R1 and R2 Retrotransposable Elements of Drosophila Evolve at Rates Similar to Those of Nuclear Genes

The non-long-terminal repeat retrotransposable elements, R1 and R2, insert at unique locations in the 28S ribosomal RNA genes of insects. Based on the nucleotide sequences of these elements in the eight members of the melanogaster species subgroup of the genus Drosophila, they have been maintained by vertical germline transmission for the 17-20 million year history of this subgroup. The stable inheritance of R1 and R2 within these species has enabled a determination of their nucleotide substitution rates. The sequence of the R1 and R2 elements from D. ambigua, a member of the obscura species group, has also been determined to enable an extrapolation of this rate over an estimated 45-60 million years. The mean rate of substitutions at synonymous sites (K(s)) was 6.6 and 9.6 times the rate at replacement sites (K(a)) in the R1 and R2 elements, respectively. Both elements appear to have been under selective pressure to maintain their open reading frames and thus their ability to retrotranspose for most of their evolution in these lineages. Using the rate of change at synonymous sites (K(s)) as the best indicator of the nucleotide substitution rate, the mean K(s) values for R1 and R2 were 2.3 and 2.2 times that of the alcohol dehydrogenase (Adh) genes. However, this faster rate is a result of the lower codon usage bias of R1 and R2 compared with that of Adh. When the K(s) rates of R1 and R2 were compared with that of a larger number of nuclear genes available from at least two of the nine species under investigation, R1 and R2 were found to evolve in most lineages at rates similar to that of nuclear genes with low codon bias. The ability of R1 and R2 to maintain their presence in this species subgroup by retrotransposition while exhibiting rates of nucleotide evolution similar to nuclear genes suggests these transposition events are rare or not as error prone as that of retroviruses.     

Evolution of R1 and R2 in the rDNA units of the genus Drosophila

R1 and R2 are non-long terminal repeat (non-LTR) retrotransposable elements that specifically insert in the 28S ribosomal RNA (rRNA) genes of insects. Using the Drosophila genus, which includes some of the best characterized insect taxa, we have conducted a number of studies on the evolution of these elements. We find that R1 and R2 are subject to the same recombinational forces that give rise to the concerted evolution of the rDNA units. The turnover of R1 and R2 elements can be readily documented in different strains of D. melanogaster using 5′ truncated elements as restriction-length polymorphisms. This turnover leads to uniform populations of elements with nucleotide sequence divergence of different copies averaging only 0.23% for the R2 and 0.47% for the R1 elements. Molecular phylogenetic analysis of elements from 16 different species of Drosophila suggests that these elements have been stable components of the rDNA locus for the 50–70 million year history of the Drosophila genus. Using changes at synonymous positions within the protein-encoding regions as estimates of the baseline substitution rate, it could be shown that R1 and R2 are evolving at rates similar to that of typical protein encoding genes provided corrections are made for the low codon bias of the elements. R1 and R2 are clearly well-adapted for their existence in the rDNA units of their host. This revised version was published online in August 2006 with corrections to the Cover Date.     

Turnover of R1 (Type I) and R2 (Type Ii) Retrotransposable Elements in the Ribosomal DNA of Drosophila Melanogaster

R1 and R2 are distantly related non-long terminal repeat retrotransposable elements each of which inserts into a specific site in the 28S rRNA genes of most insects. We have analyzed aspects of R1 and R2 abundance and sequence variation in 27 geographical isolates of Drosophila melanogaster. The fraction of 28S rRNA genes containing these elements varied greatly between strains, 17-67% for R1 elements and 2-28% for R2 elements. The total percentage of the rDNA repeats inserted ranged from 32 to 77%. The fraction of the rDNA repeats that contained both of these elements suggested that R1 and R2 exhibit neither an inhibition of nor preference for insertion into a 28S gene already containing the other type of element. Based on the conservation of restriction sites in the elements of all strains, and sequence analysis of individual elements from three strains, nucleotide divergence is very low for R1 and R2 elements within or between strains (less than 0.6%). This sequence uniformity is the expected result of the forces of concerted evolution (unequal crossovers and gene conversion) which act on the rRNA genes themselves. Evidence for the role of retrotransposition in the turnover of R1 and R2 was obtained by using naturally occurring 5' length polymorphisms of the elements as markers for independent transposition events. The pattern of these different length 5' truncations of R1 and R2 was found to be diverse and unique to most strains analyzed. Because recombination can only, with time, amplify or eliminate those length variants already present, the diversity found in each strain suggests that retrotransposition has played a critical role in maintaining these elements in the rDNA repeats of D. melanogaster.     

