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.     

Characterization of active R2 retrotransposition in the rDNA locus of Drosophila simulans

The rRNA gene (rDNA) loci of all arthropod lineages contain non-LTR retrotransposable elements that have evolved to specifically insert into the 28S rRNA genes. Extensive in vitro experiments have been conducted to investigate the mechanism of R2 retrotransposition but little is known of the insertion frequency or cellular factors that might regulate R2 activity. In this article, isofemale lines obtained from a population of Drosophila simulans were surveyed for recent R2 insertions. Within most lines, all individuals showed the same collection of R2 insertions, providing no evidence for recent R2 activity. However, in a few of the isofemale lines, virtually all individuals differed in their R2 insertion profiles. The descendants of individual pairs of flies from these "active lines" rapidly accumulated new insertions. The frequent insertion of new R2 elements was associated with the elimination of old R2 elements from the rDNA locus. The existence of lines in which R2 retrotransposes frequently and lines in which the elements appear dormant suggests that cellular mechanisms that can regulate the activity of R2 exist. Retrotransposition activity was correlated with the number of full-length R2 elements but not with the size of the rDNA locus or the number of uninserted units.     

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.     

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.     

A single lineage of r2 retrotransposable elements is an active, evolutionarily stable component of the Drosophila rDNA locus

R2 elements are non-long-terminal-repeat (non-LTR) retrotransposons that insert specifically in the 28S rRNA genes of many insects. Previous reports concerning this element in the genus Drosophila have suggested that R2 elements are absent from many species of this genus, particularly those species from the subgenus Drosophila. In this report, we present an extensive study of the distribution and evolution of R2 elements in Drosophila. A PCR survey of 59 species from 23 species groups of the two major Drosophila subgenera found that R2 elements are present in all but two species of the melanogaster species subgroup. Phylogenetic analysis based on partial nucleotide sequences of R2 elements from 23 species demonstrates that the relationships of R2 elements are congruent with those of the Drosophila species phylogeny, suggesting that these elements have been vertically inherited since the divergence of this genus some 60 MYA. Sequence variation between different copies of R2 elements within each species was less than 0.16%, indicating that these elements are undergoing concerted evolution similar to that of the 28S genes. Several properties of the R2 sequences suggest that these elements depend on retrotransposition in addition to simple recombination to remain within the rDNA locus: the rates of synonymous substitutions averaged 4.8 times the rate of replacement substitutions, 82 of 83 R2 copies partially sequenced contained intact open reading frames, and, finally, length variation associated with the poly(A) 3' tails indicated that many R2 copies are the direct result of retrotransposition.      

Monitoring the mode and tempo of concerted evolution in the Drosophila melanogaster rDNA locus

Non-LTR retrotransposons R1 and R2 have persisted in rRNA gene loci (rDNA) since the origin of arthropods despite their continued elimination by the recombinational mechanisms of concerted evolution. This study evaluated the short-term evolutionary dynamics of the rDNA locus by measuring the divergence among replicate Drosophila melanogaster lines after 400 generations. The total number of rDNA units on the X chromosome of each line varied from 140 to 310, while the fraction of units inserted with R1 and R2 retrotransposons ranged from 37 to 65%. This level of variation is comparable to that found in natural population surveys. Variation in locus size and retrotransposon load was correlated with large changes in the number of uninserted and R1-inserted units, yet the numbers of R2-inserted units were relatively unchanged. Intergenic spacer (IGS) region length variants were also used to evaluate changes in the rDNA loci. All IGS length variants present in the lines showed significant increases and decreases of copy number. These studies, combined with previous data following specific R1 and R2 insertions in these lines, help to define the type and distribution, both within the locus and within the individual units, of recombinational events that give rise to the concerted evolution of the rDNA locus.     

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.