Identification and characterization of dynein genes in Tetrahymena.
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.
Wilkes
David E
DE
Department of Biology, Harvey Mudd College, Claremont, California 91711, USA.
Bennardo
Nicole
N
Chan
Clarence W C
CW
Chang
Yu-Loung
YL
Corpuz
Elizabeth O
EO
DuMond
Jennifer
J
Eboreime
Jordan A
JA
Erickson
Julianna
J
Hetzel
Jonathan
J
Heyer
Erin E
EE
Hubenschmidt
Mark J
MJ
Kniazeva
Ekaterina
E
Kuhn
Hallie
H
Lum
Michelle
M
Sand
Andrea
A
Schep
Alicia
A
Sergeeva
Oksana
O
Supab
Natt
N
Townsend
Caroline R
CR
Ryswyk
Liesl Van
LV
Watson
Hadley E
HE
Wiedeman
Alice E
AE
Rajagopalan
Vidyalakshmi
V
Asai
David J
DJ
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*
D021122:Protein Subunits; D004398:Dyneins