INDUCIBLE-TARGETED TAGGING SYSTEM FOR LOCALIZED INSERTIONAL MUTAGENESIS IN ARABIDOPSIS THALIANA BINDU NISHAL THE NATIONAL UNIVERSITY OF SINGAPORE 2005 INDUCIBLE-TARGETED TAGGING SYSTEM FOR LOCALIZED INSERTIONAL MUTAGENESIS IN ARABIDOPSIS THALIANA BINDU NISHAL (B.Sc., M.Sc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES, NATIONAL UNIVERSITY OF SINGAPORE, RESEARCH LINK, SINGAPORE, 117604 2005 To my parents, siblings and their families, teachers, and all the good friends whose inspiration, prayers and love sustained me during these past few years. ii ACKNOWLEDGEMENTS Patience, endurance and optimism are prerequisites not only in the person directly involved in research but also in those that constitute their surroundings. This long journey involves help, guidance, discussions and even argument/s. Therefore it becomes imperative to thank them, because without their direct or indirect involvement, this work would not have been possible. I would like to begin by thanking Prof. Venkatesan Sundaresan for his direction, assistance, and guidance throughout the course of this study. I am grateful to him for allowing me to deviate “just enough” to perform experiments that could have been uninformative, for patiently listening to my theories, hypotheses and literature updates. Our weekly discussions have broadened my horizons to think for which I am immensely thankful to him. My sincere gratitude to Dr’s KK Tan, Mohan Balasubramanian and William Chia for making allowances and arrangements so I could follow Prof. Sundaresan at The University of California Davis to finish a part of this work. I would also like to acknowledge Dr.’s Prakash Kumar, Srinivasan Ramachandran, Benedict Kost, WeiCai Yang and Xie Qi for serving as members of my thesis committee. Special acknowledgements to Dr. Prakash Kumar for being my overseas supervisor during the time when I was working at UC Davis while registered at The National University of Singapore. Heaps of thanks to friends and colleagues Cameron, Megan and John Alvarez for useful discussions, arguments (and coffee!) and to all past and current laboratory members of “The Sundaresan lab”, both at IMA (now TLL) Singapore and at University of California Davis. It was marvelous working in his laboratory in both the continents and the lab members were great coworkers. iii I sincerely acknowledge Dr. John L Bowman, Prof. Charles Gasser and their laboratory members at UC Davis for discussions that we had during our joint group meetings. I thank Dr. Bo Liu also at UC Davis for ordering seed stocks from the SAIL database for some part of this work. I would like to acknowledge Prof. Nam Hai Chua for providing me laboratory space; his laboratory members at TLL and Yang Sun for being warm and generous. I owe them the credit for my rudimentary Mandarin! I am grateful to Dr. Frederick Berger and his “team” for allowing me to work on my thesis and take print outs while working in his laboratory and for the Monday group meetings, some of which would be “imprinted” in my mind. I thank my friends Ventris, Deepika, Wricha, Yezdi, Anat and Jill for sailing along with me these past years. Thanks to Aditya, Jayanth, Trevyen, Jensen, Christiana, Jenny, Agustin, and Santiago who were therapeutic during stressful times. I thank Dr. Saroja Subrahmanyan for allowing me to stay with her family during my initial days at Davis and for making me feel at home. I would like to thank Sebastien for helping me take final print outs of the thesis. I gratefully acknowledge Temasek holdings for their financial support. To conclude, I would like to thank my family members and especially my absolutely adorable parents for being supportive during these past years. Without their continued presence and encouragement, it would have been impossible to this work. Their unflinching courage and conviction has inspired me tremendously. Thanks to my sisters, Renu, Manju, and Anju, my brother Hanumant, my three wonderful brothers’s in law for unconditionally loving me, my amazing nephews Rahul, Harshil and Divyaansh and niece Yashwi with whom I have had the greatest time story telling and playing. I love you all and thank you for being such an integral part of my life. iv TABLE OF CONTENTS Title page i Acknowledgements iii Table of contents v Summary xi List of Tables xiii List of Figures xiv List of Abbreviations xvii List of Publications xix Chapter 1: Introduction 1.1 Plants and mankind 1.2 Why Arabidopsis thaliana? 1.3 Genomics in Arabidopsis 1.4 Post-sequencing era, what next? 1.5 Insertional mutagens: T-DNA versus Transposons 1.6 Targeted disruption of genes in Arabidopsis 1.7 Transposon tagging 1.7.1 Random Tagging (gene trap) 1.7.2 Targeted Tagging 1.8 Behavior of Transposable elements 1.9 Aim of the project 10 Chapter 2: 2.1 Materials and Methods Plant material and growth conditions 11 v 2.1.1 Plant Material 11 2.1.2 Plating and sterilization of seeds 11 2.1.3 Stratification of seeds on filter paper 11 2.1.4 Germination of seeds on soil 12 2.2 Media composition, antibiotic and hormone preparation 12 2.3 Construction of Starter Lines by root transformation 15 2.4 DNA gel blot hybridization 15 2.5 Mapping of Insertion Lines by TAIL-PCR 16 2.6 TAIL-PCR program 16 2.6.1 Plant DNA Extraction 16 2.6.2 Reagents/Primers used 16 2.6.3 Primary TAIL-PCR 17 2.6.4 Secondary TAIL-PCR 18 2.6.5 Tertiary TAIL-PCR 