Patent Abstract:
the present invention relates to a transgenic c . elegans which expresses an amyloid precursor protein or a part thereof , to the transgene itself , to the protein encoded by the transgene , and also to a process for preparing the transgenic c . elegans and to its use .

Detailed Description:
in the present study , the nematode caenorhabditis elegans ( c . elegans ) was chosen as the model organism for identifying secretases which are involved in processing app into the aβ peptide . this worm is outstandingly suitable for genetic studies and has therefore in the past been employed on many occasions for investigating universally important processes such as programmed cell death , neuronal guidance and ras / map kinase signaling ( riddle , d . l . et al . ( 1997 ) ). the important points which make c . elegans especially appropriate for such studies include the following ( c . kenyon , science ( 1988 ) 240 , 1448 ; p . e . kuwabara ( 1997 ), tig , 13 , 454 ): its small genome , which is composed of about 19 , 000 genes or 97 mb and which was sequenced completely in december 1998 . ( the c . elegans sequencing consortium , science ( 1998 ), 282 , 2012 ). its reproduction by self fertilization . in the case of the two sexes of c . elegans , a distinction is made between males and hermaphrodites , i . e . hermaphroditic animals which fertilize their eggs themselves before laying . a crucial advantage of this type of reproduction is that , after a transgene has been introduced into the germ line , a hermaphrodite can automatically generate homozygous transgenic descendants . there is therefore no need for any further crossing steps , as in the case of drosophila , for example , for preparing transgenic lines . its easy handling in the laboratory due to its small size ( about 1 mm in length ) and its relatively undemanding growth conditions . as a result , a large number of worms can be handled routinely in the laboratory . its short generation time of 3 days , which makes it possible to obtain large quantities of biological material for analysis within a very short time . a complete cell description for the development and anatomy of c . elegans is available . detailed genetic maps and methods for genetic analysis in c . elegans are available . technologies for preparing knock - out animals are available . in the same way , technologies exist for mutagenizing the c . elegans genome ( transposon mutagenesis and ethyl methanesulfonate ( ems ) mutagenesis ). the following are possible uses of the transgenic c . elegans lines : 1 . identification of a γ - secretase - like activity in c . elegans using mutagenesis approaches . it is planned that a transposon mutagenesis , which destroys the γ - secretase - like activity , should be carried out and that the corresponding gene should be sought by detecting the worms which no longer possess this activity . such a screening method is described in the literature : korswagen h . c . et al ., ( 1996 ), 93 , 14680 proc natl acad sci usa . alternative approaches would be mutagenesis using ethyl methanesulfonate ( ems ) or else anti - sense rna approaches . in the latter case , an attempt could be made to find motifs which were common to all c . elegans proteases and to downregulate these proteases specifically using anti - sense rnas which were directed against these motifs . screening for the aβ peptide could then show whether one of the proteases was involved in aβ peptide production . 2 . identification of a γ - secretase - like activity in c . elegans , perhaps by a similar route to that described in item 1 . 3 . armed with knowledge of a γ - secretase or γ - secretase - like activity in c . elegans , it is possible to search for human γ - secretase or γ - secretase - like activity by means of a homology comparison . inhibit the activity of γ - secretase , in order to inhibit aβ production from the amyloid precursor protein directly . activate γ - secretase and thereby indirectly inhibit formation of the aβ peptide by increasing appsec production . this approach could take place in a 96 - well format since c . elegans can be maintained in suspension in 96 - well plates . since the screening is carried out on a whole organism , it is possible , to a large extent , to exclude drugs which have an unspecific toxic effect . 