Transformation of Tetrahymena thermophila by microinjection of a foreign gene.

Kahn, Robert W.; Andersen, Bo H.; Brunk, Clifford F.

doi:10.1073/pnas.90.20.9295

Cited by 51 publications

(33 citation statements)

References 31 publications

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“…To test whether the MTT1 promoter can induce the expression of other coding regions in response to cadmium, a reporter construct was made by replacing the MTT1 coding region with the neo1 coding region which confers paromomycin (pm) resistance when expressed in Tetrahymena macronuclei (11). Because this reporter gene is flanked by MTT1 noncoding sequences, it integrates into the MTT1 locus by homologous recombination when biolistically transformed into Tetrahymena.…”

Section: Resultsmentioning

confidence: 99%

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A robust inducible-repressible promoter greatly facilitates gene knockouts, conditional expression, and overexpression of homologous and heterologous genes in Tetrahymena thermophila

Shang

Song

Bowen

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

210

239

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The Cd 2؉ -inducible metallothionein (MTT1) gene was cloned from Tetrahymena thermophila. Northern blot analysis showed that MTT1 mRNA is not detectable in the absence of Cd 2؉ , is induced within 10 min of its addition, is expressed in proportion to its concentration, and rapidly disappears upon its withdrawal. Similarly, when the neo1 gene coding region flanked by the MTT1 gene noncoding sequences was used to disrupt the MTT1 locus, no transformants were observed in the absence of Cd 2؉ , and the number of transformants was proportional to increased Cd 2؉ concentration. The neo3 cassette, in which the MTT1 promoter replaced the histone gene HHF1 promoter of the previously used neo2 cassette, transformed cells at much higher frequencies than neo2 and produced germ-line knockouts where neo2 had failed. Rescuing the progeny of a mating of ␥-tubulin gene, GTU1, knockout heterokaryons with a GTU1 gene inserted into the MTT1 locus yielded >75 times more transformants than rescuing with the wild-type GTU1 gene itself. When cells rescued with the MTT1-GTU1 chimeric gene were transferred to medium lacking Cd 2؉ , they stopped growing and had phenotypic changes indistinguishable from cells containing only disrupted GTU1 genes. Thus, it is now possible to create conditional lethal mutants and study the terminal phenotypes of null mutations for essential genes by replacing the endogenous gene with one under the control of the MTT1 promoter. The MTT1 promoter also resulted in Ϸ30 times more overexpression of the IAG48[G1] surface antigen gene of the ciliate fish parasite Ichthyophthirius multifiliis than the highly expressed BTU1 promoter, accounting for Ϸ1% of the total cell protein. Thus, the MTT1 promoter should enable routine over-expression of endogenous and foreign genes in Tetrahymena.

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Section: Resultsmentioning

confidence: 99%

“…Coupled with the introduction of heterologous drug-resistance gene markers (11,12), these methods enabled facile gene replacement, allowing gene disruption (13) and insertion of mutant genes with flanking selectable markers (14). They also led to techniques for transformation of the germ-line micronucleus which, again, occurs by homologous integration (15).…”

mentioning

confidence: 99%

A robust inducible-repressible promoter greatly facilitates gene knockouts, conditional expression, and overexpression of homologous and heterologous genes in Tetrahymena thermophila

Shang

Song

Bowen

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

210

239

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“…H2A1 and H2A2 proteins are not detectable in the HTA1-and HTA2-disruptive transformants, respectively. The unlabeled, fastestmigrating band comigrates with histone H4, whose amino-terminal sequence is similar to that of hv1 (2 However, in this report, using newly developed transformation and gene replacement techniques (13)(14)(15)21), we demonstrated that, although the HTA1 or HTA2 gene, encoding the major H2A subtypes, can be completely knocked out, the HTA3 gene can be only partially replaced. Thus, in T. thermophila, the HTA3 gene encoding H2A variant hv1 is essential for vegetative growth.…”

Section: Discussionmentioning

confidence: 95%

“…They were then transferred to media containing increasingly higher concentrations of drug. The highest concentration of paromomycin tolerated by these transformants is defined as the maximum drug concentration at which cells can divide at least once each day, as described elsewhere (21). Table 1 summarizes these results.…”

Section: Transformation In T Thermophilamentioning

confidence: 87%

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Essential and Nonessential Histone H2A Variants in Tetrahymena thermophila

Liu

GorovskyMA

1996

Molecular and Cellular Biology

115

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Although variants have been identified for every class of histone, their functions remain unknown. We have been studying the histone H2A variant hv1 in the ciliated protozoan Tetrahymena thermophila. Sequence analysis indicates that hv1 belongs to the H2A.F/Z type of histone variants. On the basis of the high degree of evolutionary conservation of this class of histones, they are proposed to have one or more distinct and essential functions that cannot be performed by their major H2A counterparts. Considerable evidence supports the hypothesis that the hv1 protein in T. thermophila and hv1-like proteins in other eukaryotes are associated with active chromatin. In T. thermophila, simple mass transformation and gene replacement techniques have recently become available. In this report, we demonstrate that either the HTA1 gene or the HTA2 gene, encoding the major H2As, can be completely replaced by disrupted genes in the polyploid, transcriptionally active macronucleus, indicating that neither of the two genes is essential. However, only some of the HTA3 genes encoding hv1 can be replaced by disrupted genes, indicating that the H2A.F/Z type variants have an essential function that cannot be performed by the major H2A genes. Thus, an essential gene in T. thermophila can be defined by the fact that it can be partially, but not completely, eliminated from the polyploid macronucleus. To our knowledge, this study represents the first use of gene disruption technology to study core histone gene function in any organism other than yeast and the first demonstration of an essential gene in T. thermophila using these methods. When a rescuing plasmid carrying a wild-type HTA3 gene was introduced into the T. thermophila cells, the endogenous chromosomal HTA3 could be completely replaced, defining a gene replacement strategy that can be used to analyze the function of essential genes.

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