19 2.7 Verification of insertion site by PCR 20 2.8 Heat Shock and Ds Transposition 21 2.9 Selection procedure for detection of somatic and germinal excision events 21 2.10 Staining for GUS activity 25 2.11 Construction of double mutants 25 2.12 Software used for bioinformatics analysis 26 2.13 Microscopy and image processing 26 2.14 Construction of phylogenetic trees using clustalx 27 2.15 Sequences of primers used in this study 28 vi Chapter 3: Development of Targeted Tagging System 3.1 Introduction 33 3.2 Design of the T-DNA vector 33 3.3 Generation of Starter Lines 35 3.4 DNA gel blot hybridization 36 3.5 Distribution of starter lines 36 3.6 Experimental heat-shock treatment 41 3.7 Strategy for Pooled PCR screen 43 3.8 Number of insertions obtained per gene 45 3.9 Hot spots of Ds insertion within a gene 54 3.10 GUS expression 57 3.11 Chapter Discussions 57 Chapter 4: Detailed analysis of the saturated region of Chromosome 4.1 Introduction 61 4.2 Genes selected as target genes 61 4.3 At2g30810 4.3.1 Introduction 62 4.3.2 Number of insertions obtained 64 4.3.3 Phylogenetic analysis 64 4.3.4 GUS expression 67 4.3.5 Changes in GUS expression after fertilization and GA3 treatment 67 4.3.6 Analysis of double and triple mutants 69 4.3.6.1 Knockouts for likely redundant partner 69 4.3.6.2 Verification of knockouts 69 vii 4.4 4.5. 4.6 4.3.7 Histochemical analysis 70 4.3.8 Remobilization of the Ds element in L360 70 4.3.9 Conclusions 72 At2g30830 and At2g30840 4.4.1 Introduction 73 4.4.2 Number of insertions obtained 76 4.4.3 Phylogenetic analysis 76 4.4.4 GUS expression patterns 79 4.4.5 Identification of likely redundant partners 79 4.4.6 Conclusions 82 At2g30910 4.5.1 Introduction 83 4.5.2 Number of insertions obtained 85 4.5.3 Phylogenetic analysis 85 4.5.4 Identification and verification of insertion lines 88 4.5.5 Generation of double mutants and their analysis 88 4.5.6 Conclusions 90 At2g30730 and At2g30740 4.6.1 Introduction 90 4.6.2 Number of insertions obtained 92 4.6.3 Phylogenetic analysis 92 4.6.4 GUS expression analysis 95 4.6.5 Analysis of double mutants 95 4.6.6 Conclusions 95 viii 4.7 4.8 4.9 4.10 At2g30770 and At2g30750 4.7.1 Introduction 97 4.7.2 Protein structure and domains present 98 4.7.3 Number of insertions obtained and GUS expression analysis 98 4.7.4 Phylogenetic analysis 100 4.7.5 Identification and verification of homozygous insertion lines 103 4.7.6 Analysis of At2g30770 At2g30750 double mutant 103 4.7.7 Conclusions 104 At2g30580 4.8.1 Introduction 104 4.8.2 Number of insertions obtained 106 4.8.3 Phylogenetic analysis 108 4.8.4 Identification and verification of homozygous insertion lines 108 4.8.5 Analysis of At2g30580 At1g06770 double mutant 108 4.8.6 Conclusions 111 At2g30940 4.9.1 Introduction 111 4.9.2 Number of insertions obtained 113 4.9.3 Phylogenetic analysis 113 4.9.4 Conclusions 115 At2g30790 4.10.1 Introduction 115 4.10.2 Number of insertions obtained 117 4.10.3 Phylogenetic analysis 117 ix ROLES OF CASPA2 AND HGC1 IN MORPHOLOGICAL CONTROL AND VIRULENCE IN CANDIDA ALBICANS ZHENG XINDE NATIONAL UNIVERSITY OF SINGAPORE 2005 ROLES OF CASPA2 AND HGC1 IN MORPHOLOGICAL CONTROL AND VIRULENCE IN CANDIDA ALBICANS ZHENG XINDE A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2005 TABLE OF CONTENTS ACKNOWLEDGEMENTS LIST OF CONTENTS LIST OF FIGURES LIST OF TABLES ABBREVIATIONS SUMMARY ii iii vi viii ix xi CHAPTER Introduction 1.1. Candida albicans: a polymorphic fungal pathogen 1.2. Transcriptional regulation of hyphal growth in C. albicans 1.2.1. The MAP kinase pathway 1.2.2. The cAMP-dependent protein kinase A pathway 1.2.3. Hyphal specific genes 1.2.4. CaTup1-mediated repression of hyphal development 1.2.5. pH responsive pathway 1.2.6. Other factors involved in hyphal growth 3 10 12 14 1.3. Morphological control in C. albicans 1.3.1. Actin and polarized growth 1.3.2. Morphological machinery controlling polarized growth 16 16 17 1.3.3. 1.3.4. 20 22 Cell cycle and morphological control in C. albicans Septin ring and morphological control Chapter Materials and Methods 2.1. 2.2. 2.3. 2.4. 2.5. Reagents Strains and culture conditions Oligonucleotide primers 25 25 27 2.3.1. 27 Primers used in the study of CaSPA2 2.3.2. Primers used in the study of HGC1 Recombinant DNA methods 28 29 2.4.1. 2.4.2. 2.4.3. 2.4.4. 2.4.5. 29 30 31 32 32 Preparation of electrocompetent E. coli cells Plasmid preparation and analysis Preparation of DNA probes Southern blot Northern blot C. albicans manipulations 2.5.1. Transformation 33 33 iii 2.6. 2.5.2. Preparation of C. albicans genomic DNA 33 2.5.3. Preparation of C. albicans RNA 34 2.5.4. Cell synchronization (Centrifugal elutriation) Gene disruption and integration 35 35 2.6.1. 2.6.2. CaSPA2, CaTUP1, CaNRG1, HGC1 gene deletion Plasmid constructs for GFP tagging 35 36 2.6.3. CaSPA2 domain-deletion constructs 37 2.6.4. Constructs in characterization of HGC1 Microscopy and fluorescence studies 2.7.1. Calcofluor and phalloidin staining Flow cytometric analysis Protein work 37 38 39 39 40 2.9.1. C. albicans protein extract preparation 2.9.2. Western blot 2.9.3. Immunoprecipitation and kinase assays 2.10. Virulence test in mice 40 40 41 41 2.7. 2.8. 2.9. CHAPTER The role of CaSPA2 in polarity establishment and maintenance in C. albicans 3.1. 3.2. 