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Experimental investigation of the gene of interest §Overexpression § Antisense inhibition § Germline knockout or gene replacement § Heterokaryon analysis § Somatic knockout or gene replacement (phenotypic assortment) § Gene ‘knock-in’ § Inducible promoter
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The purpose of this article is to showcase the tools that are available for functional genomic analysis in Tetrahymena, and to illustrate how different approaches can be harnessed to address a wide range of biological problems. At the heart of these approaches is a variety of methods for efficient DNA mediated cell transformation. An appreciation of the spectrum of possibilities requires a brief review of the unusual genetic organization of this orgathis complexity, the presence of features that are characteristic of metazoans but that are absent or less easily studied in other simple eukaryotic systems, that makes Tetrahymena so well suited for a wide range of fundamental problems including germline or somatic differentiation, programmed DNA rearrangements, distribution of mitotic chromosomes, histone modifications, microtubule diversity and tubulin modifications, basal body duplication, phagocytosis, function of rRNA, and regulated polypeptide secretion. These biological features and the functional genomics tools described in this article will greatly enhance the value of the genome sequencing project, currently under
． T. thermophila, and ciliated protozoa in general,have long captured the interests and imaginations of geneticists, because these unicellular organisms maintain two functionally distinct nuclei within the same cytoplasm
Fig. 1. A pair of conjugating Tetrahymena, at the completion of meiosis but before nuclear exchange. One of the meiotic products in each cell will generate two gamete pronuclei that will be involved in reciprocal fertilization across the mating junction. Nuclei are colored green (with Sytox); the cilia (blue) were labeled using an antibody that recognizes β-tubulin (courtesy of Joe Frankel, University of Iowa, IA, USA); the cortex (magenta) is labeled using an antiserum that recognizes a cortically-localized calcium-binding of Osamu Numata, Uniprotein (courtesy versity of Tsukuba, Japan). Photograph is courtesy of Eric Cole (St Olaf College, MN, USA).
Functional genomics: the coming of age for Tetrahymena
thermophila
Aaron P. Turkewitz, Eduardo Orias and Geoffrey Kapler
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☞ Tetrahymena thermophila has been proven to be a
valuable biological model for molecular and genetic studies of eukaryotic cells. Some investigations on the ciliated protozoan have provided the insights into the mechanism of ribozymes, self-splicing RNA, telomere structure and function, DNA sequence reorganization and so on. Compartmentalization of the genome into two functionally distinct nuclei, the silent micronucleus and the transcriptionally active macronucleus, provides a powerful means for controlling the expression of transgenes.