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Insertional Mutagenesis as a Tool to Study Genes/Functions in Chlamydomonas

  • Aurora Galván
  • David González-Ballester
  • Emilio Fernández
Part of the Advances in Experimental Medicine and Biology book series (volume 616)

Abstract

The unicellular alga Chlamydomonas reinhardtii has emerged during the last decades as a model system to understand gene functions, many of them shared by bacteria, fungi, plants, animals and humans. A powerful resource for the research community is the availability of complete collections of stable mutants for studying whole genome function. In the meantime other strategies might be developed; insertional mutagenesis has become currently the best strategy to disrupt and tag nuclear genes in Chlamydomonas allowing forward and reverse genetic approaches. Here, we outline the mutagenesis technique stressing the idea of generating databases for ordered mutant libraries, and also of improving efficient methods for reverse genetics to identify mutants defective in a particular gene.

Keywords

Insertional Mutagenesis Reverse Genetic Mutant Library Nitrate Assimilation Nuclear Transformation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. 1.
    Harris EH. Chlamydomonas as a model organism. Annu Rev Plant Physiol Plant Mol Biol 2001; 52:363–406.PubMedCrossRefGoogle Scholar
  2. 2.
    Gutman BL, Nigyogi KK. Chlamydomonas and Arabidopsis: A dynamic duo. Plant Physiol 2004; 135:607–610.PubMedCrossRefGoogle Scholar
  3. 3.
    Kathir P, LaVoie M, Brazelton WJ et al. Molecular map of the Chlamydomonas reinhardtii nuclear genome. Eukaryot Cell 2003; 2:362–379.PubMedCrossRefGoogle Scholar
  4. 4.
    Li JB, Lin SP, Jia HG et al. Analysis of Chlamydomonas reinhardtii genome structure using large-scale sequencing of regions on linkage groups I and III. J Eukaryotic Microbiology 2003; 50:145–155.PubMedCrossRefGoogle Scholar
  5. 5.
    Boyton JE, Gillham NW, Harris EH et al. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 1988; 240:1534–1538.CrossRefGoogle Scholar
  6. 6.
    Kindle KL. Nuclear transformation: Technology and applications. In: Rochaix JD et al, eds. The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, 1998:41–61.Google Scholar
  7. 7.
    Randolph-Anderson BL, Boynton JE, Gillham NW et al. Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation. Mol Gen Genet 1993; 236:235–244.PubMedCrossRefGoogle Scholar
  8. 8.
    Remacle C, Cardol P, Coosemans N et al. High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes. Proc Natl Acad Sci USA 2006; 103:4771–4776.PubMedCrossRefGoogle Scholar
  9. 9.
    Dent R, Haglund C, Chin B et al. Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii. Plant Physiol 2005; 137:545–556.PubMedCrossRefGoogle Scholar
  10. 10.
    González-Ballester D, de Montaigu A, Higuera JJ et al. Functional genomics of the regulation of the nitrate assimilation pathway in Chlamydomonas. Plant Physiol 2005; 137:522–533.PubMedCrossRefGoogle Scholar
  11. 11.
    Harris EH. The Chlamydomonas Sourcebook. New York: Academic Press, 1989.Google Scholar
  12. 12.
    Rochaix JD. Chlamydomonas reinhardtii as the photosystthetic yeast. Annu Rev Genet 1995; 29:209–230.PubMedCrossRefGoogle Scholar
  13. 13.
    Rochaix JD. Chlamydomonas, a model system for studying the assembly and dynamics of photo-synthetic complexes. FEBS Lett 2002; 529:34–38.PubMedCrossRefGoogle Scholar
  14. 14.
