Patent Application: US-24633002-A

Abstract:
disclosed are various genes encoding proteins that are shown to play a role , direct or indirect , in microbial resistance of an organism in a biofilm and homologs thereof . also disclosed are methods of identifying a compound that modulates microbial resistance of an organism in a biofilm , and methods of identifying genes that encode proteins that play a role , direct or indirect , in biofilm resistance .

Description:
we posit that biofilm resistance to antimicrobial agents is part of a regulated developmental process and thus would require an identifiable set of genetic determinants . based upon this hypothesis , a screen was designed to identify genes which , when mutated , would affect the ability of biofilm cells to resist the effects of an antimicrobial agent , while having no substantial effect on the sensitivity of planktonic cells growing in the exponential phase cells to the same antimicrobial agent . this screen was based on a modification of the microtiter plate assay that yielded surface attachment mutants ( sad ) of pseudomonas aeruginosa and p . fluorescens ( o &# 39 ; toole , g . a ., and r . kolter . mol . microbiol . 30 ( 2 ): 295 - 304 , 1998 ; o &# 39 ; toole , g . a ., and r . kolter , mol . microbiol . 28 : 449 - 461 , 1998 ). in the present studies , bacteria were cultured on the same minimal m63 medium expect arginine ( 0 . 4 %) was used as the sole source of carbon and energy , and cultures were incubated for 24 hours . other screens and techniques for generating mutations in cell components that function in biofilm resistance to antimicrobial agents are well known in the art , for example , the use of transposon insertion and chemical mutagenesis . the microtiter plate assay was modified to measure the increase in resistance developed by the wild type strain when growing in a biofilm . the wells of the microtiter dish are inoculated with bacteria and biofilms are allowed to form on the walls of the wells for 24 hrs in the absence of any shear force . after the biofilms had formed on the wells of the microtiter dish , the spent medium was replaced with the same media containing an antimicrobial agent . in this case , we used the aminoglycoside antibiotic tobramycin ( tb ), an antibiotic that targets protein synthesis . tb was selected because it is the primary antibiotic used to treat cystic fibrosis patients with chronic p . aeruginosa lung infections ( banerjee , d ., and d . stableforth , drugs , 60 ( 5 ): 1053 - 64 , 2000 ; bonsignore , c . l ., pediatr nurs . 24 ( 3 ): 258 - 9 , 1998 ; ratjen , f , int j antimicrob agents 17 ( 2 ): 93 - 6 , 2001 ). after exposing the biofilms to tb for 24 hours , the antibiotic - containing medium is removed and replaced with fresh antibiotic - free medium . any bacteria surviving in the biofilm outgrow and repopulate the planktonic phase of the wells . viable cells were detected by plating on rich medium . using this assay , we determined that the minimal bacteriocidal concentration ( mbc ) of tb for the wt biofilm grown cells is 0 . 4 mg / ml . the mbc of planktonic cells was determined by adding the antibiotic to cells at the time they were inoculated into the microtiter dish , incubating the cells in the presence of antibiotic for 24 hrs , and assessing cell viability by plating on rich medium . using this assay , the planktonic mbc was shown to be 0 . 008 mg / ml , a 50 - fold decrease relative to the biofilm - grown bacteria . using the assay for biofilm - related antibiotic resistance described above , a library of random p . aeruginosa pa14 transposon insertion mutants was screened for the inability to develop characteristic increase in resistance of biofilm - grown cells . the concentration of tb used in the screen was 0 . 2 mg / ml , a concentration 25 - fold greater than the planktonic mbc , but still below the concentration that will kill biofilm grown cells . from a library of 4 , 320 transposon mutants , forty - three putative mutants defective in biofilm - related tb resistance were identified . the goal of the screen was to identify mutants with a biofilm - related defect in the development of antimicrobial agent resistance . therefore , the 43 candidate mutants were subjected to a series of secondary tests to confirm the biofilm - related phenotype . of the original 43 candidate mutants from the initial screen , two mutants grew as well as the wild type in liquid culture , formed a wild - type biofilm in the microtiter plates , and had a planktonic mbc indistinguishable from the parent strain . these mutants , designated 45e7 and 30 b1 , were characterized further . in addition to the decrease in sensitivity to tb , we determined the mbc of biofilm and planktonic cells growing in the expotential phase for gentamycin ( gm ) and ciprofloxacin ( cip ). gm , like tb , is an aminoglycoside protein synthesis inhibitor while cip is a fluoroquinolone that targets dna gyrase . table 1 shows the results of these studies . for all antibiotics , there was no difference in the mbc of planktonic cells growing in the exponential phase between the wild type and 45e7 mutant . the mcb of biofilm grown 45e7 was lower for all three antibiotics when compared to the wt : tb ( 16 - fold ), gm ( 8 - fold ) and cip ( 8 - fold ). one of the characteristics of biofilm grown bacteria is their distinctive architecture . currently , it is not clear what relationship , if any , exists between the architecture of the biofilm and resistance to antimicrobial agents . early studies of biofilm grown cells suggested that inhibition of antibiotic diffusion through the biofilm could account for the increased resistance of these communities ( costerton et al ., microbial biofilms , p . 711 - 745 . in l . n . omston , a . balows , and e . p . greenberg ( ed . ), annu . rev . microbiol . , vol . 49 . annual reviews , inc ., palo alto , calif ., 1995 ). therefore , it was possible that altering biofilm architecture could increase diffusion of tb through the biofilm , thereby increasing sensitivity to this agent . in support of this idea , a quorum - sensing mutant of p . aeruginosa that exhibited altered architecture was reported to be abnormally sensitive to sds ( davies et al ., science 280 ( 5361 ): 295 - 298 , 1998 ). however , the same mutant was as resistant to killing by the antibiotic ofloxacin as the wild type biofilm cells ( brooun et al ., antimicrob agents chemother . 44 ( 3 ): 640 - 6 , 2000 ). we utilized flow cells to analyze the architecture of the wild type and mutant strains . a flow cell allows a continuous supply of fresh medium to be delivered to a biofilm that is formed on the walls of a small , enclosed chamber . one side of this chamber is a glass cover slip , and the chamber can be mounted on to a microscope to allow for the non - destructive imaging of the biofilm . gfp - tagged wild type and 45e7 strains were inoculated into different chambers of a flow cell and the architecture of the biofilms produced by these strains was analyzed by epifluorescence microscopy . the reconstructed architecture of the wild - type and 45e7 mutant is shown in fig1 . this analysis showed that is no discernible difference in the architecture of these strains . currently , there are no standard methods for determining biofilm resistance to antimicrobial agents . thus , we utilized two other assays of biofilm antibiotic resistance to demonstrate that the phenotype observed in the microtiter plate was robust and could be observed across a number of experimental models . we chose to analyze this mutant in flow cell and colony biofilm assays . in order to document the sensitivity phenotype of the biofilms in the flow cell , the biofilm of the wild type and 45e7 strains were allowed to form . after the biofilm had developed for 24 hrs 0 . 2 mg / ml tb was added to the medium . after 24 hours of exposure to tb , flow through the cell was stopped and the baclight viability stain was injected into the flow chambers . baclight differentiates between cells with intact membranes ( considered “ live ”) and those with damaged membranes ( considered “ dead ”). after 15 minutes of staining , flow through the chamber was resumed and following a 15 minute wash the cells were examined by epifluorescent microscopy ( fig2 ). the left - hand panels show phase contrast images — there was no difference in biofilm architecture between these strains ( see also fig1 ). the center and right panel shows that there were more live than dead cells in the tb - treated biofilm of the wild type strain . conversely , there were more dead cells than live ones in the tb - treated biofilm of the 45e7 mutant strain . this result confirmed the drug sensitivity phenotype first observed in the microtiter plate assay . we also used the quantitative colony biofilm assay to document the 45e7 mutant phenotype . it has been reported that bacterial colonies develop some of the properties associated with biofilms , including increased resistance to biocides ( anderl et al ., antimicrob agents chemother . 