5 . investigation of the aggregation behavior , and of a possible neurotoxic effect , of the aβ peptide in c . elegans . screening for drugs which inhibit aggregation of the aβ peptide . 6 . investigation of the modulation of app processing by other proteins ( e . g . presenilins or apoe ) as a result of their overexpression or knock - out . since the presenilins are alzheimer - associated proteins and apoe constitutes a risk factor in alzheimer &# 39 ; s disease , these proteins could have an effect on formation of the aβ peptide and , as a consequence , their role in the app processing pathway could be investigated . 7 . where appropriate , validation of an α - secretase and / or γ - secretase activity which has been found using other experimental approaches known to the skilled person . [ 0059 ] fig1 : fig1 shows the amyloid precursor protein ( app695 isoform and app770 and app751 isoforms ) and secretase cleavage products . [ 0060 ] fig2 : fig2 describes the construction of the transgenic vector “ unc - 119 - sp - c100 ”, which contains an unc - 119 promoter , an app signal peptide and the c100 fragment from app , with “ unc - 119 ” being a neuron - specific c . elegans promoter , the app signal peptide corresponding to amino acids 1 to 24 of app and c100 corresponding to the 100 c - terminal amino acids of app (= c100 ). c100 is composed of the aβ sequence and the c terminus of app ( shoji , m et al ., ( 1992 ) science 258 , 126 ). the vector unc - 119 - sp - c100 possesses 5112 base pairs . the following examples are illustrative of some of the products and compositions and methods of making and using the same falling within the scope of the present invention . two vectors , i . e . psklc1 - 99 , which encodes sp - c100 , and pby103 , which contains the unc - 119 promoter , were used for the cloning , with the sp - c100 - encoding dna being cloned into the pby103 vector behind the unc - 119 promoter . the basic vector pby103 is composed of the vector backbone ppd49 . 26 , which is described in “ caenorhabditis elegans : modern biological analysis of an organism ” ( 1995 ) ed . epstein et al ., vol 48 , pp . 473 , into which the unc - 119 promoter ( maduro et al . genetics ( 1995 ) , 141 , p . 977 ) has been cloned by way of the hindiii / bamhi sites . the plasmid unc - 119 - sp - c100 was prepared by kpni / saci digestion of psklc1 - 99 and cloning of the lc99 fragment into pby103 ( shoji et al . ( 1992 ). the method of microinjection was used for preparing the transgenic c . elegans lines ( mello et al ., ( 1991 ) embo j . 10 ( 12 ) 3959 ; c . mello and a . fire , methods in cell biology , academic press vol . 48 , pp . 451 , 1995 ; c . d . link , proc natl acad sci usa ( 1995 ) 92 , 9368 ). two different c . elegans strains , i . e . wild - type n2 and him - 8 ( high incidence of males ), were used . the unc - 119 - sp - c100 construct was microinjected into the gonads of young adult hermaphrodites using a microinjection appliance . the dna concentration was about 20 ng / μl . a marker plasmid was injected together with the unc - 119 - sp - c100 construct . this marker plasmid is the plasmid ttx3 - gfp , which encodes the green fluorescent protein under the control of the ttx3 promoter . the activity of the ttx3 promoter is specific for particular neurons of the c . elegans head , the so - called aiy neurons , which play a role in the thermotaxis of the worm . when plasmid dna is microinjected , it is assumed that long tandem arrays , which are composed of many copies of plasmid dna ( in our case , of the ttx3 - gfp plasmid and the unc - 119 - sp - c100 plasmid ), are formed by recombination . a certain percentage of these arrays integrate into the c . elegans genome . however , the arrays are more likely to be present extrachromosomally . worms which had been injected successfully exhibit a green fluorescence in the aiy neurons of the head region when stimulated with light of a wavelength of about 480 nm . it was possible to detect such nematodes . following stimulation with light of a wavelength of 480 nm , c100 - transgenic worms