酿酒酵母rDNA的结构及其介导整合影响因素研究进展
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摘要
为提高外源基因在酿酒酵母基因组中的拷贝数,将rDNA重复序列作为整合载体同源重组位点的研究日益被关注.本文介绍了酿酒酵母rDNA组成元件的结构,综述了以rDNA重复序列作为同源重组位点的研究进展.分析表明,hot1片段和一些蛋白因子(Fob1,RAD52和MRX,Sir2p,Sgs1和Mus81等)对rDNA单元的拷贝数目有调控作用,Smc5-Smc6复合体、RAD52的SUMO化和Mms21对rDNA的同源重组严格调控,对其稳定性有重要影响.以rDNA介导整合的外源基因在酿酒酵母细胞有丝分裂中的稳定性与整合位点在rDNA单元中的位置和性质以及整合载体的大小有重要关系.根据酿酒酵母较易发生同源重组的特点,使用rDNA重复序列作为整合载体同源重组位点可以通过提高外源基因在酿酒酵母基因组中拷贝数使外源基因在细胞中获得高效稳定表达.
In order to improve the copy number of heterologous genes in yeast genome,rDNA repeat units used as integration sites of homologous recombination has increased the concern of researchers.This paper introduced the structure and components of rDNA locus in Saccharomyces cerevisiae,and summarized the research progress of rDNA repetitive sequence as homologous recombination sites.A lot of research showed that hot1and some factors(Fob1, RAD52,MRX complexes,Sir2p,Sgs1and Mus81)regulated the copy number of rDNA unit,Smc5/Smc6complexes and the sumoylation of RAD52and Mms21strictly regulated homologous recombination of rDNA and had important influence on the stability of rDNA.In addition,the stability of heterologous genes by rDNA mediated integration in mitotic phase had important relationship with the location and the characteristic of integration sites in rDNA unit and the size of the integrated vectors.Because homologous recombination occurred easily in Saccharomyces cerevisiae,the use of rDNA repetitive sequence as homologous recombination site could improve the copy number of heterologous genes in Saccharomyces cerevisiae,as the results heterologous genes would exist more stably and express efficiently in cells.
In order to improve the copy number of heterologous genes in yeast genome,rDNA repeat units used as integration sites of homologous recombination has increased the concern of researchers.This paper introduced the structure and components of rDNA locus in Saccharomyces cerevisiae,and summarized the research progress of rDNA repetitive sequence as homologous recombination sites.A lot of research showed that hot1and some factors(Fob1, RAD52,MRX complexes,Sir2p,Sgs1and Mus81)regulated the copy number of rDNA unit,Smc5/Smc6complexes and the sumoylation of RAD52and Mms21strictly regulated homologous recombination of rDNA and had important influence on the stability of rDNA.In addition,the stability of heterologous genes by rDNA mediated integration in mitotic phase had important relationship with the location and the characteristic of integration sites in rDNA unit and the size of the integrated vectors.Because homologous recombination occurred easily in Saccharomyces cerevisiae,the use of rDNA repetitive sequence as homologous recombination site could improve the copy number of heterologous genes in Saccharomyces cerevisiae,as the results heterologous genes would exist more stably and express efficiently in cells.
引文
[1]Martin K.Damage-induced recombination in the yeast Saccharomyces cerevisiae[J].Mutation Research,2000,451(2):91-105.
[2]Aguilera A,Rothstein R.MolecularGeneticsof Recombination[M].Berlin-Heidelberg:Springer Verlag,2007.
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[5]Husnik J I,Volschenk H,Bauer J,et al.Metabolic engineering of malolactic wine yeast[J].Metabolic Engineering,2006,8(4):315-323.
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[9]杨金莹,吕建仁,党宏月.短小芽孢杆菌C-9葡聚糖内切酶基因的克隆及其在酿酒酵母中的表达[J].中国石油大学学报,2011,35(3):144-147.Yang J Y,LüJ R,Dang H Y.Cloning of glucanase gene from Bacillus pumilus C-9and its expression in Saccharomyces cerevisiae[J].Journal of China University of Petroleum,2011,35(3):144-147(Ch).
