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Retrotransposons Provide an Evolutionarily Robust Non-Telomerase Mechanism to Maintain Telomeres
Abstract
Telomere molecular biology is far more complex than originally thought. Understanding biological systems is aided by study of evolutionary variants, and Drosophila telomeres are remarkable variants. Drosophila lack telomerase and the arrays of simple repeats generated by telomerase in almost all other organisms; instead, Drosophila telomeres are long tandem arrays of two non-LTR retrotransposons, HeT-A and TART. These are the first transposable elements found to have a bona fide role in cell structure, revealing an unexpected link between telomeres and what is generally considered to be parasitic DNA. In addition to providing insight into the cellular functions performed by telomeres, analysis of HeT-A and TART is providing insight into the evolution of chromosomes, retrotransposons, and retroviruses. Recent studies show that retrotransposon telomeres constitute a robust system for maintaining chromosome ends. These telomeres are now known to predate the separation of extant Drosophila species, allowing ample time for elements and hosts to coevolve interesting mechanisms.
... Somatic cells and cells with low proliferative capacity lack enzyme activity, this is the reason why telomerase activity is extensively studied as a potential target for antitumor therapy [9,10]. However, apart from telomerase there are other mechanism used to maintain chromosome length; in some organisms, such as the fruit fly Drosophila melanogaster, retrotransposon-like elements are alternatively used to replenish the DNA at the ends of chromosomes [11]. As reported by Miriam Aparecida Giardini et al. [12], under certain circumstances, yeast and human cells that lack telomerase activity, as well as some telomerase-negative tumor lineages, are able to maintain their telomeres using a recombination-based DNA replication mechanism known as alternative lengthening of telomeres (ALT) [13,14]. ...
... There exist some alternative mechanisms which are activated to maintain telomere length in the absence of telomerase activity. These mechanisms are principally based on recombination events that come into play to amplify or reorganize previously existing telomeric sequences [73,74], and the mechanisms seem to be complementary to both the telomerase method and the method occupied in "retro transposition" [11]. The alternative mechanisms were first observed in budding yeasts that were able to survive and achieve telomere elongation despite lack of a functional telomerase [12]. ...
... Other telomere-lengthening mechanisms also exist in the absence of telomerase activity. These mechanisms have been reviewed in details in previous reports [11]. The mosquito fly Anopheles gambiae,thevinegarflyDrosophila melanogaster, and some species of plants are other examples of organisms that use alternative telomere elongation mechanism by using recombination [11]. ...
... However, not all adaptive molecular domestication events necessitate TE immobilization; in rare cases, essential host functions rely on retention of the mobilization machinery that promotes recurrent TE insertions into host DNA. The noncanonical telomere elongation mecha-nism of Drosophila is exemplary (Pardue and DeBaryshe 2003;Casacuberta 2017). ...
... The most extreme example of this cooption is found in Drosophila melanogaster, whose telomeres harbor no telomerase-added repeats. In fact, the 220-Myr-old "true fly" insect Order, Diptera, completely lacks the genes encoding the telomerase holoenzyme (Pardue and DeBaryshe 2003;Casacuberta 2017). Instead, D. melanogaster harbors three telomere-specialized retrotransposons-HeT-A, TART, and TAHRE-that preserve distal, unique sequence (Pardue and DeBaryshe 2011). ...
... Candidate telomere-specialized retrotransposons identified in the melanogaster species group D. melanogaster encodes telomere-specialized, non-LTR retrotransposons that increase in copy number by a copy-and-paste mechanism (Pardue and DeBaryshe 2011). Transcripts encoded by these elements are localized, reverse-transcribed, and integrated at the terminal nucleotides of chromosome ends, resulting in stereotypical head-to-tail arrays (Pardue and DeBaryshe 2003). Full-length, autonomous retrotransposons typically contain two open reading frames (ORFs) between the variable 5 ′ and 3 ′ UTRs. ...
