Identification and characterization of dynein genes in Tetrahymena.
| Autor: | Wilkes DE; Department of Biology, Harvey Mudd College, Claremont, California 91711, USA., Bennardo N, Chan CW, Chang YL, Corpuz EO, DuMond J, Eboreime JA, Erickson J, Hetzel J, Heyer EE, Hubenschmidt MJ, Kniazeva E, Kuhn H, Lum M, Sand A, Schep A, Sergeeva O, Supab N, Townsend CR, Ryswyk LV, Watson HE, Wiedeman AE, Rajagopalan V, Asai DJ |
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| Jazyk: | angličtina |
| Zdroj: | Methods in cell biology [Methods Cell Biol] 2009; Vol. 92, pp. 11-30. Date of Electronic Publication: 2009 Nov 21. |
| DOI: | 10.1016/S0091-679X(08)92002-1 |
| Abstrakt: | 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. (2009 Elsevier Inc. All rights reserved.) |
| Databáze: | MEDLINE |
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