A comprehensive phylogenetic analysis was conducted of non-long-terminal-repeat (non-LTR) retrotransposons based on an extended sequence alignment of their reverse transcriptase (RT) domain. The 440 amino acid positions used included a region proposed to be similar to the "thumb" of the right-handed RT structure found in retroviruses. All identified non-LTR elements could be grouped into 11 distinct clades. Using the rates of sequence change derived from studies of the vertical inheritance of R1 and R2 elements in arthropods as a comparison, we found no evidence for the horizontal transmission of non-LTR elements. Assuming vertical descent, the phylogeny suggested that non-LTR elements are as old as eukaryotes, with each of the 11 clades dating back to the Precambrian era. The analysis enabled us to propose a simple chronology for the acquisition of different enzymatic domains in the evolution of the non-LTR class of retrotransposons. The first non-LTR elements were sequence specific by virtue of a restriction-enzyme-like endonuclease located downstream of the RT domain. Evolving from this original group were elements (eight clades) that acquired an apurinic-apyrimidic endonuclease-like domain upstream of the RT domain. Finally, four of these clades have inherited an RNase H domain downstream of the RT domain. The phylogenies of the AP endonuclease and RNase H domains were also determined for this report and are consistent with the monophyletic acquisition of these domains. These studies represent the most comprehensive effort to date to trace the evolution of a major class of transposable elements.

R2 elements are non-LTR retrotransposons that insert in the 28S rRNA genes of arthropods. Partial sequence data from many species have previously suggested that these elements have been vertically inherited since the origin of this phylum. Here, we compare the complete sequences of nine R2 elements selected to represent the diversity of arthropods. All of the elements exhibited a uniform structure. Identification of their conserved sequence features, combined with our biochemical studies, allows us to make the following inferences concerning the retrotransposition mechanism of R2. While all R2 elements insert into the identical sequence of the 28S gene, it is only the location of the initial nick in the target DNA that is rigidly conserved across arthropods. Variation at the R2 5' junctions suggests that cleavage of the second strand of the target site is not conserved within or between species. The extreme 5' and 3' ends of the elements themselves are also poorly conserved, consistent with a target primed reverse transcription mechanism for attachment of the 3' end and a template switch model for the attachment of the 5' end. Comparison of the approximately 1,000-aa R2 ORF reveals that it can be divided into three domains. The central 450-aa domain can be folded by homology modeling into a tertiary structure resembling the fingers, palm, and thumb subdomains of retroviral reverse transcriptases. The carboxyl terminal end of the R2 protein appears to be the endonuclease domain, while the amino-terminal end contains zinc finger and c-myb-like DNA-binding motifs.

A large number of insect species have been screened for the presence of the retrotransposable elements RI and R2. These elements integrate independently at specific sites in the 28S rRNA genes. Genomic blots indicated that 43 of 47 insect species from nine orders contained insertions, ranging in frequency from a few percent to >50% of the 28S genes. Sequence analysis of these insertions from 8 species revealed 22 elements, 21 of which corresponded to RI or R2 elements.Surprisingly, many species appeared to contain highly divergent copies of RI and R2 elements. For example, a parasitic wasp contained at least four families of RI elements; the Japanese beetle contained at least five families of R2 elements. The presence of these retrotransposable elements throughout Insecta and the observation that single species can harbor divergent families within its rRNA-encoding DNA loci present interesting questions concerning the age of these elements and the possibility of cross-species transfer.Transposable elements probably exist in the genome of every eukaryote (1). Among the most abundant types of these elements are the retrotransposable elements that, like retroviruses, move by means of an RNA intermediate (2). Retrotransposable elements can be divided into two major classes (3, 4). One class is similar to the retroviruses in that they contain long terminal repeats (LTRs) and their encoded proteins have amino acid similarity to those of the retroviruses. The second class, termed Line 1-like or the non-LTR retrotransposable elements, lacks any type ofterminal repeat and has lower levels of amino acid similarity to the retroviruses.RI and R2 (formerly called type I and type II insertions) are non-LTR retrotransposable elements, each found at a precise location in a fraction of the 28S rRNA genes ofBombyx mori and several dipteran species (5-12). The insertion sites for RI and R2 are 74 base pairs (bp) apart in a highly conserved region of the 28S gene, the large subunit rRNA gene of eukaryotes (Fig. 1A). The presence of either RI or R2 within an rRNA-encoding DNA (rDNA) unit inactivates that unit (13)(14)(15). Only a few copies of RI and R2 are located outside the rDNA units and they appear to be nonfunctional (6,16,17). The high insertion specificity of R2 elements can be explained by an encoded endonuclease activity specific to its 28S gene insertion site (18). Individual copies of RI and R2 have the same 3' end but may be truncated at their 5' end, a feature common to other non-LTR retrotransposable elements (6,7,11,17).Although transposable elements as a group are widespread, it has been difficult to study the distribution of a particular element across broad taxonomic groups due to their dispersed genomic locations and rapid sequence divergence that limits their detection by DNA hybridization. Here we have taken advantage of the remarkable insertion specificities ofRI and R2 to determine their distribution in species throughout the class Insecta. We present evidence that RI and R2 are present in the rRNA ge...