In most eukaryotes, telomerase counteracts chromosome erosion by adding repetitive sequence to terminal ends. Drosophila melanogaster instead relies on specialized retrotransposons that insert exclusively at telomeres. This exchange of goods between host and mobile element-wherein the mobile element provides an essential genome service and the host provides a hospitable niche for mobile element propagation-has been called a "genomic symbiosis." However, these telomere-specialized, jockey family retrotransposons may actually evolve to "selfishly" overreplicate in the genomes that they ostensibly serve. Under this model, we expect rapid diversification of telomere-specialized retrotransposon lineages and, possibly, the breakdown of this ostensibly symbiotic relationship. Here we report data consistent with both predictions. Searching the raw reads of the 15-Myr-old melanogaster species group, we generated de novo jockey retrotransposon consensus sequences and used phylogenetic tree-building to delineate four distinct telomere-associated lineages. Recurrent gains, losses, and replacements account for this retrotransposon lineage diversity. In Drosophila biarmipes, telomere-specialized elements have disappeared completely. De novo assembly of long reads and cytogenetics confirmed this species-specific collapse of retrotransposon-dependent telomere elongation. Instead, telomere-restricted satellite DNA and DNA transposon fragments occupy its terminal ends. We infer that D. biarmipes relies instead on a recombination-based mechanism conserved from yeast to flies to humans. Telomeric retrotransposon diversification and disappearance suggest that persistently "selfish" machinery shapes telomere elongation across Drosophila rather than completely domesticated, symbiotic mobile elements.
... Drosophila telomeres were the first to be named as such, but life is full of surprises and Muller's first-described telomeres would turn out to be an exception to the otherwise highly-conserved eukaryote telomeres [4]. Drosophila telomeres are composed of repeated sequences like its telomerase counterparts, but in this case, the repeats expand from six to twelve kilobases (kb) in length and have a complex internal structure. ...
... This phenomenon allows, therefore, an unexpected plasticity to those sequences occupying this genome location. In addition, the study of the similarities between telomerase and retrotransposons has brought to light the mechanistic connections that are also supported from an evolutionary point of view [4]. Because TEs and viral genomes keep structural and evolutionary similarities, it is not unreasonable to consider possible TE strategies for telomere and genome integration in general to obtain clues to better understand viral integration at telomeres. ...
... In the case of HeT-A and TAHRE, both 5′ and 3′UTRs contain sequences working as sense promoters. Sequential deletions from the entire 3′ and 5′UTR were used to define the strongest promoter of the element [4,21]. This apparently shocking combination makes complete sense if, instead of looking at the individual TEs, one pictures the telomere sequence in Drosophila. ...
Drosophila and extant species are the best-studied telomerase exception. In this organism, telomere elongation is coupled with targeted retrotransposition of Healing Transposon (HeT-A) and Telomere Associated Retrotransposon (TART) with sporadic additions of Telomere Associated and HeT-A Related (TAHRE), all three specialized non-Long Terminal Repeat (non-LTR) retrotransposons. These three very special retroelements transpose in head to tail arrays, always in the same orientation at the end of the chromosomes but never in interior locations. Apparently, retrotransposon and telomerase telomeres might seem very different, but a detailed view of their mechanisms reveals similarities explaining how the loss of telomerase in a Drosophila ancestor could successfully have been replaced by the telomere retrotransposons. In this review, we will discover that although HeT-A, TART, and TAHRE are still the only examples to date where their targeted transposition is perfectly tamed into the telomere biology of Drosophila, there are other examples of retrotransposons that manage to successfully integrate inside and at the end of telomeres. Because the aim of this special issue is viral integration at telomeres, understanding the base of the telomerase exceptions will help to obtain clues on similar strategies that mobile elements and viruses could have acquired in order to ensure their survival in the host genome.
... We propose that the highly repetitive, gene-poor, transcriptionally silent, and fast-evolving primary telomeric sequence (Linardopoulou et al. 2005;Riethman et al. 2005;Villasante et al. 2007;Anderson et al. 2008;Teytelman et al. 2008) is this constantly changing selection regime. Drosophila's unique mechanism of telomere length maintenance-domesticated transposable elements rather than telomerase-based repeat addition to chromosome ends-offers manifold opportunities for conflict with telomeric proteins (Pardue and DeBaryshe 2003;Silva-Sousa et al. 2012a). However, our data suggest that transposable elements are only one of many evolutionary pressures that may select for telomere protein innovation. ...
... First, recombination between sister, homologous, or possibly non-homologous chromosome ends can extend telomeres ("terminal conversion"; Mikhailovsky et al. 1999;Kahn et al. 2000). Second, and probably most frequently, specialized telomeric retrotransposons insert at the chromosome termini (Levis et al. 1993;Pardue and DeBaryshe 2003). Telomere lengthening by both mechanisms can be detected via Southern Blot or qPCR on genomic DNA prepared from mutant flies (Biessmann and Mason 1997). ...
