The Ty5 retrotransposons of Saccharomyces cerevisiae integrate preferentially into regions of silent chromatin at the telomeres and silent mating loci (HMR and HML). We define a Ty5-encoded targeting domain that spans 6 amino acid residues near the C terminus of integrase (LXSSXP). The targeting domain establishes silent chromatin when it is tethered to a weakened HMR-E silencer, and it disrupts telomeric silencing when it is overexpressed. As determined by both yeast two-hybrid and in vitro binding assays, the targeting domain interacts with the C terminus of Sir4p, a structural component of silent chromatin. This interaction is abrogated by mutations in the targeting domain that disrupt integration into silent chromatin, suggesting that recognition of Sir4p by the targeting domain is the primary determinant in Ty5 target specificity.The long terminal repeat (LTR) retrotransposons are a large and ubiquitous class of mobile genetic elements. Like their cousins the retroviruses, they replicate by reverse transcribing an element mRNA and then integrating the cDNA product into their host's chromosomes. LTR retrotransposons are typically abundant components of nuclear genomes, constituting a few percentages of the Saccharomyces cerevisiae genome to over 50% of the genomes of some plants such as maize (31, 45). As the genome sequencing projects progress, it is apparent that most retrotransposons are not randomly distributed on chromosomes. In Drosophila melanogaster and Arabidopsis thaliana, for example, retrotransposons are highly enriched in pericentromeric heterochromatin (19,44). This nonrandom distribution may be the result of preferential integration to these sites. It has been suggested that the low gene density of heterochromatin may offer a safe haven for transposition, which ensures persistence of retrotransposons by avoiding the harmful consequences of mutations that might occur if integration were random (5). Because repetitive sequences can form heterochromatin in some species, the accumulation of retrotransposons in certain regions of the genome may, in turn, contribute to the formation of chromatin domains (18).How is it that retrotransposons identify certain chromosomal regions during integration? One model suggests that the integration apparatus recognizes specific chromatin states or DNA-bound protein complexes (7). This interaction tethers the integration machinery to target sites and results in the observed target site biases. This model is best supported by studies of the S. cerevisiae retrotransposons. Over 90% of native Ty1, Ty2, Ty3, and Ty4 insertions are located upstream of genes transcribed by RNA polymerase III (RNAP III) (31).These regions are often gene poor and, like heterochromatin, may provide a safe haven for transposition within the streamlined S. cerevisiae genome (5). For Ty1 and Ty3, the association with sites of RNAP III transcription is due to targeted integration. Targeting requires assembly of the RNAP III transcription complex, and promoter mutations in target genes that pre...

The yeast Ty5 retrotransposon preferentially integrates into heterochromatin at the telomeres and silent mating loci. Target specificity is mediated by a small domain of Ty5 integrase (the targeting domain, TD), which interacts with the heterochromatin protein Sir4 and tethers the integration complex to target sites. Here we demonstrate that TD is phosphorylated and that phosphorylation is required for interaction with Sir4. The yeast cell, therefore, through posttranslational modification, controls Ty5's mutagenic potential: when TD is phosphorylated, insertions occur in gene-poor heterochromatin, thereby minimizing deleterious consequences of transposition; however, in the absence of phosphorylation, Ty5 integrates throughout the genome, frequently causing mutations. TD phosphorylation is reduced under stress conditions, specifically starvation for amino acids, nitrogen, or fermentable carbon. This suggests that Ty5 target specificity changes in response to nutrient availability and is consistent with McClintock's hypothesis that mobile elements restructure host genomes as an adaptive response to environmental challenge.

Knowledge of the role of RNA in affecting gene expression has expanded in the past several years. Small RNAs serve as homology guides to target messenger RNAs for destruction at the post-transcriptional level in the experimental technique known as RNA interference and in the silencing of some transgenes. These small RNAs are also involved in sequence-specific targeting of chromatin modifications for transcriptional silencing of transgenes, transposable elements, heterochromatin and some cases of Polycomb-mediated gene silencing. RNA silencing processes in Drosophila are described.

In Drosophila melanogaster, small RNAs homologous to transposable elements (TEs) are of two types: piRNA (piwi-interacting RNA) with size 23-29nt and siRNA (small interfering RNA) with size 19-22nt. The siRNA pathway is suggested to silence TE activities in somatic tissues based on TE expression profiles, but direct evidence of transposition is lacking. Here we developed an efficient FISH (fluorescence in Situ hybridization) based method for polytene chromosomes from larval salivary glands to reveal new TE insertions. Analysis of the LTR-retrotransposon 297 and the non-LTR retroposon DOC shows that in the argonaut 2 (Ago2) and Dicer 2 (Dcr2) mutant strains, new transposition events are much more frequent than in heterozygous strains or wild type strains. The data demonstrate that the siRNA pathway represses TE transposition in somatic cells. Nevertheless, we found that loss of one functional copy of Ago2 or Dcr2 increases somatic transpositions of the elements at a lower level depending on the genetic background, suggesting a quantitative role for RNAi core components on mutation frequency.

A common modulation of gene expression in aneuploids is an inverse correlation of the monitored gene with the dosage of another segment of the genome. Such effects can be reduced to the action of single genes. One gene previously found to modulate leaky alleles of the white eye color gene in Drosophila is Inverse regulator-a (Inr-a). Heterozygotes of mutations increase the expression of white about 2-fold, and trisomic regions surrounding the gene reduce the expression to about two-thirds of the normal diploid level. Further cytological definition of the location of this gene on the second chromosome led to a candidate pre-mRNA cleavege complex II protein (Pcf11) as the only gene in the remaining region whose mutations exhibit recessive lethality as do alleles of Inr-a. The product of Pcf11 has been implicated in transcriptional initiation, elongation, and termination reactions. Four mutant alleles showed molecular lesions predicted to lead to nonfunctional products of Pcf11. The identification of the molecular lesion of Inr-a provides insight into the basis of this common aneuploidy effect.