3.3. Introduction Comparison of Spa2 and CaSpa2 amino acid sequence Subcellular localization of CaSpa2 in yeast and hyphal cells 43 44 45 3.4. Construction of Caspa2∆ 49 3.5. Defects of Caspa2∆ cells in polarized growth 50 3.6. Actin localization in Caspa2∆ cells 55 3.7. Multinucleate Caspa2∆ cells 55 3.8. Defects in microtubule structures in Caspa2∆ cells 59 3.9. The role of different domains of CaSpa2 in C. albicans growth 61 3.10. 3.11. Caspa2∆ exhibited no virulence Discussion 3.11.1. Persistent and cell cycle phase independent tip localization of CaSpa2 3.11.2. Functions domains of CaSpa2 3.11.3. Function of CaSpa2 in nuclear movement 62 62 63 64 65 CHAPTER Functional characterization of HGC1 4.1. Introduction 4.2. 67 Identification of a G1 cyclin-related protein in C. albicans 68 iv 4.3. The expression pattern of CLN21 71 4.4. hgc1∆ was defective in hyphal growth 73 4.5. HGC1 is not required for the expression of HWP1, HYR1 and ECE1 79 4.6. HGC1 expression is regulated by cAMP/PKA pathway and CaTup1 80 4.7. Constitutive overexpression of HGC1 alone is not sufficient to induce hyphal growth Physical and functional interaction between Hgc1 and CaCdc28 Hgc1 is required to maintain hyphal tip localization of actin and CaSpa2 Hgc1 is required for virulence Discussion 4.11.1. Role of Hgc1 in hyphal morphogenesis 4.11.2. The unknown factors in germ tube formation 4.11.3. Role of Hgc1 in virulence 81 83 86 87 88 88 90 90 4.8. 4.9. 4.10. 4.11. REFERENCE PUBLICATIONS 92 105 v List of Figures Figure 1.1 Multiple sigacgtgaatgtagacacgtcgaaataaagatttccgaattagaataatttgtttattgctttcgcct ataaatacgacggatcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattg aaaaaaaattggtaattactctttctttttctccatattgaccatcatactcattgctgatccatgtagatttcccggacatgaag ccatttacaattgaatattggttttgcacatGTTTGGTTCGGTTAGAGAAACCCGGTTTAAGA ACAGACAAGTCTTGGGCTTGAAATTAGATTCGGAGCCCAATTTTGATTTT 141 40 (1) L 44 (5) R 45 (1) R 48 (3) L 49 (3) R GGATTTTAAATTTTAGACCCACGAAATTTCTCCCGCCTTCCCGGTCCACA ACTTCGATTCAAACGACTTTCCTCT aaatgaagtgatttggmgtgatgtagacacgtcgaataaagatttccgaattaaataatttgtttattgctttcgcctataaata cgacggatcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattgaaaaaa aattggtaattactctttctttttctccatattgaccatcatactcattgctgatccatgtagatttcccggacatgaagccmttta CAARGATTAGGTTTACTAGGGTTCGTCTTCTCTCACTTTAATCAAACGTTT TGTGATATATTTAATCATATTTTTGTTTCGTTATATGTTGATTCATAGATT TGAAATCCCAAAAAAAAAAACACCTGCTTCTAAAGACTATAGCCTTATT CGAGGAGCCCTTCARATACTCTCRACGATTTCAAAAAARAKGGAATCAT GTCATTGGCCCTKGTCMCCCCTC acggcgcttmgtggwgwgctgmtggtatgACCGAACAACACACCTGAATTTAGCAGTC ACCTGCATAATAAGCATCAGTAAAACATCAATTCAAGAAAGTTTTCACT ATACTCTTTTCCTACAACTTATGATAAATCCAAGGTTTTTGGATACATTA CATTGTGATAGGTCAGGATGTCCATCAGAGAAACAAATGGAGCATCGTC ATGGGCAACCCCTAGAAAAGATTAAACCATAAGTATTATATTAAACGTT ATGTCAAATTTCCACAATTCCAAACAATTAGCTGGTTATTACCTGTGATT TCAGAATGTGAACGACTAATGCAACGCGCGATTGGGTTCCATCTCGGTG AAAACTTCTCCCCACACATTCTGAAATGGTAAGAAAGTGTGTTTTAAACT GAAGAACAAACGGACGAAGCAGAACATCAACTCACAGGATAAGAAAAG GTGTCAGCAGAAACCTTTGAGGACTAAAGGCTTCCATCTCTCTGCTCATT GTGTACTGCATCTTTGTAGAGGGAGTAAAAGTGGCGTTCCATAATCCCAT ATTTACTTGAAAGAAAAGGTTCAGAAGCTTCACGTGTTGACCAGGTTGA AGACAGTGAATGGCCTGCTTCTCACTAACTCTATCTACTATGATCCTCAA AACGGAACCAAAAGTAGGGAAACTTACGTAACATGTATGTAGGCAACAT CTYGYYYTCTYTCAC tttggtgtgatgatgctgACTNGTATATGTATACGTAGTAAAAGTAAAATGTTAAGA AGATACTAAAAATAGTTGAGTTTGAGGNTAATCCCCAATATATAAGNTC ATAACCAGATATTAGANCTGGAATTGAAAACCCTCNAAAAAAGTTGTNA ACCCTAGTGAAAAATTAACCTGTAACTGAGTGAGAGAGGAGAAGAACA GATCTGCTTTTTTGCGAGNAGANCTTCNATTACATACACNAAATCATGTC TTCATCACTGTACACTTAACCATTTTCTTGGTAACCACATCTCTTACACCA TTAATATCTCTATNGTTTTTTTCTTTTCTTTCTTTTTTTGCTGGTGTGAGAA ATTGTACGCTTACTATCTCTCTCTCTTTGGGGGNAANNNCCCCAAA cactagcccccatttggacgtgaatgtagacacgtcgaaataaagatttccgaattagaataatttgtttattgctttcgcctat aaatacgacggatcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattgaa aaaaaattggtaattactctttctttttctccatattgaccatcatactcattgctgatccatgtagatttcccggacatgaagcc atttacaattgaatataagccaTTGGAACTCCGGCGACTAACGTTTACATGGTGCTCGA CACAGGAAGCGACGTCGTTTGGCTCCAATGCTCTCCTTGCAAAGCCTGTT ACAACCAGACCGACGCCATTTTTGACCCGAAGAAATCCAAAACTTTCGC CACCGTACCATGTGGATCTCGCCTTTGCCGGAGACTAGACGACTCGTCGG AATGTGTCACTCGTCGAAGCAAGACTTGTCTCTACCAAGTATCGTACGGT GACGGATCATTCACCGAAGGAGATTTCTCAACTGAAACGCTGACGTTTC ATGGAGCGCGNGTGTNCCACTCTCTACAAAN ttggtgtgatgATATTGCGACGGGTTATTTCTTTATGCAACCTGTTATGGGATTG GGATTTGCAACCCATGGCTGAGACAGATTAGATGGTTCAAATCCAGTTA TGAAGGTTATGATTTCAGTGGGATGGGATACGACAATAGTAGACAGGAC AAGCACTACAAGATCTTAGGGTCTTACTGTACAAATACAACCATGAAAG CTAGTGTCACCGAGTTGGGATCCGATGCATGGAAATCTTACGAATTTGCA TTCCATAGTTGGAATTTATCTATGTCGCCTTATAGTGTATCATTGAATGG AAATTTGTATTGGGTTGCTTATAATCATGAGTCTCGTGATTATTTTATTCA AAGCTTCGACTTTTCTACGGTGAGCTTCAAACCCTATTGTATCTTGCCTA CTAAAAACGGGCATCGTCAATGCGATGGTAGATCACTTGCGATATTTAG GGAAGATCGATTTTCGTTTTTAGAGCAAGAGATCTATAACACAAGGAAT ATAGAGATTTGGGTGACAAAAGAAACTATTAAAAATGGGGATGGAGAA GCTGTAGAGTGGGTGAATTTAGAANNNGGGGGGTTAAANNCCCCCAAA GGGCCAAGNTTTAAGNGCCACCTATTACCCGCCAAGTTACTTTCGTCGAT 142 58 (1) L 59 (1) L 60 (1) b R GAAGATAAAGTTGGTTTGACTCTTGTAATATGTTGNTATAACCAAGGAG GGAAAAAGCTTATATTTATATTGNCAAGGGAGATAAGTTCCATGAAATT GAAATAAAAGATTTGGTTGAGTATAATCCTCGTCACCGTACCTATTTTCC CAATTTGATCCAAGTTCCTACGTTTACGATGAGCGGNAGGAGTATANCC CACATCAGGTTGAATCTCGCTTCNCCCCCCCAGGGGATACAGTGANCGTT GGATTGGAGGAACCAANGGGACCATGGTTTCTCCACAATGNAAGGAAAT ATTNTTCT aaactgctagccatttggmgtgatgtagacacgtcgaataaagatttccgaattagaataatttgtttattgctttcgcctata aatacgacggatcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattgaaa aaaaattggtaattactctttctttttctccatattgaccatcatactcattgctgatccaataaagggaacataaaaccaacat