Rates of R1 and R2 retrotransposition and elimination from the rDNA locus of Drosophila melanogaster

R1 and R2 elements are non-LTR retrotransposons that insert specifically into the 28S rRNA genes of arthropods. The process of concerted evolution of the rDNA locus should give rise to rapid turnover of these mobile elements compared to elements that insert at sites throughout a genome. To estimate the rate of R1 and R2 turnover we have examined the insertion of new elements and elimination of old elements in the Harwich mutation accumulation lines of Drosophila melanogaster, a set of inbred lines maintained for >350 generations. Nearly 300 new insertion and elimination events were observed in the 19 Harwich lines. The retrotransposition rate for R1 was 18 times higher than the retrotransposition rate for R2. Both rates were within the range previously found for retrotransposons that insert outside the rDNA loci in D. melanogaster. The elimination rates of R1 and R2 from the rDNA locus were similar to each other but over two orders of magnitude higher than that found for other retrotransposons. The high rates of R1 and R2 elimination from the rDNA locus confirm that these elements must maintain relatively high rates of retrotransposition to ensure their continued presence in this locus.     

HeT-A and TART, two Drosophila retrotransposons with a bona fide role in chromosome structure for more than 60 million years

Drosophila telomeres have been maintained by retrotransposition for at least 60 MY, which predates the separation of extant species of this genus. Studies of D. melanogaster, D. yakuba, and D. virilis show that, in Drosophila, telomeres are composed of two non-LTR retrotransposons, HeT-A and TART. Far from being static, HeT-A and TART evolve faster than Drosophila euchromatic genes. In spite of their high rate of sequence change, HeT-A and TART maintain their basic structures and unusual individual features. The maintenance of their separate identities suggests that HeT-A and TART cooperate either in the process of retrotransposition onto the chromosome end, or in the formation of telomere chromatin by transposed DNA copies. The telomeric retrotransposons and the Drosophila genome constitute an example of a robust symbiotic relationship between mobile elements and the genome.     

Rapid R2 retrotransposition leads to the loss of previously inserted copies via large deletions of the rDNA locus

R2 non-long terminal repeat retrotransposable elements insert specifically into the 28S rRNA genes of a wide range of animals. These elements maintain long-term stable relationships with the host genome. By scoring the variation present at the 5' ends of individual R2 copies, lines of Drosophila simulans have been identified with high rates of R2 retrotransposition. Comparing the R2 elements present in the parents with that of their progeny after 1 or 30 generations in this report revealed that retrotransposition rates were higher through the female germ line compared with the male germ line. In addition, most events in females occur late in germ line development. Surprisingly, the gain of new R2 insertions by retrotranspositions was counterbalanced by deletions of preexisting R2 insertions. These deletions occurred by the loss of large segments of the rDNA units that contained on average an estimated 15 R2 elements. When monitored over single generations, the rate of loss of preexisting elements was higher than the rate of new insertions. However, the chromosomes with the largest deletions appear to be eliminated from the population because the rates of R2 insertions and deletions after 30 generations were approximately equal. These findings suggest that high rates of R2 retrotransposition do not necessarily lead to dramatic increases in the level of R2 insertions in the rDNA locus but can lead to a more rapid turnover of rDNA units.     