... Who are the cryptic selfish elements found in telomeric DNA and how do they hijack telomere biology for their own evolutionary gain? The repeat-rich DNA sequence at and adjacent to Drosophila telomeres encodes mostly satellite elements and three jockey-like non-LTR TEs charged with maintaining Drosophila telomere length (Pardue and DeBaryshe 2003;Capkova Frydrychova et al. 2008). Both these telomere-specialized TEs (see below) and telomereembedded satellite repeats may antagonize telomeric proteins to gain an evolutionary advantage. ...
Telomeres are nucleoprotein complexes at the ends of linear chromosomes. These specialized structures ensure genome integrity and faithful chromosome inheritance. Recurrent addition of repetitive, telomere-specific DNA elements to chromosome ends combats end-attrition, while specialized telomere-associated proteins protect naked, double-stranded chromosome ends from promiscuous repair into end-to-end fusions. Although telomere length homeostasis and end-protection are ubiquitous across eukaryotes, there is sporadic but building evidence that the molecular machinery supporting these essential processes evolves rapidly. Nevertheless, no global analysis of the evolutionary forces that shape these fast-evolving proteins has been performed on any eukaryote. The abundant population and comparative genomic resources of Drosophila melanogaster and its close relatives offer us a unique opportunity to fill this gap. Here we leverage population genetics, molecular evolution, and phylogenomics to define the scope and evolutionary mechanisms driving fast evolution of genes required for telomere integrity. We uncovered evidence of pervasive positive selection across multiple evolutionary timescales. We also document prolific expansion, turnover, and expression evolution in gene families founded by telomeric proteins. Motivated by the mutant phenotypes and molecular roles of these fast-evolving genes, we put forward four alternative, but not mutually exclusive, models of intra-genomic conflict that may play out at very termini of eukaryotic chromosomes. Our findings set the stage for investigating both the genetic causes and functional consequences of telomere protein evolution in Drosophila and beyond.
... Transposons or elements moving and moving along the genome are mobile pieces of DNA and can move along the genome, these pieces were rst discovered in corn by Barbara McClintock. Transposon elements are in two categories, the rst category is the elements that can be moved in the form of DNA and the second category is the elements that are transferred by an RNA intermediate (Pardue and Debarysh, 2003) Transposons with LTRs exist almost throughout the plant genome, although each of these types of retrotransposons exists in separate families in a speci c part of the genome. Most retrotransposons are mostly located in euchromatin regions (Deininger et al., 2003). ...
Wheat is an annual plant from the cereal family, this plant is the most important agricultural plant used in the human food chain. In plant breeding programs, identifying genes resistant to biotic and non-biotic stresses is of particular importance to transfer them to crop species to increase production. The most important goal in breeding programs is the existence or creation of diversity to select superior plants. One of the ways to enrich genetic resources is to know the level of diversity in germplasm and genetic treasures, which is one of the important sources of diversity and identification of useful genes in wild relatives. In this research, the genetic diversity of 125 Aegilops tauschii genotypes was investigated using SSR, ISSR, and retrotransposon markers. In this research, based on the presented results and the changes in ΔK and its maximum curve at K = 7, the number of 125 studied Aegilops tauschii genotypes were divided into 7 subpopulations with a probability of more than 70%. In general, the results obtained from this study showed that retrotransposon markers were better markers than SSR and ISSR in terms of useful indicators. However, the average PIC index in the other two systems was higher than retrotransposon, but they did not differ much from each other. Among the three marker systems, retrotransposons and SSRs were able to show a high level of intraspecies diversity.
... Drosophila telomeres are maintained by retrotranspositions of specialized telomeric retrotransposons to the chromosome ends; the two major retrotransposons are HeT-A and TART [30]. Despite the absence of telo-merase, many components and molecular mechanisms for the telomere maintenance in Drosophila are similar to those in the species that use telomerase [31]. ...
