Length polymorphism of integrated copies of R1 and R2 retrotransposons in the German cockroach (Blattella germanica) as a potential marker for population and phylogenetic studies

Using polymerase chain reaction technique with primers flanking target sites of retrotransposons R1 and R2, integrated copies of these transposable elements were amplified in various cockroach species (Blattodea). It was shown that each species has a unique pattern of “5′-truncated copies” with the definite set of amplified fragments of different lengths. Intraspecies polymorphism was revealed in analysis of German cockroach specimens obtained upon individual mating. This is the first report providing results of identifying, cloning, and sequencing extended fragments (5′-truncated copies) of Blattella germanica R1 and R2 retrotransposons. It may be assumed that patterns of 5′-truncated copies of R1 and R2 elements can be used as markers in population and phylogenetic studies. Moreover, cloned and sequenced fragments will be employed in our further studies for screening of the German cockroach genomic library in order to detect full-length copies in this class transposable elements.     

R1 and R2 retrotransposons of German cockroach Blattella germanica: comparative analysis of 5' truncated copies integrated into genome

This is the first report providing results on identification, cloning, and sequencing of extended fragments (5'-truncated copies) of R1 and R2 retrotransposons integrated into Blattella germanica genome. Comparative structural analysis of the received clones revealed two distinct subfamilies of R1 elements. However, all B. germanica R1 clones have two common features: poly(T) tails and similar target site duplications. Nucleotide structure and organization of five sequenced R2 fragments was similar. Analysis of R2 nucleotide sequences revealed typical deletions at the 3'end of target sites and lack of homopolynucleotides tails.     

Analysis of the inheritance patterns of 5'-truncated copies of the German cockroach R2 retroposons in individual crosses

The inheritance patterns of the 5'-truncated copies of R2 retroposons were analyzed in individual crosses of the German cockroach. The recombination level within the cluster of ribosomal RNA genes was determined. It was demonstrated that only the frequencies of individual variants of 5'-truncated retroposon copies are appropriate for population analysis rather than the patterns characterizing individual X chromosomes. The methodical approach used in the work is convenient for studying the genetic variation in ribosomal DNA multigene families.     

Domain organization of the ORF2 C-terminal region of the German cockroach retroposon R1

Using cosmid vector, a gene library of German cockroach Blattella germanica was constructed. From this library, clones containing full-length copies of two subfamilies of R1 retroposons were selected. Retroposons R1 of German cockroach belonging to different subfamilies were shown to be different in domain organization of the ORF2 C-terminal region. For the first time, retroposons transmembrane domains were identified in the sequences of R1. It was demonstrated that two retroposon R1 subfamilies of German cockroach arose as a result of intragenomic divergence rather than via horizontal transfer of alien mobile element into cockroach genome. The differences in domain organization appeared not as a result of saltatory recombination processes, but as a consequence of gradual mutation accumulation, which led to either degeneration, or to domain formation.     

R1 and R2 retrotransposons of German cockroach <Emphasis Type="Italic">Blatella germanica</Emphasis>: A comparative study of 5′-truncated copies integrated into the genome

For the first time, extended fragments (5′-truncated copies) of R1 and R2 retrotransposons integrated into the Blattella germanica genome were identified, cloned, and sequenced. Structural comparison of the clones revealed two distinct R1 subfamilies. However, all R1 clones had two common features: poly(T) tails and similar target site duplications. R1 retrotransposons are the first known mobile elements with poly(T) tails on the 3′-ends. The structure and nucleotide sequences of five sequenced R2 fragments were similar to each other. Nucleotide sequence analysis of R2 retrotransposons revealed typical deletions at the 3′ ends of the target sites and the lack of homopolynucleotide tails.     

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.     

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.     

Analysis of the inheritance patterns of 5′-truncated copies of the German cockroach R2 retroposons in individual crosses

The inheritance patterns of the 5′-truncated copies of R2 retroposons were analyzed in individual crosses of the German cockroach. The recombination level within the cluster of ribosomal RNA genes was determined. It was demonstrated that only the frequencies of individual variants of 5′-truncated retroposon copies are appropriate for population analysis rather than the patterns characterizing individual X chromosomes. The methodical approach used in the work is convenient for studying the genetic variation in ribosomal DNA multigene families.     

The first complete <Emphasis Type="Italic">Mag</Emphasis> family retrotransposons discovered in <Emphasis Type="Italic">Drosophila</Emphasis>

A retrotransposon of the Mag family was found in the Drosophila simulans genome for the first time. We also identified novel transposable elements representing the Mag family in seven Drosophila species. The high similarity between the 3’ and 5’ long terminal repeats in the found copies of transposable elements indicates that their retrotransposition has occurred relatively recently. Thus, the Mag family of retrotransposons is quite common for the genus Drosophila.     

Evolutionary forces generating sequence homogeneity and heterogeneity within retrotransposon families

Genome projects allow us to sample copies of a retrotransposon sequence family residing in a host genome. The variation in DNA sequence between these individual copies will reflect the evolutionary process that has spread the sequences through the genome. Here I review quantitatively the expected diversity of elements belonging to a transposable genetic element family. I use a simple neutral model for replicative mobile DNAs such as retrotransposons to predict the extent of sequence variability between members of a single family of transposable elements, both within and between species. The effects of horizontal transfer are also explored. I also consider the impact on these distributions of an increase in transposition rate arising from a mutational change in copy of the sequence. In addition, I consider the question of the interaction between retrotransposons and their hosts, and the causes of the abundance of transposable elements in the genomes that they occupy.     

Molecular identification of the active ninja retrotransposon and the inactive aurora element in Drosophila simulans and D. melanogaster.

How transposable elements evolve is a key facet in understanding of spontaneous mutation and genomic rearrangements in various organisms. One of the best ways to approach this question is to study a newly evolved transposable element whose presence is restricted to a specific population or strain. The retrotransposons ninja and aurora may provide insights into the process of their evolution, because of their contrasting characteristics, even though they show high sequence identity. The ninja retrotransposon was found in a Drosophila simulans strain in high copy number and is potent in transposition. On the other hand, aurora elements are distributed widely among the species belonging to the Drosophila melanogaster species complex, but are immobile at least in D. melanogaster. In order to distinguish the two closely resembled retrotransposons by molecular means, we determined and compared DNA sequence of the elements, and identified characteristic internal deletions and nucleotide substitutions in 5'-long terminal repeats (LTR). Analyses of the structure of ninja homologs and LTR sequences amplified from both genomic and cloned DNA revealed that the actively transposable ninja elements were present only in D. simulans strains, but inactive aurora elements exist in both D. melanogaster and D. simulans.     

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.