Search

To facilitate studies of rDNA molecular genetics in Tetrahymena thermophila, we attempted the detection of polymorphisms in the nontranscribed spacers (NTSs) using polymerase chain reaction (PCR), starting with minute amounts of DNA. The targeted polymorphic regions are 85% adenine-thymine (AT). We found conditions of efficient and specific in vitro amplification of targeted segments in the replication domain of the 5'NTS and in the subtelomeric segment of the 3'NTS. The identity of the amplified segments was confirmed by restriction enzyme digestion and DNA sequence analysis. Digestion of the template DNA at restriction sites upstream and downstream of the targeted region increased the efficiency of amplification, presumably because the targeted segments are in a palindromic molecule. Starting from total cell DNA corresponding to as little as 0.03 picogram (equivalent to the DNA content of 0.003 cells or about 30 rDNA molecules), we observed the amplified band after agarose gel electrophoresis and ethidium bromide staining. The yield indicated more than 10-billion-fold amplification. Amplification of the subtelomeric fragment yielded homogeneous product of minimum possible length even though the telomeric-specific primer can bind, at least initially, at a multiplicity of GGGGTT repeats. Amplified 5'NTS product also was detected in an ethidium-bromide-stained gel when PCR was started with a single cell.

We have determined the nucleotide sequence of the 26S large subunit (LSU) rRNA genes for two Tetrahymena species, T. thermophila and T. pyriformis. The inferred rRNA sequences are presented in their most probable secondary structures based on compensatory mutations, energy, and conservation criteria. The majority of the nucleotide changes between the two Tetrahymena LSU rRNAs and the positions of a relatively large deletion and of the processing cleavage sites resulting in the generation of the hidden break are all located within the so-called divergent domains or expansion segments. These are regions within the common core of secondary structure where expansions have taken place during the evolution of the rRNA of higher eukaryotes. The dispensable nature of some of the expansion segments has been taken as evidence of their non-functionality. However, our data show that a considerable selective constraint has operated to preserve the secondary structure of these segments. Especially in the case of the D2 and D8 segments, the presence of a considerable number of compensatory base changes suggests that the secondary structure of these regions is of functional importance. Alternatively, these expansion segments may have maintained characteristic folding patterns because only such structures are being tolerated within otherwise functionally important regions.

The nucleotide sequences of 5S rRNAs from three ciliated protozoa, Paramecium tetraurelia, Tetrahymena thermophila and Blepharisma japonicum have been determined. All of them are 120 nucleotides long and the sequence of probable tRNA binding site of position 41-44 is GAAC which is characteristic of the plant 5S rRNAs. The sequence similarity percents are 87% (Paramecium/Tetrahymena), 86% (Paramecium/Blepharisma) and 79% (Tetrahymena/Blepharisma), suggesting a close relationship of these three ciliates.

The 5'-terminus of 35S pre-rRNA and mature 17S rRNA of Tetrahymena thermophila was mapped on cloned rDNA fragments by S1 nuclease protection experiments. A single site for transcription initiation was observed when pre-rRNA prepared by three different methods was used as RNA probe. These mapping results were unambiguously confirmed by sequencing the 5'-terminal region of in vitro capped 35S pre-rRNA. DNA sequence analysis of about 520 nucleotides upstream of the transcription initiation site revealed several distinct sets of highly conserved repeat sequences. In addition, the 840 nucleotides downstream of the transcription initiation site (+ 1) was determined and shown to include the 5'-terminus of the 17S rRNA coding region at position + 647. A region surrounding the position + 195 contains an inverted repeat sequence which could be the structural basis for the recently described premature transcription termination event in this organism (Kister et al. (1983) Nucl. Acids Res. 11, 3487-3502).

A cloned rDNA fragment containing the transcriptional start point of Tetrahymena pyriformis was transcribed in vitro with a crude extract from homologous Tetrahymena cells. When a KpnI-HindIII fragment from the cloned rDNA plasmid was used as the template, the runoff transcription gave rise to two major RNA products about 490 and 460 nucleotides (nt) in length. An RNA of about 490 nt long was found to correspond to the expected transcript starting from the in vivo start point determined at our laboratory [Saiga et al., Nucl. Acids Res. 10 (1982) 4223-4235]. Precise size determination was performed using an RNA size marker prepared by hybridizing the template DNA with in vitro-capped 35S pre-rRNA followed by treatment with single-strand specific P1 nuclease. The size of the runoff transcript changed as predicted, according to downstream truncation points. The effects of alpha-amanitin and actinomycin D showed the transcription to be dependent on the added template DNA and to be catalyzed by form-I RNA polymerase. Possible reasons for the discrepancy between our determination of the size of the transcription product and that of Sutiphong et al. [Biochemistry 23 (1984) 6319-6326] are discussed.