exhibit a green fluorescence in the aiy neurons of the head region . since it was also possible to detect green fluorescence in the head neurons once again in the descendants of the worms , it can be assumed that the plasmids are able to pass down through the germ line . however , the penetrance is not 100 %, which makes it possible to conclude that the long tandem arrays composed of ttx3 - gfp marker dna and unc - 119 - sp - c100 are present extrachromosomally rather than being integrated into the genome . six different transgenic c100 c . elegans lines ( three in an n2 wt background and three in a him 8 background ) were examined in a western blot for expression of the c100 fragment using a polyclonal antiserum directed against the c terminus of app . a band having the appropriate molecular weight of about 10 kda was detectable in all the six lines . in an aβ sandwich elisa , signals which were above the background level , and which were statistically significant in two cases , were detected in cell extracts from transgenic animals . this indicates that c . elegans could possess a γ - secretase - like activity . in the aβ sandwich elisa assay , 96 - well plates are first of all incubated with the monoclonal antibody clone 6e10 ( senetek plc ., mo , usa ), which reacts specifically with the aβ peptide ( amino acids 1 - 17 ), and then coated with worm extracts from transgenic worms or control worms . the aβ peptide is detected using the monoclonal aβ antibody 4g8 ( senetek plc ., mo , usa ), which recognizes amino acids 17 - 24 in the aβ peptide and is labeled with biotin . the detection is effected by way of the alkaline phosphatase reaction using an appropriate antibody which is directed against biotin . disruption of the worms involves detergent treatment , nitrogen shock freezing , sonication and rupture of the cells using glass beads . the elisa signal from the above - described experiment can be based either on weak expression of the aβ peptide or on expression of the c100 precursor protein , since the appropriate epitopes are present in both proteins . expression of the aβ peptide could , for example , also be specifically detected in an analogous manner : for this , aβ - specific antibodies which do not react with the c100 precursor would have to be employed in an aβ sandwich elisa . an aβ - specific antibody could , for example , be a monoclonal antibody which specifically recognizes the c - terminal end of the aβ form , which is composed of 40 or 42 amino acids . in parallel , the aβ peptide could be detected in a western blot using the monoclonal antibodies 4g8 and 6e10 and then be distinguished from the larger c100 precursor by its molecular weight of 4 kd . the vectors can be obtained from andrew fire ( department of embryology , carnegie institution of washington , baltimore , md . 21210 , usa ) in the case of ppd49 . 26 and lc99 ( amyloid precursor protein ), which is deposited under atcc number 106372 . the unc - 119 promoter can be obtained from maduro , m . ( department of biological science , universitiy of alberta edmonton , canada ), while unc - 54 and unc - 16 . 2 can be obtained from andrew fire . the above description of the invention is intended to be illustrative and not limiting . various changes or modifications in the embodiments described may occur to those skilled in the art . these can be made without departing from the spirit or scope of the invention . seq id no . 1 : nucleotide sequence of c100 ctggatgc agaattccga catgactcag gatatgaagt tcatcatcaaaaattggtgt tctttgcaga agatgtgggt tcaaacaaag gtgcaatcat tggactcatggtgggcggtg ttgtcatagc gacagtgatc gtcatcacct tggtgatgct gaagaagaaacagtacacat ccattcatca tggtgtggtg gaggttgacg ccgctgtcac cccagaggagcgccacctgt ccaagatgca gcagaacggc tacgaaaatc caacctacaa gttctttgagcagatgcaga actag seq id no . 2 : nucleotide sequence of sp atg ctgcccggtt tggcactgtt cctgctggcc gcctggacgg ctcgggcg seq id no . 3 : nucleotide sequence of sp + c100 atg ctgcccggtt tggcactgtt cctgctggcc gcctggacgg ctcgggcgct g gatgc agaattccga catgactcag gatatgaagt tcatcatcaa aaattggtgt tctttgcaga agatgtgggt tcaaacaaag gtgcaatcat tggactcatg gtgggcggtg ttgtcatagc gacagtgatc gtcatcacct tggtgatgct gaagaagaaa cagtacacat ccattcatca tggtgtggtg gaggttgacg ccgctgtcac cccagaggag cgccacctgt ccaagatgca gcagaacggc tacgaaaatc caacctacaa gttctttgag cagatgcaga actag seq id no . 