Over 190 independent insertions into target plasmids of the retrovirus-like element Ty3 were recovered and mapped. Ty3 was shown to insert upstream of tRNA, 5S, and U6 genes, all of which are transcribed by RNA polymerase III. Integration sites were within 1-4 nucleotides of the position of transcription initiation, even for one mutant gene where the polymerase III initiation site was shifted to a completely new context. Mutagenesis of a SUP2 tRNA gene target showed that integration required functional promoter elements but that it did not correlate in a simple way with target transcription. This is the first report directly linking a discrete genomic function with preferential insertion of a retrotransposon.

Abundant ∼28-nucleotide RNAs that are thought to direct histone H3 lysine 9 (H3K9) methylation and promote the elimination of nearly 15 Mbp of DNA from the developing somatic genome are generated during Tetrahymena thermophila conjugation. To identify the protein(s) that generates these small RNAs, we studied three Dicer-related genes encoded within the Tetrahymena genome, two that contain both RNase III and RNA helicase motifs, Dicer 1 (DCR1) and DCR2, and a third that lacks the helicase domain, Dicer-like 1 (DCL1). DCL1 is expressed upon the initiation of conjugation, and the protein localizes to meiotic micronuclei when bidirectional germ line transcription occurs and small RNAs begin to accumulate. Cells in which we disrupted the DCL1 gene (ΔDCL1) grew normally and initiated conjugation as wild-type cells but arrested near the end of development and eventually died, unable to resume vegetative growth. These ΔDCL1 cells failed to generate the abundant small RNAs but instead accumulated germ line-limited transcripts. Together, our findings demonstrate that these transcripts are the precursors of the small RNAs and that DCL1 performs RNA processing within the micronucleus. Postconjugation ΔDCL1 cells die without eliminating the germ line-limited DNA sequences from their newly formed somatic macronuclei, a result that shows that this Dicer-related gene is required for programmed DNA rearrangements. Surprisingly, ΔDCL1 cells were not deficient in overall H3K9 methylation, but this modification was not enriched on germ line-limited sequences as it is in wild-type cells, which clearly demonstrates that these small RNAs are essential for its targeting to specific loci.

A large number of DNA segments are excised from the chromosomes of the somatic nucleus during development of Tetrahymena thermophila. How these germline-limited sequences are recognized and excised is still poorly understood. We have found that many of these noncoding DNAs are transcribed during nuclear development. Transcription of the germline-limited M element occurs from both DNA strands and results in heterogeneous transcripts of < 200 b to > 1 kb. Transcripts are most abundant when developing micro- and macronuclei begin their differentiation. Transcription is normally restricted to unrearranged DNA of micronuclei and/or developing nuclei, but germline-limited DNAs can induce their own transcription when placed into somatic macronuclei. Brief actinomycin D treatment of conjugating cells blocked M-element excision, providing evidence that transcription is important for efficient DNA rearrangement. We propose that transcription targets these germline-limited sequences for elimination by altering chromatin to ensure their accessibility to the excision machinery.

Ciliated protozoa extensively remodel their somatic genomes during nuclear development, fragmenting their chromosomes and removing large numbers of internal eliminated sequences (IESs). The sequences eliminated are unique and repetitive DNAs, including transposons. Recent studies have identified transposase proteins that appear to have been domesticated and are used by these cells to eliminate DNA not wanted in the somatic macronucleus. This DNA elimination process is guided by meiotically produced small RNAs, generated in the germline nucleus, that recognize homologous sequences leading to their removal. These scan RNAs are found in complexes with PIWI proteins. Before they search the developing genome for IESs to eliminate, they scan the parental somatic nucleus and are removed from the pool if they match homologous sequences in that previously reorganized genome. In Tetrahymena, the scan RNAs target heterochromatin modifications to mark IESs for elimination. This DNA elimination pathway in ciliates shares extensive similarity with piRNA-mediated silencing of metazoans and highlights the remarkable ability of homologous RNAs to shape developing genomes.

Research using ciliates revealed early examples of epigenetic phenomena and continues to provide novel findings. These protozoans maintain separate germline and somatic nuclei that carry transcriptionally silent and active genomes, respectively. Examining the differences in chromatin within distinct nuclei of Tetrahymena identified histone variants and established that transcriptional regulators act by modifying histones. Formation of somatic nuclei requires both transcriptional activation of silent chromatin and large-scale DNA elimination. This somatic genome remodeling is directed by homologous RNAs, acting with an RNA interference (RNAi)-related machinery. Furthermore, the content of the parental somatic genome provides a homologous template to guide this genome restructuring. The mechanisms regulating ciliate DNA rearrangements reveal the surprising power of homologous RNAs to remodel the genome and transmit information transgenerationally.

Ty3, a retrotransposon of Saccharomyces cerevisiae, is found within 20 base pairs (bp) of the 5' ends of different tRNA genes. Determination of the complete nucleotide sequence of one Ty3 retrotransposon (Ty3-2) shows that the element is composed of an internal domain 4,748 bp long flanked by long terminal repeats of the 340-bp sigma element. Three open reading frames (ORFs) longer than 100 codons are present in the sense strand. The first ORF, TYA3, encodes a protein with a motif found in the nucleic acid-binding protein of retroviruses. The second ORF, TYB3, has homology to retroviral pol genes. The deduced amino acid sequence of the reverse transcriptase domain shows the greatest similarity to Drosophila retrotransposon 17.6, with 43% identical residues. The inferred order of functional domains within TYB3--protease, reverse transcriptase, and endonuclease--resembles the order in Drosophila element 17.6 and in animal retroviruses but is different from that found in yeast elements Ty1 and Ty2. A second Ty3 element (Ty3-1) from a standard laboratory strain was overexpressed and shown to transpose.