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[12]Kobayashi T.The replication fork barrier site forms a unique structure with Fob1p and inhibits the replication fork[J].Mol Cell Biol,2003,23(24):9178-9188.
[13]Vogelauer M,Cioci F,Camilloni G.DNA Protein-interactions at the Saccharomyces cerevisiae 35SrRNA promoter and in its surrounding region[J].Mol Biol,1998,275(2):197-209.
[14]Hontz R D,French S L,Oakes M L,et al.Transcription of multiple yeast ribosomal DNA genes requires targeting of UAF to the promoter by Uaf30[J].Mol Cell Biol,2008,28(21):6709-6719.
[15]Chuwattanakul V,Kim Y H,Sugiyama M,et al.Construction of a Saccharomyces cerevisiae strain with a high level of RNA[J].Biosci Bioeng,2011,112(1):1-7.
[16]Voelkel-Meiman K,Keil R L,Roeder G S.Recombination stimulating sequences in yeast ribosomal DNA correspond to sequences regulating transcription by RNA polymeraseⅠ[J].Cell,1987,48(6):1071-1079.
[17]Ward T R,Hoang M L,Prusty R,et al.Ribosomal DNA replication fork barrier and hot1recombination hot spot:shared sequences but independent activities[J].Mol Cell Biol,2000,20(13):4948-4957.
[18]Kobayashi T,Heck D J,Nomura M,et al.Expansion and contraction of ribosomal DNA repeats in Saccharomyces cerevisiae:Requirement of replication fork blocking(Fob1)protein and the role of RNA polymeraseⅠ[J].Genes Dev,1998,12(24):3821-3830.
[19]Johzuka K,Horiuchi T.Replication fork block protein,Fob1,acts as an rDNA region specific recombinator in S.cerevisiae[J].Genes to Cells,2002,7(2):99-113.
[20]Feng Q,Düring L,de Mayolo A A,et al.Rad52and Rad59exhibit both overlapping and distinct functions[J].DNA Repair,2007,6(1):27-37.
[21]Wolner B,van Komen S,Sung P,et al.Recruitment of the recombinational repair machinery to a DNA double-strand break in yeast[J].Mol Cell,2003,12(1):221-232.
[22]Fortin G S,Symington L S.Mutations in yeast Rad51that partially bypass the requirement for Rad55and Rad57in DNA repair by increasing the stability of Rad51-DNA complexes[J].Embo,2002,21(12):3160-3170.
[23]Pannunzio N R,Manthey G M,Bailis A M.RAD59and RAD1cooperate in translocation formation by single-strand annealing in Saccharomyces cerevisiae[J].Curr Genet,2010,56(1):87-100.
[24]Lewis L K,Storici F,Van Komen S,et al.Role of the nuclease activity of Saccharomyces cerevisiae Mre11in repair of DNA double-strand breaks in mitotic cells[J].Genetics,2004,166(4):1701-1713.
[25]Chan C Y,Zhu J,Schiestl R H.Effect of rad50mutation on illegitimate recombination in Saccharomyces cerevisiae[J].Mol Genet Genomics,2011,285(6):471-484.
[26]Giunta S,Belotserkovskaya R,Jackson S P.DNA damage signaling in response to double-strand breaks during mitosis[J].J Cell Biol,2010,190(2):197-207.
[27]Westmoreland J,Ma W,Yan Y,et al.RAD50is required for efficient initiation of resection and recombinational repair at random,gamma-induced doublestrand break ends[J].PLoS Genet,2009,5(9):e1000656.
[28]Kobayashi T,Horiuchi T,Tongaonkar P,et al.SIR2regulates recombination between different rDNA repeats,but not recombination within individual rRNA genes in yeast[J].Cell,2004,117(4):441-453.
[29]Kobayashi T.A new role of the rDNA and nucleolus in the nucleus-rDNA instability maintains genome integrity[J].Bioessays,2008,30(3):267-272.
[30]Rustchenko E P,Curran T M,Sherman F.Variations in the number of ribosomal DNA units in morphological mutants and normal strains of Candida albicans and in normal strains of Saccharomyces cerevisiae[J].Bacteriol,1993,175(22):7189-7199.