4 : nucleotide sequence of the 42bp intron gtatgtttcgaatgatactaacataacatagaacattttcag seq id no . 5 : nucleotide sequence of intron + sp + c100 gtatgtttcgaatgatactaacataacatagaacattttcaggaggacccttggctagcgtcgacggt accgggccccccctcgaggtcgacggtatcgataaccttcacagcagcgcactcggtgccccgcg cagggtcgcgatg ctgcccggtt tggcactgtt cctgctggccgcctggacgg ctcgggcgct ggatgc agaattccgaatgactcaggatatgaagtcatcatcaaaaattggtgt tctttgcaga agatgtgggt tcaaacaaag gtgcaatcat tggactcatg gtgggcggtg ttgtcatagc gacagtgatc gtcatcacct tggtgatgct gaagaagaaa cagtacacat ccattcatca tggtgtggtg gaggttgacg ccgctgtcac cccagaggagcgccacctgt ccaagatgca gcagaacggc tacgaaaatc caacctacaa gttctttgag cagatgcaga actag seq id no . 6 : nucleotide sequence of unc - 119 aagcttcagtaaaagaagtagaattttatagttttttttctgtttgaaaaattctccccatcaatgttct ttcaaataaatacatcactaatgcaaagtattctataacctcatatctaaattcttcaaaatcttaacat atc ttatcattgctttaagtcaacgtaacattaaaaaaaatgttttggaaaatgtgtcaagtctctcaaaatt cagttttttaaaccactcctatagtcctatagtcctatagttacccatgaaatccttatatattactgta aaatgtttcaaaaaccattggcaaattgccagaactgaaaatttccggcaaattggggaaccggcaa attgccaatttgctgaatttgccggaaacggtaattgccgaaagtttttgacacgaaaatggcaaatt gtggttttaaaatttttttttttggaaatttcagaatttcaattttaatcggcaaaactgtaggcatcct aagaatgttcctacatctattttgaaaagtaagcgaattaattctatgaaaatgtctaaagaaaatggg gaaacaatttcaaaaaggcacagtttcaatggtttccgaattatactaaatccctctaaaaacttccgg caaattgatatccgtaaaagagcaaatccgcatttttgccgaaaattaaaatttccgacaaatcggca aaccggcaatttggcgaaatttgccggaacgattgccgcccacccctgttccagaggttcaaactg gtagcaaagctcaaaatttctcaaattctccaatttttttttgaattttggcagtgtaccaaaatgaca ttcagtcatattggtttattatagatttatttagataaaatcctaaatgattctacctttaaagatgccc actttaaaagtaatgactcaaacttcaaattgctctaagattctattgaattaccatcttttcctctcat tttctctcactgtctatttcatcacaaattcatccctctctcctctcttctctctccctctctctctctt tctctttgctcatcatctgtcattttgtccgttcctctctctgcgccctcagcgttccccacactctc tcgcttctcttttcctagacgtcttcttttttcatcttcttcagcctttttcgccattttccatctctgt caatcattacggacgacccccattatcgat seq id no . 7 : nucleotide sequence of unc - 119 + intron + sp + c100 aagcttcagtaaaagaagtagaattttatagttttttttctgtttgaaaaattctccccatca atgttctttcaaataaatacatcactaatgcaaagtattctataacctcatatctaaattcttcaaaatc ttaacatatcttatcattgctttaagtcaacgtaacattaaaaaaaatgttttggaaaatgtgtcaagtc tctcaaaattcagttttttaaaccactcctatagtcctatagtcctatagttacccatgaaatccttata tattactgtaaaatgtttcaaaaaccattggcaaattgccagaactgaaaatttccggcaaattgggg aaccggcaaattgccaatttgctgaatttgccggaaacggtaattgccgaaagtttttgacacgaaaa tggcaaattgtggttttaaaatttttttttttggaaatttcagaatttcaattttaatcggcaaaactgt aggcatcctaagaatgttcctacatctattttgaaaagtaagcgaattaattctatgaaaatgtctaaa gaaaatggggaaacaatttcaaaaaggcacagtttcaatggtttccgaattatactaaatccctctaa aaacttccggcaaattgatatccgtaaaagagcaaatccgcatttttgccgaaaattaaaatttccga caaatcggcaaaccggcaatttggcgaaatttgccggaacgattgccgcccacccctgttccagag gttcaaactggtagcaaagctcaaaatttctcaaattctccaatttttttttgaattttggcagtgtac caaaatgacattcagtcatattggtttattatagatttatttagataaaatcctaaatgattctaccttt aaagatgcccactttaaaagtaatgactcaaacttcaaattgctctaagattctattgaattaccatct tttcctctcattttctctcactgtctatttcatcacaaattcatccctctctcctctcttctctctccct ctctctctctttctctttgctcatcatctgtcat tttgtccgttcctctctctgcgccctcagcgttccccacactctctcgcttctcttttcctagacgtc ttcttttttcatcttcttcagcctttttcgccattttccatctctgtcaatcattacggacgaccccca ttatcgataagatctccacggtggccgcgaattcctgcagcccgggggatccccgggattggccaa aggacccaaaggtatgtttcgaatgatactaacataacatagaacattttcaggaggacccttggcta gcgtcgacggtaccgggccccccctcgaggtcgacggtatcgataaccttcacagcagcgcactc ggtgccccgcgcagggtcgcgatgctgcccggtt tggcactgttcctgctggccgcctggacggctcgggcgctggatgcagaattccga catgactcaggatatgaagttcatcatcaaaaattggtgttctttgcagaagatgtgggttcaaacaa ag gtgcaatcat tggactcatggtgggcggtgttgtcatagcgacagtgatcgtcatcacct tggtgatgct gaagaagaaacagtacacat ccattcatca tggtgtggtg gaggttgacg ccgctgtcac cccagaggagcgccacctgt ccaagatgca gcagaacggc tacgaaaatc caacctacaa gttctttgagcagatgcaga actag seq id no . 