Current Genetics

, Volume 49, Issue 2, pp 106–124

Generation of an oligonucleotide array for analysis of gene expression in Chlamydomonas reinhardtii

  • Stephan Eberhard
  • Monica Jain
  • Chung Soon Im
  • Steve Pollock
  • Jeff Shrager
  • Yuan Lin
  • Andrew S. Peek
  • Arthur R. Grossman
Research Article


The availability of genome sequences makes it possible to develop microarrays that can be used for profiling gene expression over developmental time, as organisms respond to environmental challenges, and for comparison between wild-type and mutant strains under various conditions. The desired characteristics of microarrays (intense signals, hybridization specificity and extensive coverage of the transcriptome) were not fully met by the previous Chlamydomonas reinhardtii microarray: probes derived from cDNA sequences (~300 bp) were prone to some nonspecific cross-hybridization and coverage of the transcriptome was only ~20%. The near completion of the C. reinhardtii nuclear genome sequence and the availability of extensive cDNA information have made it feasible to improve upon these aspects. After developing a protocol for selecting a high-quality unigene set representing all known expressed sequences, oligonucleotides were designed and a microarray with ~10,000 unique array elements (~70 bp) covering 87% of the known transcriptome was developed. This microarray will enable researchers to generate a global view of gene expression in C. reinhardtii. Furthermore, the detailed description of the protocol for selecting a unigene set and the design of oligonucleotides may be of interest for laboratories interested in developing microarrays for organisms whose genome sequences are not yet completed (but are nearing completion).