[31]French S L,Osheim Y N,Cioci F,et al.In exponentially growing Saccharomyces cerevisiae cells,rRNA synthesis is determined by the summed RNA polymeraseⅠloading rate rather than by the number of active genes[J].Mol Cell Biol,2003,23(5):1558-1568.
[32]Burkhalter M D,Sogo J M.rDNA enhancer affects replication initiation and mitotic recombination:Fob1mediates nucleolytic processing independently of replication[J].Mol Cell,2004,15(3):409-421.
[33]Ii M,Ii T,Brill S J.Mus81functions in the quality control of replication forks at the rDNA and is involved in the maintenance of rDNA repeat number in Saccharomyces cerevisiae[J].Mutation Research,2007,625(1-2):1-19.
[34]Kobayashi T,Ganley A R D.Recombination regulation by transcription-induced cohesion dissociation in rDNA Repeats[J].Science,2005,309:1581-1584.
[35]Mullen J R,Nallaseth F S,Lan Y Q,et al.Yeast Rmi1/Nce4controls genome stability as a subunit of the Sgs1-Top3complex[J].Mol Cell Biol,2005,25(11):4476-4487.
[36]OgˇrünM,Sancar A.Identification and characterization of human MUS81-MMS4structure-specific endonuclease[J].Biol Chem,2003,278(24):21715-21720.
[37]Eckert-Boulet N,Lisby M.Regulation of rDNA stability by sumoylation[J].DNA Repair,2009,8(4):507-516.
[38]Torres-Rosell J,Machin F,Farmer S,et al.SMC5and SMC6genes are required for the segregation of repetitive chromosome regions[J].Nature Cell Biology,2005,7(4):412-419.
[39]Takahashi Y,Dulev S,Liu X,et al.Cooperation of sumoylated chromosomal proteins in rDNA maintenance[J].PLoS Genet,2008,4(10):e1000215.
[40]Montpetit B,Hazbun TR,Fields S,et al.Sumoylation of the budding yeast kinetochore protein Ndc10is required for Ndc10spindle localization and regulation of anaphase spindle elongation[J].J Cell Biol,2006,174(5):653-663.
[41]Heideker J,Perry J J P,Boddy M N.Genome stability roles of SUMO-targeted ubiquitin ligases[J].DNA Repair,2009,8(4):517-524.
[42]Zhao X,Blobel G.A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization[J].Proc Natl Acad Sci,2005,102(13):4777-4782.
[43]Lopes T S,Klootwijk J,Veenstra A E,et al.Highcopy-number integration into the ribosomal DNA of Saccharomyces cerevisiae:a new vector for high-level expression[J].Gene,1989,79(2):199-206.
[44]Lopes T S,de Wijs I J,Steenhauer S I,et al.Factors affecting the mitotic stability of high-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae[J].Yeast,1996,12(5):467-477.
[45]Ganley A R D,Kobayashi T.Monitoring the rate and dynamics of concerted evolution in the ribosomal DNA repeats of Saccharomyces cerevisiae using experimental evolution[J].Mol Biol Evol,2011,28(10):2883-2891.
[46]刘向勇,沈煜,郭亭,等.rDNA介导的多拷贝整合表达载体的构建及其在酿酒酵母工业菌株中的应用[J].山东大学学报,2005,40(3):105-109.Liu X Y,Shen Y,Guo T,et al.Construction of a ribosomal DNA multi-copy integration vector and application in the industrial Saccharomyces cerevisiae strain[J].Journal of Shandong University,2005,40(3):105-109(Ch).
[47]姜勇,张学成,孙平楠,等.以rDNA为同源重组位点酵母表达鲑鱼降钙素基因多拷贝整合载体的构建[J].中国海洋大学学报,2009,39(3):443-447.Jiang Y,Zhang X C,Sun P N,et al.Construction of a ribosomal DNA multi-copy integration vector for Saccharomyces cerevisiae expressing salmon calcitonin[J].Periodical of Ocean University of China,2009,39(3):443-447(Ch).
[48]张桂敏,曾毓芳,陈雅兰,等.rDNA介导的酿酒酵母稳定表达载体的构建[J].菌物学报,2011,30(1):39-45.Zhang G M,Zeng Y F,Chen Y L,et al.Construction of rDNA-mediated stable expression vectors in Saccharomyces cerevisiae[J].Mycosystema,2011,30(1):39-45(Ch).
