R2 Elements Research Articles

The Nurse-Implemented Chronotherapeutic Bundle in Critically Ill Children, RESTORE Resilience (R2): Pilot Testing in a Two-Phase Cohort Study, 2017-2021.

Pilot test the nurse-led chronotherapeutic bundle in critically ill children, RESTORE Resilience (R2). A two-phase cohort study was carried out from 2017 to 2021. Two similarly sized and organized PICUs in the United States. Children 6 months to 17 years old who were mechanically ventilated for acute respiratory failure. R2 seven-item chronotherapeutic bundle, including: 1) replication of child's pre-hospital daily routine (i.e., sleep/wake, feeding, activity patterns); 2) cycled day-night light/sound modulation; 3) minimal effective sedation; 4) night fasting with bolus enteral daytime feedings; 5) early progressive mobility; 6) nursing care continuity; and 7) parent diaries. Children underwent environmental (light, sound) and patient (actigraphy, activity log, salivary melatonin, electroencephalogram) monitoring. Parents completed the Child's Daily Routine and Sleep Survey (CDRSS) and Family-Centered Care Scale. The primary outcome was post-extubation daytime activity consolidation (Daytime Activity Ratio Estimate [DARE]). Twenty baseline-phase (2017-2019) and 36 intervention-phase (2019-2021) participants were enrolled. During the intervention phase, nurses used the CDRSS to construct children's PICU schedules. Overall compliance with nurse-implemented R2 elements 1-5 increased from 18% (interquartile range, 13-30%) at baseline to 63% (53-68%) during the intervention phase (p < 0.001). Intervention participants were exposed to their pre-hospitalization daily routine (p = 0.002), cycled day-night light/sound modulation (p < 0.001), and early progressive mobility on more PICU days (p = 0.02). Sedation target identification, enteral feeding schedules, and nursing care continuity did not differ between phases. Parent diaries were seldom used. DARE improved during the intervention phase and was higher pre-extubation (median 62% vs. 53%; p = 0.04) but not post-extubation (62% vs. 57%; p = 0.56). In the PICU, implementation of an individualized nurse-implemented chronotherapeutic bundle is feasible. Children who received the R2 bundle had increased pre-extubation daytime activity consolidation compared to children receiving usual care. Given variation in protocol adherence, further R2 testing should include interprofessional collaboration, pragmatic trial design, and implementation science strategies.

All-RNA-mediated targeted gene integration in mammalian cells with rationally engineered R2 retrotransposons

All-RNA-mediated targeted gene integration methods, rendering reduced immunogenicity, effective deliverability with non-viral vehicles, and a low risk of random mutagenesis, are urgently needed for next-generation gene addition technologies. Naturally occurring R2 retrotransposons hold promise in this context due to their site-specific integration profile. Here, we systematically analyzed the biodiversity of R2 elements and screened several R2 orthologs capable of full-length gene insertion in mammalian cells. Robust R2 system gene integration efficiency was attained using combined donor RNA and protein engineering. Importantly, the all-RNA-delivered engineered R2 system showed effective integration activity, with efficiency over 60% in mouse embryos. Unbiased high-throughput sequencing demonstrated that the engineered R2 system exhibited high on-target integration specificity (99%). In conclusion, our study provides engineered R2 tools for applications based on hit-and-run targeted DNA integration and insights for further optimization of retrotransposon systems.

Branched Continued Fraction Expansions of Horn’s Hypergeometric Function H3 Ratios

The paper deals with the problem of construction and investigation of branched continued fraction expansions of special functions of several variables. We give some recurrence relations of Horn hypergeometric functions H3. By these relations the branched continued fraction expansions of Horn’s hypergeometric function H3 ratios have been constructed. We have established some convergence criteria for the above-mentioned branched continued fractions with elements in R2 and C2. In addition, it is proved that the branched continued fraction expansions converges to the functions which are an analytic continuation of the above-mentioned ratios in some domain (here domain is an open connected set). Application for some system of partial differential equations is considered.