Vertical Transmission of the Retrotransposable Elements R1 and R2 during the Evolution of the Drosophila Melanogaster Species Subgroup

R1 and R2 are non-long-terminal repeat retrotransposable elements that insert into specific sequences of insect 28S ribosomal RNA genes. These elements have been extensively described in Drosophila melanogaster. To determine whether these elements have been horizontally or vertically transmitted, we characterized R1 and R2 elements from the seven other members of the melanogaster species subgroup by genomic blotting and nucleotide sequencing. Each species was found to have homogeneous families of R1 and R2 elements with the exception of erecta and orena, which have no R2 elements. The DNA sequences of multiple R1 and R2 copies from each species indicated nucleotide divergence within each species averaged only 0.48% for R1 and 0.35% for R2, well below the level of divergence among the species. Most copies of R1 and R2 (40 of 47) sequenced from the seven species were potentially functional, as indicated by the absence of premature termination codons or translational frameshifts that would destroy the open reading frame of the element. The sequence relationships of both the R1 and R2 elements from the various members of the melanogaster subgroup closely followed that of the species phylogeny, suggesting that R1 and R2 have been stably maintained by vertical transmission since the origin of this species subgroup 17-20 million years ago. The remarkable stability of R1 and R2, compared to what has been suggested for transposable elements that insert at multiple locations in these same species, may be due to their unique specificity for sites in the rRNA gene locus. Under low copy number conditions, when it is essential for any mobile element to transpose, the insertion specificities of R1 and R2 ensure uniform developmentally regulated target sites that can be occupied with little or no detrimental effect on the host.     

Repetitive DNA and chromosome evolution in plants

Most higher plant genomes contain a high proportion of repeated sequences. Thus repetitive DNA is a major contributor to plant chromosome structure. The variation in total DNA content between species is due mostly to variation in repeated DNA content. Some repeats of the same family are arranged in tandem arrays, at the sites of heterochromatin. Examples from the Secale genus are described. Arrays of the same sequence are often present at many chromosomal sites. Heterochromatin often contains arrays of several unrelated sequences. The evolution of such arrays in populations is discussed. Other repeats are dispersed at many locations in the chromosomes. Many are likely to be or have evolved from transposable elements. The structures of some plant transposable elements, in particular the sequences of the terminal inverted repeats, are described. Some elements in soybean, antirrhinum and maize have the same inverted terminal repeat sequences. Other elements of maize and wheat share terminal homology with elements from yeast, Drosophila, man and mouse. The evolution of transposable elements in plant populations is discussed. The amplification, deletion and transposition of different repeated DNA sequences and the spread of the mutations in populations produces a turnover of repetitive DNA during evolution. This turnover process and the molecular mechanisms involved are discussed and shown to be responsible for divergence of chromosome structure between species. Turnover of repeated genes also occurs. The molecular processes affecting repeats imply that the older a repetitive DNA family the more likely it is to exist in different forms and in many locations within a species. Examples to support this hypothesis are provided from the Secale genus.     

Genome size as a mutation-selection-drift process

A novel method for estimating neutral rates and patterns of DNA evolution in Drosophila takes advantage of the propensity of non-LTR retrotransposable elements to create nonfunctional, transpositionally inactive copies as a product of transposition. For many LINE elements, most copies present in a genome at any one time are nonfunctional "dead-on-arrival" (DOA) copies. Because these are off-shoots of active, transpositionally competent "master" lineages, in a gene tree of a LINE element from multiple samples from related species, the DOA lineages are expected to map to the terminal branches and the active lineages to the internal branches, the primary exceptions being when the sample includes DOA copies that are allelic or orthologous. Analysis of nucleotide substitutions and other changes along the terminal branches therefore allows estimation of the fixation process in the DOA copies, which are unconstrained with respect to protein coding; and under selective neutrality, the fixation process estimates the underlying mutational pattern. We have studied the retroelement Helena in Drosophila. An unexpectedly high rate of DNA loss was observed, yielding a half-life of unconstrained DNA sequences approximately 60-fold faster in Drosophila than in mammals. The high rate of DNA loss suggests a straightforward explanation of the seeming paradox that Drosophila has many fewer pseudogenes than found in mammalian species. Differential rates of deletion in different taxa might also contribute to the celebrated C-value paradox of why some closely related organisms can have very different DNA contents. New data presented here rule out the possibility that the transposition process itself is highly mutagenic, hence the observed linear relation between number of deletions and number of nucleotide substitutions is most easily explained by the hypothesis that both types of changes accumulate in unconstrained sequences over time.     