[49]黄贞杰,陈玲,张积森,等.ScINO1基因克隆及酵母多基因多拷贝整合表达载体的构建[J].福建师范大学学报,2012,28(6):100-105.Huang Z J,Chen L,Zhang J S,et al.Cloning of ScINO1Gene and Construction of Yeast Multi-copy Integration Expression Vector[J].Journal of Fujian Normal University,2012,28(6):100-105(Ch).
[50]Leite F C B,Anjos R S G,Basilio A C M,et al.Construction of integrative plasmids suitable for genetic modification of industrial strains of Saccharomyces cerevisiae[J].Plasmid,2013,69(1):114-117.
[2]Aguilera A,Rothstein R.MolecularGeneticsof Recombination[M].Berlin-Heidelberg:Springer Verlag,2007.
[3]Daley J M,Palmbos P L,Wu D,et al.Nonhomologous end joining in yeast[J].Annu Rev Genet,2005,39:431-451.
[4]何诚,朱运松.甲醇营养型酵母表达系统的研究进展[J].生物工程进展,1998,18(3):1-4.He C,Zhu Y S.Advances in the studies of methylotrophic yeast as a foreign gene expression system[J].Progress in Biotechnology,1998,18(3):1-4(Ch).
[5]Husnik J I,Volschenk H,Bauer J,et al.Metabolic engineering of malolactic wine yeast[J].Metabolic Engineering,2006,8(4):315-323.
[6]Jacobs E,Rutgers T,Voet P,et al.Simultaneous synthesis and assembly of various hepatitis B surface proteins in Saccharomyces cerevisiac[J].Gene,1989,80(2):279-291.
[7]武志强,贾耐兵,李娜,等.酵母整合型载体的构建及其功能分析[J].生物学通报,2008,43(5):47-50.Wu Z Q,Jia N B,Li M,et al.The construction and functional analysis of the integrated vector in yeast[J].Bulletin of Biology,2008,43(5):47-50(Ch).
[8]葛菁萍,曹喜生,宋刚,等.木酮糖激酶基因整合表达载体构建及在酿酒酵母中的过表达[J].微生物学报,2010,50(6):762-767.Ge J P,Cao X S,Song G,et al.Construction of integrative vector for xylulokinase gene and its overexpression in Saccharomyces cerevisiae[J].Acta Microbiologica Sinica,2010,50(6):762-767(Ch).
[9]杨金莹,吕建仁,党宏月.短小芽孢杆菌C-9葡聚糖内切酶基因的克隆及其在酿酒酵母中的表达[J].中国石油大学学报,2011,35(3):144-147.Yang J Y,LüJ R,Dang H Y.Cloning of glucanase gene from Bacillus pumilus C-9and its expression in Saccharomyces cerevisiae[J].Journal of China University of Petroleum,2011,35(3):144-147(Ch).
[10]Kobayashi T.Strategies to maintain the stability of the ribosomal RNA gene repeats[J].Genes&Genetic Systems,2006,81(3):155-161.
[11]Ganley A R D,Kobayashi T.Highly efficient concerted evolution in the ribosomal DNA repeats:Total rDNA repeat variation revealed by whole-genome shotgun sequence data[J].Genome Res,2007,17:184-191.
[12]Kobayashi T.The replication fork barrier site forms a unique structure with Fob1p and inhibits the replication fork[J].Mol Cell Biol,2003,23(24):9178-9188.
[13]Vogelauer M,Cioci F,Camilloni G.DNA Protein-interactions at the Saccharomyces cerevisiae 35SrRNA promoter and in its surrounding region[J].Mol Biol,1998,275(2):197-209.
[14]Hontz R D,French S L,Oakes M L,et al.Transcription of multiple yeast ribosomal DNA genes requires targeting of UAF to the promoter by Uaf30[J].Mol Cell Biol,2008,28(21):6709-6719.
[15]Chuwattanakul V,Kim Y H,Sugiyama M,et al.Construction of a Saccharomyces cerevisiae strain with a high level of RNA[J].Biosci Bioeng,2011,112(1):1-7.