    Dutcher SK. Flagellar assembly in two hundred and fifty easy-to-follow steps. Trends Genet 1995; 11:398–404.PubMedCrossRefGoogle Scholar
  15. 15.
    Rosembaum JL, Cole DG, Diener DR. Intraflagellar transport; the eyes have it. J Cell Biol 1999; 144:385–388.CrossRefGoogle Scholar
  16. 16.
    Silflow C, Lefebvre PA. Assembly and motility of eukaryotic cilia and flagella: Lessons from Chlamydomonas reinhardtii. Plant Physiol 2001; 127:1500–1507.PubMedCrossRefGoogle Scholar
  17. 17.
    Grossman A, Takahashi H. Macronutrient utilization by photosynthetic eukaryotes and the fabric of interactions. Annu Rev Plant Physiol Plant Mol Biol 2001; 52:163–210.PubMedCrossRefGoogle Scholar
  18. 18.
    Galván A, Fernández E. Eukaryotic nitrate and nitrite transport. Cell Mol Life Sci 2001; 58:225–233.PubMedCrossRefGoogle Scholar
  19. 19.
    Mittag M, Wagner V. The circadian clock of the unicellular eukaryotic model organism Chlamydomonas reinhardtii. Biol Chem 2003; 384:689–695.PubMedCrossRefGoogle Scholar
  20. 20.
    Breton G, Kay SA. Circadian rhythms lit up in Chlamydomonas. Genome Biol 2006; 7:215.PubMedCrossRefGoogle Scholar
  21. 21.
    Ferris PJ, Goodenough UW. Mating type in Chlamydomonas is specified by mid, the minus-dominance gene. Genetics 1997; 146:859–869.PubMedGoogle Scholar
  22. 22.
    In: Rochaix JD, Goldschmidt-Clermont M, Merchant S, eds. The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. Dordrecht: Kluwer, 1998.Google Scholar
  23. 23.
    Fernández E, Galván A, Quesada A. Nitrogen assimilation and its regulation. In: Rochaix JD, Goldschmidt-Clermont M, Merchant S, eds. The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. Dordrecht: Kluwer, 1998:637–659.Google Scholar
  24. 24.
    Moseley JL, Chang CW, Grossman AR. Genome-based approaches to understanding phosphorus deprivation responses and PSR1 control in Chlamydomonas reinhardtii. Eukaryot Cell 2006; 5:26–44.PubMedCrossRefGoogle Scholar
  25. 25.
    Zhang Z, Shrager J, Jain M et al. Insights into the survival of Chlamydomonas reinhardtii during sulfur starvation based on microarray analysis of gene expression. Eukaryotic Cell 2004; 3:1331–1348.PubMedCrossRefGoogle Scholar
  26. 26.
    Pollock SV, Pootakham W, Shibagaki N et al. Insights into the acclimation of Chlamydomonas reinhardtii to sulfur deprivation. Photosynth Res 2005; 86:475–489.PubMedCrossRefGoogle Scholar
  27. 27.
    Rexach J, Fernández E, Galván A. The Chlamydomonas reinhardtii Nar1 gene encodes a chloroplast membrane protein involved in nitrite transport. Plant Cell 2000; 12:1441–1453.PubMedCrossRefGoogle Scholar
  28. 28.
    Mariscal V, Moulin P, Orsel M et al. Differential Regulation of the Chlamydomonas Nar1 gene family by carbon and nitrogen. Protist 2006; 157:421–433.PubMedCrossRefGoogle Scholar
  29. 29.
    Miura K, Yamano T, Yoshioka S et al. Expression profiling-based identification of CO2-responsive genes regulated by CCM1 controlling a carbon-concentrating mechanism in Chlamydomonas reinhardtii. Plant Physiol 2004; 135:1595–1607.PubMedCrossRefGoogle Scholar
  30. 30.
    Quesada A, Galván A, Schnell R et al. Five nitrate assimilation related loci are clustered in Chlamydomonas reinhardtii. Mol Gen Genet 1993; 240:387–394.PubMedGoogle Scholar
  31. 31.
    Quesada A, Galván A, Fernández E. Identification of nitrate transporter genes in Chlamydomonas reinhardtii. Plant J 1994; 5:407–419.PubMedCrossRefGoogle Scholar
  32. 32.