gaggattctaactgaaatgttttcttatgtatatcgaaattcgtgaacaaaggtgataggtatctgtttgtcacctaacatagtc ggtatcagaatcttcttcttcgtcttcttcttcttcttcACCTTGCWCAATGGTCTTTACGTCAAGGT TGAGAGCCAAATCCTCAACTGAGACCTTAATGACGTCGCTTCTAACACC AATATCGGTAGCATAGCGACTCATACAGTACATACCAGCAGTAACTGTT GCCACACCAATGGGACTTGGAATAAGCAATTTGAAAGGAGAAGAGAGG AGTGCAGCCAAAGGAGCACCAACAATTGAAGAAGCTTGACCGCTTCTCC CTACACCGAGCTTATCCATTATAAGCAAACCGGTCTTGCAATAGAGCGC GAAATCCTCACAGTTGTTCTTGAAAATGTCGTAGTTGCCAAATCCGTTTT GGAGAAGGTACATTGCCCTGTGGATGACTGAATCAGTTGTATCAGATTG CGCGGTTGKGCAAGTCCCTCCGCGG tttggacgtgaatgtagactcctcgaaataaagatttccgaattagaataatttgtttattgctttcgcctataaatacgacgga tcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattgaaaaaaaattggtaa ttactctttctttttctccatattgaccatcatactcattgctgatccatgtaGATTTCTTACTTTGATTATTG ATATGCATCTGAAGTATTTCCTTACAACGTTTTTTACATGTTTTTGTACTG TCTAGTGTCTGTGTCAACACCTGATATTATTCTAATATGTATGAAATTTA CTTAATCCATTGGATTTCAGTTTATCATTCGATAATAATATGTTTTTTCCT TGGATCCCATTCTTGACCAAATTTTGTTTAAAATTTCCCCACATTATACGT AGCTGCTAGCTAGGGTTTATGTGGAGTTACCAGGAGTCAGTGCATTGCTA CTTTTGGTGTTGCCTTTAGCAAATGACGTGCTCCATCATGATTTTTATTAC TTTATGCACCGACCATATCTAAATCCACGTACATCACCTTTTCTTCTTCTT TAAAGAATGCACATATAACATTTTGTATACTTTTGTCTTTAGAACTGCTTT CTTTTTGGGGNATGGNAANN tttggtgtgatgatgctgactggtgaagttgttaaaacgccgccgttttggttttttttgtttctctcgttttctggcgtcagactct caccctttgtgcaagaaaccttgagagcttaaaaattcttcacttgggtctcttctctctctcttctctTGCGACCATT CCGTTGTTCGATCCATATATCAAAGGTATGTTGCTTTGATTGAATGGGTT TCTTTTGTTGAATCTAAATTGATAACTTTAAATATATTGAATTTGGGGTTT AAGAAAAGTTTTCAATTTTCGTCTCTGAGTTTGAGCTAAGAGCTGATCTT TGAGTTTGAGTTTGTGTTTGATTTGATTCACTCACAATTCTTAATTAGGGT TGGTATATCTTGTCTTTTTCATCGTTTTCCTTACACATTGCCCATGGCGAT GAGACTCTTCCGGGTTTTTAAGCTCTGTCAGATTTGAACGCTTCAGTCTA CTTACTTGCTCAGACGTGGTTTCTTTTCATTTAAGTTTGAACATTGTTGAT GAAGAACAGATTTAGCTTCCACGTTTCTAAATTTCCAGAGAAGACACTAT TTCGCATTTTTTGCAGCTTGATTTAGTTGGTTTTCAATGGGGGACAGTCA GTACTCGTTTTCTCTCACCACCTTCAGGTATTAACTTTGTTTATCATATCA ATGTTATCTCTTTCTGAGCATTTGGAACGGTTCTCGGGGTTTGATTCTGG NGCCCCCCCCCNNCCNCCCCAAAA L cactagcccccatttggacgtgaatgtagacacgtcgaaataaagatttccgaattagaataatttgtttattgctttcgcctat aaatacgacggatcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattgaa aaaaaattggtaattactctttctttttctccatattgaccatcatactcattgctgatccatgtagatttcccggacatgaagcc atttacaattgaagccattgtcgtgagttcgttgtcgcgcccagcctggcagacgaggcgtccaactatggatgattggaa cgggaggcgcgagcgaaTGAGAATGAAAATTTTGCGGCCGAAGTTTTTAAGTGGT CATATATGATATTTCATCCTTCACAAATGAGTGCATTGATGAGCATTTGT TTTTCGCATCACAGCTAATTTTGTAAAATAGGGTTTTACTCCTACTGAAA AAGGATTTTATTTGGAATGAACTTACCATTTTAATTAAACCCAAAAAATG AAAACAAATTCCGCCTAAATTTTAAATATCACACACATTTCAACCAAGAT 143 61 (4) R 62 (1) L 69 (1) L 71 (5) L 72 (3) R GTACATTCACATTTCTTAAATAGAATTTACTTTGTTGAGTTATTATTAGCA CATTCACATTTTCAACTATTACCCCNTTTTGTCAGACCTTTCTTCATTCGT TTGTCAAACCCCTNCATTTTTTATTTTTTATTTNAANATNAAAACTGNGG AANCATATATGTCCCANGATCATTAAAAAAACGGTGATCAAATNGAGGG AAACTGTGTAGGGATANATCTTANGANCCNGANAGTATNAAGCNATCGA TGACAGA tttggtgtgatgatgctgactgTAGGCCCATNANATGTTTTTGACTCTTTAATAATTCC CTCTCTTTTGTCTTTTATTGTAAAATGCACACAATTTCGAAGAAACTGCC AATGGAGTCTCCATTTTCCAGCGCCATGCCAAGCTTGACTCTACGATGAT AGCTTATTTGACTGCTTCAAAAAAAAAACTTCANANAATNAATTNCNAN AATTTNCCCAATTNCANATCANCGGAAAATNCTANCNATAANCTTTCTN ATNTCGCTNCNCTCAGTNNCTTCAACGTTNNGGNNAGTTNCGGANATAA ANGTTTNAATTNCTNAGTTCCCGTTCCTCCTCCTCCTNCTTTTTTTTTTTTT NNNGGNCCNGNAAANNGGGGGNAAANNAAAAAAAGGGNNNAAANCNN NTTTTTTTNNNNNNGGGGCCCCNTNNAAAAAAAAAAATTTNNCNNNGGG NTTTNGGNCCNNAAAAANNGGNNNGGGGGGGGGNNGNNTTNTTTTTNA AANCCCNGNTTTTTTTTTTTTTNAAAANNNTTTNCCNGNAAAANNNNGG GNNNNNNGNTNNNCCAAAAAAATTTTTTTTTGGNGCCCCCCCCCCCCCC CCCCNNAAAA ctagcccccatttggacgtgaatgtagacacgtcgaaataaagatttccgaattagaataatttgtttattgctttcgcctataa atacgacggatcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattgaaaa aaaattggtaattactctttctttttctccatattgaccatcatactcattgctgatccatgtagatttcccggacatgaagccatt tacaattgAATAGATACGGGTACAAACTCTGATGACACGACACAGCTTTCGTT AATTTCCCACCGTTGATTCCGTTGACTTTCCCGATCACTACCTTTTCCTTT GACCCGGCTACGTNTCNNNNCGGGGGAAAAANAANNN aggtgagctaggktgwgwgctgmtattcgggcctatttcccgcGCCGGTTCAATATTTGATATCA TTTCGGATCAAACAGAAATTAGACAAGCTCTTAGCCCAAGTATTGGGCC TAAGAAGCCCATTGGGCTAACACTAAACAAAACCCTAGGGACTCTTGTG GGTCATTTATATAGTCCATCCCTAACCTCGCCGCGGGAAGATTTTCATCT TTTCGAGCCAACGTCTAATCTAAGCAAGAGAGTTTCATTCAATTCAATCT CTGTAGGAAGATGGCTCGAGTAGGGGCCAAGTCGAGTGGAGCTGGTGCT AAGAAGAAAGGAGTTTCTTTTGTGATCGACTGCTCTAAACCAGTTGATG ATACGATCTTGGAGATAGCCACGCTTGAGAAGTTTCTTCAGGAACGAAT CAAGGTCAGAGGTAAAGCCGGTGCTCTTGGTAACTCCGTCTCCATCACCC GATATAATGGAaAGATCAATGTCaACGCCaATTCCaATTTCTCCGAACGGT AAACTTCTTTTCATCTCTCYTCGACKAC ttcactagcccccatttggacgtgaatgtagacacgtcgaaataaagatttccgaattagaataatttgtttattgctttcgcct ataaatacgacggatcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattg aaaaaaaattggtaattactctttctttttctccatattgaccatcatactcattgctgatccatgtagatttcccggacatgaag ccatttacaattgaatatagttgtgaatatatAGTATAGTTGTGAATATATAGTATAAGATGTT AGAAAGGGCGGTTTGACATACGATTACTTGTCAATGATTTTATTTGAATA GAAAATTTCAAAGAGAGTGGAAGTCAAATCAAATGCGATTGCATGGAAT CGACGGTTGGCTTACTAACCAAATTACAAATGAATTAAGCGTTGACAAT TAAATTAATTGTCTAATCTCTTGATGGATAAAACTTAAATAAAATTAAAT GTAATGGTGGTTCACGTAGTATTTTACAACAGAAGACAGTCATGTTCCTG CTTTTCTAGTGCACCGCCTAAAGATTTCACAAATCTTTTTCCATGTGATTT TTTTGTTTTCTTAGTCAATGNTGGGAATTTGCAGTCCTATTAGTTATTAAT CTTTGCTTTGNAAACTAGAATTAAATGTCACTACCACATACNCCNACTTN ANTAAAAGTCCCTNTCCCCCCCCNAAA cgagcttggtgtgwtgwgctactaattgcatgacagtctaacaagaaaacaagccctagtttccttacaatctcttcaaaa cagactgagacaaaaacaggTGAATGTTTGATAAGTTAAGGTGATTCGCCAATGTA GAGTCTCCAAGGCTCTGTGCGTCTGCAATGTATTGGTTCATTAGCATGTG