Targeted gene knockin in zebrafish using the 28S rDNA-specific non-LTR-retrotransposon R2Ol

BackgroundAlthough most of long interspersed elements (LINEs), one class of non-LTR-retrotransposons, are integrated into the host genome randomely, some elements are retrotransposed into the specific sequences of the genomic regions, such as rRNA gene (rDNA) clusters, telomeric repeats and other repetitive sequenes. Most of the sequence-specific LINEs have been reported mainly among invertebrate species and shown to retrotranspose into the specific sequences in vivo and in vitro systems. Recenlty, 28S rDNA-specific LINE R2 elements are shown to be distributed among widespread vertebrate species, but the sequence-specific retrotransposition of R2 has never been demonstrated in vertebrates.ResultsHere we cloned a full length unit of R2 from medaka fish Oryzias latipes, named R2Ol, and engineered it to a targeted gene integration tool in zebrafish. By injecting R2Ol-encoding mRNA into zebrafish embryos, R2Ol retrotransposed precisely into the target site at high efficiency (98%) and was transmitted to the next generation at high frequency (50%). We also generated transgenic zebrafish carrying the enhanced green fluorescent protein (EGFP) reporter gene in 28S rDNA target by the R2Ol retrotransposition system.ConclusionsSequence-specific LINE retrotransposes into the precise sequence using target primed reverse transcription (TPRT), possibly providing an alternative and effective targeted gene knockin method in vertebrates.

R1 retrotransposons in the nucleolar organizers of Drosophila melanogaster are transcribed by RNA polymerase I upon heat shock

ABSTRACTThe ribosomal RNA genes (rDNA) of Drosophila melanogaster reside within centromere-proximal nucleolar organizers on both the X and Y chromosomes. Each locus contains between 200–300 tandem repeat rDNA units that encode 18S, 5.8S, 2S, and 28S ribosomal RNAs (rRNAs) necessary for ribosome biogenesis. In arthropods like Drosophila, about 60% of the rDNA genes have R1 and/or R2 retrotransposons inserted at specific sites within their 28S regions; these units likely fail to produce functional 28S rRNA. We showed earlier that R2 expression increases upon nucleolar stress caused by the loss of the ribosome assembly factor, Nucleolar Phosphoprotein of 140 kDa (Nopp140). Here we show that R1 expression is selectively induced by heat shock. Actinomycin D, but not α-amanitin, blocked R1 expression in S2 cells upon heat shock, indicating that R1 elements are transcribed by Pol I. A series of RT-PCRs established read-through transcription by Pol I from the 28S gene region into R1. Sequencing the RT-PCR products confirmed the 28S-R1 RNA junction and the expression of R1 elements within nucleolar rDNA rather than R1 elements known to reside in centromeric heterochromatin. Using a genome-wide precision run-on sequencing (PRO-seq) data set available at NCBI-GEO, we show that Pol I activity on R1 elements is negligible under normal non-heat shock conditions but increases upon heat shock. We propose that prior to heat shock Pol I pauses within the 5ʹ end of R1 where we find a consensus “pause button”, and that heat shock releases Pol I for read-through transcription farther into R1.

Globular domain structure and function of restriction-like-endonuclease LINEs: similarities to eukaryotic splicing factor Prp8