    Galván A, Quesada A, Fernández E. Nitrate and nitrite are transported by different specific transport systems and by a bispecific transporter in Chlamydomonas reinhardtii. J Biol Chem 1996; 271:2088–2092.PubMedCrossRefGoogle Scholar
  33. 33.
    Zhou JJ, Fernández E, Galván A et al. A high affinity nitrate transport system from Chlamydomonas requires two gene products. FEBS Lett 2000; 466:225–227.PubMedCrossRefGoogle Scholar
  34. 34.
    Tong Y, Zhou JJ, Li ZS et al. A two-component high-affinity nitrate uptake system in barley. Plant J 2005; 41:442–450.PubMedCrossRefGoogle Scholar
  35. 35.
    Okamoto M, Kumar A, Li W et al. High-affinity nitrate transport in roots of Arabidopsis depends on expression of the NAR2-like gene AtNRT3.1. Plant Physiol 2006; 140:1036–1046.PubMedCrossRefGoogle Scholar
  36. 36.
    Zein LE, Omran H, Bouvagnet P. Lateralization defects and ciliary dyskinesia: Lessons from algae. Trends Genet 2003; 19:162–167.PubMedCrossRefGoogle Scholar
  37. 37.
    Snell WJ, Pan J, Wang Q. Cilia and flagella revealed: From flagellar assembly in Chlamydomonas to human obesity disorders. Cell 2004; 117:693–697.PubMedCrossRefGoogle Scholar
  38. 38.
    Pazour G, Agrin N, Leszyk J et al. Proteomic analysis of a eukaryotic cilium. J Cell Biol 2005; 170:103–113.PubMedCrossRefGoogle Scholar
  39. 39.
    Gfeller RP, Gibbs M. Fermentative metabolism of Chlamydomonas reinhardtii: I. Analysis of fermentative products from starch in dark and light. Plant Physiol 1984; 75:212–218.PubMedCrossRefGoogle Scholar
  40. 40.
    Hemschemeier A, Happe T. The exceptional photofermentative hydrogen metabolism of the green alga Chlamydomonas reinhardtii. Biochem Soc Trans 2005; 33:39–41.PubMedCrossRefGoogle Scholar
  41. 41.
    León-Bañares R, González-Ballester D, Galván A et al. Transgenic microalgae as green cell-factories. Trends Biotechnol 2004; 22:45–52.PubMedCrossRefGoogle Scholar
  42. 42.
    Im CS, Zhang Z, Shrager J et al. Analysis of light and CO2 regulation in Chlamydomonas reinhardtii using genome-wide approaches. Photosynth Res 2003; 75:111–125.PubMedCrossRefGoogle Scholar
  43. 43.
    Kucho K, Okamoto K, Tabata S et al. Identification of novel clock-controlled genes by cDNA macroarray analysis in Chlamydomonas reinhardtii. Plant Mol Biol 2005; 57:889–906.PubMedCrossRefGoogle Scholar
  44. 44.
    Abe J, Kubo T, Takagi Y et al. The transcriptional program of synchronous gametogenesis in Chlamydomonas reinhardtii. Curr Genet 2004; 46:304–315.PubMedCrossRefGoogle Scholar
  45. 45.
    Prieto R, Fernández E. Toxicity of and mutagenesis by chlorate are independent of nitrate reductase activity in Chlamydomonas reinhardtii. Mol Gen Genet 1993; 237:429–438.PubMedGoogle Scholar
  46. 46.
    Ranum LPW, Thompson MD, Schloss JA et al. Mapping flagellar genes in Chlamydomonas using restriction fragment length polymorphisms. Genetics 1988; 120:109–122.PubMedGoogle Scholar
  47. 47.
    Rymarquis LA, Handley JM, Thomas M et al. Beyond complementation: Map-based cloning in Chlamydomonas reinhardtii. Plant Physiol 2005; 137:557–566.PubMedCrossRefGoogle Scholar
  48. 48.
    Dykxhoorn DM, Novina CD, Sharp PA. Killing the messenger: Short RNAs that silence gene expression. Nat Rev Mol Cell Biol 2003; 4:457–467.PubMedCrossRefGoogle Scholar
  49. 49.
    Waterhouse PM, Helliwell CA. Exploring plant genomes by RNA-induced gene silencing. Nat Rev Genet 2003; 4:29–38.PubMedCrossRefGoogle Scholar
  50. 50.