AGTAGCACGTGACTGGCCGGTTCTTATTCTTTGAATCTGAAGTCTGAACC ATATAGTTAGTTGAGTTTTTTTTTTATTTCTTAAAATCAGTAAAGGTCTCA AGTCAACTTTAAAgAAGGAAGTACATATAAATCGAGTCAACTTAAGTTTT TCAATTTTGGTTCACTTATCTCGAC 144 73 (4) R 74 (5) R 77 (5) L 86 (3) L gcrgttagctagacgtcttggcctgtgaatcattacatagctttcgtgractcrcgcatccaatrttaagctctargcatcttctt tgcattatcttatttaggatcgagattttgaagattatggcctcwttttggaactttttttctcwgctaaacccatcwaagactt aacacatgtgattatttttacaagcttacaataatattagcwgAAGTATCTCATTCCTAGTGGTTCCT GATACTCWGAAGGCTTTGCRGTCCCCTTAAGGGGACTGTATTGCCTTCW TAGAAACCATTATTATACCCAATTTATTATTTTTTGCTCATACTACTTCGC CATAGAAACCATGCATGTGTGTGTTCTCTCWCTGTGAGGAATAYTTATA GAAAAATTGTWTAAACTGGACGCTAKWGACCTCATTCACTAATCTTCCG GATCGGAATGCWTAGTAGCTTCCTATTGCTTTAATTTTWTTTGTCTCCTG CGTGGACATCTTCTATCGCTTTCRWTTCACGTCGACTTGGGACCTCTACA CTCWGCCCTTGWWACTCACTCGCGGGTGCCCCCCCCCCAAATTCTKSCG TCTTCCCCYCCYCCTTCGKCTTCGTCGTTAWTAACTCTCCTCTCTCTCYCC TTGATCMCTCCTCTACAAT gggctgttggngtaagtttntcganaanngggnGATACTTTAAACTGTTNACTTGTTGAGTG CAAACAGTTGTTATACATTTTGTCTATGATAACACCTAATGGGCCTGACC TAAACTCTGATGGGCTGATAAGGTTAATGTCGCAGCTGATTATATTTGAA CTTTTTTCATTTAAATATTTGTACTTGCTAGTAAATACCTTTGCTTCAGCA TGCAAACAAGCTACAAGAGCCAAATGCTACCAATTGTGATCTTAAAACG TAATCGGTAACACAAATGAATAATTTGACAACATCGGCGGAAAAAGATA AACTATGGTCTCAATACGACTGATGATTGTTACAAAAATGACTAATTTAT TTTGTTCGGATATATCCAGATTTTTAATAACTGCAAAAACAAGATTGGCC GGTATGAATCTTAAACTACACTATCGCTGACCACATTGTCAATCGATCAA AGCTGTCATATAAATGCATAGACAAAACATTGATGATTAATCCCTTTTTG TCAACGAATCTTGNAGGTATTTATTTATTTTGTCACCAAAACATCAAATC ACACTAGATACAAGTTTGGTCCAGATCGTATAAGTTCGTTTCCNCCTCCT CCACNAAAACNNATNANCCACNNTAAACCATNNANAANGNNCNANCCC CNCNNNACCNTNNNTGNNCTNTATTTCNNNTCCCNNANANNCTNCANCA TCNNNCCANCNNCNCTTNACANCCCNNCNNNCCTNNNGTNCTCCTNNCN NNCNNCCAGGNTATATNCNCGACGCCGCNCCNTCCCNCCCTTCNCCCNC CNCNNTACTTNGNCTTNTTNNNCACAACNNCNCCCAACNCNNAGCCTNN NCNNACCCCTACCCNANTCCNNNNNCACTTCATTACCCNATNTTTTNCCC NCCCACATCCNNCNCCTCNCCCNNCTCCCCNTCNCANCGCNNGNTAGNC CCCNCAAATCTNCCATG tactagccccatttggacgtgaaatgtagacacgtcgaaataaagatttccgaattagaataatttgtttattgctttcgcctat aaatacgacggatcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattgaa aaaaaattggtaattactctttctttttctccatattgaccatcatactcattgctgatccatgtagCCACGGCGGTTA CATTGTCTCTTTTACCAGAGACGCCTGAGAAACGAGTTTTTCCAGATCTG AATGCTTTTCCGGTGGAGAAGCAGAAGCGAAACGGTCCTTTATGGCTGA GTTTCAACGGCGGTGGTGAGATTTTAACGCCGTACAAAACGGCGGAGAT ATCACGAAGGACGGTGGTGGTTTCGTCGTGTGTGACGGTTGAGCGTGTG ACTGACGCTTGGATCGACGGTTATGGATTAGGGGAGACAAATCAGGAGA GGAAGATGAATCTAGTGGAAGACACGTGTCCTGGTTTTATATCGGACGG TGTAGGGAGAGTCACGTGGACCAATGAGGCGTATAAGAAGATGGCTAG AGAAGATATTCATATTCCGATGGAGGAAGGTGTACCGGAGGATATTAGT TACGATAATTTTCACGTGAACGTACGGTTAGTGATGAAGGAAAGCCCAT GCNNACGTACCCAGCTTTCCATGCNGAGNGNGATNCNGTACACGTGTCA GATCGAAAANAGATCNTCCNGTGCNTGTGACNGTGAAAAGGACGGNGT GNTTTNNNGGAGCTTGACTTAGGCNCTTGG ctctctagcccccatttggacgtgaatgtagacacntcgaaataaagatttccgaattagaataatttgtttattgctttcgcct ataaatacgacggatcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattg aaaaaaaattggtaattactctttctttttctccatattgaccatcatactcattgctgatccatgtagatttcccggacatgaag ccatttactattaaatttaaaactgacattttcacttggatAAAATCAGAGGAGATTAATTATAAGA ATGGAAAAAGAATCTTATAGCAAAAGTTGGTGTTAGTTGCACTTTTGTCT GCTTCGTAGCTTTCGGCTTAGTTGTTGTTATCTTTATTTTGGGGAAACGTT ACACTATCCTTATCTTCACATTTTTCGTATTTTTCTGCTCTAATTTATCTAA ATGCCGGTGTGGCCGAGTTTATCTATTCATGGTAACAGAAAAAACTCGG TGGTAAATTCATACTATTAATTATGAGATTTAAAACTAGTTGTGGAGTTA 145 101 (5) L 112 (5) L 122 (1) R 124 (1) R 132 (2) R TGGTACGTCTAATTATCTCGAATAATTTATCAACGGGATTGTGTGTACCT AACTAGAAATGTAATACTAAGAAATTGACAGAATATAAGACAAGAGTCC CCACGGTCACGGATATAAGGAATTAGGTAATGGTTCTCCTTTTCCTATCA CACCGAAAAACTCGNGAACCGTAATTGATGGNGATCTTTGACACTAATT CGGNTTATCACATTGTCGAACATGGTCTCATGAATTCATCAAAGTGTTGA NAAAAANTTTAATGTTCCACANATGGGGAAACGGTGGATCACNGGAATG CANGNTTGGAGGATCCAAGGGGAGGCCGCCCANGGGCCCTGCTTTCAAA CGATNAGNTTCCCCCCCCCC cactagcccccatttggacgtgaatgtagacacgtcgaaataaagatttccgaattagaataatttgtttattgctttcgcctat aaatacgacggatcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattgaa aaaaaattggtaattactctttctttttctccatattgaccatcatactcattgctgatccatgtagatttcccggacatgaagcc atttacaattgaaagagggattaggggccggtgtcgaattcgcctctgatcccggtctgagcagaggctgaatgtccgtg gatcgggctgtaatatgccgtcgccgtagcattgcccaaggcgtcgttgacgaagatgtcggtggagagagattcctga gaatagcctcggtaaccggcgccgccatagatggtccatgatggtccaccgatccacgggtcgataatgataaattacC GTTAAGGAAACCTAATCCGTCATCATCTTCGTCGGAGCTGCCTCCGTAGT AGCCAACTTCAGCTGATTCTTCGAAGAACTGAAAAACCTCAGGGTTTTTG CGCTTCTTAATCCTCAACCTCNNNNNCNCTCGAACAAA cactagcccccatttggacgtgaatgcagacacgntcgaaataaagatttccgaattacaataatttgtttattgctttcgcct ataaatacgacggatcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattg aaaaaaaattggtaattactctttctttttctccatattgaccatcatactcattgctgatccatgtagatttcccggacatgaag ccatttacaatttaacaaaaaaaTAATTAGTAGTTTAGTTTAGACAAATCTAGTACAAA CCTCGACCAAATTTGTACCACATCTTCATCTAAGGGTTATATCTATCAAT ACCTGCAATTAATCTGGTGGAGGTTTGGCACATGCCAACTTCAACATATA TCTGAATGATAAAAATGAGTTTGTGAAAAATCATTTAATGGTCAAATGTT TCTAAATCTTCTAGAAGATGAGCTAAGCGATATCATATTCCACGTGAGTG CATCTTAACCTTGTGAGAGACTTTTTGAATCTTTCAGATTGATATTCAAC AATGTGATAGTATCTTTGTATGTAGGGAAATTCTGCTGGCCTTAGNACTT TTTTTTTTTTTTTNGGCCCCCCCCCCCNTTTTTTNNCCAAAANAAAAAAAN CNNGGGGGNAGNNCCTTTTTNAAAANCCCCCTNNTCNNTANANNGGGNC NGGNGANAAACCCCCCCCAANGGGGTNANAAANNTTTNNTCCCCCAAAT CCCCCNNGNGNCNAAAATTCCCCAAANTTTTTTTTNAAAANCCCCCNCC NAGNCNNGCA acctccctgtggwgwgctgaacgagacccaaatgataaatttgaccgcattccttggtatggaagctgctccattcagag tgactactttaaagtcgttgactttttcatagggcccactagtggactattttaggccacatataatatacgtaccgctttaaaat cttcgataaaacaaaaaacaaaaaTAATACCTGGGAGATTTCATGCATTGAGGCCCAGT