44 ( 7 ): 1818 - 24 , 2000 ; stewart , p . s ., biotechnol bioeng . 59 ( 3 ): 261 - 72 , 1998 ). colony biofilms were formed on polycarbonate filters for 48 hrs then transferred to solid media containing tb . viable cell numbers in the colony was determined after 4 , 24 and 48 hours of exposure to tb ( fig3 ). the 45e7 biofilm cells remained as resistant to the effects of tb as the wild type at the first two time points assayed . however , at 48 hours , while the wild type biofilm cell viability was only reduced by 100 - fold , no viable cells were detected for the 45e7 mutant . in the absence of tb , there was difference in viability between the two strains tested . taken together with the data presented above , we concluded that the 45e7 mutant is less resistant to the antimicrobial effects of tb when growing in a biofilm . the transposon insertion carried by the 45e7 strain was cloned and the dna flanking the transposon sequenced and compared to the published sequence of p . aeruginosa pao1 . the gene disrupted in 45e7 , pa1163 ( genbank accession no . ______ ) is 58 % identical to a bradyrhizobium japonicum gene ndvb . the ndvb gene of bradyrhizobium japonicum codes for a glucosyltransferase that is required for the synthesis of cyclic - b -( 1 , 3 ), b -( 1 , 6 )- glucans ( bhagwat et al ., j bacteriol . 178 ( 15 ): 4635 - 42 , 1996 ). located in the periplasm and extracellular media , cyclic glucans have been shown to play a role in growth in low osmotic media and in plant infection ( breedveld , m . w ., and k . j . miller , microbiol rev . 58 ( 2 ): 145 - 61 , 1994 ). in b . japonicum , another gene , ndvc acts in concert with ndvb to form the b -( 1 , 3 ), b -( 1 , 6 ) linkages . ndvc mutants produce glucans with only b -( 1 , 3 ) linkages ( bhagwat et al ., plant physiol . 119 ( 3 ): 1057 - 64 , 1999 ). furthermore , in sinorhizobium meliloti , the ndva gene product is thought to be required for the export of cyclic glucans from the periplasm to the extracellular medium ( breedveld , m . w ., and k . j . miller . microbiol rev . 58 ( 2 ): 145 - 61 , 1994 ). upon investigation of the p . aeruginosa genome , we noticed that there is no ndvc homolog present , but there are three genes with ˜ 50 % homology to s . meliloti ndva . thus , it seemed likely that p . aeruginosa produces periplasmic and extracellular b -( 1 , 3 )- glucans . to confirm that cyclic glucans were made by the wild type strain and that these glucans were altered or not present in the ndvb pa1163 mutant we characterized the periplasmic and extracellular polysaccharides produced by these strains . ethanol extracts from both the periplasm and the extracellular medium of these strains were fractionated by gel filtration chromatography to estimate the molecular weight of the material ( wang et al ., j bacteriol . 181 ( 15 ): 4576 - 83 , 1999 ). fractions were assayed for polysaccharides using the colorometric anthrone - sulfuric acid method . the anthrone assay measures the total concentration of carbohydrate , or monosaccharide equivalents , in a sample ( loewus , f . a ., anal . chem . 24 : 219 , 1952 ). anthrone - positive extracellular material from the wild type eluted from the sizing column in fractions that corresponded to a molecular weight ( mw ) of approximately 1500 , while the mutant produced no anthrone positive fractions in the 1500mw range . in contrast , anthrone - positive material from the 45e7 mutant extracts eluted in the column void volume , indicates a mw of greater than 80 , 000 , possibly indicating an aggregate of polysaccharides . the material extracted from the periplasm was also fractionated by gel filtration chromatography and it was found that the anthrone - positive material produced by the wild type and mutant strains also differed ( fig4 ). while the wild type strain produced material that eluted across the molecular weight range of the column , the mutant strain lacked anthrone - positive material in fractions where cyclic glucans typically elute ( wang et al ., j bacteriol . 181 ( 15 ): 4576 - 83 , 1999 ). based on the current understanding of the roles of cyclic glucans in b . japonicum , s . meliloti and agrobacterium tumefaciens , we envisioned three possible models to describe the role of these molecules in the development of resistance in biofilm populations . cyclic glucans may be required to : i ) maintain the osmotic balance within biofilms , ii ) sequester antimicrobial agents in biofilm - grown cells , or iii ) act as signaling molecules required for the development of resistance to antimicrobial agents . to test the hypothesis that cyclic glucans are important for hypo - osmotic adaptation , we examined the wild type and - pa1163 strains for their ability to survive and grow in low osmotic strength medium . as a first step , we diluted overnight cultures into water and monitored the survival of the wild type and mutant strains over 24 hrs . there was no difference in the survival of these strains in water . we assessed the growth of both strains in liquid { fraction ( 1 / 10 )} strength minimal salts m63 medium ( a hypo - osmotic medium compared to full strength m63 ) and on solid gym media with no salt . the ndvb mutant of s . meliloti grows very slowly on gym media , while the wild type shows no growth defect ( bhagwat et al ., fems microbiol lett . 114 ( 2 ): 139 - 44 , 1993 ; cangelosi et al ., j bacteriol . 172 ( 4 ): 2172 - 4 , 1990 ; lelpi et al ., j biol chem . 265 ( 5 ): 2843 - 51 , 1990 ). there was no difference in the growth of the wild type and pa1163 mutant of p . aeruginosa on these media , suggesting that pa1163 may not be involved in hypo - osmotic adaptation in p . aeruginosa . a number of reports suggest that cyclic glucans can bind or sequester a range of chemically unrelated compounds [ reviewed in ( breedveld , m . w ., and k . j . miller . microbiol rev . 58 ( 2 ): 145 - 61 , 1994 )]. this suggested the possibility that glucans in the extracellular and / or periplasmic space might sequester antimicrobial agents and thus prevent them from entering into the cytoplasm of the bacterial cell . to test this idea , we utilized the putative cyclic glucan fractions isolated by gel filtration chromatography from the periplasm of the wild - type strain ( see above ). this material was incubated in the presence of tb , then spotted on a filter disk placed on a freshly spread lawn of e . coli . filter disks spotted with tb alone , glucans alone , and water were also included as controls . preliminary studies with the disk - diffusion assay indicated that glucan - treated tb was decreased in its zone of killing as compared to tb alone . glucans had no antimicrobial activity at the concentrations used in these assays . these data indicate that the presence of cyclic glucans can decrease the antimicrobial activity of tb , and is consistent with a role for glucans in the sequestering of antimicrobial agents . pa1163 mrna is expressed in biofilm grown cells , but not in planktonic cells in order to investigate the expression pattern of pa1163 we used a continuous flow system to grow planktonic and biofilm bacteria . total mrna was harvested from both biofilm and planktonic cells and rt - pcr used to detect the presence of pa1163 transcript . in this study expression of rplu , a gene known to be expressed in both planktonic cells and biofilm grown cells , was used as a control . chromosomal dna was used to identify the expected size of the pa1163 mrna . this study revealed that pa1163mrna is expressed in biofilm grown cells and is undetectable in planktonic cells . these data indicate that pa1163 may be poorly expressed ( or not expressed at all ) under planktonic conditions . the fact that pa1163 appears to be expressed only in biofilm - grown cells is consistent with our model that pa1163 plays a role in biofilm - related antibiotic resistance . [ 0147 ] p . aeruginosa pa1163 can