Transformation of Tetrahymena to cycloheximide resistance with a ribosomal protein gene through sequence replacement.

Abstract

A method for transforming Tetrahymena has been established earlier, but its application has been limited because of the lack of selectable markers other than the rRNA-encoding DNA (rDNA). Mutations in the yeast ribosomal protein L29 gene (CYH2) are known that confer cycloheximide resistance. We have cloned and sequenced the homologue of this gene from both a wild-type and a cycloheximide-resistant (ChxA) strain of Tetrahymena. Surprisingly, a comparison shows that the ChxA mutation is not present in the CYH2 homologue. We therefore created the yeast mutations in the Tetrahymena gene by site-directed mutagenesis and used them to transform Tetrahymena either with or without linking to an rDNA vector. All clones transformed by the rDNA vector also became resistant to cycloheximide when the rDNA contained the engineered mutant genes. Without the rDNA vector, the mutant genes transform ='1 % of iu'ected cells to become resistant to cycloheximide. DNA analysis indicates that transformation occurs by replacement of the host sequence and not by random integration of the iu'ected sequence. The replacement occurs to some but not all copies of this gene in the polyploid macronuclear genome. Thus, transformation in Tetrahymena occurs by specific sequence replacement, and the injected mutant genes can serve as dominant selectable transformation markers in this organism.DNA-mediated transformation is an important tool for genetic studies of an organism. The ciliated protozoan Tetrahymena thermophila has been a favored model system for the studies of many interesting problems, including RNA splicing, telomere structure, gene regulation, and DNA rearrangements. To facilitate these and other studies, a method for transforming Tetrahymena was developed in this laboratory several years ago (1). It exploited the unique features of the ribosomal RNA genes (rDNA) of this organism, and used its drug-resistant alleles as selectable markers and its replication origin for maintenance in the cell. To date all reported transformation studies in Tetrahymena rely on the use of the rDNA. While the method has been instrumental in many studies, it also has serious limitations. For instance, the rDNA exists in the macronucleus as high-copy-number extrachromosomal molecules. In addition, they are transcribed actively by RNA polymerase I and are localized in nucleoli (reviewed in ref.2). The effects of these properties on the expression of other genes linked to it are not known, causing concerns over its use as a general vector.Our efforts to adapt foreign drug-resistant genes to serve as selectable markers in Tetrahymena had only limited success (unpublished results). Thus, we decided to explore the possibility of using native Tetrahymena genes for this purpose.

Temporal and spatial association of histone H2A variant hv1 with transcriptionally competent chromatin during nuclear development in Tetrahymena thermophila.

“…With the development of techniques for gene replacement (Yao and Yao 1991;Kahn et al 1993) and mass transformation of Tetrahymena (Gaertig and Gorovsky 1992), additional studies aimed directly at determining the in vivo function of this histone variant should now be possible.…”

Tetrahymena IFT122Ais not essential for cilia assembly but plays a role in returning IFT proteins from the ciliary tip to the cell body

“…For selecting the transformant, a rpl29(K40M) gene conferring cycloheximide-resistance (Yao and Yao, 1991) inserted into a MTT1 promoter-driven cassette with a BTU2 3Ј terminator (J. Bowen and M.A.G., personal communication) was included between the GFP and the IFT88 3Ј flanking region. The tagged construct was transformed into wild type or IFT122A knockout cells.…”

Identification of novel chromatin-associated proteins involved in programmed genome rearrangements inTetrahymena

“…The S65T GFP variant was PCR amplified and cloned in place of the rpL29 coding sequence in a vector containing a 3.4 kbp HindIII genomic fragment containing this gene (Yao and Yao, 1991). The GFP sequence replaced rpL29 nucleotides 341-2214 of the published sequence (Acc.…”