AAATTATGTAACAAASACCATGAACGGTGAAAAGGGCCATGACCATGTG GCCGAGCCATATATGGYATTTGATGCTTGACTCCSAGGKTAATCCCATTG CCGGTAGCAACGATGACCCACGAGCCACCGGTAAGAACAAGAACGCCR AACATATGTTTCCGATCAGTCCTAACCTTAGCGCCGYTGATTCCAGCTTC GCTTGCCACCTTAATTCATATGTGATCAACCGATTAACTATGTTAAGTAT TTTACAAAGAAAATTGTTCCTTTTTATTAATAATTTGTCAGGAGATTTTA AAAAGAAATCAAGCACAGCAYTACCMMCAGT gagacgtcgtggwgwgctgatggcagtctAATCCAAGTAGAAAACTGAAAATCACAAA AGTCAGCAACCACAAGTCCCCATTGTGTTGACCAAAGCCAATTCAACTCT AGCTCTCGTAACACACAACCGCAATCTCCAGTAGAGCAAGAGCAATGTA GTGCGAAACACTCACCTGTTATAGCACAGMACTACCAACAGRTATWCM ACGCTGAKAGTATTGAACARTTCCATTAACATTATCTTAAACCTTAATAG GTTCATTACCTGCACAGCACTACCAAC gcctgctagttgwgwgctgatCATTTCACTAGCTTTCTTATTTTTGGTCTCACACTTTT CAAGCATGTCCTTATGTGTGTTTCACTAAAATCTAACTGCCTTTTCTCCAT CTTTTTGTTTGCATTGGCCTTATGTATTATATGATTCTTTACCAATTTTTG AAACATCAATTCAAGCCAAAGATTCTATTTACATGTCATGTCTTCTTATG ATCATTTTTGTGCATATATGTGTTTTAGGAAGAAGGATGGGTGGTTTGTA GAGCATTCAAAAAAAGAGCTACAGGGCAAGCCAAGAACACGGAAACTT GGAGCTCAAGTTACTTTTACGATGAAGTTGCACCGAATGGAGTTAACTC GGTTATGGACCCCATTGATCACYTCTCTACWA 146 134 (1) L 135 (4) R 136 (5) R 137 (3) L 138 (1) L cactagcccccatttggacgtgaatgctagacacgtcgaaataaagatttccgaattngaataatttgtttattgctttcgcct ataaatacgacggatcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattg aaaaaaaattggtaattactctttcttttcctccatattgaccatcatactcattgctgatccatgtagatttcccggacatgaag ccatttacaattgaatatatcctgtttctcatatacgatccatgtagatacgatcttcgtttcccttcttctctcattgctctgtttctc atatacaatttctcatgtgccgacttcgttctcatcttactcttcttcttctccgtcttcgttttctcttttcctttctcCGACGA CTTCACCACACCACTCAATCTCGCCGCCGTAGACGACGTCGACGATGTA GATGAAGAGCCGTTGCTTAAAAACACAAACGCATCTTTTCCATCACTGTT ACTTCTCNAAACAAGATCTCTAAATCTCCATAGTTTTGAAAACCNNGTCG AGTTACTTTTCCCNAAAGTCNCCGGTGATGCTTGCTCCACCGTACGATTT GTNCATGNNCAGNANNNANCAAAAAAA Tttggtgtgatgatgctgactggactactcttcaattcggtctctgacacattattcttcagttttcgtaacttaatgctccataa cattttaccaatcacattagattatatcgtttacataagaaaaaaaaatcatACTATTTTTGTTTTCTTGTGT TTAAGATAAATTTTTTGGAATAATGCTATCACTCGATAAATTAACGTGTT TCGGTTTATTTATTTACGTTGAATTGAGAATTCTTTATTGGTACATTAAAC TATGTATGAAACCAAAGAAAAGAAACACAAACTACTAATTAGATCTCAA TACATTATACATACTACTAGTTAAATGCAAAAGAAATCCGAATATTATA ATAAATTACACGTATAATATAATGTATGGNGTTGATATCGCATTAGTTCC CTAATCACATTCACAAATTACCAAAGAAATATAAGTAGGACCCATGTTTT GAATATCTTTAACTAAATCAAACGGTGGAGGGAGGAGTTGTGTCCATGA TCGTCATGGNGGACGGTGACTTTACATACCATTTCTTTCTTTTTAACTTTG TTTTTTTTATGACACATATTTATCAATAATTTTAGTCTTTTCTTTGTTATAA NAAAAATATATATACCCCACTTAACAAAAATGTTTATTCTACCAGCATAG TATTGTCACACATGTAAAAAAATAAGCAAAAATAACCTAATCTCTTAAT CTAGTCGTTGACAAAACTTTAGTATTTTCTAAGTTATAAATAAATAAACA AGAACCCAATTATAAACCTTATTCAGCAAATTCAAAGGNATGAATAAAT CTACCAATCGTACGGATTNAANCCGATGNNATTTTTTNT attgctgmkctwgtgttwagccacATTGATGCCTTAATTGTAaAAGTATATGGTTTTAT ATGAATATGATTTTGTAAATATGGCTTAAGTAGGATTTGTTATTTTCCTAT TCTAAAGCTTTCACAATTAGATGTTAAGAGCTGGTTTTGTTTTTAGCTGCT TAAATCGCATTAGGCTAAGAATAAAAGGCACACAAAAAGTGAAGATTCT AGAACTGATTCCTTAGTTTGTTATAGATAGTAAGTATTGTTCAAACGAGA ATCCAATTTTAATGTAGAGGACTTGCATATAGGATGAAACTAATATATTC GTGAATGAAATTCCAGAATCACTATGGTTGGAGTCTTGGAGAAGAAATA CATGAAAATACAAAAACTGTAGAACTTCTTTTTCTTGGTAAACATTATTC GGATAAAAAGGAATAAAAGAGCTTGGACCTATTATTTTAGCTATTTATTT ATTTATTTAGTGTTGTCATTTTGTGTTGTAGTGGTTGTGTTTTATTAGCAT GAATGTATGAATTTTAAGCAGATCATCATATGATATACTTTGTTCACCWC CATCAMCAA cactagcccccatttggacgtgaatgnagacacgtcgaaataaagatttccgaattagaataatttgtttattgctttcgccta taaatacgacggatcgtaatttgtcgttttatcaaaatgtgctttcattttataataacgctgcggacatctacatttttgaattga aaaaaaattggtaattactctttctttttctccatattgaccatcatactcattgctgatccatgtagatttcccggacatgaagc catttacaatTGAATAAAACCAGATTGATTCAAGTAAAGGCCAGACAAACCTA TATATGTAAATCAGGTTCAGCTTTCAGATTATAGATCTACATAAACATAA ACGAACAAGTGAAAGAATTGCATCAAATCAAGTAACATGCAAAATATAT AAGAGAATCTGGATAGACATAATCCCTGATTGTAGAACATTGGTTCCTTT GTCTAAACAAGTGAAATTCATAAAAAAAACTTAAACTGTCAATCANACG ATCAAAAGATCTACTCTTCGACACCAGCATTCTCGCCACTCTTGACGTTG TGCTTCCTTGCATACCTCTGGTTTCTTAGGAATTTAGGATCCATCTGCAAC ATTTCACAAAAATAGATTAAATATCAAACTTGGNAGGTAACATCAGAAT CTGAAANTGTTTATAGCTTTAAAAGGTACATCANAANNGNAATCGTTTA TAGGTTTAAAANGAACAGAAACTCTTACCCCNTGGGTGGGGNGNGACGG GGTCCCCTGGNNTCTTGATCCNTTCTTATGCCCCTGGCAAAAGAATT cactagcccccatttggacgtgaaatgtagacacgtcgaaataaagatttccgaattagaataatttgtttattgctttcgcct ataaatacgacggatcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattg aaaaaaaattggtaattactctttctttttctccatattgaccatcatactcattgctgatccatgtagatttcccgGCAATG 147 143 (3) L 145 (5) L 147 (5) R b ACTGCAACTACAACTCCTCCCAACTGATCCAAACTAGTCAAGGTATTGTT GTTTTTCCCCTGCATTCCCAAAAAAGTTGGTCTGGTTTAAGATATAGTCA TATAGTCTCAAAGCAAATGTGGATTAGATTCTCACATCATACACACCTCT AGCATCTTTATAATGTTGAGTCCTAAGCTATAGATTCCCTGAAGATTAAG AGAGTGAGAGTACTTTAGATGATTCAGAAACGTTTCACTCATTATGACG AGTATCTAGAGAGTCCTTACAGCAAATACAGGCACTCGGAGCACCAACC CAAAGTATGTAGTAACTTTTGGGTAAAGGAGCATATCGAGAAGCGTAAT GGCATACACAGGTCCATTAGCACCCTGTACACAAATTGGAAAAGAGTTT GACCATCCAAAACATGAGCGTGTGCATCTCTGTTCCAGTCNGNTAATGCT TTTAAAAATAAATTTANCTGGATGAGTGAAAAACATACCAGCTGCCCAA NGGGGACTTTTNATTGATCCTTAGGGACAACTCTTTGGCCCNNTNNNTTG NTTAACNAATAAAAAGGGNAAGCATTTCGTCTA ctagcccccatttggacgtgaatgtagacacgtcgaaataaagatttccgaattagaataatttgtttattgctttcgcctataa atacgacggatcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattgaaaa aaaattggtaattactctttctttttctccatattgaccatcatactcattgctgatccatgtagatttcccggacatgaanccatn tacaactgnatacanatggaccagcntacncantgctgatccatgtagatttcccggannatgaagccatttacaattaaa aacngatanntgttgnggaggangataataatTGTTACTGTGTCTAAGTAAGGAATACAAA ANGAGAGAANGATTAGNTAAGGAGGAGGACAANGGCATCNCCAACCCT CAGGTTGGTAATTNGCATAATGAGGAGATNGGTGAGGGATNGAANAAA TGGTAGGGAATTGGATTGCTTCACATGNCTCACATCCATAGCATTTTTTC