BackgroundR2 elements are a clade of early branching Long Interspersed Elements (LINEs). LINEs are retrotransposable elements whose replication can have profound effects on the genomes in which they reside. No crystal or EM structures exist for the reverse transcriptase (RT) and linker regions of LINEs.ResultsUsing limited proteolysis as a probe for globular domain structure, we show that the protein encoded by the Bombyx mori R2 element has two major globular domains: (1) a small globular domain consisting of the N-terminal zinc finger and Myb motifs, and (2) a large globular domain consisting of the RT, linker, and type II restriction-like endonuclease (RLE). Further digestion of the large globular domain occurred within the RT. Mapping these RT cleavages onto an updated model of the R2Bm RT indicated that the thumb of the RT was largely protected from proteolytic cleavage. The crystal structure of the large globular domain of Prp8, a eukaryotic splicing factor, was a major template used in building the R2Bm RT model, particularly the thumb region. The large fragment of Prp8 consists not only of a RT similar to R2Bm, but also an RLE and a linker connecting the two regions. The linker sequences adjacent to the RLE in LINEs and Prp8 share a set of two important α-helices and a (presumptive) knuckle/ββα structural motif that are closely associated with the thumb. The RLEs of LINEs and Prp8 share a unique catalytic core residue spacing as well as other key residues.ConclusionsThe protein encoded by RLE LINEs consists of two major globular domains. The larger of the two globular domain contains the RT, linker, and RLE and is similar to the large fragment of the spliceosomal protein Prp8. The similarities are suggestive of possible common ancestry.

The Structure and Methylation Level of the McMYB10 Promoter Determine the Leaf Color of Malus Crabapple

Anthocyanins are protective pigments that accumulate in plant organs such as fruits and leaves, and are nutritionally valuable components of the human diet. The MYB10 transcription factor (TF) plays an important role in regulating anthocyanin biosynthesis in Malus crabapple leaves. However, little is known about how the promoter regulates McMYB10 expression and influences the substantial variation in leaf anthocyanin accumulation and coloration that is observed in different crabapple cultivars. In this study, we analyzed leaf coloration, anthocyanin levels, and the expression levels of McMYB10 in the leaves of 15 crabapple cultivars with three leaf colors at various development stages, and showed that the expression of McMYB10 correlates positively with anthocyanin accumulation. We also examined the relationship between the number of R6 and R1 elements in the McMYB10 promoters of the different cultivars and the pigmentation of the new buds of spring-red cultivars, as well as the methylation level of the McMYB10 promoters at different development stages in three representative crabapple cultivars. The ratio of R6/R1 minisatellites in the promoters correlated with the color and anthocyanin accumulation in new crabapple buds, and we concluded that the differences in promoter structure and methylation level of the McMYB10 promoters coordinately affect the leaf color of crabapple cultivars.

Hybridogenesis and a potential case of R2 non-LTR retrotransposon horizontal transmission in Bacillus stick insects (Insecta Phasmida)

Horizontal transfer (HT) is an event in which the genetic material is transferred from one species to another, even if distantly related, and it has been demonstrated as a possible essential part of the lifecycle of transposable elements (TEs). However, previous studies on the non-LTR R2 retrotransposon, a metazoan-wide distributed element, indicated its vertical transmission since the Radiata-Bilateria split. Here we present the first possible instances of R2 HT in stick insects of the genus Bacillus (Phasmida). Six R2 elements were characterized in the strictly bisexual subspecies B. grandii grandii, B. grandii benazzii and B. grandii maretimi and in the obligatory parthenogenetic taxon B. atticus. These elements were compared with those previously retrieved in the facultative parthenogenetic species B. rossius. Phylogenetic inconsistencies between element and host taxa, and age versus divergence analyses agree and support at least two HT events. These HT events can be explained by taking into consideration the complex Bacillus reproductive biology, which includes also hybridogenesis, gynogenesis and androgenesis. Through these non-canonical reproductive modes, R2 elements may have been transferred between Bacillus genomes. Our data suggest, therefore, a possible role of hybridization for TEs survival and the consequent reshaping of involved genomes.

