Cloning of Flagellar Genes in Chlamydomonas reinhardtii by DNA Insertional Mutagenesis

INVESTIGATIONS

Cloning of Flagellar Genes in Chlamydomonas reinhardtii by DNA Insertional Mutagenesis

L. W. Tam and P. A. Lefebvre
Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108

Chlamydomonas is a popular genetic model system for studying many cellular processes. In this report, we describe a new approach to isolate Chlamydomonas genes using the cloned nitrate reductase gene (NIT1) as an insertional mutagen. A linearized plasmid containing the NIT1 gene was introduced into nit1 mutant cells by glass-bead transformation. Of 3000 Nit(+) transformants examined, 74 showed motility defects of a wide range of phenotypes, suggesting that DNA transformation is an effective method for mutagenizing cells. For 13 of 15 such motility mutants backcrossed to nit(-) mutant strains, the motility phenotype cosegregated with the Nit(+) phenotype, indicating that the motility defects of these 13 mutants may be caused by integration of the plasmid. Further genetic analysis indicated that three of these mutants contained alleles of previously identified loci: mbo2 (move backward only), pf13 (paralyzed flagella) and vfl1 (variable flagellar number). Three other abnormal-flagellar-number mutants did not map to any previously described loci at which mutations produce similar phenotypes. Genomic sequences flanking the integrated plasmid in the mbo2 and vfl1 mutants were isolated and used as probes to obtain wild-type genomic clones, which complemented the motility defects upon transformation into cells. Our results demonstrate the potential of this new approach for cloning genes identified by mutation in Chlamydomonas.

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				R. L. Nguyen, L.-W. Tam, and P. A. Lefebvre The LF1 Gene of Chlamydomonas reinhardtii Encodes a Novel Protein Required for Flagellar Length Control Genetics, March 1, 2005; 169(3): 1415 - 1424. [Abstract] [Full Text] [PDF]

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				D. Gonzalez-Ballester, A. de Montaigu, J. J. Higuera, A. Galvan, and E. Fernandez Functional Genomics of the Regulation of the Nitrate Assimilation Pathway in Chlamydomonas Plant Physiology, February 1, 2005; 137(2): 522 - 533. [Abstract] [Full Text] [PDF]

				C. Schonfeld, L. Wobbe, R. Borgstadt, A. Kienast, P. J. Nixon, and O. Kruse The Nucleus-encoded Protein MOC1 Is Essential for Mitochondrial Light Acclimation in Chlamydomonas reinhardtii J. Biol. Chem., November 26, 2004; 279(48): 50366 - 50374. [Abstract] [Full Text] [PDF]

				N. F. Wilson and P. A. Lefebvre Regulation of Flagellar Assembly by Glycogen Synthase Kinase 3 in Chlamydomonas reinhardtii Eukaryot. Cell, October 1, 2004; 3(5): 1307 - 1319. [Abstract] [Full Text] [PDF]

				E. E. Dymek, P. A. Lefebvre, and E. F. Smith PF15p Is the Chlamydomonas Homologue of the Katanin p80 Subunit and Is Required for Assembly of Flagellar Central Microtubules Eukaryot. Cell, August 1, 2004; 3(4): 870 - 879. [Abstract] [Full Text] [PDF]

				K. Matsuura, P. A. Lefebvre, R. Kamiya, and M. Hirono Bld10p, a novel protein essential for basal body assembly in Chlamydomonas: localization to the cartwheel, the first ninefold symmetrical structure appearing during assembly J. Cell Biol., June 7, 2004; 165(5): 663 - 671. [Abstract] [Full Text] [PDF]

				L. M. DiBella, E. F. Smith, R. S. Patel-King, K.-i. Wakabayashi, and S. M. King A Novel Tctex2-related Light Chain Is Required for Stability of Inner Dynein Arm I1 and Motor Function in the Chlamydomonas Flagellum J. Biol. Chem., May 14, 2004; 279(20): 21666 - 21676. [Abstract] [Full Text] [PDF]

				B. N. Gomperts, X. Gong-Cooper, and B. P. Hackett Foxj1 regulates basal body anchoring to the cytoskeleton of ciliated pulmonary epithelial cells J. Cell Sci., March 15, 2004; 117(8): 1329 - 1337. [Abstract] [Full Text] [PDF]

				A. R. Grossman, E. E. Harris, C. Hauser, P. A. Lefebvre, D. Martinez, D. Rokhsar, J. Shrager, C. D. Silflow, D. Stern, O. Vallon, et al. Chlamydomonas reinhardtii at the Crossroads of Genomics Eukaryot. Cell, December 1, 2003; 2(6): 1137 - 1150. [Full Text] [PDF]

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				G. Rupp and M. E. Porter A subunit of the dynein regulatory complex in Chlamydomonas is a homologue of a growth arrest-specific gene product J. Cell Biol., July 7, 2003; 162(1): 47 - 57. [Abstract] [Full Text] [PDF]

				P. Kathir, M. LaVoie, W. J. Brazelton, N. A. Haas, P. A. Lefebvre, and C. D. Silflow Molecular Map of the Chlamydomonas reinhardtii Nuclear Genome Eukaryot. Cell, April 1, 2003; 2(2): 362 - 379. [Abstract] [Full Text] [PDF]

				H. Zhao, M. Lu, R. Singh, and W. J. Snell Ectopic expression of a Chlamydomonas mt+-specific homeodomain protein in mt- gametes initiates zygote development without gamete fusion Genes & Dev., October 15, 2001; 15(20): 2767 - 2777. [Abstract] [Full Text] [PDF]

				D. G. W. Roberts, M. R. Lamb, and C. L. Dieckmann Characterization of the EYE2 Gene Required for Eyespot Assembly in Chlamydomonas reinhardtii Genetics, July 1, 2001; 158(3): 1037 - 1049. [Abstract] [Full Text] [PDF]

				C. D. Silflow, M. LaVoie, L.-W. Tam, S. Tousey, M. Sanders, W.-c. Wu, M. Borodovsky, and P. A. Lefebvre The Vfl1 Protein in Chlamydomonas Localizes in a Rotationally Asymmetric Pattern at the Distal Ends of the Basal Bodies J. Cell Biol., March 26, 2001; 153(1): 63 - 74. [Abstract] [Full Text] [PDF]