[16]Voelkel-Meiman K,Keil R L,Roeder G S.Recombination stimulating sequences in yeast ribosomal DNA correspond to sequences regulating transcription by RNA polymeraseⅠ[J].Cell,1987,48(6):1071-1079.
[17]Ward T R,Hoang M L,Prusty R,et al.Ribosomal DNA replication fork barrier and hot1recombination hot spot:shared sequences but independent activities[J].Mol Cell Biol,2000,20(13):4948-4957.
[18]Kobayashi T,Heck D J,Nomura M,et al.Expansion and contraction of ribosomal DNA repeats in Saccharomyces cerevisiae:Requirement of replication fork blocking(Fob1)protein and the role of RNA polymeraseⅠ[J].Genes Dev,1998,12(24):3821-3830.
[19]Johzuka K,Horiuchi T.Replication fork block protein,Fob1,acts as an rDNA region specific recombinator in S.cerevisiae[J].Genes to Cells,2002,7(2):99-113.
[20]Feng Q,Düring L,de Mayolo A A,et al.Rad52and Rad59exhibit both overlapping and distinct functions[J].DNA Repair,2007,6(1):27-37.
[21]Wolner B,van Komen S,Sung P,et al.Recruitment of the recombinational repair machinery to a DNA double-strand break in yeast[J].Mol Cell,2003,12(1):221-232.
[22]Fortin G S,Symington L S.Mutations in yeast Rad51that partially bypass the requirement for Rad55and Rad57in DNA repair by increasing the stability of Rad51-DNA complexes[J].Embo,2002,21(12):3160-3170.
[23]Pannunzio N R,Manthey G M,Bailis A M.RAD59and RAD1cooperate in translocation formation by single-strand annealing in Saccharomyces cerevisiae[J].Curr Genet,2010,56(1):87-100.
[24]Lewis L K,Storici F,Van Komen S,et al.Role of the nuclease activity of Saccharomyces cerevisiae Mre11in repair of DNA double-strand breaks in mitotic cells[J].Genetics,2004,166(4):1701-1713.
[25]Chan C Y,Zhu J,Schiestl R H.Effect of rad50mutation on illegitimate recombination in Saccharomyces cerevisiae[J].Mol Genet Genomics,2011,285(6):471-484.
[26]Giunta S,Belotserkovskaya R,Jackson S P.DNA damage signaling in response to double-strand breaks during mitosis[J].J Cell Biol,2010,190(2):197-207.
[27]Westmoreland J,Ma W,Yan Y,et al.RAD50is required for efficient initiation of resection and recombinational repair at random,gamma-induced doublestrand break ends[J].PLoS Genet,2009,5(9):e1000656.
[28]Kobayashi T,Horiuchi T,Tongaonkar P,et al.SIR2regulates recombination between different rDNA repeats,but not recombination within individual rRNA genes in yeast[J].Cell,2004,117(4):441-453.
[29]Kobayashi T.A new role of the rDNA and nucleolus in the nucleus-rDNA instability maintains genome integrity[J].Bioessays,2008,30(3):267-272.
[30]Rustchenko E P,Curran T M,Sherman F.Variations in the number of ribosomal DNA units in morphological mutants and normal strains of Candida albicans and in normal strains of Saccharomyces cerevisiae[J].Bacteriol,1993,175(22):7189-7199.
[31]French S L,Osheim Y N,Cioci F,et al.In exponentially growing Saccharomyces cerevisiae cells,rRNA synthesis is determined by the summed RNA polymeraseⅠloading rate rather than by the number of active genes[J].Mol Cell Biol,2003,23(5):1558-1568.
[32]Burkhalter M D,Sogo J M.rDNA enhancer affects replication initiation and mitotic recombination:Fob1mediates nucleolytic processing independently of replication[J].Mol Cell,2004,15(3):409-421.
[33]Ii M,Ii T,Brill S J.Mus81functions in the quality control of replication forks at the rDNA and is involved in the maintenance of rDNA repeat number in Saccharomyces cerevisiae[J].Mutation Research,2007,625(1-2):1-19.
[34]Kobayashi T,Ganley A R D.Recombination regulation by transcription-induced cohesion dissociation in rDNA Repeats[J].Science,2005,309:1581-1584.