In the parasitic wasp, Nasonia vitripennis, males are haploid and usually develop from unfertilized eggs, whereas females are diploid and develop from fertilized eggs. Some individuals in this species carry a genetic element, termed psr (paternal sex ratio), which is transmitted through sperm and causes condensation and subsequent loss of paternal chromosomes in fertilized eggs, thus converting diploid females into haploid males. In this report the psr trait was shown to be caused by a supernumerary chromosome. This B chromosome contains at least three repetitive DNA sequences that do not cross-hybridize to each other or to the host genome. The psr chromosome apparently produces a trans-acting product responsible for condensation of the paternal chromosomes, but is itself insensitive to the effect. Because the psr chromosome enhances its transmission by eliminating the rest of the genome, it can be considered the most "selfish" genetic element yet described.

R2 retrotransposable elements exclusively insert into a conserved region of the tandemly organized 28S rRNA genes. Despite inactivating a subset of these genes, R2 elements have persisted in the ribosomal DNA (rDNA) loci of insects for hundreds of millions of years. Controlling R2 proliferation was addressed in this study using lines of Drosophila simulans previously shown to have either active or inactive R2 retrotransposition. Lines with active retrotransposition were shown to have high R2 transcript levels, which nuclear run-on transcription experiments revealed were due to increased transcription of R2-inserted genes. Crosses between R2 active and inactive lines indicated that an important component of this transcriptional control is linked to or near the rDNA locus, with the R2 transcription level of the inactive parent being dominant. Pulsed-field gel analysis suggested that the R2 active and inactive states were determined by R2 distribution within the locus. Molecular and cytological analyses further suggested that the entire rDNA locus from the active line can be silenced in favor of the locus from the inactive line. This silencing of entire rDNA loci represents an example of the large-scale epigenetic control of transposable elements and shares features with the nucleolar dominance frequently seen in interspecies hybrids.Eukaryotic genomes have evolved elaborate surveillance and regulatory mechanisms to control the spread of transposable elements (38). The success of a transposable element is thus dependent upon its ability to elude these cellular controls. One ingenious approach used by transposable elements is to target locations within the genome that cannot be completely silenced. The most successful known examples of this approach are the numerous mobile elements that insert specifically into the rRNA genes of animals (10). Eukaryotic genomes encode hundreds to thousands of rRNA genes organized in tandem arrays within one or more chromosomal loci. Transcription of the rRNA gene arrays, also termed nucleolar organizer regions (NORs), is tightly coupled to the growth status of the cell. Significant progress has been made in understanding the transcription of the DNA encoding the rRNA genes (ribosomal DNA [rDNA]) as well as the many subsequent steps involved in ribosome biogenesis (14, 16). The level of regulatory complexity added by the presence of transposable elements is largely unknown.The nonlong terminal repeat (non-LTR) retrotransposable elements, R1 and R2, insert into a conserved central region of the 28S gene (Fig. 1A). R1 and R2 are present in most lineages of arthropods (2), and R2 elements have been found in a number of other divergent animal groups (20, 21). Phylogenetic analyses suggest that R2 elements have been a stable component of genomes throughout the evolution of arthropods (2, 27) and possibly since the origin of multicellular animals (20, 21). As such, they represent the longest known stable relationship of a mobile element and its host. During their long history, R2 elements ...