complement a ndvb s . meliloti mutant the rm8519 ndvb mutant of s . meliloti exhibits a hypo - osmotic growth phenotype . in order to investigate whether p . aeruginosa pa1163 can complement this mutant , and is thus likely to encode a glucosyltransferase , we introduced a vector psmc654 containing the p . aeruginosa pa1163 gene into the ndvb mutant of s . meliloti . as a control , wild type strain and the ndvb mutant of s . meliloti were transformed with empty vector psw213 alone . the pa1163 containing vector ( but not the empty vector ) complemented the ndvb mutant of s . meliloti for its hypo - osmotic growth phenotype . therefore , the pa 1163 gene of p . aeruginosa can functionally substitute for s . meliloti ndvb , strongly suggesting that p . aeruginosa pa1163 protein is a glucosyltransferase . wild - type p . aeruginosa , but not pa1163 mutants produce glucans . we compared carbohydrates produced by wild - type p . aeruginosa to purified cyclic glucan from s . meliloti using chromatographic analysis . this analysis revealed that glucans produced by wild - type p . aeruginosa elute in same fractions as purified cyclic glucan from s . meliloti . similarly eluting material was not detectable in carbohydrates produced by a p . aeruginosa pa1163 mutant . moreover , analysis of the pooled glucan - containing fractions from the wild - type p . aeruginosa strain showed that glucose is the predominate sugar as judged by a glucose standard and consistent with their identification as glucans . the same fractions from the pa1163 mutant did not contain glucose . if periplasmic glucans were indeed sequestering tb , we predicted that these molecules should interact in vitro . to test this possibility , we examined in vitro interactions of purified glucans with tb . tb was loaded onto a c18 column and its elution profile was monitored by a bioassay on a lawn of sensitive bacteria . a zone of clearing indicated the presence of tb in a fraction . tb was detected in the column flow - through and first water wash , but not in any subsequent fractions . in contrast , when a crude periplasmic carbohydrate extracted from the wild type strain was pre - loaded onto the column , a portion of the tb was retained on the column and eluted with 25 % acetonitrile . thus , the presence of the wild type extract on the column retarded the elution of tb , suggesting that periplasmic glucans interacted with tb . to determine which components of the crude extract were responsible for the tb retention on the c18 column , we performed the same experiment as above except the c - 18 column was pre - loaded with partially pure glucan - containing fractions from a g - 75 gel filtration sizing column . tb was retained on the column pre - loaded with material from the wild type extract and eluted from the column with 25 % acetonitrile . in contrast , no tb activity was present in the 25 % acetonitrile fraction from the column preloaded with the corresponding fractions isolated from the p . aeruginosa pa1163 mutant . only the material present in glucan - containing fractions derived from the wild type interacted with and changed the chromatographic behavior of tb . a second mutant strain defective in biofilm specific tb resistance , designated 30b1 , was isolated and characterized . like 45e7 , this strain grew as well as the wild type in liquid culture , formed a wild - type biofilm in the microtiter plates , and had an exponential phase planktonic mbc indistinguishable from the parent strain . this mutant is also less resistant to the antibiotics gm and cip . in all cases , the decrease in resistance of this mutant is less than the decrease observed for the 45e7 mutant . these results suggest that the functions disrupted in this strain debilitate biofilm - related antibiotic resistance via a different mechanism . we also examined the architecture of this mutant and showed that it was identical to the wild - type strain as judged by the flow cell assay and analysis of the biofilms by fluorescence microscopy ( data not shown ). the colony biofilm assay was also utilized to assess the antibiotic resistance of the 30b1 mutant strain as described above . as was observed for the 45e7 mutant , the 30b1 strain showed a marked decrease in resistance to tb as compared to the wild - type strain ( fig5 ). after 48 hrs of exposure to tb , the viability of the wild type had dropped ˜ 100 - fold , while there were no viable cells detected for the 30b1 mutant ( a drop in viable count of ˜ 10 9 ). this experiment confirms the results obtained in the microtiter dish assay . the transposon insertion carried by the 30b1 strain was cloned and the dna flanking the transposon was sequenced and compared to the published sequence of p . aeruginosa pao1 . the open reading frame ( orf ) disrupted by the transposon , pa1874 , encodes a predicted outer membrane protein with sequence similarity to lapa of p . putida ( 42 % similarity ) and bap of staphylococcus aureus ( 46 % similarity ). both of these proteins are important for biofilm development . lapa is required for the colonization of seeds by p . putida and bap was identified in a screen for mutants unable to make a biofilm ( cucarella et al ., j bacteriol . 183 ( 9 ): 2888 - 2896 , 2001 ; espinosa - urgel et al ., j bacteriol . 182 ( 9 ): 2363 - 2369 , 2000 ). in p . aeruginosa , pa1874 is the first gene in a predicted four gene operon that , in addition to pa1874 , includes : pa1875 , an oprn - like outer membrane protein that bears some similarity to outer membrane proteins in the rnd family ; pa1876 , a protein that is similar to an abc family atpase ; and pa1877 , a protein that appears to be a abc family membrane fusion protein . as noted above , pa1875 is similar to oprn . the oprn protein is part of the mexef - oprn multidrug efflux pump , a rnd - type efflux pump which is involved in fluoroquinolone resistance ( nikaido , h . 1994 . prevention of drug access to bacterial targets : permeability barriers and active efflux . science . 264 ( 5157 ): 382 - 8 ; piddock , l . j . 1999 . mechanisms of fluoroquinolone resistance : an update 1994 - 1998 . drugs . 58 ( suppl 2 ): 11 - 8 . thus , the pa1874 - pa1877 operon encodes components of both multi - drug efflux pumps and abc transporters . although other rnd efflux pumps include an outer membrane protein and a membrane fusion protein , they do not typically include a abc family cytoplasmic membrane - located atpase and they do not typically require two outer membrane proteins ( poole , k ., j mol microbiol biotechnol . 3 ( 2 ): 255 - 64 , 2001 ). thus , the pa1874 - pa1877 operon appears to encode anew type of hybrid efflux pump that combines features of a rnd multidrug efflux pumps and abc transporters . we analyzed the sequence of the p . aeruginosa genome in a effort to identify other genetic loci that resemble pa1874 - pa1877 . we identified two other genetic loci , pa4142 - pa4143 and pa2389 - pa2391 , that are similar to the pa1874 - pa1877 operon in both sequence and organization . each of these loci appears to encode three , rather than four polypeptides . these loci encode putative hybrid efflux pumps that are predicted to play a role in biofilm resistance . table 3 summarizes information for each of the proteins in the three identified hybrid efflux pump operons . putative expression control sequences upstream of pa1874 , pa4142 , and 2389 are shown in fig1 , 18 and 19 respectively . in order to determine whether the putative hybrid efflux pump encoded by pa4144 - pa4146 ) plays a role antibiotic resistance , we inserted a nucleotide sequence that includes pa4144 - pa4146 into a medium copy plasmid psmc32 and used the resulting vector psmc51 to transform p . aeruginosa strain pa14 . neither the pa4144 - pa4146 encoding vector nor the parent plasmid had any effect on the resistance of planktonic p . aeruginosa to tb . we also investigated the resistance of the transformed cells to tb when grown as a biofilm . interestingly , wild - type p . aeruginosa carrying the pa4144 - pa4146 encoding vector became hypersensitive to antibiotics in the colony biofilm assay for reasons that are not clear , but may be due to the increased metabolic burden of carrying a plasmid ( see fig2 ). therefore , we created a “ synthetic sensitivity ” to antibiotics by having the wild type p . aeruginosa strain carry a parent plasmid ( no pa4144 - pa4146 encoding sequences ). as shown in fig2 , a p . aeruginosa strain carrying both this parent