Chromatin spatial organization in the nucleus is essential for the genome functioning and regulation of gene activity. The nuclear lamina and lamina-associated proteins, lamins, play a key role in this process. Lamin dysfunction leads to the decompaction and transcriptional activation of heterochromatin, which is associated with the premature aging syndrome. In many cell types, telomeres are located at the nuclear periphery, where their replication and stability are ensured by the nuclear lamina. Moreover, diseases associated with defects in lamins and telomeres have similar manifestations and resemble physiological aging. Understanding molecular changes associated with aging at the organismal level is especially important. In this study, we compared the effects caused by the mutation in lamin B and physiological aging in the germline of the model organism Drosophila melanogaster . We have shown that the impaired localization of lamin B leads to the heterochromatin decompaction and transcriptional activation of some transposable elements and telomeric repeats. Both DNA damage and activation of homologous recombination in the telomeres were observed in the germ cells of lamin B mutants. The instability of repeat-enriched heterochromatin can be directly related to the genome destabilization, germ cell death, and sterility observed in lamin B mutants. Similar processes were observed in Drosophila germline in the course of physiological aging, which indicates a close link between the maintenance of the heterochromatin stability at the nuclear periphery and mechanisms of aging.
... Examples of the occurrence of non-canonical "insect" telomeric motif are observed in Coleoptera (beetles), with the description of the motif (TCAGG) n on chromosome termini in representatives of Tenebrionidae (Mravinac et al., 2011) and Cleridae (Prušáková et al. 2021), and the recently discovered motif (TTGGG) n on the Geotrupidae Anoplotrupes stercorosus (Prušáková et al., 2021). Remarkably among Diptera, the transposition of the non-long terminal repeat (non-LTR) retrotransposon was co-opted to maintain the telomere of Drosophila (Mason and Biessmann, 1995;Pardue and DeBaryshe, 2003; Figure 1G), and in basal dipterans, as chironomid midges, the recombination of long terminal repeats (LTR), i.e., satellite DNAs, maintains the telomeres (Nielsen and Edstrom, 1993). ...
Telomere has a central role in chromosomal stability events. Chromosome ends organized in telomere-loop prevent activation of DNA damage response (DDR) mechanisms, thus keeping the chromosome structure organized. On the other hand, free chromosome ends, dysfunctional telomeres, and interstitial telomeric sequences (ITS) can trigger chromosome rearrangements. Here, the telomere organization, function, and maintenance mechanisms, in addition to ITS types and their involvement in chromosome changes, were revisited. Despite a general (TTAGGG)n sequence being present in vertebrate telomeres, insects show more diversification of their telomere motif. The relation between ITS and chromosome rearrangements was observed in insects and vertebrates, demonstrating different types of genome organization and distribution. Some ITS cannot be considered relicts of chromosome rearrangements because probable they were inserted during a double-strand break repair mechanism. On the other hand, the involvement of telomere sequences participating or triggering chromosome rearrangements or organizing satellite DNA components in several species groups is evident. The genomic assembling advances and applying other methodologies over ITS, and their flanking regions, can help to understand the telomere participation in the chromosomal evolution in species groups with highly diversified karyotypes.
... Telomeres are the regions located at each end of the chromosomes, that comprise hundreds of repeats of the {TTAGGG} DNA sequence, highly conserved in eukaryotes (Meyne et al. 1989;Moyzis et al. 1988). Interestingly, not all organisms possess particular telomeric sequences as the Drosophila melanogaster for which chromosome ends consist of an array of non-LTR retrotransposons (Pardue and Debaryshe 2003). Telomeric tandem repeats assemble into a lariatlike structure (T-Loop) which shields the DNA end ( Figure 18A) Griffith et al. 1999). ...
Most of mammalian genomes are composed of DNA repeated elements. Pericentromeric heterochromatin (PCH) regions are formed of large arrays of major satellite repeats. DNA repeats are often silenced via similar mechanisms such as DNA methylation, H3K9me3 modification and HP1. Loss of heterochromatin silencing is commonly observed in different cancers and can lead to genomic instability. PCH of different chromosome can assemble into large DAPI-dense foci called chromocenters. Formation of chromocenter arise during the early embryogenesis when the level of DNA methylation is low. Such reorganization is essential for further development of the embryo, yet, the molecular mechanisms regulating its maintenance in the early pluripotent cells remain unknown. This work focuses on the role of the histone variant H3.3 and its chaperone DAXX in heterochromatin organization in embryonic stem cells (ESCs). Firstly, I observed that the loss of Daxx alters the organization of chromocenters in pluripotent cells. ESCs can be converted toward the ground-state of pluripotency which displays a dramatic loss of DNA methylation. I uncovered that DAXX is essential for proper growth of ESC upon ground-state conversion. Furthermore, I observed that active loss of DNA demethylation can generate DNA damages at PCH, which induces a strong accumulation of DAXX. Dissecting the function of DAXX, I discovered that the DAXX-H3.3 interaction was implicated in the recruitment of SETDB1 at chromocenters to reform the heterochromatin state. Finally, I noticed that the function of DAXX extend beyond pericentromeric heterochromatin. Indeed, the absence of Daxx impairs the proper silencing of different heterochromatin regions including the lamina-associated domains. Altogether, this work provides evidence that DAXX is a regulator of heterochromatin maintenance in pluripotent cells.