[35]Mullen J R,Nallaseth F S,Lan Y Q,et al.Yeast Rmi1/Nce4controls genome stability as a subunit of the Sgs1-Top3complex[J].Mol Cell Biol,2005,25(11):4476-4487.
[36]OgˇrünM,Sancar A.Identification and characterization of human MUS81-MMS4structure-specific endonuclease[J].Biol Chem,2003,278(24):21715-21720.
[37]Eckert-Boulet N,Lisby M.Regulation of rDNA stability by sumoylation[J].DNA Repair,2009,8(4):507-516.
[38]Torres-Rosell J,Machin F,Farmer S,et al.SMC5and SMC6genes are required for the segregation of repetitive chromosome regions[J].Nature Cell Biology,2005,7(4):412-419.
[39]Takahashi Y,Dulev S,Liu X,et al.Cooperation of sumoylated chromosomal proteins in rDNA maintenance[J].PLoS Genet,2008,4(10):e1000215.
[40]Montpetit B,Hazbun TR,Fields S,et al.Sumoylation of the budding yeast kinetochore protein Ndc10is required for Ndc10spindle localization and regulation of anaphase spindle elongation[J].J Cell Biol,2006,174(5):653-663.
[41]Heideker J,Perry J J P,Boddy M N.Genome stability roles of SUMO-targeted ubiquitin ligases[J].DNA Repair,2009,8(4):517-524.
[42]Zhao X,Blobel G.A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization[J].Proc Natl Acad Sci,2005,102(13):4777-4782.
[43]Lopes T S,Klootwijk J,Veenstra A E,et al.Highcopy-number integration into the ribosomal DNA of Saccharomyces cerevisiae:a new vector for high-level expression[J].Gene,1989,79(2):199-206.
[44]Lopes T S,de Wijs I J,Steenhauer S I,et al.Factors affecting the mitotic stability of high-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae[J].Yeast,1996,12(5):467-477.
[45]Ganley A R D,Kobayashi T.Monitoring the rate and dynamics of concerted evolution in the ribosomal DNA repeats of Saccharomyces cerevisiae using experimental evolution[J].Mol Biol Evol,2011,28(10):2883-2891.
[46]刘向勇,沈煜,郭亭,等.rDNA介导的多拷贝整合表达载体的构建及其在酿酒酵母工业菌株中的应用[J].山东大学学报,2005,40(3):105-109.Liu X Y,Shen Y,Guo T,et al.Construction of a ribosomal DNA multi-copy integration vector and application in the industrial Saccharomyces cerevisiae strain[J].Journal of Shandong University,2005,40(3):105-109(Ch).
[47]姜勇,张学成,孙平楠,等.以rDNA为同源重组位点酵母表达鲑鱼降钙素基因多拷贝整合载体的构建[J].中国海洋大学学报,2009,39(3):443-447.Jiang Y,Zhang X C,Sun P N,et al.Construction of a ribosomal DNA multi-copy integration vector for Saccharomyces cerevisiae expressing salmon calcitonin[J].Periodical of Ocean University of China,2009,39(3):443-447(Ch).
[48]张桂敏,曾毓芳,陈雅兰,等.rDNA介导的酿酒酵母稳定表达载体的构建[J].菌物学报,2011,30(1):39-45.Zhang G M,Zeng Y F,Chen Y L,et al.Construction of rDNA-mediated stable expression vectors in Saccharomyces cerevisiae[J].Mycosystema,2011,30(1):39-45(Ch).
[49]黄贞杰,陈玲,张积森,等.ScINO1基因克隆及酵母多基因多拷贝整合表达载体的构建[J].福建师范大学学报,2012,28(6):100-105.Huang Z J,Chen L,Zhang J S,et al.Cloning of ScINO1Gene and Construction of Yeast Multi-copy Integration Expression Vector[J].Journal of Fujian Normal University,2012,28(6):100-105(Ch).
[50]Leite F C B,Anjos R S G,Basilio A C M,et al.Construction of integrative plasmids suitable for genetic modification of industrial strains of Saccharomyces cerevisiae[J].Plasmid,2013,69(1):114-117.