plasmid and the pa4144 - pa4146 encoding vector regained a level of antibiotic resistance similar to the of an untransformed strain ( no parent plasmid and no pa4144 - pa4146 encoding vector ). these indirect data suggest that the hybrid efflux pump encoded by pa4144 - pa4146 has the ability to confer antibiotic resistance in biofilm - grown bacteria and supports our hypothesis that these novel , hybrid efflux pumps play a role in biofilm - related antibiotic resistance . we used sequence homology searching to identify genes encoding proteins that are likely homologs . the proteins encoded by these genes , like ndvb , are expected to play a role in biofilm resistance . the genes are : b . japonicum ndvb ( genbank accession no . aac62210 ; fig2 ; seq id no : 27 ); agrobacterium tumefaciens unannotated sequence ( genbank accession no . np 357541 ; fig2 ; seq id no : 28 ); pseudomonas putida kt2440 ( fig2 a - 24 c ; seq id nos : 29 and 30 ); and a pseudomonas syringae gene ( fig2 a - 25 c ; seq id nos : 31 and 32 ). the data described herein suggest two novel mechanisms for biofilm - related antimicrobial resistance . we have shown that a mutant unable to acquire biofilm - related resistance to the antibiotic tb is defective in glucan synthesis . the p . aeruginosa ndvb mutant had increased biofilm - related sensitivity to gm and cip . we propose that these glucans sequester tb and thereby prevent access of this antibiotic to the cytoplasm of the bacteria . this observation is consistent with previous reports that glucans can bind a range of chemically distinct molecules ( breedveld , m . w ., and k . j . miller . microbiol rev . 58 ( 2 ): 145 - 61 , 1994 ). the ability to bind a range of biocides is also consistent with the reported ability of biofilms to develop broad resistance to antimicrobial agents [ reviewed in ( mah , t .- f ., and g . a . o &# 39 ; toole . tims 9 : 34 - 39 , 2001 )]. based on our data , we submit that compounds that modulate the expression of ndvb or the function of an ndvb polypeptide , such that there is a decrease in ndvb gene transcription , ndvb mrna translation , or ndvb polypeptide function , are expected to promote a decrease in microbial resistance to antimicrobial agents , possibly due to the loss of , or lower levels of , glucan synthesis . compounds which alter the activity of pa1163 can be identified using an assay for ndvb activity , e . g ., the assay described by bhagwat et al . ( j . bact . 178 : 4635 - 42 , 1994 ). compounds that modulate function of the identified homologs of ndvb are also expected to promote a decrease in microbial resistance to antimicrobial agents , possibly due to the loss of , or lower levels of , glucan synthesis . this decrease in resistance is predicted to occur in biofilms , but may also occur in other physiological states as well as in cells having genetic changes leading to increased resistance . we have also identified a novel efflux pump that is required for full biofilm - related antibiotic resistance . this pump appears to be a hybrid between known efflux pumps of the rnd superfamily and abc transporters . efflux pumps typically have broad substrate specificity , which is consistent with the decrease in resistance of the 30b1 mutant to tb , gm and cip . compounds that decrease either the expression of pa1874 or one or more of the following : pa1875 , pa1876 , and pa1877 , or the function of the corresponding polypeptides , such that there is a decrease in gene transcription , mrna translation , or polypeptide function of one or more of pa1874 , pa1875 , pa1876 , or pa1877 are expected to promote a decrease in microbial resistance to antimicrobial agents . similarly , compounds that decrease either the expression of any of pa4142 - pa4144 and pa2389 - pa2391 , or the function of the corresponding polypeptides , such that there is a decrease in gene transcription , mrna translation , or polypeptide function of one or more of pa4142 - pa4144 and pa2389 - pa2391 are expected to promote a decrease in microbial resistance to antimicrobial agents . this decrease in resistance is predicted to occur in biofilms , but may also occur in other physiological states as well as in cells having genetic changes leading to increased resistance . compounds which alter the expression of one or more of the hybrid efflux pump genes described herein can be identified using a reporter construct in which a reporter gene is operably linked to an expression control region located upstream of pa1873 , pa4142 or pa2389 ( see table 3 ). the reporter construct is introduced into a cell , e . g ., bacterial cell such as a p . aeruginosa cell . the cell is exposed to a test compound and the expression of the reporter gene is monitored . one method to further examine the function of the novel efflux pumps is to express the components of the efflux pump in a variety of cell types , prokaryotic and eukaryotic , and use it to screen for compounds which overcome ( inhibit ) the action of the efflux pump . bacteria have developed several different mechanisms to overcome the action of antibiotics . these mechanisms of resistance can be specific for a molecule or a family of antibiotics , or can be non - specific and be involved in resistance to unrelated antibiotics . several mechanisms of resistance can exist in a single bacterial strain , and those mechanisms may act independently or they may act synergistically to overcome the action of an antibiotic or a combination of antibiotics . specific mechanisms include degradation of the drug , inactivation of the drug by enzymatic modification , and alteration of the drug target ( b . g . spratt , science 264 : 388 ( 1994 )). there are , however , more general mechanisms of drug resistance , in which access of the antibiotic to the target is prevented or reduced by decreasing the transport of the antibiotic into the cell or by increasing the efflux of the drug from the cell to the outside medium . both mechanisms can lower the concentration of drug at the target site and allow bacterial survival in the presence of one or more antibiotics which would otherwise inhibit or kill the bacterial cells . some bacteria utilize both mechanisms , combining a low permeability of the cell wall ( including membranes ) with an active efflux of antibiotics . ( h . nikaido , science 264 : 382 - 388 ( 1994 )). different pumps can efflux specifically a drug or group of drugs , such as the nora system that transports quinolones , or tet a that transports tetracyclines , or they can efflux a large variety of molecules , such as certain efflux pumps of p . aeruginosa . in general , efflux pumps have a cytoplasmic component and energy is required to transport molecules out of the cell . some efflux pumps have a second cytoplasmic membrane protein that extends into the periplasm . at least some efflux pumps of p . aeruginosa have a third protein located in the outer membrane . efflux pumps are involved in antibiotic resistance since , in some cases , they can remove a significant fraction of the antibiotic molecules which manage to enter the cells , thereby maintaining a very low intracellular antibiotic concentration . to illustrate , p . aeruginosa laboratory - derived mutant strain 799 / 61 which does not produce any measurable amounts of efflux pump is 8 to 10 fold more susceptible to tetracycline and ciprofloxacin than the parent strain p . aeruginosa 799 , which synthesizes efflux pumps . also , null mutants of mexa , the cytoplasmic component of a p . aeruginosa efflux pump , are more susceptible to antibiotics than the wild type . the physiological role of efflux pumps has not been clearly defined yet . they are involved in drug resistance but they also are involved in the normal physiology of the bacterial cell . the efflux pump coded in the mexa operon of p . aeruginosa has been shown to be regulated by the iron content of the medium , and it is co - regulated with the synthesis of the receptors of siderophores . siderophores are molecules that are needed for bacterial growth under iron starvation conditions , such as during infection of an animal . they are synthesized in the cytoplasm and exported when the bacterial cell needs iron . siderophores scavenge iron within the infected animal and return the iron to the microbe to be used for essential microbial processes . since there is essentially no free iron in the bodies of animals , including the human body , the production of siderophores by infecting bacteria