TCACAACTTGGTGGCTTTGATCCTATTCTTCTTCTTCNTNNTCACTCGACN AA catctagcccccatttggacgtgaatgtagacacgtcgaaataaagatttccgaattagaataatttgtttattgctttcgccta taaatacgacggatcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattga aaaaaaattggtaattactctttctttttctccatattgaccatcatactcattgctgatccatgtagatttcccggacatgaagc cATTGACAAATTGACAGAAGGGAAACAGAAAATTCCTTGGATAAGCATA AACAGGGCCAAAATATAAAGTTAAAGAGGGATGGGCTTCATGGATTTGA AAGAATAGGGCCCTTCTAAACCAAAGAATAATGGAGATTGATTGGTCAA ATTCAAGAGAGTCTGTTTTCTTTCTTTTTTTTACAAATCAAAACAGTCTAN TGAACTGAGTCCTGAAAGAAAGAAAAAGATTCTAAAATTGGTTCTCTGT TAACCTCAAAGTTATTGAATAAAACCCATCTCATGTACTTGGTAATTTGA AGATCTTTGCTTACATGAAGATATCCATTAAAGCACGGACTTGTATATAT ATATCATATTAGATTGGTTCATATGCGGATCCNNCNCCCCTCGAAAAA tacgacctgttgwgwgctgmtgccaTAGTTTGTGTTATTTTAGTAACTAACTTAGACA ATTATGTAATGTCATGTGTATATTTTAACAGAATTTGAGTGTCAATTCTCT TCCCAGATATGGCAATGAGTCACTACCATGCTGCATATTTATAGTTGGCT CCTTCATCTTGGAATCAACTTCTCTTGGTTGCTGCATGATACAACAGATA AAGCTTGAGTCTGCTCCAAGGATGGCGATTTATGAAACTTGGAAGGAAC ATAATCACCATTATCACCTTGACATTATACACTACAACCTGATGTTACCG TTGTCCCAAAAGACCAAGAAGATCTCTCTAAACAAAGCTTAGGCTCTGC TTCGATTGGGGTCCCTTACTTACTTAGTGATGTTGGTTTGATCAGTAGTTA TCAACTTACCACTTATTAGTTTGGTGAAACGATGGTTGAGAATTTGTATA TTTCTTTGAATGCTACATTTATAGGGTGTTTATTTTCTCTTCTATTTTTTGG TTGATATGGTTCATGTTTAATTCTTTTATAAACTTTATTTTGTTATTGCAA ACAAATCCTTTGCCAATAAAAGAAAAATGTCAAAGTGTACATATTACTG GTCACTGTCACGTTTGAGTTTTATGTTTTGGTCCATATATCGTTTGAGTTT TATGTTTTGGTCCATATATCACCTTTACTTTCGACTTGTGAGATTAAACAC TTGTACCCC R tttggtgtgatgatgctgactgGTATGAACCGAACAACACACCTGAATTTAGCAGTCA CCTGCATAATAAGCATCAGTAAAACATCAATTCAAGAAAGTTTTCACTAT ACTCTTTTCCTACAACTTATGATAAATCCAAGGTTTTTGGATACATTACA TTGTGATAGGTCAGGATGTCCATCAGAGAAACAAATGGAGCATCGTCAT GGGCAACCCCTAGAAAAGATTAAACCATAAGTATTATATTAAACGTTAT GTCAAATTTCCACAATTCCAAACAATTAGCTGGTTATTACCTGTGATTTC AGAATGTGAACGACTAATGCAACGCGCGATTGGGTTCCATCTCGGTGAA 148 148 (5) L 151 (1) L 152 (2) L 153 (3) R 156 (4) R AACTTCTCCCCACACATTCTGAAATGGTAAGAAAGTGTGTTTTAAACTGA AGAACAAACGGACGAAGCAGAACATCAACTCACAGGATAAGAAAAGGT GTCAGCAGAAACCTTTGAGGACTAAAGGCTTCCATCTCTCTGCTCATTGT GTACTGCATCTTTGTAGAGGGAGTAAAAGTGGCGTTCCATAATCCCATAT TTACTTGAAAGAAAAGGTTCAGAAGCTTCACGTGTTGACCAGGTTGAAG ACAGTGAATGGCCTGCTTCTCACTAACTCTATCTACTATGATCCTCAAAA CGGAACCAAAAGTAGGAAACTTACGTANTGATGAGACNCCNNNNNNNN NNNNNNNNNAAAAAAAAAAAAAAAAAA ttcactagcccccatttggacgtgaatgtagacacgtcgaaataaagatttccgaattagaataatttgtttattgctttcgcct ataaatacgacggatcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattg aaaaaaaattggtaattactctttctttttctccatattgaccatcatactcattgctgatccatgtagatttcccggacatgaag ccaTTTTCAAGATTCCGTTAATGAAATTAGATATATAAGATTATTTTATTC ATTGACAAAAGAAAAGGTAGTCAGGTTATTTTAATTTGTATATTTTGGTT TATTCTCCTCACTCGACCAATTCNNCATCNNTNCCNTNACNNNNAA tttggacgtgaatgtagacacgtcgaaataaagatttccgaattagaataatttgtttattgctttcgcctataaatacgacgg atcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattgaaaaaaaattggta attactctttctttttctccatattgaccatcatactcattgctgatccatgtagatttcccggacatgaagccatttacaattgaat ataacctgaacaaagcatatatgagtttaaactgattcacaaagtacaagttttcaagaaaatgcggatcgtatctgccatA CCTGGCCAGCAAAATAGCCTTGAAGCTTGGTTTTCCCACTGCGGTACTGC TCAAGCTGCTTTGGGTTCTCAGAGACCACTTGAATAACCATTTTCNAAAT CCCTNTGNAGGATCAGTTATCTACAGAGAAGCAAAAGTCATTTCTCAATT AAAAATGGCATGTTGGAAAGTGAAAGATGTATCCCCCCCCCCCCCNACC AAA ctagcccctttggacgtgaatgtagacncgtcgaaataaagatttccgaattagaataatttgtttattgctttcgcctataaat acgacggatcgtaatttgtcgtttcgcctataaATACTACAGGATATTGGTGCCCTGACACCT CAGGAAGAGCTGACAGAACTTGGAGAGAAATTTGGTCATCTCCCAGTTC ATCCTCTGATTAGCAAAATGCTATTCTTCGCTGTGTTAGTAAACTGTCTG GATCCAGCACTTACTCTGGCATGTGCAGCAGACTACAAGGAACCATTTA CGATGCCCATGTCGCCTGTTGAAAGACAGAAAGCTGCTGCTGCAAAACT TGAACTTGCATCACTTTGTGGAGGTGACAGTGATCACCTTGCAGTTGTTG CTGCCTTTGAGTGCTGGAAAAACGCAAAAGGAAGAGGTCTTTCAGCAGA GTTTTGCTCTCAGTACTTTGTCTCTCCAAGTGCTATGAAGATGTTGGATC AAATGCGTAGCCAACTTGAGTCGGAACTTAAAAGACATGGGATTATTCC TAACGACATTTCAAGTTGTAGCCAAAATTCGCGTGATCCCGGTATACTCC GTGCTGTGCTAGCAGTAGGATTGTATCCTATGGTGGGAAGATTGTGCCCA GCTTTTGGTAATAATCGAAGAACTATAGTAGAACTGCTAGGGGTAAAGT CNTCNNN agaatcgmgatagcacgattatttgatgtaatgctcatcataactccgatgatctacgtaaacacaaactgcgaggtataa gaaattctgtttatacaaaaggtgccaaattttctttaaaataacgacagtACATCATTACATGACACCTA TAGAAGCTAAGAGCCACCACATCAAGAGGAAGAGAAGGTATCGATAAT GGTGGTGTGAAGTGGGTCACTAAGGTGAGTCGCCCAGTTGTTGAGTGGT CCTTTACCGGTTGCAGCCGCCTGAACCGCAAAACCCAAGAACCCAACCA TGGCTAAACGAGCATGTTTGATCTCAGCTAGCTGAAGCTGAGCCTTCWT CTCCWCTCG ttttttggtgngatgttgctgnctggngccctccttncgtcatcgaggcctggctcttgccccgacggccgggtataggtc gcgcgcttaagcgccatccattttcggggctagttgattcggcaggtgagttgttacacactccttagcggatttcgacttcc atgaccaccgtcctgctgtcttaatcgaccaacggtggtgcaacggcgcaacctcagacaaagcctaaagtccgagcta ggagaggtcaagccactgatcctcacagtatcgccgaacgggttcgatttttttatttaacttttcctcattattcgcttttcagt taccgagaaaaacgtataaagttatttatgaaaacgtataaagTCATGTGAACAATACAAGCCATA AGGTTGGGTTTCAAGTTCACACGATCACTAAACATTGCCATTACTTCTCA TGTTTCAACCACCGCAACTTCTAGCCGGTAATAACGCAAAACTAAAACC AAGCACGGTTTACTAAAGACTACAATGATTTACTGAAGATTCTTAATCTT AGGTTACAACAGTCCAAGACCAGAAGAAAATATTGAAACTTTAAAAAAG AGGTCCNAGGGAACAAAATCAAACATACCCTCCATTCTATGAGGCAATC CCTGNAAAATGTGGATCCTTCTGAAGGCTCACCCTTCACCTTATCCAAGT TCCTATAGTTGCTGANGNTNTCAATTTCATTTGTTCGTNGAAAAACAAAN 149 162 (1) L 165 (4) R TTGNACTNCNNCCNAAAGTAAAAAGCNCAANCNGCCNTNTTNAAGNNCC NACNAAAAAATAANTGNGCTTT ccccatttggacgtgaatgtagacacntcgaaataaagatttccgaattagaataatttgtttattgctttcgcctataaatacg acggatcgtaatttgtcgttttatcaaaatgtactttcattttataataacgctgcggacatctacatttttgaattgaaaaaaaat tggtaattactctttctttttctccatattgaccatcatactcattgctgatccatgtagatttcccggacatgaagccatttAC ATTGGAATCAATTTCTCTATGTTTTGATTCTATTTTAAAGATCCAATATTC TTTCTCCATTGAGATTAAGCTTCTTTCCTCGCTGTCGTCTCTCTATAGATC TGTCCCCCCCCNNNNNNAACAAAAA tttggtgtgatgatgctgactgGAAAGTTTTAAAAGAACTTTTACCGACCGAGATCAC GAAAGCTATTGAAGTGTTAAACTTTTTGAAAAAGTTTGAAGGTTGTTATC CAAATACATGGATTGCGTTTAGAGTGATGCTAACAGTTCCAGTTTCAGTC GCCTCAGCCGAAAGAAGTTTTTCCAAGCTAAAGTTGATAAAGTCATACTT AGGATCAACAATGTCAGAAGAGAGATTGAATGCCTTGGCGATATTGTCA ATTGAAAGAGATTTAGTTGGAGAGCTCGACTATATAAGTTTGATAAACG ACTTTGCGGCAAAAACTGCAAGAAGATCTATTTTCGAAATCCGAGATGA TGATGAGTAGCAAGTGTAGTTTCAGTTTTTTGAAAAACTTGTGTTTCATT TACGGTTTATCATTGGTTTTGTGCTTTTTCTTNGGGGGCCATGCAACNNT GACCACNTTGNCAATCGATCAAAGCTGTCATATAAATGCATAGACAAAA CATTGATGATTAATCCCTTTTTGTCAACGAATCTTGNAGGTATTTATTTAT TTTGNCACCAAAACATCAAATCACACTAGATACAAGTTGGNCCAGATCG TATAAGTTCGTTTCCN 150 [...]