Physical-Chemical Properties of Bi13-XMexMo5O34±δ (Me = Mg, Ca, Sr, Ba) Solid Solutions

Single phase samples of Bi13-xMexMo5O34± δ, Me = Mg, Ca, Sr, Ba (0.0 £ x £ 1.0) series were indexed according to triclinic Bi13Mo5O34.5 structure (if the concentration of the dopant was small) or monoclinic Bi13Mo5O34.5 structure (if the concentration of dopant was large). These ranges are various for different dopants because of ratio difference of ionic radii of the dopants and the Bi ion: rMg << rBi+3; rCa, rSr≈ rBi+3; rBa> rBi+3 (the values of ionic radii with coordination number = 8 are: rMg= 0.89Å, rCa= 1.12 Å, rSr = 1.26 Å, rBa= 1.42 Å; rBi+3= 1.17 Å. For all substituting metals the increase of dopant concentration results in the rise of symmetry and in stabilization of the monoclinic form at room temperature. HTXPRD was used for investigation of thermal properties of Bi13-xMexMo5O34± δ, Me = Mg, Ca, Sr, Ba solid solutions. The dependences of volume and unit cell parameters vs. temperature were obtained. The phase transition to the monoclinic modification was detected for the samples corresponding to the triclinic modification at room temperature. This phase transition causes stepwise change of the unit cell parameters and unit cell compression in the moment of transition. No impurities were detected within the whole temperature range, the unit cell parameters curves coincided at heating and cooling. The unit cell parameters vs. temperature curves are satisfactorily fitted by the linear equation. The decrease of dopant concentration leads to the phase transition temperature (Tt à m) decrease, for example Tt à m=~593 K for Bi12.9Ca0.1Mo5O34± δ and Tt à m=~523 K for Bi12.6Ca0.4Mo5O34± δ. The EDX- and AAS- analysis detected that the concentration of dopants is close to theoretical; concentration of bismuth and molybdenum couldn’t be determined because of analytical peaks overlapping. The concentration of all dopants confirms the theoretical formula within the experimental errors. Hydrostatic weighting showed high density of the sintered pellets, experimental density reaches values more than 97% of theoretical (X-ray) density. The impedance spectroscopy was used for investigation of electro-conductive properties of the ceramic samples of substituted Bi13Mo5O34±δ. For analysis of impedance plots the equivalent electrical circuits method was used (Zview software, Version 2.6b, Scribner Associates, Inc.). At high temperatures (higher than~ 823-873 K) the impedance plots of all investigated complex oxides correspond to non-central semicircle or two separated semicircles (Fig.8 a), the low intercept is non-equal to zero. The equivalent electrical circuits for high temperature region is shown in Fig.8 a. It can be described as R1 – R2(CPE1) – R3(CPE2) serial connection, where R2 (CPE1) and R3 (CPE2) fragments are parallel connections of resistor (R) and constant phase element (CPE). The “capacitance” of CPE1 and CPE2 is about 10-5-10-6 F, which is typical for oxide systems. Therefore R2(CPE1) and R3(CPE2) parallel connections correspond to electrochemical processes at the electrodes, and R1 describes total resistance of the sample. At low temperatures the complex plane plot and the equivalent electrical circuits change (Fig.8 b). The impedance plot in this case consists of one separated and two adjacent semi-circles. The low intercept of the left (high-frequency) semicircle is equal to zero. The equivalent electrical circuits can be described as R1(C1)–R2(CPE2)–R3(CPE3) serial connection, where R2 (CPE2) and R3 (CPE3) fragments are parallel connections of resistor (R) and constant phase element (CPE) and R1(C1) is a parallel connection of resistor and capacitor. The R1(C1) connection describes the smallest left semi-circle, the C1 capacitance value is about 10-11 F, therefore R1 can be attributed to the total conductivity of the sample. R2(CPE2) element can be attributed to electrochemical processes at the electrodes (the “capacitance” value of CPE2 is about 10-6 F), R3 (CPE3) can describe complicated diffusion processes at low temperatures (the “capacitance” value of CPE2 is about 10-5 F). The present work concerns investigation of the Bi13-xMexMo5O34±δ, Me=Mg, Ca, Sr, Ba complex oxides series. The solid solutions ranges and modifications limits for mentioned solid solutions are defined by ratio of ion radii of isolated Bi and dopant ions. The increase of the dopant concentration leads to the unit cell compression and change of crystal system from triclinic to monoclinic. The deviation from linear function of unit sell parameters and volume vs temperature are observed above 850-900 K. It agrees with activation energy changing at ~850 K detected for samples with unchanging monoclinic structure. In general the modification of electroconductive properties depend on radius of the dopant ion. The assumption of the presence of two forms of monoclinic modifications with different oxygen sublattice structure was suggested. The work was financially supported by Russian Fund of Fundamental Research, grant 14-03-92605.