is an important virulence factor for the progress of the infection . one aspect of this invention concerns the identification of compounds that are inhibitors of a hybrid efflux pump described herein . such efflux pumps export substrate molecules from the cytoplasm in an energy - dependent manner , and the exported substrate molecules can include antibacterial agents or other antimicrobial agents . such efflux pump inhibitors are useful , for example , for treating microbial infections by reducing the export of a co - administered antimicrobial agent or by preventing the export of a compound synthesized by microbes ( e . g ., bacteria ) to allow or improve their growth . an example of reducing the export of such a compound is inhibiting iron availability for the microbe by reducing the export of siderophores . thus , this invention provides methods to identify compounds that are efflux pump inhibitors . one recent hypothesis to explain biofilm - related antibiotic resistance invoked the development of “ persistors ”, or a subset of bacteria that develop high level resistance to antimicrobial agents . as is the case for the development of biofilm architecture we propose that entry into this persistent state requires a specific set of genes and their gene products . the isolation of mutants defective in biofilm - related resistance , such as pa1163 , supports the hypothesis that there is a distinct genetic basis for this biofilm - related resistance and our approach has begun to identify these components . the resistance of biofilms to traditional antibiotic therapy in the clinical setting is an ongoing problem . our invention , however , provides new strategies to block the development of this resistance by identifying the genes and gene products responsible for resistance . in addition to providing a means for inhibiting biofilms , we provide a co - therapeutic approach where traditional antibiotics are combined with a drug that interferes with biofilm - related resistance to render biofilms , and possibly related physiological states , more susceptible to treatment . atg tct tca cgc aag atc ggg ctc aac ctg gtg gtc atc gtc gcc ctg 48 gcc gcc ctc ttc acc ggc atc tgg gcc ctg tac aac cgt ccg gtc agc 96 ala ala leu phe thr gly ile trp ala leu tyr asn arg pro val ser gta ccg gac tgg ccg gaa cgc atc tcc ggc ttc tcc ttc tcg ccg ttc 144 cgc ctc aac cag aac ccg cag agc ggc cgc tac ccc agc gcc gaa cag 192 atg cgc acc gac ctg gaa ctg gtc gcc cgg cac acc cac agc atc cgc 240 met arg thr asp leu glu leu val ala arg his thr his ser ile arg acc tat tcg gtc cag ggc gcg ctc ggc gac atc ccg gcg ctg gcc gag 288 thr tyr ser val gln gly ala leu gly asp ile pro ala leu ala glu gcg ttc ggc ctg cgc gtc agc ctg ggc atc tgg ctc ggc ccg gac ctg 336 ala phe gly leu arg val ser leu gly ile trp leu gly pro asp leu tcg ccg agc gtg gtg cga gtg ata gtc ggc aac gag gcg ctg ttc cgc 432 ser pro ser val val arg val ile val gly asn glu ala leu phe arg cgc gag gtg acg gcg gaa cag ttg atc gcc tac ctc gac cgg gtc cgc 480 gcg gcg gtc aag gtt ccg gtg acc acc gcc gaa cag tgg cac gtc tac 528 cgc gaa cac ccg gaa ctg gcg caa cac gtc gac ctg atc gcc gcc cac 576 gtc ctg ccc tac tgg gag gcc acg ccg gtg gcc gac gcg gtg gac ttc 624 gtg ctc gaa cgc gcg cgc gaa ctc aag gcc gcc ttc ccg agg aag ccg 672 ctg ctg ctc gcc gag gtc ggc tgg ccg agc aac ggg cgc atg cgc ggc 720 leu leu leu ala glu val gly trp pro ser asn gly arg met arg gly agc gcc gag gcg aca ccc gcg gac cag gcc atc tac ctg cgg cgc ctg 768 ser ala glu ala thr pro ala asp gln ala ile tyr leu arg arg leu acc aac gcg ctc aac ggc gaa ggc tac agc tac ttc gtc atc gaa gcc 816 thr asn ala leu asn gly glu gly tyr ser tyr phe val ile glu ala ttc gac cag ccc tgg aag gtc agc gcc gaa ggc tcg gtg ggc gcc tac 864 phe asp gln pro trp lys val ser ala glu gly ser val gly ala tyr tgg ggc gtc tac aac gcc gac cgc aag gcc aag ttc aac ttc acc ggg 912 trp gly val tyr asn ala asp arg lys ala lys phe asn phe thr gly ccg gtg gtg ccg att ccc aag tgg cgc gcc ctg gcc atc gcc tcg gcg 960 gta ctc gcg gta ctc gcc ttc acc ctg ctg ctg atc gac agt tcc tcg 1008 ctg cgc cag cgc ggg agg acc ttc ctc gcc gtg gtc tcg ttc gcc tgc 1056 gcc tcg gtg ctg gtg tgg atc gcc tac gac tac agc cag cag tac agc 1104 acc tgg ttc agc ctg acc gtc ggc gcg ttg ctg ggc gtc ggc gcg cta 1152 ggg gtg gtc atc gtg ctg ttc acc gag gcc cac gag ctg gcc gag gcg 1200 gtc tgg acg cgc aag cgg cgc cgg cca ttc ctg ccg atc acc gcc gcg 1248 cgg gcc tat cgg ccc aag gtg tcg atc cac gtg ccc tgc tac aac gag 1296 arg ala tyr arg pro lys val ser ile his val pro cys tyr asn glu ccg ccg gaa ctg ctg aag cag acc ctc gac gcc ctt gcc cgc ctc gac 1344 tac ccg gac tac gaa gtc ctg gtg atc gac aac aac acc cgc gac ccg 1392 gcc gtc tgg cag ccg gtc gag gcg cac tgc gcg cgc ctg ggc gag cgc 1440 ala val trp gln pro val glu ala his cys ala arg leu gly glu arg ttc cgc ttc ttc cac gtt gcc ccg ctg gaa ggc ttc aag gcc ggc gcg 1488 ctg aac ttc gcc ctg ggc cac gtg gcg gcg gac gtc gag gtg gtc gcg 1536 gtg atc gac gcc gac tac tgc gtc gac ccc gac tgg ctc agg cac atg 1584 val ile asp ala asp tyr cys val asp pro asp trp leu arg his met gtg ccg cac ttc ggc gac ccg cgg atc gcc gtg gtg cag tcg ccg cag 1632 val pro his phe gly asp pro arg ile ala val val gln ser pro gln gac tac cgc gac cag cac gag agc gcc ttc aag cgg ctc tgc tac gcc 1680 asp tyr arg asp gln his glu ser ala phe lys arg leu cys tyr ala gag tac aag ggc ttc ttc cac atc ggc atg gtc acc cgc aac gac cgc 1728 glu tyr lys gly phe phe his ile gly met val thr arg asn asp arg gac gcg atc atc gag cac ggc acc atg acc atg atc cgg cgc agc gtg 1776 asp ala ile ile glu his gly thr met thr met ile arg arg ser val ctg gac gag ctg aga tgg ccg gaa tgg tgc atc acc gag gac gcc gag 1824 ctg ggc ctg cgg gtg ttc gag aag ggc ctg tcg gcc gcc tac ttc gag 1872 cgc agc tac ggc aag ggg gtg atg ccc gat acc ttc atc gat ttc aag 1920 arg ser tyr gly lys gly val met pro asp thr phe ile asp phe lys aag cag cgc ttc cgc tgg gcc tac ggc gcg atc cag atc atg aag cgg 1968 cat acc gac gcc ctg ctg cgc ggc cgc ggt ccc gac ggc agc cgc ctg 2016 acc cgc ggc cag cgc tac cac ttc gtg gcc ggc tgg ctg ccg tgg atc 2064 thr arg gly gln arg tyr his phe val ala gly trp leu pro trp ile gcc gac ggc ctg aac atc ttc ttc acc ctc ggc gcg ctg ctc tgg tcg 2112 gcg gcg atg atc atc gtg ccc aag cgc gtc gac ccg ccg ctg ctg atc 2160 ttc gcg atc ctg ccg ctg gcc ctg ttc gtc ttc aag gtc ggc aag atc 2208 ctc ttc ctc tac cgg cgc acc gtc ggc gtc gac ctg cgc gac tcg ttc 2256 ttc gcc gcc ctc gcc ggc ctg tcg ctc tcg cac acc att gcc aag gcg 2304 gtg ctg tac ggc ttc gtc acc cgc ggc atc ccg ttc ttc cgc acg ccg 2352 aag atg cgc tcc agc cac ggc ctg ctg gtg gcc ctg gcg gag gcc cgc 2400 gag gaa gtc ttc gtg atg ctc ctg ctg tgg ggc gcg gcg gcc ggc atc 2448 gtg gcg gtt cag ggc gtg ccg agc cgc gac ctg ctg atc tgg gtc gcc 2496 val ala val gln gly val pro ser arg asp leu leu ile trp val ala atg ctc ctg gtg caa tcg ctg ccc tac ctg gcg gcg ctg gtc atg gcc 2544 ttg ctc tcg tcg ctg ccg aaa ccg cgc gag gaa ctg gcc ggc ggc gcc 2592 ala ala leu phe thr gly ile trp ala leu tyr asn arg pro val ser met arg thr asp leu glu leu val ala arg his thr his ser ile arg thr tyr ser val gln gly ala leu gly asp ile pro ala leu ala glu ala phe gly leu arg val ser leu gly ile trp leu gly pro asp leu ser pro ser val val arg val ile val gly asn glu ala leu phe arg leu leu leu ala glu val gly trp pro ser asn gly arg met arg gly ser ala glu ala thr pro ala asp gln ala ile tyr leu arg arg leu thr asn ala leu asn gly glu gly tyr ser tyr phe val ile glu ala phe asp gln pro trp lys val ser ala glu gly ser val gly ala tyr trp gly val tyr asn ala asp arg lys ala lys phe asn phe thr gly arg ala tyr arg pro lys val ser ile his val pro cys tyr asn glu ala val trp gln pro val glu ala his cys ala arg leu gly glu arg val ile asp ala asp tyr cys val asp pro asp trp leu arg his met val pro his phe gly asp pro arg ile ala val val gln ser pro gln asp tyr arg asp gln his glu ser ala phe lys arg leu cys tyr ala glu tyr lys gly phe phe his ile gly met val thr arg asn asp arg asp ala ile ile glu his gly thr met thr met ile arg arg ser val arg ser tyr gly lys gly val met pro asp thr phe ile asp phe lys thr arg gly gln arg tyr his phe val ala gly trp leu pro trp ile val ala val gln gly val pro ser arg asp leu leu ile trp val ala atg tcg atc cag gcg aaa gtt acc cct atc gat cag agt att tct tct 48 gcg gct gcc gtc gag gtt ccg gaa aac ggg ata ctc aaa ctc tcc cag 96 ala ala ala val glu val pro glu asn gly ile leu lys leu ser gln agc agt aat gtc gcg ctc gat gtc