8 : nucleotide sequence of the expression vector acccccgccacagcagcctctgaagttggacacggatccactagttctagagcggccgccaccgc ggtggagctccgcatcggccgctgtcatcagatcgccatctcgcgcccgtgcctctgacttctaag tccaattactcttcaacatccctacatgctctttctccctgtgctcccaccccctatttttgttattat caaaaaaacttcttcttaatttctttgttttttagcttcttttaagtcacctctaacaatgaaattgtgt agattcaaaaatagaattaattcgtaataaaaagtcgaaaaaaattgtgctccctccccccattaataa taattctatcccaaaatctacacaatgttctgtgtacacttcttatgttttttttacttctgataaatttt ttttgaaacatcatagaaaaaaccgcacacaaaataccttatcatatgttacgtttcagtttatgaccg caatttttatttcttcgcacgtctgggcctctcatgacgtcaaatcatgctcatcgtgaaaaagtttt ggagtatttttggaatttttcaatcaagtgaaagtttatgaaattaattttcctgcttttgctttttggg ggtttcccctattgtttgtcaagagtttcgaggacggcgtttttcttgctaaaatcacaagtattgat gagcacgatgcaagaaagatcggaagaaggtttgggtttgaggctcagtggaaggtgagtagaagt tgataatttgaaagtggagtagtgtctatggggtttttgccttaaatgacagaatacattcccaatata ccaaacataactgtttcctactagtcggccgtacgggccctttcgtctcgcgcgtttcggtgatgac ggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccggg agcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgc ggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaag gagaaaataccgcatcaggcggccttaagggcctcgtgatacgcctatttttataggttaatgtcat gataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgt ttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataat attgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcatttt gccttcctgtttttgctc acccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcg aactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgag cacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggt cgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacgg atggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaactt acttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgta actcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacg atgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttccc ggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttc cggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcag cactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaacta tggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcaga ccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaag atcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccc cgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaa aaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggt aactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccac ttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgcca gtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggt cgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgaga tacctacagcgtgagcattgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccg gtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatct ttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagggggg cggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttg ctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagct gataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagc gcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggt ttccggactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcac cccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcac acaggaaacagctatgaccatgattacgccaagctt seq id no . 9 : amino acid sequence of sp mlpglalfll aawtara seq id no . 10 : amino acid sequence of the fusion protein mlpglalfll aawtaralda efrhdsgyev hhqklvffae dvgsnkgaii glmvggvvia tvivitlvml kkkqytsihh gvvevdaavt peerhlskmq qngyenptyk ffeqmqn seq id no . 11 : nucleotide sequence of the vector unc - 119 - sp - c100 atgaccatgattacgccaagcttcagtaaaagaagtagaattttatagttttttttctgtttgaaaaat tctccccatcaatgttctttcaaataaatacatcactaatgcaaagtattctataacctcatatctaaat tcttcaaaatcttaacatatcttatcattgctttaagtcaacgtaacattaaaaaaaatgttttggaaaa tgtgtcaagtctctcaaaattcagttttttaaaccactcctatagtcctatagtcctatagttacccat gaaatccttatatattactgtaaaatgtttcaaaaaccattggcaaattgccagaactgaaaatttccg gcaaattggggaaccggcaaattgccaatttgctgaatttgccggaaacggtaattgccgaaagttt ttgacacgaaaatggcaaattgtggttttaaaatttttttttttggaaatttcagaatttcaattttaat cggcaaaactgtaggcatcctaagaatgttcctacatctattttgaaaagtaagcgaattaattctat gaaaatgtctaaagaaaatggggaaacaatttcaaaaaggcacagtttcaatggtttccgaattatac taaatccctctaaaaacttccggcaaattgatatccgtaaaagagcaaatccgcatttttgccgaaaa ttaaaatttccgacaaatcggcaaaccggcaatttggcgaaatttgccggaacgattgccgcccacc cctgttccagaggttcaaactggtagcaaagctcaaaatttctcaaattctccaatttttttttgaatt ttggcagtgtaccaaaatgacattcagtcatattggtttattatagatttatttagataaaatcctaaat gattctacctttaaagatgcccactttaaaagtaatgactcaaacttcaaattgctctaagattctatt