    Cerutti H. RNA interference: Traveling in the cell and gaining functions? Trends Genet 2003; 19:39–46.PubMedCrossRefGoogle Scholar
  51. 51.
    Schroda M. RNA silencing in Chlamydomonas: Mechanisms and tools. Curr Genet 2006; 49:69–84.PubMedCrossRefGoogle Scholar
  52. 52.
    Fuhrmann M, Stahlberg A, Govorunova E et al. The abundant retinal protein of the Chlamydomonas eye is not the photoreceptor for phototaxis and photophobic responses. J Cell Sci 2001; 114:3857–3863.PubMedGoogle Scholar
  53. 53.
    Rohr J, Sarkar N, Balenger S et al. Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas. Plant J 2004; 40:611–621.PubMedCrossRefGoogle Scholar
  54. 54.
    Nelson JA, Lefebvre PA. Targeted disruption of the NIT8 gene in Chlamydomonas reinhardtii. Mol Cell Biol 1995; 15:5762–5769.PubMedGoogle Scholar
  55. 55.
    Sodeinde OA, Kindle KL. Homologous recombination in the nuclear genome of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 1993; 90:9199–9203.PubMedCrossRefGoogle Scholar
  56. 56.
    Zorin B, Hegemann P, Sizova I. Nuclear-gene targeting by using single-stranded DNA avoids illegitimate DNA integration in Chlamydomonas reinhardtii. Eukaryotic Cell 2005; 4:1264–1272.PubMedCrossRefGoogle Scholar
  57. 57.
    Cenkci B, Petersen JL, Small GD. REX1, a novel gene required for DNA repair. J Biol Chem 2003; 278:22574–22577.PubMedCrossRefGoogle Scholar
  58. 58.
    Tarn LW, Lefebvre PA. Cloning of flagellar in Chlamydomonas reinhardtii by DNA insertional mutagenesis. Genetics 1993; 135:375–384.Google Scholar
  59. 59.
    Pazour GJ, Sineshchekov OA, Witman GB. Mutational analysis of the phototransduction pathway of Chlamydomonas reinhardtii. J Cell Biol 1995; 131:427–440.PubMedCrossRefGoogle Scholar
  60. 60.
    Prieto R, Dubus A, Galván A et al. Isolation and characterization of two regulatory mutants for nitrate assimilation in Chlamydomonas reinhardtii. Mol Genet Genom 1996; 251:461–471.Google Scholar
  61. 61.
    Davies JP, Yildiz FH, Grossman A. Sacl, a putative regulator that is critical for survival of Chlamydomonas reinhardtii during sulfur deprivation. EMBO J 1996; 15:2150–2159.PubMedGoogle Scholar
  62. 62.
    Yoshioka S, Taniguchi F, Miura K et al. The novel Myb transcription factor LCR1 regulates the CO2-responsive gene Cah1, encoding a periplasmic carbonic anhydrase in Chlamydomonas reinhardtii. Plant Cell 2004; 16:1466–1477.PubMedCrossRefGoogle Scholar
  63. 63.
    Zabawinski C, van den Koornhuyse N, d’Hulst N et al. Starchless mutants of Chlamydomonas reinhardtii lack the small subunit of a heterotetrameric ADP-glucose pyrophosphorylase. J Bacteriol 2001; 183:1069–1077.PubMedCrossRefGoogle Scholar
  64. 64.
    Posewitz MC, Smolinski SL, Kanakagiri S et al. Hydrogen photoproduction is attenuated by disruption of an isoamylase gene in Chlamydomonas reinhardtii. Plant Cell 2004; 16:2151–2163.PubMedCrossRefGoogle Scholar
  65. 65.
    Horst CJ, Fishkind DJ, Pazour GJ et al. An insertional mutant of Chlamydomonas reinhardtii with defective microtubule positioning. Cell Motil Cytoskeleton 1999; 44:143–154.PubMedCrossRefGoogle Scholar
  66. 66.
    Pazour GJ, Witman GB. Forward and reverse genetic analysis of microtubule motors in Chlamydomonas. Methods 2000; 22:285–298.PubMedCrossRefGoogle Scholar
  67. 67.
    Kindle KL, Schnell RA, Fernández E et al. Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J Cell Biol 1989; 109:2589–2601.PubMedCrossRefGoogle Scholar
  68. 68.