gca ccg gag tcg gtg gcg gga tac 144 tcg aag agc ggt tcg gac ctg atc gtc cag ctg aag acc ggg gaa agc 192 gtc cgg atc gcc aac ttc tat gcg gaa ggc cag cct tcc agc caa ctg 240 val arg ile ala asn phe tyr ala glu gly gln pro ser ser gln leu ttc ctg gcc gac aag gac aag ctg gtg gcg gta gat ctg ccg ccg gtc 288 gct gcc gac ggg ccg ctg atg gcc ggc tac atc ccg cag gaa agc ctg 336 ala ala asp gly pro leu met ala gly tyr ile pro gln glu ser leu gcc ggt ttc gag tcg ctg acc ggc gcc ggt gtg ctc ggt ggc atg agc 384 gca ggg act gcg ctg ctg gtc ggt gcg gcg gcc atc ggc gcc ggg gtg 432 gcg att tcc aac agc agc ggc ggc ggt ggc ggc ggc ggt tct tcg gtg 480 ccc ccg gac acc act ccg ccg aag gcg gcc agc ggc ctg aag ata gcg 528 cct gac ggc agc agc atc agc ggc cag gcc gag gcc ggc gcg agc gtc 576 ggc atc gat acc aat ggc gac ggc aag ccg gac ctc acc gtg atc gcc 624 gat gcc aac ggc aat ttc acc gct ccg ctg aac ccg ccg ctg acc aat 672 ggc cag acg gtc acc gtg gtg gtc acc gac ccg gct ggc aac gcc agc 720 ccg ccg gcc cag gtc acc gct ccg gac act acc gcc ccg gcg ccg gct 768 acc gac gtg cag gtg gcg ccg gac ggc agc agc gtc acc ggc aag gcc 816 gaa ccc ggc tcg acg gtg ggc gtc gat acc gac ggc gac ggc cag ccg 864 gac acc acc gtg gtg gtc ggc ccc ggc ggc agc ttc gag gtt ccg ctg 912 aac ccg ccg ctg acc aat ggc gag acg gtg acg gtg atc gtt acc gac 960 ccg gcc ggc aac aac agc acc ccg gtg acc gtc gag gcg ccg gac acc 1008 acc gcc ccg gcg ccg gcc acc gac gtg cag gtg gcg ccg gac ggc agc 1056 agc gtc acc ggc aac gca gag ccg ggc gcc acc gtc ggt gtc gac acc 1104 gat ggc gac ggc cag ccg gac acc acc gtg gtg gtc ggt ccc ggc ggc 1152 agc ttc gag gtt ccg ctg aac ccg ccg ctg acc aat ggc gag acg gtg 1200 acg gtg atc gtt acc gac ccg gcc ggc aac agc agc acc ccg gtc acc 1248 gcc gaa gcc ccc gac ttc ccc gac gcg ccc cag gtc aat gcc agc aac 1296 ggc agc gtc ctc agt ggt acg gcg gaa gcg ggc gtg acc atc gtg atc 1344 acc gac ggc aac ggc aat ccg atc ggc cag acc agc gcc gat gcc aac 1392 ggc aac tgg agc ttc acc ccc ggt agc caa ctg ccg gat ggc acc gtg 1440 gly asn trp ser phe thr pro gly ser gln leu pro asp gly thr val gtc aat gtg gtg gcc agg gac gcc gcc ggc aac agc agc ccg gcg acc 1488 tcc atc acc gtc gac ggc gtg gcg ccg aac gcg ccg gtg gtc gag ccg 1536 agc aac ggc agc gaa ctc agc ggg act gcc gaa ccg ggc agc agc gtg 1584 acc ctg acc gac ggc aat ggc aat ccg atc ggc cag acc acc gcc gat 1632 gcc aac ggc aac tgg tct ttc acg ccg tcc acc ccg ttg ccg gac ggt 1680 acc gtg gtc aac gtg gtg gcc agg gat gcc gcc ggc aac agc agt ccg 1728 ccg gcc agc gtt acc gtg gat gcc gtc gcg ccg gcc acg ccc acc gtc 1776 gat ccg agc aac ggt acg acc ctc agc ggc acc gcc gag ccg ggc agt 1824 agc gtg acc ctg acc gac ggc aac ggt aac ccg ata ggg cag gtc acc 1872 gcc gac ggc agc ggc aac tgg acc ttc acc ccg agc acg ccg ttg ccc 1920 aac ggc acg gtg gtc aac gcc acg gct acc gac ccg tcc ggc aac gcc 1968 agt tcg ccg gcc agc gtc acc gtg gac gcc gtg gca ccg gcc acg cca 2016 gtg gtc aac ccg agc aac ggc acc acg ctc agc ggc acc gcc gag ccg 2064 ggc gcc acc gtg acc ctg acc gat ggc aac ggc aat ccc atc ggg cag 2112 gtc acc gcc gat ggc agc ggc aac tgg agc ttc act ccg acc acg ccg 2160 ttg ccc aac ggc acc gtg gtc aac gcc acg gcc acc gac gcc tcc ggc 2208 aac acc agt gcg ggc agc agt gtc acc gtg gac tcg gta gcc ccg gcc 2256 acg cca gtg atc aac ccc agc aac ggc acc acg ctc agc ggc acc gcc 2304 gag ccg ggc agc agc gtg act ctg acc gat ggc aac ggc aac ccg att 2352 ggc cag gtc acc gcc gac ggc agc ggc aac tgg agc ttc acc ccg tcc 2400 gly gln val thr ala asp gly ser gly asn trp ser phe thr pro ser acg ccg ctg gcg gat gga acc gtg gtc aac gcc acg gcc acc gat ccg 2448 gcg ggc aac acc agc ggc cag ggc agc acc acc gtc gat ggc gtg gcg 2496 ccg acc acg ccg acc gtc aac ctg agc aac ggc agc agc ctc agc ggc 2544 act gcg gaa ccg ggc agc acg gtg atc ctc acc gac ggc aac ggc aat 2592 thr ala glu pro gly ser thr val ile leu thr asp gly asn gly asn ccg atc gcc gag gtc acc gcc gac ggc agc ggc aac tgg acc tac acc 2640 pro ile ala glu val thr ala asp gly ser gly asn trp thr tyr thr ccg tcc acg ccg atc gcc aac ggc acc gtg gtc aac gtg gtg gcc cag 2688 gac gcc gcc ggc aat agc agc ccg ggc gcc agc gtc acc gtg gac tcg 2736 cag gcc ccg gcg gct ccg gtg gtc aac ccg agc aac ggc act acg ctc 2784 agc ggc acc gcc gag ccg ggc gct acc gtg acc ctg acc gac ggc aac 2832 ggc aac ccg att ggc cag gtc acc gcc gac ggc agc ggc aac tgg agc 2880 gly asn pro ile gly gln val thr ala asp gly ser gly asn trp ser ttc aca ccg ggc acg ccg ctg gcc aac ggc acc gtg gtc aac gcc acg 2928 gcc agc gac ccg acc ggc aat acc agc gct ccg gcc agc acc acc gtg 2976 gac tcg gtg gcg ccg gcc gcg ccg gtg gtc aat ccg agc aac ggc gcg 3024 gag atc agc ggc acc gcc gaa ccg ggc gcc acc gtg acc ctg acc gat 3072 ggc agc ggc aat ccg atc ggg cag gtc acc gcc gac ggc agc ggc aac 3120 tgg agc ttc acc ccg tcc acg ccg ctg gcg gat gga acc gtg gtc aac 3168 trp ser phe thr pro ser thr pro leu ala asp gly thr val val asn gcc acc gct acc gac ccg gcc ggc aat acc ggc ggc cag ggc agc acc 3216 acc gtg gac gcc atc gcg ccg gcc acg ccg acc gtc aac ctg agc aat 3264 ggc agc agc ctc agc ggc acc gcc gag ccg ggc agc acg gtg att ctc 3312 acc gac ggc aac ggc aat ccg atc gcc gag gtc acc gcc gac ggc agc 3360 ggc aac tgg acc tac acc ccg tcc acg ccg atc gcc aac ggt act gtg 3408 gtc aac gtg gtg gcc cag gac gcc tcc ggt aac agc agc ccg ccg gcg 3456 acg gtg acc gtc gat tcc agc gcg ccg ccg gcg ccg gtg atc aac ccg 3504 agc aac ggc gtc gtc atc agc ggc acc gcc gag gcc ggt gcc acg gtg 3552 acc ctc acc gat gcc ggc ggc aac ccg ata ggg cag gtc acc gcc gac 3600 ggc agc ggc aac tgg agc ttc acg ccg ggc acc ccg ctg gcc aac ggc 3648 acg gtg atc gtc gcc acg gcc acc gac ccg acc ggc aat acc ggc ccg 3696 cag gcc gcc acc acg gtg gac gcg gtg gcg ccg ccg gcg ccg gtg atc 3744 gat ccg agc aac ggc acg acc atc agc ggc acc gcg gag gcc ggg gcc 3792 aag gtg atc ctc acc gac ggc aac ggc aac ccg atc ggc gaa acc acc 3840 gcc gac ggc agc ggc aac tgg agc ttc acg ccc ggc acg ccg ctg gcc 3888 aac ggc acg gtg gtc aac gcc gtg gcc cag gac cct gcg ggc aat acc 3936 ggc ccg cag ggc agc act acc gtg gac gcg gtg gcg ccg aac acg cct 3984 gtg gtc aat ccg agc aac ggc aac ctg ctc aac ggt acc gcc gag ccg 4032 ggc agc acc gtg acc ttg acc gac ggc aac ggc aac ccg atc ggc cag 4080 acc acc gcc gat ggc agc ggc aac tgg agc ttc acg ccc ggc tcg caa 4128 ctg ccc aac ggc acc gtg gtc aac gtg acc gcg agc gac gcc gcc ggc 4176 aat acc agc ctt ccc gct acc acg acg gtg gat tcc tcg ctg ccg tcg 4224 atc ccg cag gtg gat ccg agc aac ggt tcg gtg atc agc ggc acc gcg 4272 gac gcc ggc aac acc atc atc atc acc gat ggc aac ggc aac ccg att 4320 ggc cag gtc acc gcc gac ggc agc ggc aac tgg tcc ttc act cca ggc 4368 gly gln val thr ala asp gly ser gly asn trp ser phe thr pro gly atc ccg ctg ccg gat ggc acg gtg gtc aac gtg gtg gcg cgc agc cca 4416 ile pro leu pro asp gly thr val val asn val val ala arg ser pro agc aat gtc gac agt gcg ccg gcg gtg atc act gtg gat ggc gtg gcc 4464 ccg gcg gcg ccg gtg atc gat ccg agc aac ggc acc gag ata agc ggt 4512 acc gcg gag gcc ggc gcg acg gtg atc ctc acc gat ggc ggc ggc aac 4560 ccg atc ggc cag gcc acc gcc gac ggc agc ggc aac tgg acg ttc acc 4608 pro ile gly gln ala thr ala asp gly ser gly asn trp thr phe thr ccg agc acc ccg ctg gcc aac ggc acc gtg atc aac gcc gtg gcc cag 4656 gac ccg gcc ggc aat acc agc ggt ccg gcc agc gtc acc gtc gat gcc 4704 atc gcc ccg ccg gcg ccg gtg atc aat ccg agc aat gga gtc gtc atc 4752 agc ggt acg gcg gaa gcc ggg gcc acg gtg atc ctc acc gac ggc aac 4800 ggc aac ccg atc ggc cag gtc acc gcc gac ggc agc ggc aac tgg agc 4848 gly asn pro ile gly gln val thr ala asp gly ser gly asn trp ser ttc acg ccc ggc acg ccg ctg gcc aac ggc tcg gtg atc aat gcg ctg 4896 gcc cag gac gcc gcc ggc aac aac agc agt ccc acc agc gcc acc gtc 4944 gac tcg ctg gcg cca gca gcc ccg gtg atc gat ccg agc aac ggt agc 4992 gtg atc gcc ggt acc gcc gag gct ggt gcc acg gtg atc ctc acc gac 5040 ggc aac ggc aac ccg atc ggc cag gtc acc gcc gat ggc agc ggc aac 5088 tgg agc ttc acg ccc ggc acg ccg ctg tcc aat ggc acg gtg gtc aat 5136 gcg gtg gcc cag gac gct gcc ggc aac acc agc ggc ccg gtc agc acc 5184 acg gtg gac gcg gtg gcc ccg gcc acc ccg gtg atc gac ccg agc aac 5232 ggt gtc gaa ctc agc ggc acc gcc gaa ccc ggc gtc cgg gtg atc ctc 5280 acc gat ggc aat ggc aat ccg atc ggc cag acc ctt gcc gac ggc agc 5328 ggc aac tgg agc ttc acg ccg ggc acg ccg ctg gcc aac ggc acg gtg 5376 gtc aat gcc gtg gcc cag gac ccg gcc ggc aat acc agc ggc ccg gcc 5424 agc acc acg gtg gac acg gtg gct ccg gcc acg ccg gtg atc aat ccc 5472 agc aac ggc agc gtg atc acc ggc acc gcc gag gtc ggc gcc aag gtg 5520 atc ctc acc gat ggc aac