttagac attatttgcc gactaccttg   4200 

gtgatctcgc ctttcacgta gtggacaaat tcttccaact gatctgcgcg cgaggccaag   4260 

cgatcttctt cttgtccaag ataagcctgt ctagcttcaa gtatgacggg ctgatactgg   4320 

gccggcaggc gctccattgc ccagtcggca gcgacatcct tcggcgcgat tttgccggtt   4380 

actgcgctgt accaaatgcg ggacaacgta agcactacat ttcgctcatc gccagcccag   4440 

tcgggcggcg agttccatag cgttaaggtt tcatttagcg cctcaaatag atcctgttca   4500 

ggaaccggat caaagagttc ctccgccgct ggacctacca aggcaacgct atgttctctt   4560 

gcttttgtca gcaagatagc cagatcaatg tcgatcgtgg ctggctcgaa gatacctgca   4620 

agaatgtcat tgcgctgcca ttctccaaat tgcagttcgc gcttagctgg ataacgccac   4680 

ggaatgatgt cgtcgtgcac aacaatggtg acttctacag cgcggagaat ctcgctctct   4740 

ccaggggaag ccgaagtttc caaaaggtcg ttgatcaaag ctcgccgcgt tgtttcatca   4800 

agccttacgg tcaccgtaac cagcaaatca atatcactgt gtggcttcag gccgccatcc   4860 

actgcggagc cgtacaaatg tacggccagc aacgtcggtt cgagatggcg ctcgatgacg   4920 

ccaactacct ctgatagttg agtcgatact tcggcgatca ccgcttccct catgatgttt   4980 

aactttgttt tagggcgact gccctgctgc gtaacatcgt tgctgctcca taacatcaaa   5040 

catcgaccca cggcgtaacg cgcttgctgc ttggatgccc gaggcataga ctgtacccca   5100 

aaaaaacagt cataacaagc catgaaaacc gccactgcgc cgttaccacc gctgcgttcg   5160 

gtcaaggttc tggaccagtt gcgtgagcgc atacgctact tgcattacag cttacgaacc   5220 

gaacaggctt atgtccactg ggttcgtgcc ttcatccgtt tccacggtgt gcgtcacccg   5280 

gcaaccttgg gcagcagcga agtcgaggca tttctgtcct ggctggcgaa cgagcgcaag   5340 

gtttcggtct ccacgcatcg tcaggcattg gcggccttgc tgttcttcta cggcaagtgc   5400 

tgtgcacgga tctgccctgg cttcaggaga tcggaagacc tcggccgtcc gggcgcttgc   5460 

cggtggtgct gaccccggat gaagtggttc gcatcctcgg ttttctggaa ggcgagcatc   5520 

gtttgttcgc ccagcttctg tatggaacgg gcatgcggat cagtgagggt ttgcaactgc   5580 

gggtcaagga tctggatttc gatcacggca cgatcatcgt gcgggagggc aagggctcca   5640 

aggatcgggc cttgatgtta cccgagagct tggcacccag cctgcgcgag cagggatcga   5700 

tccaacccct ccgctgctat agtgcagtcg gcttctgacg ttcagtgcag ccgtcttctg   5760 

aaaacgacat gtcgcacaag tcctaagtta cgcgacaggc tgccgccctg cccttttcct   5820 

ggcgttttct tgtcgcgtgt tttagtcgca taaagtagaa tacttgcgac tagaaccgga   5880 

gacattacgc catgaacaag agcgccgccg ctggcctgct gggctatgcc cgcgtcagca   5940 

ccgacgacca ggacttgacc aaccaacggg ccgaactgca cgcggccggc tgcaccaagc   6000 

tgttttccga gaagatcacc ggcaccaggc gcgaccgccc ggagctggcc aggatgcttg   6060 

accacctacg ccctggcgac gttgtgacag tgaccaggct agaccgcctg gcccgcagca   6120 

cccgcgacct actggacatt gccgagcgca tccaggaggc cggcgcgggc ctgcgtagcc   6180 

tggcagagcc gtgggccgac accaccacgc cggccggccg catggtgttg accgtgttcg   6240 

ccggcattgc cgagttcgag cgttccctaa tcatcgaccg cacccggagc gggcgcgagg   6300 

ccgccaaggc ccgaggcgtg aagtttggcc cccgccctac cctcaccccg gcacagatcg   6360 

cgcacgcccg cgagctgatc gaccaggaag gccgcaccgt gaaagaggcg gctgcactgc   6420 

ttggcgtgca tcgctcgacc ctgtaccgcg cacttgagcg cagcgaggaa gtgacgccca   6480 

ccgaggccag gcggcgcggt gccttccgtg aggacgcatt gaccgaggcc gacgccctgg   6540 

cggccgccga gaatgaacgc caagaggaac aagcatgaaa ccgcaccagg acggccagga   6600 

cgaaccgttt ttcattaccg aagagatcga ggcggagatg atcgcggccg ggtacgtgtt   6660 

cgagccgccc gcgcacgtct caaccgtgcg gctgcatgaa atcctggccg gtttgtctga   6720 

tgccaagctg gcggcctggc cggccagctt ggccgctgaa gaaaccgagc gccgccgtct   6780 

aaaaaggtga tgtgtatttg agtaaaacag cttgcgtcat gcggtcgctg cgtatatgat   6840 

gcgatgagta aataaacaaa tacgcaaggg gaacgcatga aggttatcgc tgtacttaac   6900 

cagaaaggcg ggtcaggcaa gacgaccatc gcaacccatc tagcccgcgc cctgcaactc   6960 

gccggggccg atgttctgtt agtcgattcc gatccccagg gcagtgcccg cgattgggcg   7020 

gccgtgcggg aagatcaacc gctaaccgtt gtcggcatcg accgcccgac gattgaccgc   7080 

gacgtgaagg ccatcggccg gcgcgacttc gtagtgatcg acggagcgcc ccaggcggcg   7140 

gacttggctg tgtccgcgat caaggcagcc gacttcgtgc tgattccggt gcagccaagc   7200 

ccttacgaca tatgggccac cgccgacctg gtggagctgg ttaagcagcg cattgaggtc   7260 

acggatggaa ggctacaagc ggcctttgtc gtgtcgcggg cgatcaaagg cacgcgcatc   7320 

ggcggtgagg ttgccgaggc gctggccggg tacgagctgc ccattcttga gtcccgtatc   7380 