    Kindle KL. High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 1990; 87:1228–1232.PubMedCrossRefGoogle Scholar
  69. 69.
    Shimogawara K, Fujiwara S, Grossman A et al. High-efficiency transformation of Chlamydomonas reinhardtii by electroporation. Genetics 1998; 148:1821–1828.PubMedGoogle Scholar
  70. 70.
    Alonso JM, Stepanova AN, Leisse TH et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 2003; 301:653–657.PubMedCrossRefGoogle Scholar
  71. 71.
    Prieto R, Pardo JM, Niu X et al. Salt-sensitive mutants of Chlamydomonas reinhardtii isolated after insertional tagging. Plant Physiol 1996; 112:99–104.PubMedGoogle Scholar
  72. 72.
    Cerutti H, Johnson AM, Gillham NW et al. A eubacterial gene conferring spectinomycin resistance on Chlamydomonas reinhardtii: Integration into the nuclear genome and gene expression. Genetics 1997; 145:97–110.PubMedGoogle Scholar
  73. 73.
    Sizova I, Fuhrmann M, Hegemann P. A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene 2001; 277:221–229.PubMedCrossRefGoogle Scholar
  74. 74.
    Hecht SM. Bleomycin: New perspectives on the mechanism of action. J Nat Prod 2000; 63:158–168.PubMedCrossRefGoogle Scholar
  75. 74.
    Stevens DR, Rochaix JD, Purton S. The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas. Mol Gen Genet 1996; 251:23–30.PubMedGoogle Scholar
  76. 76.
    Liu YG, Whittier RF. Thermal asymmetric interlaced PCR: Automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 1995; 25:674–681.PubMedCrossRefGoogle Scholar
  77. 77.
    Liu YG, Mitsukawa N, Oosumi T et al. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 1995; 8:457–463.PubMedCrossRefGoogle Scholar
  78. 78.
    González-Ballester D, de Montaigu A, Galván A et al. Restriction enzyme site directed amplification (RESDA)-PCR: A tool to identify regions flanking a marker DNA. Anal Biochem 2005; 340:330–335.PubMedCrossRefGoogle Scholar
  79. 78.
    Harrison RW, Miller JC, D’Souza MJ et al. Easy gene walking. Biotechniques 1997; 22:650–653.PubMedGoogle Scholar
  80. 79.
    González-Ballester D. Genómica functional de la señalizaciń por amonio y nitrato, y caracterización de genes para el transporte de amonio en Chlamydomonas. PhD Thesis 2005, (Universidad de C órdoba, Spain).Google Scholar
  81. 80.
    Peréz-Alegre M, Dubus A, Fernández E. REM1, a new type of long terminal repeat retrotransposon in Chlamydomonas reinhardtii. Mol Cell Biol 2005; 25:10628–10638.PubMedCrossRefGoogle Scholar
  82. 81.
    Thyssen C, Hermes M, Sultemeyer D. Isolation and characterisation of Chlamydomonas reinhardtii mutants with an impaired CO2-concentrating mechanism. Planta 2003; 217:102–112.PubMedGoogle Scholar
  83. 82.
    Polle JE, Kanakagiri SD, Melis A. Tla1, a DNA insertional transformant of the green alga Chlamydomonas reinhardtii with a truncated light-harvesting chlorophyll antenna size. Planta 2003; 217:49–59.PubMedGoogle Scholar

Copyright information

© Landes Bioscience and Springer Science+Business Media 2007

Authors and Affiliations

  • Aurora Galván
    • 1
    • 2
  • David González-Ballester
    • 2
    • 3
  • Emilio Fernández
    • 2
  1. 1.Departamento de Bioquímica y Biología Molecular, Facultad de CienciasUniversidad de CórdobaCórdobaSpain
  2. 2.Departamento de Bioquímica y Biología Molecular Campus de RabanalesUniversidad de CódobaCódobaSpain
  3. 3.Department of Plant Biology The Carnegie Institution of WashingtonUniversity of StanfordStanfordUSA

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