Dead Element Replicating: Degenerate R2 Element Replication and rDNA Genomic Turnover in the Bacillus rossius Stick Insect (Insecta: Phasmida)

R2 is an extensively investigated non-LTR retrotransposon that specifically inserts into the 28S rRNA gene sequences of a wide range of metazoans, disrupting its functionality. During R2 integration, first strand synthesis can be incomplete so that 5’ end deleted copies are occasionally inserted. While active R2 copies repopulate the locus by retrotransposing, the non-functional truncated elements should frequently be eliminated by molecular drive processes leading to the concerted evolution of the rDNA array(s). Although, multiple R2 lineages have been discovered in the genome of many animals, the rDNA of the stick insect Bacillus rossius exhibits a peculiar situation: it harbors both a canonical, functional R2 element (R2Brfun) as well as a full-length but degenerate element (R2Brdeg). An intensive sequencing survey in the present study reveals that all truncated variants in stick insects are present in multiple copies suggesting they were duplicated by unequal recombination. Sequencing results also demonstrate that all R2Brdeg copies are full-length, i. e. they have no associated 5' end deletions, and functional assays indicate they have lost the active ribozyme necessary for R2 RNA maturation. Although it cannot be completely ruled out, it seems unlikely that the degenerate elements replicate via reverse transcription, exploiting the R2Brfun element enzymatic machinery, but rather via genomic amplification of inserted 28S by unequal recombination. That inactive copies (both R2Brdeg or 5'-truncated elements) are not eliminated in a short term in stick insects contrasts with findings for the Drosophila R2, suggesting a widely different management of rDNA loci and a lower efficiency of the molecular drive while achieving the concerted evolution.

Identification of RNA binding motifs in the R2 retrotransposon-encoded reverse transcriptase.

R2 non-LTR retrotransposons insert at a specific site in the 28S rRNA genes of many animal phyla. R2 elements encode a single polypeptide with reverse transcriptase, endonuclease and nucleic acid binding domains. Integration involves separate cleavage of the two DNA strands at the target site and utilization of the released 3′ ends to prime DNA synthesis. Critical to this integration is the ability of the protein to specifically bind 3′ and 5′ regions of the R2 RNA. In this report, alanine mutations in two conserved motifs N-terminal to the reverse transcriptase domain were generated and shown to result in proteins that retained the ability to cleave the first strand of the DNA target, to reverse transcribe RNA from an annealed primer and to displace annealed RNA when using DNA as a template. However, the mutant proteins had greatly reduced ability to bind 3′ and 5′ RNA in mobility shift assays, use the DNA target to prime reverse transcription and conduct second-strand DNA cleavage. These motifs thus appear to participate in all activities of the R2 protein known to require specific RNA binding. The similarity of these R2 RNA binding motifs to those of telomerase and group II introns is discussed.

Preferential Occupancy of R2 Retroelements on the B Chromosomes of the Grasshopper Eyprepocnemis plorans