... transpositions with similar underlying principle (Zhang et al., 2003; Tissier et al., 1999; Muskett et al., 2003) 1.7.2 Targeted tagging This study describes a system of inducible insertional mutagenesis based on the Ac-Ds family of transposons for targeted tagging in Arabidopsis thaliana In this system, the Ac and Ds elements are carried within the same T-DNA and a heat shock inducible transposase fusion... two main insertional mutagens, used widely for gene disruption in Arabidopsis While T-DNA insertions are easily generated in Arabidopsis, it is difficult to generate large collections of independent T-DNA lines in plant species for which transformation methods are more laborious In contrast, transposon mutagenesis can be accomplished using a limited number of “starter lines” generated by transformation... saturation mutagenesis in Arabidopsis This system is very efficient and much less laborious than conventional crossing schemes, and may be generally applicable to other plant species for which large scale T-DNA tagging is not currently feasible Further studies were carried out on insertions obtained for the targeted genes Since the Ds element is a gene trap, GUS staining assays were performed Interesting... mutants In conclusion, this study provides an efficient inducible system for targeted tagging of genes using transposons in Arabidopsis with the possibility of using it in other plants also xii LIST OF TABLES Table 1: List of 40 independent starter lines 39 Table 2: Somatic excision frequency for 19 starter lines that were heatshocked 55 Table 3: Details of the genes selected as targets 58 Table 4: Pooling... X-Gluc 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid xviii LIST OF PUBLICATIONS Nishal B, Tantikanjana T, and Sundaresan V (2005) An inducible targeted tagging system for localized saturation mutagenesis in Arabidopsis thaliana Plant Physiology 137:3-12 Parinov S, Nishal B, Oliferenko S, and Sundaresan V (2005) MISSBEAN is required for gametophyte development in Arabidopsis thaliana (manuscript under... cartoon depicting the random tagging approach has been illustrated in Figure 2 Several laboratories have used different versions of the binary 6 Figure 2: Random Tagging approach of gene tagging: Transposon tagging with selection for unlinked events The open triangle on chromosome 3 indicates the original site of starter T-DNA while the filled-black triangles indicate new Ds insertion sites 7 system to... development and application of a novel system of inducible insertional mutagenesis based on the Ac-Ds family of transposons for targeted tagging in Arabidopsis thaliana that aids identification of gene function by their pattern of expression during different stages of development In this approach, the Ac and Ds elements are carried within the same T-DNA and a heat shock inducible transposase fusion is utilized... Phylogenetic tree for At2g30580 109 Figure 41: Alignment of the two closely related Zinc finger proteins of Arabidopsis 110 Figure 42: Domains present in At2g30940 112 Figure 43: Phylogenetic tree for At2g30940 114 Figure 44: Domains present in At2g30790 116 Figure 45: Phylogenetic tree for At2g30790 118 Figure 46: Protein alignment of At2g30790 119 Figure 47: GUS staining for L491; an insertion for At2g30790... subunits of the Arabidopsis ARP2/3 complex 86 Figure 29: WD40 domains present in At2g30910 87 Figure 30: Phylogenetic tree for the Arabidopsis ARP2/3 gene family members 89 Figure 31: Domains present in the kinases; At2g30730 and At2g30740 90 Figure 32: Alignment of the three most closely related serine/threonine protein kinases 93 Figure 33: Phylogenetic tree for Serine/Threonine protein kinases 94 Figure... be exploited for targeted (i.e localized) transposon mutagenesis of a chromosomal region, which has been used in endogenous systems (reviewed in Sundaresan, 1996) as well as in heterologous systems (Smith et al., 1996; Zhang et al., 2003; Muskett et al., 2003) 8 Figure 3: Targeted tagging approach of gene tagging: This method is useful for saturation of known genomic regions For example, in this representative . THE NATIONAL UNIVERSITY OF SINGAPORE 2005 INDUCIBLE- TARGETED TAGGING SYSTEM FOR LOCALIZED INSERTIONAL MUTAGENESIS IN ARABIDOPSIS THALIANA BINDU NISHAL (B.Sc., M.Sc.) . INDUCIBLE- TARGETED TAGGING SYSTEM FOR LOCALIZED INSERTIONAL MUTAGENESIS IN ARABIDOPSIS THALIANA BINDU NISHAL . transformation. This study describes the development and application of a novel system of inducible insertional mutagenesis based on the Ac-Ds family of transposons for targeted tagging in Arabidopsis