Chlamydomonas Gene expression Oligo-array Genomics 

Supplementary material

294_2005_41_MOESM1_ESM.pdf (26 kb)
Supplementary material


  1. Asamizu E, Nakamura Y, Sato S, Fukuzawa H, Tabata S (1999) A large scale structural analysis of cDNAs in a unicellular green alga, Chlamydomonas reinhardtii I Generation of 3433 non-redundant expressed sequence tags. DNA Res 6:369–373PubMedCrossRefGoogle Scholar
  2. Asamizu E, Miura K, Kucho K, Inoue Y, Fukuzawa H, Ohyama K, Nakamura Y, Tabata S (2000) Generation of expressed sequence tags from low-CO2 and high-CO2 adapted cells of Chlamydomonas reinhardtii. DNA Res 7:305–307PubMedCrossRefGoogle Scholar
  3. Barrett JC, Kawasaki ES (2003) Microarrays: the use of oligonucleotides and cDNA for the analysis of gene expression. Drug Discov Today 8(3):134–141PubMedCrossRefGoogle Scholar
  4. Choudhuri S (2004) Microarrays in biology and medicine. J Biochem Mol Toxicol 18:171–179PubMedCrossRefGoogle Scholar
  5. Davies J, Yildiz F, Grossman AR (1996) Sac1, a putative regulator that is critical for survival of Chlamydomonas reinhardtii during sulfur deprivation. EMBO J 15:2150–2159PubMedGoogle Scholar
  6. Dent RM, Han M, Niyogi KK (2001) Functional genomics of plant photosynthesis in the fast lane using Chlamydomonas reinhardtii. Trends Plant Sci 6:364–371PubMedCrossRefGoogle Scholar
  7. Dharmadi Y, Gonzalez R (2004) DNA microarrays: experimental issues, data analysis, and application to bacterial systems. Biotechnol Prog 20:1309–1324PubMedCrossRefGoogle Scholar
  8. Dutcher SK (2000) Chlamydomonas reinhardtii: biological rationale for genomics. J Eukaryot Microbiol 47:340–349PubMedCrossRefGoogle Scholar
  9. Dutcher SK (2003) Elucidation of basal body and centriole functions in Chlamydomonas reinhardtii. Traffic 4:443–451PubMedCrossRefGoogle Scholar
  10. Elrad D, Grossman AR (2004) A genome’s-eye view of the light-harvesting polypeptides of Chlamydomonas reinhardtii. Curr Genet 45:61–75PubMedCrossRefGoogle Scholar
  11. Grossman AR (2000) Chlamydomonas reinhardtii and photosynthesis: genetics to genomics. Curr Opin Plant Biol 3:132–137PubMedCrossRefGoogle Scholar
  12. Grossman AR, Harris EE, Hauser C, Lefebvre PA, Martinez D, Rokhsar D, Shrager J, Silflow CD, Stern D, Vallon O, Zhang Z (2003) Chlamydomonas reinhardtii at the crossroads of genomics. Eukaryot Cell 2:1137–1150PubMedCrossRefGoogle Scholar
  13. Grossman AR, Lohr M, Im CS (2004) Chlamydomonas reinhardtii in the landscape of pigments. Annu Rev Genet 38:119–173PubMedCrossRefGoogle Scholar
  14. Harris EH (2001) Chlamydomonas as a model organism. Annu Rev Plant Physiol Plant Mol Biol 52:363–406PubMedCrossRefGoogle Scholar
  15. He Z, Wu L, Fields MW, Zhou J (2005) Use of microarrays with different probe sizes for monitoring gene expression. Appl Environ Microbiol 71(9):5154–5162PubMedCrossRefGoogle Scholar
  16. Hollingshead D, Lewis DA, Mirnics K (2005) Platform influence on DNA microarray data in postmortem brain research. Neurobiol Dis 18(3):649–655PubMedCrossRefGoogle Scholar
  17. Huang K, Merkle T, Beck CF (2002) Isolation and characterization of a Chlamydomonas gene that encodes a putative blue-light photoreceptor of the phototropin family. Physiol Plant 115:613–622PubMedCrossRefGoogle Scholar
  18. Im CS, Grossman AR (2002) Identification and regulation of high light-induced genes in Chlamydomonas reinhardtii. Plant J 30:301–313PubMedCrossRefGoogle Scholar
  19. Kamiya R (2002) Functional diversity of axonemal dyneins as studied in Chlamydomonas mutants. Int Rev Cytol 219:115–155PubMedCrossRefGoogle Scholar
  20. Kane MD, jatkoe TA, Stumpf CR, Lu J, Thomas JD, Madore SJ (2000) Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays. Nucleic Acids Res 28(22):4552–4557PubMedCrossRefGoogle Scholar
  21. Kateriya S, Nagel G, Bamberg E, Hegemann P (2004) “Vision” in single-celled algae. News Physiol Sci 19:133–137PubMedGoogle Scholar
  22. Kim HL (2003) Comparison of oligonucleotide-microarray and serial analysis of gene expression (SAGE) in transcript profiling analysis of megakaryocytes derived from CD34+ cells. Exp Mol Med 35:460–466PubMedGoogle Scholar