R2 non-LTR retrotransposons exclusively insert into the 28S rRNA genes of their host, and are expressed by co-transcription with the rDNA unit. The grasshopper Eyprepocnemis plorans contains transcribed rDNA clusters on most of its A chromosomes, as well as non-transcribed rDNA clusters on the parasitic B chromosomes found in many populations. Here the structure of the E. plorans R2 element, its abundance relative to the number of rDNA units and its retrotransposition activity were determined. Animals screened from five populations contained on average over 12,000 rDNA units on their A chromosomes, but surprisingly only about 100 R2 elements. Monitoring the patterns of R2 insertions in individuals from these populations revealed only low levels of retrotransposition. The low rates of R2 insertion observed in E. plorans differ from the high levels of R2 insertion previously observed in insect species that have many fewer rDNA units. It is proposed that high levels of R2 are strongly selected against in E. plorans, because the rDNA transcription machinery in this species is unable to differentiate between R2-inserted and uninserted units. The B chromosomes of E. plorans contain an additional 7,000 to 15,000 rDNA units, but in contrast to the A chromosomes, from 150 to over 1,500 R2 elements. The higher concentration of R2 in the inactive B chromosomes rDNA clusters suggests these chromosomes can act as a sink for R2 insertions thus further reducing the level of insertions on the A chromosomes. These studies suggest an interesting evolutionary relationship between the parasitic B chromosomes and R2 elements.

ReducedrDNACopy Number Does Not Affect “Competitive” Chromosome Pairing inXYYMales ofDrosophila melanogaster

The ribosomal DNA (rDNA) arrays are causal agents in X-Y chromosome pairing in meiosis I of Drosophila males. Despite broad variation in X-linked and Y-linked rDNA copy number, polymorphisms in regulatory/spacer sequences between rRNA genes, and variance in copy number of interrupting R1 and R2 retrotransposable elements, there is little evidence that different rDNA arrays affect pairing efficacy. I investigated whether induced rDNA copy number polymorphisms affect chromosome pairing in a “competitive” situation in which complex pairing configurations were possible using males with XYY constitution. Using a common normal X chromosome, one of two different full-length Y chromosomes, and a third chromosome from a series of otherwise-isogenic rDNA deletions, I detected no differences in X-Y or Y-Y pairing or chromosome segregation frequencies that could not be attributed to random variation alone. This work was performed in the context of an undergraduate teaching program at Texas A&M University, and I discuss the pedagogical utility of this and other such experiments.

Evolution of the R2 Retrotransposon Ribozyme and Its Self-Cleavage Site

R2 is a non-long terminal repeat retrotransposon that inserts site-specifically in the tandem 28S rRNA genes of many animals. Previously, R2 RNA from various species of Drosophila was shown to self-cleave from the 28S rRNA/R2 co-transcript by a hepatitis D virus (HDV)-like ribozyme encoded at its 5' end. RNA cleavage was at the precise 5' junction of the element with the 28S gene. Here we report that RNAs encompassing the 5' ends of R2 elements from throughout its species range fold into HDV-like ribozymes. In vitro assays of RNA self-cleavage conducted in many R2 lineages confirmed activity. For many R2s, RNA self-cleavage was not at the 5' end of the element but at 28S rRNA sequences up to 36 nucleotides upstream of the junction. The location of cleavage correlated well with the types of endogenous R2 5' junctions from different species. R2 5' junctions were uniform for most R2s in which RNA cleavage was upstream in the rRNA sequences. The 28S sequences remaining on the first DNA strand synthesized during retrotransposition are postulated to anneal to the target site and uniformly prime second strand DNA synthesis. In species where RNA cleavage occurred at the R2 5' end, the 5' junctions were variable. This junction variation is postulated to result from the priming of second strand DNA synthesis by chance microhomologies between the target site and the first DNA strand. Finally, features of R2 ribozyme evolution, especially changes in cleavage site and convergence on the same active site sequences, are discussed.

On existence of Budaghyan–Carlet APN hexanomials

Budaghyan and Carlet (2008) [4] constructed a family of almost perfect nonlinear (APN) hexanomials over a field with r2 elements, and with terms of degrees r+1, s+1, rs+1, rs+r, rs+s, and r+s, where r=2m and s=2n with GCD(m,n)=1. The construction requires a certain technical condition, which was verified empirically in a finite number of examples. Bracken, Tan, and Tan (2011) [1] proved that the condition holds when m≡2 or 4(mod6). In this article, we prove that the construction of Budaghyan and Carlet produces APN polynomials for all relatively prime values of m and n.More generally, if GCD(m,n)=k⩾1, Budaghyan and Carlet showed that the nonzero derivatives of the hexanomials are 2k-to-one maps from Fr2 to Fr2, provided the same technical condition holds. We prove that their construction produces polynomials with this property for all m and n.