... Many species of the Drosophila genus use telomeric retrotransposons to maintain chromosome ends [34][35][36] and the piRNA machinery regulates these telomeric retrotrans- [37][38][39][40][41]. In Drosophila, the region subterminal to the retrotransposon array is also composed of repetitive telomere-associated sequences (TAS). ...
First discovered in maize, paramutation is a phenomenon in which one allele can trigger an epigenetic conversion of an alternate allele. This conversion causes a genetically heterozygous individual to transmit alleles that are functionally the same, in apparent violation of Mendelian segregation. Studies over the past several decades have revealed a strong connection between mechanisms of genome defense against transposable elements by small RNA and the phenomenon of paramutation. For example, a system of paramutation in Drosophila melanogaster has been shown to be mediated by piRNAs, whose primary function is to silence transposable elements in the germline. In this paper, we characterize a second system of piRNA-mediated paramutation-like behavior at the telomere of Drosophila virilis. In Drosophila, telomeres are maintained by arrays of retrotransposons that are regulated by piRNAs. As a result, the telomere and sub-telomeric regions of the chromosome have unique regulatory and chromatin properties. Previous studies have shown that maternally deposited piRNAs derived from a sub-telomeric piRNA cluster can silence the sub-telomeric center divider gene of Drosophila virilis in trans. In this paper, we show that this silencing can also be maintained in the absence of the original silencing allele in a subsequent generation. The precise mechanism of this paramutation-like behavior may be explained by either the production of retrotransposon piRNAs that differ across strains or structural differences in the telomere. Altogether, these results show that the capacity for piRNAs to mediate paramutation in trans may depend on the local chromatin environment and proximity to the uniquely structured telomere regulated by piRNAs. This system promises to provide significant insights into the mechanisms of paramutation.
... The observed Cnl1 subtelomeric dynamics mirror those observed for MoTeR transposons of the fungal plant pathogen Magnaporthe oryzae, which also localize in dynamic subtelomeric arrays 43 . The Cnl1 subtelomeric arrays identified here could also potentially overcome the requirement for telomerase, as in Drosophila telomeres, in which the functions of telomerase have been supplanted by a telomeric retrotransposon 44 . ...
Cryptococcus neoformans infections cause approximately 15% of AIDS-related deaths owing to a combination of limited antifungal therapies and drug resistance. A collection of clinical and environmental C. neoformans isolates were assayed for increased mutation rates via fluctuation analysis, and we identified two hypermutator C. neoformans clinical isolates with increased mutation rates when exposed to the combination of rapamycin and FK506. Sequencing of drug target genes found that Cnl1 transposon insertions conferred the majority of resistance to rapamycin and FK506 and could also independently cause resistance to 5-fluoroorotic acid and the clinically relevant antifungal 5-flucytosine. Whole-genome sequencing revealed both hypermutator genomes harbour a nonsense mutation in the RNA-interference component ZNF3 and hundreds of Cnl1 elements organized into massive subtelomeric arrays on each of the fourteen chromosomes. Quantitative trait locus mapping in 28 progeny derived from a cross between a hypermutator and wild-type identified a locus associated with hypermutation that included znf3. CRISPR editing of the znf3 nonsense mutation abolished hypermutation and restored small-interfering-RNA production. We conclude that hypermutation and drug resistance in these clinical isolates result from RNA-interference loss and accumulation of Cnl1 elements.