R1 and R2 retrotransposition and deletion in the rDNA loci on the X and Y chromosomes of Drosophila melanogaster

The non-LTR retrotransposons R1 and R2 insert into the 28S rRNA genes of arthropods. Comparisons among Drosophila lineages have shown that these elements are vertically inherited, while studies within species have indicated a rapid turnover of individual copies (elimination of old copies and the insertion of new copies). To better understand the turnover of R1 and R2, 200 retrotranspositions and nearly 100 eliminations have been scored in the Harwich mutation-accumulation lines of Drosophila melanogaster. Because the rDNA arrays in D. melanogaster are present on the X and Y chromosomes and no exchanges were detected in these lines, it was possible to show that R1 retrotranspositions occur predominantly in the male germ line, while R2 retrotranspositions were more evenly divided between the germ lines of both sexes. The rate of elimination of elements from the Y rDNA array was twice that of the X rDNA array with both chromosomal loci containing regions where the rate of elimination was on average eight times higher. Most R1 and R2 eliminations appear to occur by large intrachromosomal events (i.e., loop-out events) that involve multiple rDNA units. These findings are interpreted in light of the known abundance of R1 and R2 elements in the X and Y rDNA loci of D. melanogaster.     

Neutral evolution of the sex-determining gene transformer in Drosophila

The amino acid sequence of the transformer (tra) gene exhibits an extremely rapid rate of evolution among Drosophila species, although the gene performs a critical step in sex determination. These changes in amino acid sequence are the result of either natural selection or neutral evolution. To differentiate between selective and neutral causes of this evolutionary change, analyses of both intraspecific and interspecific patterns of molecular evolution of tra gene sequences are presented. Sequences of 31 tra alleles were obtained from Drosophila americana. Many replacement and silent nucleotide variants are present among the alleles; however, the distribution of this sequence variation is consistent with neutral evolution. Sequence evolution was also examined among six species representative of the genus Drosophila. For most lineages and most regions of the gene, both silent and replacement substitutions have accumulated in a constant, clock-like manner. In exon 3 of D. virilis and D. americana we find evidence for an elevated rate of nonsynonymous substitution, but no statistical support for a greater rate of nonsynonymous relative to synonymous substitutions. Both levels of analysis of the tra sequence suggest that, although the gene is evolving at a rapid pace, these changes are neutral in function.     

Competition between R1 and R2 transposable elements in the 28S rRNA genes of insects

R1 and R2 are non-LTR retrotransposons that insert in the 28S rRNA genes of arthropods. R1 elements insert into a site that is 74 bp downstream of the R2 insertion site, thus the presence of an R2 in the same 28S gene may inhibit the expression of R1. Consistent with such a suggestion, the R1 elements of Drosophila melanogaster have a strong bias against inserting into 28S genes already containing an R2 element. R2 elements, on the other hand, are only 2-3 fold inhibited from inserting into a 28S gene already containing an R1. D. melanogaster R1 elements are unusual in that they generate a 23-bp deletion of the target site upstream of the insertion. Using in vitro assays developed to study R2 integration, we show that the presence of R1 sequences 51 bp downstream of the R2 insertion site changes the nucleosomal structure that can be formed by the R2 target site. The R2 endonuclease is inhibited from cleaving these altered nucleosomes. We suggest that R1 elements have been selected to make this large deletion of the 28S gene to block the insertion of an upstream R2 element. These findings are consistent with the model that R1 and R2 are in competition for the limited number of insertion sites available within their host's genome.