Non-LTR R2 Element Evolutionary Patterns: Phylogenetic Incongruences, Rapid Radiation and the Maintenance of Multiple Lineages

Retrotransposons of the R2 superclade specifically insert within the 28S ribosomal gene. They have been isolated from a variety of metazoan genomes and were found vertically inherited even if their phylogeny does not always agree with that of the host species. This was explained with the diversification/extinction of paralogous lineages, being proved the absence of horizontal transfer. We here analyze the widest available collection of R2 sequences, either newly isolated from recently sequenced genomes or drawn from public databases, in a phylogenetic framework. Results are congruent with previous analyses, but new important issues emerge. First, the N-terminal end of the R2-B clade protein, so far unknown, presents a new zinc fingers configuration. Second, the phylogenetic pattern is consistent with an ancient, rapid radiation of R2 lineages: being the estimated time of R2 origin (850–600 Million years ago) placed just before the metazoan Cambrian explosion, the wide element diversity and the incongruence with the host phylogeny could be attributable to the sudden expansion of available niches represented by host’s 28S ribosomal genes. Finally, we detect instances of coexisting multiple R2 lineages showing a non-random phylogenetic pattern, strongly similar to that of the “library” model known for tandem repeats: a collection of R2s were present in the ancestral genome and then differentially activated/repressed in the derived species. Models for activation/repression as well as mechanisms for sequence maintenance are also discussed within this framework.

A Population Genetic Model for the Maintenance of R2 Retrotransposons in rRNA Gene Loci

R2 retrotransposable elements exclusively insert into the tandemly repeated rRNA genes, the rDNA loci, of their animal hosts. R2 elements form stable long-term associations with their host, in which all individuals in a population contain many potentially active copies, but only a fraction of these individuals show active R2 retrotransposition. Previous studies have found that R2 RNA transcripts are processed from a 28S co-transcript and that the likelihood of R2-inserted units being transcribed is dependent upon their distribution within the rDNA locus. Here we analyze the rDNA locus and R2 elements from nearly 100 R2-active and R2-inactive individuals from natural populations of Drosophila simulans. Along with previous findings concerning the structure and expression of the rDNA loci, these data were incorporated into computer simulations to model the crossover events that give rise to the concerted evolution of the rRNA genes. The simulations that best reproduce the population data assume that only about 40 rDNA units out of the over 200 total units are actively transcribed and that these transcribed units are clustered in a single region of the locus. In the model, the host establishes this transcription domain at each generation in the region with the fewest R2 insertions. Only if the host cannot avoid R2 insertions within this 40-unit domain are R2 elements active in that generation. The simulations also require that most crossover events in the locus occur in the transcription domain in order to explain the empirical observation that R2 elements are seldom duplicated by crossover events. Thus the key to the long-term stability of R2 elements is the stochastic nature of the crossover events within the rDNA locus, and the inevitable expansions and contractions that introduce and remove R2-inserted units from the transcriptionally active domain.

Ribosomal DNA Organization Before and After Magnification in Drosophila melanogaster