  23. Kothapalli R, Yoder SJ, Mane S, Loughran TP Jr (2002) Microarray results: how accurate are they? BMC Bioinform 3:22CrossRefGoogle Scholar
  24. Kuo WP, Jenssen T-K, Butte AJ, Ohno-Machado L, Kohane IS (2002) Analysis of matched mRNA measurements from two different microarray technologies. Bioinformatics 18:405–412PubMedCrossRefGoogle Scholar
  25. LaFontaine S, Quinn JM, Nakamoto SS, Page MD, Gohre V, Moseley JL, Kropat J, Merchant S (2002) Copper-dependent iron assimilation pathway in the model photosynthetic eukaryote Chlamydomonas reinhardtii. Eukaryot Cell 1:736–757CrossRefGoogle Scholar
  26. Ledford HK, Baroli I, Shin JW, Fischer BB, Eggen RI, Niyogi KK (2004) Comparative profiling of lipid-soluble antioxidants and transcripts reveals two phases of photo-oxidative stress in a xanthophyll-deficient mutant of Chlamydomonas reinhardtii. Mol Genet Genomics 272:470–479PubMedCrossRefGoogle Scholar
  27. Li JB, Gerdes JM, Haycraft CJ, Fan Y, Teslovich TM, May-Simera H, Li H, Blacque OE, Li L, Leitch CC, Lewis RA, Green JS, Parfrey PS, Leroux MR, Davidson WS, Beales PL, Guay-Woodford LM, Yoder BK, Stormo GD, Katsanis N, Dutcher SK (2004) Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene. Cell 117:541–552PubMedCrossRefGoogle Scholar
  28. Lohr M, Im CS, Grossman AR (2005) Genome-based examination of chlorophyll and carotenoid biosynthesis in Chlamydomonas reinhardtii. Plant Physiol 138:490–515PubMedCrossRefGoogle Scholar
  29. Mantripragada KK, Buckley PG, de Stahl TD, Dumanski JP (2004) Genomic microarrays in the spotlight. Trends Genet 20:87–94PubMedCrossRefGoogle Scholar
  30. Mittag M, Wagner V (2003) The circadian clock of the unicellular eukaryotic model organism Chlamydomonas reinhardtii. Biol Chem 384:689–695PubMedCrossRefGoogle Scholar
  31. Mittag M, Kiaulehn S, Johnson CH (2005) The circadian clock in Chlamydomonas reinhardtii. What is it for? What is it similar to? Plant Physiol 137:399–409PubMedCrossRefGoogle Scholar
  32. Miura K, Yamano T, Yoshioka S, Kohinata T, Inoue Y, Taniguchi F, Asamizu E, Nakamura Y, Tabata S, Yamato KT, Ohyama K, Fukuzawa H (2004) Expression profiling-based identification of CO2-responsive genes regulated by CCM1 controlling a carbon-concentrating mechanism in Chlamydomonas reinhardtii. Plant Physiol 135:1595–1607PubMedCrossRefGoogle Scholar
  33. Moseley JL, Chang, C-W, Grossman AR (2005) Genome-based approaches to understanding phosphorus deprivation responses and PSR1 Control in Chlamydomonas reinhardtii. Eukaryot Cell (in press)Google Scholar
  34. Nagel G, Szellas T, Huhn W, Kateriya S, Adeishvili N, Berthold P, Ollig D, Hegemann P, Bamberg E (2003) Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci USA 100:13940–13945PubMedCrossRefGoogle Scholar
  35. Omran H, Haffner K, Volkel A, Kuehr J, Ketelsen UP, Ross UH, Konietzko N, Wienker T, Brandis M, Hildebrandt F (2000) Homozygosity mapping of a gene locus for primary ciliary dyskinesia on chromosome 5p and identification of the heavy dynein chain DNAH5 as a candidate gene. Am J Respir Cell Mol Biol 23:696–702PubMedGoogle Scholar
  36. Park PJ, Cao YA, Lee SY, Kim JW, Chang MS, Hart R, Choi S (2004) Current issues for DNA microarrays: platform comparison, double linear amplification and universal RNA reference. J Biotech 112:225–245CrossRefGoogle Scholar
  37. Pazour GJ (2004) Intraflagellar transport and cilia-dependent renal disease: the ciliary hypothesis of polycystic kidney disease. J Am Soc Nephrol 15:2528–2536PubMedCrossRefGoogle Scholar
  38. Pazour GJ, Dickert BL, Vucica Y, Seeley ES, Rosenbaum JL, Witman GB, Cole DG (2000) Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella. J Cell Biol 151:709–718PubMedCrossRefGoogle Scholar
  39. Petersen D, Chandramouli GVR, Geoghegan J, Hilburn J, Paarlberg J, Kim CH, Munroe D, Gangi L, Han J, Puri R, Staudt L, Weinstein J, Barret JC, Green J, Kawasaki ES (2005) Three microarray platforms: an analysis of their concordance in profiling gene expression. BMC Genomics 6(1):63PubMedCrossRefGoogle Scholar
  40. Qin H, Rosenbaum JL, Barr MM (2001) An autosomal recessive polycystic kidney disease gene homolog is involved in intraflagellar transport in C. elegans ciliated sensory neurons. Curr Biol 11:457–461PubMedCrossRefGoogle Scholar
