Accurate processing and amplification of cloned germ line copies of ribosomal DNA injected into developing nuclei of Tetrahymena thermophila.

Abstract

The ciliate Tetrahymena thermophila contains a chromosomally integrated copy of the rRNA genes (rDNA) in its germinal (micronuclear) genome. These genes are excised from the chromosome through a process involving site-specific DNA breakage, become linear palindromic molecules with added telomeres, and are greatly amplified during development of the somatic nucleus (macronucleus). In this study, we cloned a 15-kilobase segment of the germ line DNA containing these genes and injected it into developing macronuclei of T. thermophila. Up to 11% of injected cells were transformed to the paromomycin-resistant phenotype specified by the injected DNA. Transformation efficiency was dependent on the developmental stages of the injected cells and the integrity of the injected DNA but not the DNA concentration or conformation. The injected DNA was apparently processed and amplified correctly to produce rDNA molecules with the expected linear palindromic structure which carried the appropriate physical markers. Thus, the 15-kilobase DNA contained aHl cis-acting sequences sufficient for the DNA-processing events leading to rDNA amplification in T.

Induction of Gene Silencing by Hairpin RNA Expression inTetrahymena thermophilaReveals a Second Small RNAPathway

“…The plasmid pIBF-1, a gift from Douglas Chalker, was used as the backbone of all hairpin constructs used in this study. This plasmid contains the MTT1 metallothionine promoter, the blue fluorescent protein gene, and the 3Ј portion of the rpL29 gene as a transcriptional terminator cloned into the NotI site of the ribosomal DNA (rDNA) vector pD5H8 (44,59). A 90-bp fragment containing a small intron was amplified from predicted gene 117.m00123, CH 445424, by use of a forward primer containing PmeI and XmaI restriction sites and a reverse primer containing XhoI and ApaI restriction sites.…”

“…This hairpin was then inserted into the polylinker of a T. thermophila rDNA vector containing the Cd-inducible metallothionine promoter MTT (44). When introduced into mating T. thermophila, this plasmid was processed and maintained as a high-copy-number autonomously replicating chromosome (59). The combination of using a strong promoter and placing the construct in a highly amplified chromosome was chosen to ensure robust expression of the hairpin RNA.…”

An rRNA variable region has an evolutionarily conserved essential role despite sequence divergence.

“…Mating T. thernophila cells can be transformed via either microinjection (42,51) or electroporation (14) with a micronuclear rDNA vector that confers paromomycin resistance (pm-r) (51). This rDNA is converted to macronuclear form and can completely replace the resident rDNA when transformants are maintained under selection (41,51).…”

A distant 10-bp sequence specifies the boundaries of a programmed DNA deletion in Tetrahymena.

“…To determine the cis-acting sequences that regulate DNA deletion, we have developed a transformation system through which a DNA construct can be introduced into the developing macronucleus of Tetrahymena, and its ability to go through rearrangement can be analyzed in the transformed cell (Tondravi and Yao 1986;Yao and Yao 1989). A cloned copy of the M-element is able to undergo rearrangement efficiently and accurately when tested by this method (Godiska and Yao 1990).…”