... The simple tandem repeat is not the only form of telomere composition. Drosophila is known to have a telomerase-independent mechanism to maintain chromosome integrity (Lundblad, 2002;Pardue & DeBaryshe, 2003;Mason et al, 2008Mason et al, , 2016, which lacks TRM. ...
Telomeres consist of highly conserved simple tandem telomeric repeat motif (TRM): (TTAGG)n in arthropods, (TTAGGG)n in vertebrates, and (TTTAGGG)n in most plants. TRM can be detected from chromosome-level assembly, which typically requires long-read sequencing data. To take advantage of short-read data, we developed an ultra-fast Telomeric Repeats Identification Pipeline and evaluated its performance on 91 species. With proven accuracy, we applied Telomeric Repeats Identification Pipeline in 129 insect species, using 7 Tbp of short-read sequences. We confirmed (TTAGG)n as the TRM in 19 orders, suggesting it is the ancestral form in insects. Systematic profiling in Hymenopterans revealed a diverse range of TRMs, including the canonical 5-bp TTAGG (bees, ants, and basal sawflies), three independent losses of tandem repeat form TRM (Ichneumonoids, hunting wasps, and gall-forming wasps), and most interestingly, a common 8-bp (TTATTGGG)n in Chalcid wasps with two 9-bp variants in the miniature wasp (TTACTTGGG) and fig wasps (TTATTGGGG). Our results identified extraordinary evolutionary fluidity of Hymenopteran TRMs, and rapid evolution of TRM and repeat abundance at all evolutionary scales, providing novel insights into telomere evolution.
... It is proposed that while invading the nucleus of primitive eukaryotes, some mobile group II introns have lost the RT ORF and became splicosomal introns; others lost the intron RNA structure and become non-LTR retrotransposons (Robart and Zimmerly 2005). Alternately, some studies in Drosophila and as well as in other higher order species suggest an evolutionary link between the telomerase complex and the reverse transcriptase domain of retrotransposons (Pardue and DeBaryshe 2003). ...
Retrotransposons are mobile genetic elements that employ an RNA intermediate and a reverse transcription step for their replication. Long INterspersed Elements-1 (LINE-1 or L1) form the only autonomously active retrotransposon family in humans. Although most copies are defective due to the accumulation of mutations, each individual genome contains an average of 100 retrotransposition-competent L1 copies, which contribute to the dynamics of contemporary human genomes. L1 integration sites in the host genome directly determine the genetic consequences of the integration and the fate of the integrated copy. Thus, where L1 integrates in the genome, and whether this process is random, is critical to our understanding of human genome evolution, somatic genome plasticity in cancer and aging, and host-parasite interactions. To characterize L1 insertion sites, rather than studying endogenous L1 which have been subjected to evolutionary selective pressure, we induced de novo L1 retrotransposition by transfecting a plasmid-borne active L1 element into HeLa S3 cells. Then, we mapped de novo insertions in the human genome at nucleotide resolution by a dedicated deep-sequencing approach, named ATLAS-seq. Finally, de novo insertions were examined for their proximity towards a large number of genomic features. We found that L1 preferentially integrates in the lowly-expressed and weak enhancer chromatin segments. We also detected several hotspots of recurrent L1 integration. Our results indicate that the distribution of de novo L1 insertions is non-random both at local and regional scales, and pave the way to identify potential cellular factors involved in the targeting of L1 insertions
... However, besides telomerase and Shelterin, other means of telomere elongation and protection are also known [8,14,15]; Drosophila, for example, have lost telomerase during evolution and solve the end replication problem by integration of three telomeric retrotransposons at chromosome ends. These 'domesticated' transposons, HeT-A, TART and TAHRE form arrays of repeats at telomeric regions [16,17]. As a consequence, in the absence of short telomeric repeats, in flies sequence-specific DNA binding of Shelterin-like proteins cannot provide telomere protection. ...
DNA end protection is fundamental for the long-term preservation of the genome. In vertebrates the Shelterin protein complex protects telomeric DNA ends, thereby contributing to the maintenance of genome integrity. In the Drosophila genus, this function is thought to be performed by the Terminin complex, an assembly of fast-evolving subunits. Considering that DNA end protection is fundamental for successful genome replication, the accelerated evolution of Terminin subunits is counterintuitive, as conservation is supposed to maintain the assembly and concerted function of the interacting partners. This problem extends over Drosophila telomere biology and provides insight into the evolution of protein assemblies. In order to learn more about the mechanistic details of this phenomenon we have investigated the intra- and interspecies assemblies of Verrocchio and Modigliani, two Terminin subunits using in vitro assays. Based on our results and on homology-based three-dimensional models for Ver and Moi, we conclude that both proteins contain Ob-fold and contribute to the ssDNA binding of the Terminin complex. We propose that the preservation of Ver function is achieved by conservation of specific amino acids responsible for folding or localized in interacting surfaces. We also provide here the first evidence on Moi DNA binding.