  41. Rochaix JD (2002) Chlamydomonas, a model system for studying the assembly and dynamics of photosynthetic complexes. FEBS Lett 529:34–38PubMedCrossRefGoogle Scholar
  42. Rochaix JD (2004) Genetics of the biogenesis and dynamics of the photosynthetic machinery in eukaryotes. Plant Cell 16:1650–1660PubMedCrossRefGoogle Scholar
  43. Scholey JM (2003) Intraflagellar transport. Annu Rev Cell Dev Biol 19:423–443PubMedCrossRefGoogle Scholar
  44. Shrager J, Hauser C, Chang CW, Harris EH, Davies J, McDermott J, Tamse R, Zhang Z, Grossman AR (2003) Chlamydomonas reinhardtii genome project. A guide to the generation and use of the cDNA information. Plant Physiol 131:401–408PubMedCrossRefGoogle Scholar
  45. Silflow CD, Lefebvre PA (2001) Assembly and motility of eukaryotic cilia and flagella. Lessons from Chlamydomonas reinhardtii. Plant Physiol 127:1500–1507PubMedCrossRefGoogle Scholar
  46. Sineshchekov OA, Jung KH, Spudich JL (2002) The rhodopsins mediate phototaxis to low- and high-intensity light in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 99:225–230CrossRefGoogle Scholar
  47. Snell WJ, Pan J, Wang Q (2004) Cilia and flagella revealed: from flagellar assembly in Chlamydomonas to human obesity disorders. Cell 117:693–697PubMedCrossRefGoogle Scholar
  48. Stauber EJ, Fink A, Markert C, Kruse O, Johanningmeier U, Hippler M (2003) Proteomics of Chlamydomonas reinhardtii light-harvesting proteins. Eukaryot Cell 2:978–994PubMedCrossRefGoogle Scholar
  49. Stears RL, Martinsky T, Schena M (2003) Trends in microarray analysis. Nature Med 9(1):140–145PubMedCrossRefGoogle Scholar
  50. Stoughton RB (2005) Applications of DNA microarrays in biology. Annu Rev Biochem 74:53–82PubMedCrossRefGoogle Scholar
  51. Takahashi H, Braby CE, Grossman AR (2001) Sulfur economy and cell wall biosynthesis during sulfur limitation of Chlamydomonas reinhardtii. Plant Physiol 127:665–673PubMedCrossRefGoogle Scholar
  52. Tan PK, Downey TJ, Spitznagel EL Jr, Xu P, Fu D, Dimitrov DS, Lempicki RA, Raaka BM, Cam MC (2003) Evaluation of gene expression measurements from commercial microarray platforms. Nucleic Acids Res 31:5676–5684PubMedCrossRefGoogle Scholar
  53. Tiquia SM, Wu L, Chong SC, Passovets S, Xu D, Xu Y, Zhou J (2004) Evaluation of 50-mer oligonucleotide arrays for detecting microbial populations in environmental samples. Bio Tech 36(4):664–675Google Scholar
  54. Wagner V, Fiedler M, Markert C, Hippler M, Mittag M (2004) Functional proteomics of circadian expressed proteins from Chlamydomonas reinhardtii. FEBS Lett 559:129–135PubMedCrossRefGoogle Scholar
  55. Wang H, He X, Band M, Wilson C, Liu L (2005) A study of inter-lab and inter-platform agreement of DNA microarray data. BMC Genomics 6(1):71PubMedCrossRefGoogle Scholar
  56. Werner R (2002) Chlamydomonas reinhardtii as a unicellular model for circadian rhythm analysis. Chronobiol Int 19:325–343PubMedCrossRefGoogle Scholar
  57. Wostrikoff K, Girard-Bascou J, Wollman FA, Choquet Y (2004) Biogenesis of PSI involves a cascade of translational autoregulation in the chloroplast of Chlamydomonas. EMBO J 23:2696–2705PubMedCrossRefGoogle Scholar
  58. Wykoff D, Grossman A, Weeks DP, Usuda H, Shimogawara K (1999) Psr1, a nuclear localized protein that regulates phosphorus metabolism in Chlamydomonas. Proc Natl Acad Sci USA 96:15336–15341PubMedCrossRefGoogle Scholar
  59. Zhang Z, Shrager J, Jain M, Chang CW, Vallon O, Grossman AR (2004) Insights into the survival of Chlamydomonas reinhardtii during sulfur starvation based on microarray analysis of gene expression. Eukaryot Cell 3:1331–1348PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Stephan Eberhard
    • 1
    • 2
  • Monica Jain
    • 1
  • Chung Soon Im
    • 1
  • Steve Pollock
    • 1
  • Jeff Shrager
    • 1
  • Yuan Lin
    • 3
  • Andrew S. Peek
    • 3
  • Arthur R. Grossman
    • 1
  1. 1.Department of Plant BiologyThe Carnegie InstitutionStanfordUSA
  2. 2.Laboratoire de Physiologie Moléculaire et Membranaire du Chloroplaste, Institut de Biologie Physico-ChimiqueUMR7141 (CNRS – Université Paris VI)ParisFrance
  3. 3.Integrated DNA TechnologiesCoralvilleUSA

Personalised recommendations