ggc aac ccg atc ggc gag acc acc gcc gac 5568 ggc agt ggt aac tgg acc ttc acc ccc ggc acg ccg ctg gcc aac ggt 5616 acg gtg atc aac gcc gtc gcc gag gac gcc gcg ggc aac gcc agc ggt 5664 ccg gcc agc acc acg gtg gac tcg gtg gcg ccg tcc gct ccg ctg ctg 5712 agc atc agc gcc gac ggc gcg ctg ctg acc ggc acc gcc gag ccg aac 5760 agc cag gtg cgc atc gtg gtc aac ggc gac acc gcc aac ccg atc acg 5808 ser gln val arg ile val val asn gly asp thr ala asn pro ile thr gtc acc gtc gac ggc gcc ggc aac ttc agc ctg ccg ttc gcg ccg ccg 5856 ctg atc acc ggc gag ctg atc gcc ggg gtc gcc gtc gac gcc gcc ggc 5904 aac gtc agc ggg ccg gcc acc atc aac gcc ccg gac ctg gcg ccg ccg 5952 acc atc agc gtg ccg gaa gcc gcc gat acc tgg atc aat gcc gcg gag 6000 atc ggc gac ggt atc cag gtc gat gtg acg gtc cgt ccg acc atg cag 6048 gtc ggc cag gtg gtc acg gtc aag ttc gcc ggg cag aac ggc tac gag 6096 gcc gag gtc agc cat acc ctc acc gcc ggc gac atc gcc gcc ggc aac 6144 ala glu val ser his thr leu thr ala gly asp ile ala ala gly asn ctg acc ctg acc ctg acg cct ccc ggc ggc atg ggc ccg ttc ccg gag 6192 ggt gcc tcg acc gtc acc gcc gac atc aac ggc ggc acc gcg tcg acc 6240 ccg gtg ccg ttc acc atc gac acc att ccg ccg gcg acc ccg gtg ctg 6288 tcc ctg gtc ggc aac atc ctg acc atc tcg gcg gag cca ggg acc gag 6336 ttg acg gtg acc gtc gac gtc ggc ggg gtg acc gcc acc gcc acg gtg 6384 acc gcc gac aac agc ggg ctg gcg tcg ctg aac ctg ctc acc gac ctg 6432 gac atc gac ttc agt tgg gac cag ttg ctc aat gcc cag gtg tcg gtg 6480 gtc gga cgc gac ccg gcc ggc aac ccg agc aac acg gcg agc atc ggc 6528 gtc ggc acc agc atc gag caa ccg gtg acc atc ggc aac ttc ggc ctc 6576 val gly thr ser ile glu gln pro val thr ile gly asn phe gly leu gac gtc agc ctc aac ccg ctg aac ccg cgt ttc ggt ttc agc gga acc 6624 acc gag cct gac tcc agc gtg gtg atc cgg gtc atc acc ccg gcg ttg 6672 aac gtc gaa ttg ctg ccg atc cag gcg gat tcg tcc gga aac ttc tcg 6720 asn val glu leu leu pro ile gln ala asp ser ser gly asn phe ser ctg aac ctg ctg agc ccg acc atc ctc acc cag ttg ggg ctg aac atc 6768 acc gac atc ctc aac ctc ggc tcg cag atc tcg ttc aac ctg gtg tcc 6816 acc gac tcc aat ggc aac gac agc gcc gcc tac ggg atc acc ctg acc 6864 ccc aac gga ctg tcg ctc aat atc ggc cag atc gat gtc aac ggt act 6912 tcc ggc gac gac gtg ctg tcc ggc gcc aac ggc agt tcg gag cac atc 6960 aac ggc ggc gac ggc agc gac ctg atc ttc aac gtg ggc acc ggc gat 7008 cac gtg gtg gcc ggc aac ggc aac gac acc atc cag atc acc gcg acc 7056 gat ttc gtc agc atc gat ggc ggc gcc ggg ttc gac acc ctg gtc ctg 7104 gcc aac ggc atc gac ctc gac tac aac gcc gtc ggc gtc ggc acg ctc 7152 agc aac ctc gag cgc atc gac ctc ggc aag ggc gat tcg ggt agc gtg 7200 ctg acc ctg acc gcg gcg gag gtg gat gcc atc acc gat gcc aac aac 7248 acg ttg cag atc acc ggc gag aac aac gac acc ctg aac gtg gtg ggc 7296 gcg gtg aat acc ggt acc acg caa ctg atc aac ggc att acc tac gac 7344 gtc tac acc ttc ggc agt acc acc ctg ctg atc gag gac aac acg gta 7392 val tyr thr phe gly ser thr thr leu leu ile glu asp asn thr val ala ala ala val glu val pro glu asn gly ile leu lys leu ser gln val arg ile ala asn phe tyr ala glu gly gln pro ser ser gln leu ala ala asp gly pro leu met ala gly tyr ile pro gln glu ser leu gly asn trp ser phe thr pro gly ser gln leu pro asp gly thr val gly gln val thr ala asp gly ser gly asn trp ser phe thr pro ser thr ala glu pro gly ser thr val ile leu thr asp gly asn gly asn pro ile ala glu val thr ala asp gly ser gly asn trp thr tyr thr gly asn pro ile gly gln val thr ala asp gly ser gly asn trp ser trp ser phe thr pro ser thr pro leu ala asp gly thr val val asn gly gln val thr ala asp gly ser gly asn trp ser phe thr pro gly ile pro leu pro asp gly thr val val asn val val ala arg ser pro pro ile gly gln ala 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ggg 432 tyr ser gly thr ala val phe ala lys cys arg tyr arg pro asp gly cgg gtc ggc gac tac gcc agc gcc ttg ccc gaa cac tgg ttc ttc ggc 480 arg val gly asp tyr ala ser ala leu pro glu his trp phe phe gly ccg ctc aag cgg ctc tgg cgt tcc tac gcc gag gtc acc gcc gcg gcg 528 pro leu lys arg leu trp arg ser tyr ala glu val thr ala ala ala ttg gtg gcc aac gtc ctg gcg gtc gcc tcg gca ctg ttc gcc atg cag 576 gtc tac gac cgc gtg gtg ccc aac gcg gcg ttc gac acc ctg tgg atc 624 val tyr asp arg val val pro asn ala ala phe asp thr leu trp ile ctc gcc agc ggc gtg gcc ctg gcg atc gtc ctc gac ggt gtc ctg cgg 672 atc atg cgc ggc cac ctg ctc aac gtg ctc ggc aag cgc ctc gac ctg 720 caa ctc tcg acc ctg ctg ttc tcc cgc gtg ctg agc acc cgg gtc gcc 768 gcc aag ccg gcg tcg atg ggc gcc ttc agt acc cag gtg cgg gag ttc 816 ala lys pro ala ser met gly ala phe ser thr gln val arg glu phe gag tcg gtg cgc gag ttc ttt acc tcg tcc agc gcg gcg ctg atc agc 864 gac ctg ccg ttc gtg gcg atc ttc ctg ctg atc atc gcc gtg atc 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ala asp arg val ile val leu gly gln gly lys val ser leu asp glu ser thr atg cgt gcc ctc gcc ggc ctg ttg tgc ggc ctg ctc ggc ctg gtt ccc 48 ggc gcc gcc gcc tac gag ccg gac gtg ttc ggc acc acc ggc cag gtc 96 gcc ggc cag gcg gtc tac gac ctc ggc ggc agc ggt ttg ccc tgc cgc 144 ala gly gln ala val tyr asp leu gly gly ser gly leu pro cys arg ggc ggg ccg cca ccg acc gag ctg agc ctg gag gaa gcc atc gag cgg 192 atc ctc tgc cac gac ccg cag acc cgc ctc gcc tgg gcc aat gcc aag 240 ile leu cys his asp pro gln thr arg leu ala trp ala asn ala lys gcc cag gcg gcc cag gtc ggg atc ggc aag tcc gcc tac ctg ccg cgc 288 ala gln ala ala gln val gly ile gly lys ser ala tyr leu pro arg ctg gac ggc cgt ctc gac gcc agt cgc ggc tac agc gac atg gat tat 336 cgc gat gcc ccc tac ctc tcc ggc gac ggc cat cgc cac cgg cgc ggc 384 gcc agc ctc caa ttg agc tgg gtg ctg ttc gac ttc ggc cgc cgc agc 432 cag gac gcg acc ctg cag aac acc ttc gcc ctc gcc gcc cag gcc tac 528 tac gac gcc ctc gcc gcc cag cgc agc ctg gcc gcc tcg cgg 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384 tac cag cgc cag cag cgg ctg gcc gcc ggc ggc gca acg cgt acc gag 432 gac gtg cag agc gcc cag gcg cag atg ctc gcc acc cag gcg cgg atc 480 gag atg tac cag gcg cag atc cgc cag gcc cag gcc tcg ttg cgc agc 528 gac gaa gcc gaa ctc ggc tat acc cgc atc tac gcg ccg atg tcc ggc 576 asp glu ala glu leu gly tyr thr arg ile tyr ala pro met ser gly acg gtg gtg gcg gtc gat gcg cgc gaa ggc cag acc ctc aat gcc cag 624 cag cag acc ccg ttg atc ctg cgg atc gcc aaa ttg tcg ccg atg acc 672 gtc tgg gcc cag gtt tcg gaa gcc gac atc ggc cgg gtc aag ccc ggc 720 val trp ala gln val ser glu ala asp ile gly arg val lys pro gly atg ccg gcc tac ttc acg acc ctc agc ggc gaa ggc cgg cgc tgg acc 768 met pro ala tyr phe thr thr leu ser gly glu gly arg arg trp thr ggc aag gtc cgg cag atc ctc ccg gtg ccg ccc aag ccg ctg gac cag 816 ggc agc ggc cgg gtg gtg ctg tat acc gtg ctg gtc gac gtg gac aac 912 ggc gac cac caa ctg atg gcg gaa atg acc gcc cag gtg ttc ttc gtc 960 gly asp his gln leu met ala glu met thr ala gln val phe phe val gcc gcc acc gca gaa aac atc ctc acc gcg ccg gtc gcc gcc atc cac 1008 gac gac ggc aag ggc ggc cag gtc gcc tgg gtg gtc ggc agc aac ggc 1056 aag ccg cag agc cgc cag atc agg acc ggc atc agc gac cgc ctg cgg 1104 gta cag gtg ctt gcc ggc ctg gag gaa ggc gac cgc ctg ttg atg gcc 1152 ser val ser ala leu gly thr leu gln pro arg arg tyr val asp val gly ala gln ala ser gly gln ile arg lys leu his val glu ala gly gln gln ala lys val asp ala gly arg tyr ser ile glu met leu lys asp glu ala glu leu gly tyr thr arg ile tyr ala pro met ser gly val trp ala gln val ser glu ala asp ile gly arg val lys pro gly met pro ala tyr phe thr thr leu ser gly glu gly arg arg trp thr gly asp his gln leu met ala glu met thr ala gln val phe phe val atg gaa aac gcc acg caa ccc gtc ccc ctg atc gaa ctg cgc gac atc 48 met glu asn ala thr gln pro val pro leu ile glu leu arg asp ile cgc aag cgc tac ggc ggc aat ggc acc ccg gaa gtc gag gta ctc aag 96 ggc gta tcg ctg tcg atc cac gcc ggc gag ttc gtc gcc atc gtc ggc 144 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gln tyr pro thr asp asp glu atg cgc cag gac ctg gag caa ctg agc aaa ctg acc gac agc atc cgt 240 atc tat acc gtg gaa ggc acc cag gcc gac gtc ccg cgc ctg gcc gag 288 ile tyr thr val glu gly thr gln ala asp val pro arg leu ala glu gag ttc ggc ctg cgg gtg acg ctg ggg ata tgg atc agc ccg gac ctg 336 glu phe gly leu arg val thr leu gly ile trp ile ser pro asp leu gag cgc aac gag cgc gaa att gcc acg gcc atc cag ctg gcc aac acg 384 tcg cgc agc gtg gtg cgg gtg gtg gtc ggc aac gag gcg ctg ttc cgt 432 gaa gaa gtc aca ccg gaa aac ctg atc aaa tac ctg gac cgc gta cgc 480 glu glu val thr pro glu asn leu ile lys tyr leu asp