acgcagcgcg tgagctaccc aggcactgcc gccgccggca caaccgttct tgaatcagaa   7440 

cccgagggcg acgctgcccg cgaggtccag gcgctggccg ctgaaattaa atcaaaactc   7500 

atttgagtta atgaggtaaa gagaaaatga gcaaaagcac aaacacgcta agtgccggcc   7560 

gtccgagcgc acgcagcagc aaggctgcaa cgttggccag cctggcagac acgccagcca   7620 

tgaagcgggt caactttcag ttgccggcgg aggatcacac caagctgaag atgtacgcgg   7680 

tacgccaagg caagaccatt accgagctgc tatctgaata catcgcgcag ctaccagagt   7740 

aaatgagcaa atgaataaat gagtagatga attttagcgg ctaaaggagg cggcatggaa   7800 

aatcaagaac aaccaggcac cgacgccgtg gaatgcccca tgtgtggagg aacgggcggt   7860 

tggccaggcg taagcggctg ggttgtctgc cggccctgca atggcactgg aacccccaag   7920 

cccgaggaat cggcgtgacg gtcgcaaacc atccggcccg gtacaaatcg gcgcggcgct   7980 

gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc gcccagcggc aacgcatcga   8040 

ggcagaagca cgccccggtg aatcgtggca agcggccgct gatcgaatcc gcaaagaatc   8100 

ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg gcgacgagca   8160 

accagatttt ttcgttccga tgctctatga cgtgggcacc cgcgatagtc gcagcatcat   8220 

ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga gctggcgagg tgatccgcta   8280 

cgagcttcca gacgggcacg tagaggtttc cgcagggccg gccggcatgg ccagtgtgtg   8340 

ggattacgac ctggtactga tggcggtttc ccatctaacc gaatccatga accgataccg   8400 

ggaagggaag ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg acgtactcaa   8460 

gttctgccgg cgagccgatg gcggaaagca gaaagacgac ctggtagaaa cctgcattcg   8520 

gttaaacacc acgcacgttg ccatgcagcg tacgaagaag gccaagaacg gccgcctggt   8580 

gacggtatcc gagggtgaag ccttgattag ccgctacaag atcgtaaaga gcgaaaccgg   8640 

gcggccggag tacatcgaga tcgagctagc tgattggatg taccgcgaga tcacagaagg   8700 

caagaacccg gacgtgctga cggttcaccc cgattacttt ttgatcgatc ccggcatcgg   8760 

ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca gatggttgtt   8820 

caagacgatc tacgaacgca gtggcagcgc cggagagttc aagaagttct gtttcaccgt   8880 

gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat ttgaaggagg aggcggggca   8940 

ggctggcccg atcctagtca tgcgctaccg caacctgatc gagggcgaag catccgccgg   9000 

ttcctaatgt acggagcaga tgctagggca aattgcccta gcaggggaaa aaggtcgaaa   9060 

aggtctcttt cctgtggata gcacgtacat tgggaaccca aagccgtaca ttgggaaccg   9120 

gaacccgtac attgggaacc caaagccgta cattgggaac cggtcacaca tgtaagtgac   9180 

tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac tctttaaaac ttattaaaac   9240 

tcttaaaacc cgcctggcct gtgcataact gtctggccag cgcacagccg aagagctgca   9300 

aaaagcgcct acccttcggt cgctgcgctc cctacgcccc gccgcttcgc gtcggcctat   9360 

cgcggccgct ggccgctcaa aaatggctgg cctacggcca ggcaatctac cagggcgcgg   9420 

acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat caaggcaccc tgcctcgcgc   9480 

gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt   9540 

gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg   9600 

ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa   9660 

ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca   9720 

cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc   9780 

gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg   9840 

gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa   9900 

ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga   9960 

cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag  10020 

ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct  10080 

taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg  10140 

ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc  10200 

ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt  10260 

aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta  10320 

tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac  10380 

agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc  10440 

ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat  10500 

tacgcgcaga aaaaaaggat ctcaagaaga tccggaaaac gcaagcgcaa agagaaagca  10560 

ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag  10620 

cgaaccggaa ttgcc                                                   10635