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

Abstract

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.

Cloning, characterization, and gene expression analysis of a novel cadmium metallothionein gene in Tetrahymena pigmentosa

“…Cd-MTs have been previously reported in T. pyriformis, T. pigmentosa, T. thermophila and T. tropicalis (Piccinni et al, 1994(Piccinni et al, , 1999Shang et al, 2002;Boldrin et al, 2003;Fu and Miao, 2006;Diaz et al, 2007, Shuja andShakoori, 2007). The Tpig-MT1 gene is unusually long (521 bp), uses TGA as the stop codon, and is intronless.…”

“…Different MT isoforms have been described in the unicellular ciliated protozoan genus Tetrahymena (T. pyriformis, T. pigmentosa, T. thermophila and T. tropicalis; Piccinni et al, 1999;Shang et al, 2002;Boldrin et al, 2003;Fu and Miao, 2006;Diaz et al, 2007, Shuja andShakoori, 2007). According to their phylogenetic relationships, the pattern of clustering of Cys residues, and which metal inducer they preferentially respond to, they were grouped into Cd-MTs (subfamily 7a) or Cu-MTs (subfamily 7b), showing little sequence identity with one another.…”

Members of the NIMA-related Kinase Family Promote Disassembly of Cilia by Multiple Mechanisms

“…We overexpressed Nrk1p and Nrk2p as GFP fusions using the cadmium-inducible MTT1 promoter (Shang et al, 2002). Nrk1p-GFP was detected 2 h after cadmium addition, and the levels remained high for at least 24 h (Supplemental Figure S1, A).…”

“…The CU522 strain (from Dr. Donna Cassidy-Hanley, Cornell University, Ithaca, NY) was used to insert transgenes as described previously , and such strains were maintained in SPP with 20 M paclitaxel. To overexpress a transgene under MTT1 promoter (Shang et al, 2002), cells were grown without paclitaxel overnight, diluted to 10 5 -2 ϫ 10 5 cells/ml, and incubated with 2.5 g/ml CdCl 2 . …”

Centromeric Histone H3 Is Essential for Vegetative Cell Division and for DNA Elimination during Conjugation in Tetrahymena thermophila

“…Cna1p-GFP localization in vegetative cells demonstrates it is a centromere protein. To study Cna1p localization and deposition, CNA1-GFP, with a GFP coding sequence inserted at the C terminus of the CNA1 open reading frame, was used to replace the MAC Cd 2ϩ -inducible metallothionein gene (MTT1 [29]) coding sequence, creating somatic MCG (MTT1-CNA1-GFP) strains ( Fig. 2A).…”