arg val arg gca gcc gtg aag gtg ccc gtg acc acc agt gaa cag tgg cac atc tgg 528 ala ala val lys val pro val thr thr ser glu gln trp his ile trp aag gaa cat cct gag ctg gcc agg cac gtc gac ctg att gcc gcg cac 576 atc ctg ccc tac tgg gag ttc gtg ccg atg aag gat tcg gtc gag ttc 624 ile leu pro tyr trp glu phe val pro met lys asp ser val glu phe gtc ctc gag cgc gcc cgt gaa ctg aag cac 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cgc gag ttc ctg ccc gtg cag gcc gac 1248 val trp ile his lys arg arg arg glu phe leu pro val gln ala asp act gcc tac cgg ccc aag gtg tcg gtg cat gtg ccg tgc tac aac gag 1296 thr ala tyr arg pro lys val ser val his val pro cys tyr asn glu cca cct gag atg gtg aaa cag acc ctg gac gcc ctg gcc gcc ctg gac 1344 tac ccc gac tac gaa gtg ctg gtg atc gac aac aac acc aag gac ccg 1392 gcc gtg tgg gag ccg ctc aag gcc cac tgc gaa aag ctt ggc gag cgc 1440 ala val trp glu pro leu lys ala his cys glu lys leu gly glu arg ttc aag ttc ttc cac gtc gcg cca ctg gcc ggc ttc aag ggt ggc gcg 1488 ctg aat tac ctg atc ccg cac acg gca aag gac gcc gaa gtg atc gcg 1536 leu asn tyr leu ile pro his thr ala lys asp ala glu val ile ala gta atc gac tcg gac tac tgc gtc gac cgc aac tgg ctc aag cac atg 1584 val ile asp ser asp tyr cys val asp arg asn trp leu lys his met gtg ccg cac ttc gcc gac ccg aaa att gcc gtg gtg cag tca ccg cag 1632 gat tac cgt gac cag cac gaa agc gcc ttc aag aag ctg tgc tac agc 1680 asp tyr arg asp gln his glu ser ala phe lys lys leu cys tyr ser gaa tac aag ggc ttc ttc cac atc ggt atg gtc acc cgc aac gac cgt 1728 glu tyr lys gly phe phe his ile gly met val thr arg asn asp arg gac gcg atc atc cag cac ggc acc atg acc atg acc cgg cgc agt gtg 1776 asp ala ile ile gln his gly thr met thr met thr arg arg ser val ctg gaa gaa ctg ggc tgg gcc gag tgg tgc atc tgc gag gac gcc gaa 1824 ctg ggc ctg cgc gtg ttc gag aaa ggc ctg tcc gcc gcc tac gcc cac 1872 leu gly leu arg val phe glu lys gly leu ser ala ala tyr ala his aac agc tac ggc aag ggc ctg atg ccc gac acc ttc atc gac ttc aag 1920 asn ser tyr gly lys gly leu met pro asp thr phe ile asp phe lys aag caa cgc ttc cgc tgg gcc tac ggc gcc atc cag atc atc aag cac 1968 cac gcc ggc gcg ctg ctg cgc ggc aaa ggc agc cag ctg acc cgt ggc 2016 cag cgc tac cac ttc ctg gcc ggc tgg cta ccg tgg atc gcc gat ggc 2064 gln arg tyr his phe leu ala gly trp leu pro trp ile ala asp gly atg aac atc ttc ttc acc atc ggc gcg ctg ttg tgg tcg gcg gcg atg 2112 atc atc gtg ccg cat cgg gtc gat ccg ccc ctg atg atc ttc gcc atc 2160 tac cgc cga gcg gtg ggg gtg aac ctc aag gat gcc ttc gca gct gcg 2256 ctg gcc ggg ctg gca ctg tcg cac acc atc gcc aag gcg gta ctg tat 2304 ggt ttc ttc acc agc agc atg ccg ttc ttc cgc acg ccg aag aac gct 2352 gac agc cat ggg ttg ctg gtg gcg att tcc gaa gcc cgt gaa gag ctg 2400 ttc atc atg gtg ctg ctg tgg ggc gcg gcg ttg ggt atc tac ctg gtg 2448 cag ggg ctg ccg agt tcg gac atg cgc ttc tgg gtg gcg atg ttg ctg 2496 gln gly leu pro ser ser asp met arg phe trp val ala met leu leu gtg cag tcg ttg cct tat gtg gca gcg ctg gtg atg gcg ttc ctg tcg 2544 tcg ctg ccc aag ccc gca gaa aag gct gcc caa gcg cag cag gct 2589 met ser ser arg lys phe gly leu asn leu val val val leu ala ile ala ala leu phe thr gly phe trp ala leu ile asn arg pro val ser arg leu gly glu ser pro gln lys gly gln tyr pro thr asp asp glu ile tyr thr val glu gly thr gln ala asp val pro arg leu ala glu glu phe gly leu arg val thr leu gly ile trp ile ser pro asp leu glu glu val thr pro glu asn leu ile lys tyr leu asp arg val arg ala ala val lys val pro val thr thr ser glu gln trp his ile trp ile leu pro tyr trp glu phe val pro met lys asp ser val glu phe val asn thr leu asn arg arg gly tyr asn tyr phe val ile glu ala tyr asp gln pro trp lys ala ser asp glu gly ser val gly ala tyr trp gly val tyr asn ala glu arg gln gln lys phe asn phe asp gly val trp ile his lys arg arg arg glu phe leu pro val gln ala asp thr ala tyr arg pro lys val ser val his val pro cys tyr asn glu ala val trp glu pro leu lys ala his cys glu lys leu gly glu arg leu asn tyr leu ile pro his thr ala lys asp ala glu val ile ala val ile asp ser asp tyr cys val asp arg asn trp leu lys his met asp tyr arg asp gln his glu ser ala phe lys lys leu cys tyr ser glu tyr lys gly phe phe his ile gly met val thr arg asn asp arg asp ala ile ile gln his gly thr met thr met thr arg arg ser val leu gly leu arg val phe glu lys gly leu ser ala ala tyr ala his asn ser tyr gly lys gly leu met pro asp thr phe ile asp phe lys gln arg tyr his phe leu ala gly trp leu pro trp ile ala asp gly gln gly leu pro ser ser asp met arg phe trp val ala met leu leu atg tcg ata tac cgc atg gag cac agt tta gac atg aat aaa aaa ata 48 met ser ile tyr arg met glu his ser leu asp met asn lys lys ile tca gac gct cca atc tgg ccg gtc aac tca ttc aaa tcc gtc gtg acc 96 ser asp ala pro ile trp pro val asn ser phe lys ser val val thr aaa gtc ccg gac tgg cct gac agc atc tcg ggc ctt gcc tat aac ccc 144 lys val pro asp trp pro asp ser ile ser gly leu ala tyr asn pro ttt cgt ccc gga caa agt ccc tac aag cac atc tat ccg acc cgc gag 192 phe arg pro gly gln ser pro tyr lys his ile tyr pro thr arg glu caa atc aaa gaa gac ttg ctg ctg atc cgc ccg ttg act cga cat gta 240 gln ile lys glu asp leu leu leu ile arg pro leu thr arg his val aga acc tac tcg gtc gag cag acg ctg gcc tgt att ccc gaa ata gcc 288 arg thr tyr ser val glu gln thr leu ala cys ile pro glu ile ala gaa gaa ctc ggc atg agt gtc aca ctc ggc ata tgg ata ggc tgg gac 336 gaa aaa cgc aat gat cgg gaa ctg atc gag ggc gtg aag ctt gcc aat 384 cag tat ccc agc gtc cgg cgt ctg atc atc gga aat gaa aca tta ctg 432 gln tyr pro ser val arg arg leu ile ile gly asn glu thr leu leu cgc aat gac gtc acc gtc agc caa ctg atc gat tac atg caa acg gca 480 arg asn asp val thr val ser gln leu ile asp tyr met gln thr ala cga caa ggt gtc aac gtt ccg att tca acc tca gag gga tgg caa cag 528 arg gln gly val asn val pro ile ser thr ser glu gly trp gln gln tgg cac gat acg ccg gaa ctg gct gat cac gca gac ttc atc gcg gcg 576 cat gtc ttg cca ttc agg gag ttc gtt cca gtc acc cag gca ggc tct 624 his val leu pro phe arg glu phe val pro val thr gln ala gly ser gca gtt ctc gca cgg gcg aac gaa ttg agg ctg atg ttt ccc gaa aaa 672 ccg ctg ata ctt tcc gag att ggc tgg cca gac aaa ggc aac ttc aga 720 pro leu ile leu ser glu ile gly trp pro asp lys gly asn phe arg aga cgc acc acc gcc tac gtc gcc gaa cag tca att tac ctg cgc agc 768 cag ctc gcg ctg ttg aac cag agt ggc ctc gac tac ttt gtc agg gag 816 gln leu ala leu leu asn gln ser gly leu asp tyr phe val arg glu gca ttt gat caa caa tgg aaa act gag gaa ggg ttg ccg ggg cct cac 864 ala phe asp gln gln trp lys thr glu glu gly leu pro gly pro his tgg ggc ctg ttc gat gcc cag cga aag ata aag tta cca ctg caa ggc 912 trp gly leu phe asp ala gln arg lys ile lys leu pro leu gln gly cca gtg aaa ata cgg gcc agc tgg cga tca gaa gtt ccg aga ttg gtc 960 gcc gat tgg cag ccc gac aac tgg cga aca acc gta ttg att ttt gct 1008 ala asp trp gln pro asp asn trp arg thr thr val leu ile phe ala gcg ttg tac aca tta ttg gta ggc gtt ggc ata agt tac gca cag ccc 1056 tta tcg atg tgg gtg gct ttg ccc atc gcc ttg gtg tgg gtg acc agc 1104 tta ctg atc ggc acg ggg ata cag ggt tac gag ttc ctc gaa tca tgc 1152 tgg gga ccg gag aaa ccg cga tct ttt cct ccg tta aga gct tac ccg 1200 trp gly pro glu lys pro arg ser phe pro pro leu arg ala tyr pro ggg ccg tta ccc aaa gtg tcc ata cac gta ccg tgc tac aac gaa cct 1248 gly pro leu pro lys val ser ile his val pro cys tyr asn glu 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ala ile ser leu tyr caa cga ttg gcc agc acc gac atc aaa gac gcc ttc gca gcc ata att 2208 gcg agc atg gcg ctg tac tcg gtt gta ggc aag gcc gtg ctt tca tcg 2256 gca ttc acc tca gga tta ccg ttc ttt cgc act ccc aag cag acc tct 2304 ggc agc ggg ctc ggc aag gcc ctg ctg gac gtc cgg gaa gat ctg tac 2352 atg gcc gtg gtc tgg tgg gtc atg acg gta tcg ctg tgc ttc cga aaa 2400 met ala val val trp trp val met thr val ser leu cys phe arg lys gaa gct atc ggt ccg gac ctt gga ttc tgg gtg gcg ata atg ttc gcc 2448 glu ala ile gly pro asp leu gly phe trp val ala ile met phe ala cag tca ttg cct tac gta gcc gcc atg atc atg gca ata ctg tcg gct 2496 ctc gca aac cgc cct tca cgc tcc aca acc tga 2529 met ser ile tyr arg met glu his ser leu asp met asn lys lys ile ser asp ala pro ile trp pro val asn ser phe lys ser val val thr lys val pro asp trp pro asp ser ile ser gly leu ala tyr asn pro phe arg pro gly gln ser pro tyr lys his ile tyr pro thr arg glu gln ile lys glu asp leu leu leu ile arg pro leu thr arg his val 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