gaattaccatcttttcctctcattttctctcactgtctatttcatcacaaattcatccctctctcctctc ttctctctccctctctctctctttctctttgctcatcatctgtcattttgtccgttcctctctctgcgc cctcagcgttccccacactctctcgcttctcttttcctagacgtcttcttttttcatcttcttcagcct ttttcgccattttccatctctgtcaatcattacggacgacccccattatcgataagatctccacggtg gccgcgaattcctgcagcccgggggatccccgggattggccaaaggacccaaaggtatgtttcgaa tgatactaacataacatagaacattttcaggaggacccttggctagcgtcgacggtaccgggccccc cctcgaggtcgacggtatcgataaccttcacagcagcgcactcggtgccccgcgcagggtcgcga tg ctgcccggtt tggcactgtt cctgctggccgcctggacgg ctcgggcgct ggatgc agaattccga catgactcag gatatgaagt tcatcatcaaaaattggtgt tctttgcaga agatgtgggt tcaaacaaag gtgcaatcat tggactcatggtgggcggtg ttgtcatagc gacagtgatc gtcatcacct tggtgatgct gaagaagaaacagtacacat ccattcatca tggtgtggtg gaggttgacg ccgctgtcac cccagaggagcgccacctgt ccaagatgca gcagaacggc tacgaaaatccaacctacaattctttgagcagatgcagaactagacccccgccacagcagcctctga agttggacacggatccactagttctagagcggccgccaccgcggtggagctccgcatcggccgct gtcatcagatcgccatctcgcgcccgtgcctctgacttctaagtccaattactcttcaacatccctac atgctctttctccctgtgctcccaccccctatttttgttattatcaaaaaaacttcttcttaatttcttt gttttttagcttcttttaagtcacctctaacaatgaaattgtgtagattcaaaaatagaattaattcgta ataaaaagtcgaaaaaaattgtgctccctccccccattaataataattctatcccaaaatctacacaat gttctgtgtacacttcttatgttttttttacttctgataaattttttttgaaacatcatagaaaaaaccg cacacaaaataccttatcatatgttacgtttcagtttatgaccgcaatttttatttcttcgcacgtctg ggcctctcatgacgtcaaatcatgctcatcgtgaaaaagttttggagtatttttggaatttttcaatca agtgaaagtttatgaaattaattttcctgcttttgctttttgggggtttcccctattgtttgtcaagag tttcgaggacggcgtttttcttgctaaaatcacaagtattgatgagcacgatgcaagaaagatcgga agaaggtttgggtttgaggctcagtggaaggtgagtagaagttgataatttgaaagtggagtagtgt ctatggggtttttgccttaaatgacagaatacattcccaatataccaaacataactgtttcctactagt cggccgtacgggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcag ctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgc gtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgag agtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcggcc ttaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcag gtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgt atccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtatt caacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaa acgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggat ctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaa agttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcat acactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatga cagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgac aacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgcctt gatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgta gcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaat taatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggct ggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggc cagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaac gaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagttta ctcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatccttttt gataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaa gatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctg cttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctt tttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagt taggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagt ggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataa ggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctaca ccgaactgagatacctacagcgtgagcattgagaaagcgccacgcttcccgaagggagaaaggcg gacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaa acgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgc tcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttt tgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgc ctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgagg aagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagct ggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctca ctcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcgg ataacaatttcacacaggaaacagct kang , j ., lemaire , h . g ., unterbeck , a ., salbaum j . m ., masters c . l ., grzeschik , k . h ., multhaupt , g ., beyreuther , k ., mueller - hill , b . 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