title: Identification and characterization of dynein genes in Tetrahymena.
abstract: We describe the protocol through which we identify and characterize dynein subunit genes in the ciliated protozoan Tetrahymena thermophila. The gene(s) of interest is found by searching the Tetrahymena genome, and it is characterized in silico including the prediction of the open reading frame and identification of likely introns. The gene is then characterized experimentally, including the confirmation of the exon-intron organization of the gene and the measurement of the expression of the gene in nondeciliated and reciliating cells. In order to understand the function of the gene product, the gene is modified-for example, deleted, overexpressed, or epitope-tagged-using the straightforward gene replacement strategies available with Tetrahymena. The effect(s) of the dynein gene modification is evaluated by examining transformants for ciliary traits including cell motility, ciliogenesis, cell division, and the engulfment of particles through the oral apparatus. The multistepped protocol enables undergraduate students to engage in short- and long-term experiments. In our laboratory during the last 6 years, more than two dozen undergraduate students have used these methods to investigate dynein subunit genes.
authors.affiliation: Department of Biology, Harvey Mudd College, Claremont, California 91711, USA.
authors.forename: David E
authors.initials: DE
authors.lastname: Wilkes
authors.forename: Nicole
authors.initials: N
authors.lastname: Bennardo
authors.forename: Clarence W C
authors.initials: CW
authors.lastname: Chan
authors.forename: Yu-Loung
authors.initials: YL
authors.lastname: Chang
authors.forename: Elizabeth O
authors.initials: EO
authors.lastname: Corpuz
authors.forename: Jennifer
authors.initials: J
authors.lastname: DuMond
authors.forename: Jordan A
authors.initials: JA
authors.lastname: Eboreime
authors.forename: Julianna
authors.initials: J
authors.lastname: Erickson
authors.forename: Jonathan
authors.initials: J
authors.lastname: Hetzel
authors.forename: Erin E
authors.initials: EE
authors.lastname: Heyer
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Hubenschmidt
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Kniazeva
authors.forename: Hallie
authors.initials: H
authors.lastname: Kuhn
authors.forename: Michelle
authors.initials: M
authors.lastname: Lum
authors.forename: Andrea
authors.initials: A
authors.lastname: Sand
authors.forename: Alicia
authors.initials: A
authors.lastname: Schep
authors.forename: Oksana
authors.initials: O
authors.lastname: Sergeeva
authors.forename: Natt
authors.initials: N
authors.lastname: Supab
authors.forename: Caroline R
authors.initials: CR
authors.lastname: Townsend
authors.forename: Liesl Van
authors.initials: LV
authors.lastname: Ryswyk
authors.forename: Hadley E
authors.initials: HE
authors.lastname: Watson
authors.forename: Alice E
authors.initials: AE
authors.lastname: Wiedeman
authors.forename: Vidyalakshmi
authors.initials: V
authors.lastname: Rajagopalan
authors.forename: David J
authors.initials: DJ
authors.lastname: Asai
mesh_terms: D000818:Animals; D001681:Biological Assay; D002923:Cilia / Q000378:metabolism; D019295:Computational Biology / Q000379:methods*; D004398:Dyneins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D018390:Gene Targeting; D017125:Genes, Protozoan / Q000235:genetics*; D010641:Phenotype; D010802:Phylogeny; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013768:Tetrahymena / Q000235:genetics*
chemical_list: D021122:Protein Subunits; D004398:Dyneins