In all eukaryotes, the ribosomal RNA genes are stably inherited redundant elements. In Drosophila melanogaster, the presence of a Ybb(-) chromosome in males, or the maternal presence of the Ribosomal exchange (Rex) element, induces magnification: a heritable increase of rDNA copy number. To date, several alternative classes of mechanisms have been proposed for magnification: in situ replication or extra-chromosomal replication, either of which might act on short or extended strings of rDNA units, or unequal sister chromatid exchange. To eliminate some of these hypotheses, none of which has been clearly proven, we examined molecular-variant composition and compared genetic maps of the rDNA in the bb(2) mutant and in some magnified bb(+) alleles. The genetic markers used are molecular-length variants of IGS sequences and of R1 and R2 mobile elements present in many 28S sequences. Direct comparison of PCR products does not reveal any particularly intensified electrophoretic bands in magnified alleles compared to the nonmagnified bb(2) allele. Hence, the increase of rDNA copy number is diluted among multiple variants. We can therefore reject mechanisms of magnification based on multiple rounds of replication of short strings. Moreover, we find no changes of marker order when pre- and postmagnification maps are compared. Thus, we can further restrict the possible mechanisms to two: replication in situ of an extended string of rDNA units or unequal exchange between sister chromatids.

28S junctions and chimeric elements of the rDNA targeting non-LTR retrotransposon R2 in crustacean living fossils (Branchiopoda, Notostraca)

The 28S rRNA genes of several metazoans are interrupted by site-specific targeting non-LTR retrotransposons, such as R2. R2 elements have been deeply analyzed but aspects of their retrotransposition mechanism and the origin of the wide diversity observed are still debated. We characterized six new R2 lineages in four tadpole shrimp species (Notostraca), samples deriving from a parthenogenetic population of Triops cancriformis (R2Tc_it) and from bisexual Lepidurus populations of L. lubbocki (R2Ll), L. couesii (R2LcA, R2LcB, R2LcC) and L. arcticus (R2La). All elements fit the canonical R2 structure but R2Ll which turned out to be a chimera with an additional ORF originating from another R2. Consistently with data on LINEs, R2Ll could be the result of recombination due to reverse transcriptase template jump. The analysis of 28S/R2 5′ end junctions further suggests aberrant homologous recombination, as observed in RNA viruses.

R2 and R2/R1 hybrid non-autonomous retrotransposons derived by internal deletions of full-length elements

BackgroundR2 is a non-long terminal repeat (non-LTR) retrotransposable element that inserts site specifically into the 28S genes of the ribosomal (r)RNA gene loci. Encoded at the 5' end is a ribozyme that generates the precise 5' end by self-cleavage of a 28S gene cotranscript. Sequences at the 3' end are necessary for the R2 protein to bind RNA and initiate the target primed reverse transcription (TPRT) reaction. These minimal RNA requirements suggested that if recombination/DNA repair conjoined the 5' and 3' ends of R2, the result would be a non-autonomous element that could survive as long as autonomous R2 elements supplied the TPRT activity.ResultsA PCR-based survey of 39 Drosophila species aided by genomic sequences from 12 of these species revealed two types of non-autonomous elements. We call these elements SIDEs (for ‘Short Internally Deleted Elements’). The first consisted of a 5' ribozyme and a 3' end of an R2 element as predicted. Variation at the 5' junctions of the R2 SIDE copies was typical for R2 insertions suggesting their propagation by TPRT. The second class of SIDE contained sequences from R1 elements, another non-LTR retrotransposon that inserts into rRNA gene loci. These insertions had an R2 ribozyme immediately upstream of R1 3' end sequences. These hybrid SIDEs were inserted at the R1 site with 14 bp target site duplications typical of R1 insertions suggesting they used the R1 machinery for retrotransposition. Finally, the survey revealed examples of U12 small nuclear (sn)RNA and tRNA sequences at the 5' end of R2 elements suggesting the R2 reverse transcriptase can template jump from the R2 transcript to a second RNA during TPRT.ConclusionsThe R2 SIDE and R2/R1 hybrid SIDEs are rare examples of non-autonomous retrotransposons in the Drosophila genome. Associated non-autonomous elements and in vivo template jumps are two additional characteristics R2 shares with other non-LTR retrotransposons such as mammalian L1s. Analysis of the hybrid SIDEs provides supporting evidence that R1 elements, like R2 elements, recognize their 3' untranslated region (UTR) sequences and, thus, belong to the stringent class of non-LTR elements.