... Importantly, proteins coded by TEs can be co-opted (i.e., domesticated) by the host as an adaptation to evolutionary conflict (32). A classic example of TE domestication is found in Drosophila, where the integrity of the chromosome telomeres is maintained not by telomerase but by two non-long terminal repeat retrotransposons (59). Eukaryotes may also acquire genes from prokaryotes via lateral gene transfer, though the occurrence may be rare and restricted (38). ...
A pan-genome is the nonredundant collection of genes and/or DNA sequences in a species. Numerous studies have shown that plant pan-genomes are typically much larger than the genome of any individual and that a sizable fraction of the genes in any individual are present in only some genomes. The construction and interpretation of plant pan-genomes are challenging due to the large size and repetitive content of plant genomes. Most pan-genomes are largely focused on nontransposable element protein coding genes because they are more easily analyzed and defined than noncoding and repetitive sequences. Nevertheless, noncoding and repetitive DNA play important roles in determining the phenotype and genome evolution. Fortunately, it is now feasible to make multiple high-quality genomes that can be used to construct high-resolution pan-genomes that capture all the variation. However, assembling, displaying, and interacting with such high-resolution pan-genomes will require the development of new tools.
Expected final online publication date for the Annual Review of Plant Biology, Volume 72 is May 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Drosophila telomeres appear to constitute a separate class of the piRNA clusters; with abundant piRNAs corresponding to telomeric and subtelomeric sequences being present in Drosophila ovarian small RNA libraries. The telomeres of D. melanogaster are maintained in the absence of telomerase by the transpositions of the specialized telomeric retrotransposons HeT-A, TART and TAHRE organized in tandem head-to-tail arrays; with HeT-A being the prevailing telomeric retrotransposon [29][30][31]. Telomere associated sequences (TAS) consist of complex satellite-like repeats and are located proximally to retrotransposon arrays. Telomeric transcripts are processed into piRNAs that regulate telomeric TE expression and their transposition rate onto chromosome ends in the germline [3,32]. ...
Telomeric small RNAs related to PIWI-interacting RNAs (piRNAs) were discovered in different species, however, their role in germline-specific telomere function remains poorly understood. Using a Drosophila model, we show that the piRNA pathway provides a strong germline-specific mechanism of telomere homeostasis. We show that telomeric retrotransposon arrays belong to a unique class of dual-strand piRNA clusters whose transcripts, required for telomere elongation, serve simultaneously as piRNA precursors and their only targets. However, the ability to produce piRNAs and bind Rhino – a germline-specific homolog of heterochromatic protein 1 (HP1) – varies along telomeres. Most likely, this heterogeneity is determined by the peculiarities of telomeric retrotransposons themselves. piRNAs play a pivotal role in the establishment and maintenance of telomeric and subtelomeric chromatin in the germline facilitating loading of HP1 and histone 3 lysine 9 trimethylation mark – highly conservative telomere components – at different telomeric regions. piRNA pathway disruption results in telomere dysfunction characterized by a loss of heterochromatic components and translocation of telomeres from the periphery to the nuclear interior but does not affect the telomere end capping.
... Other types of noncoding RNAs could also be involved. Flies maintain chromosome length through retrotranscription [84], which requires complex and specific chromatin structures [85]. Retrotransposon proliferation can promote mutagenesis [86]. ...
Individuals within populations vary enormously in mortality risk and longevity, but the causes of this variation remain poorly understood. A potentially important and phylogenetically widespread source of such variation is maternal age at breeding, which typically has negative effects on offspring longevity. Here, we show that paternal age can affect offspring longevity as strongly as maternal age does and that breeding age effects can interact over 2 generations in both matrilines and patrilines. We manipulated maternal and paternal ages at breeding over 2 generations in the neriid fly Telostylinus angusticollis. To determine whether breeding age effects can be modulated by the environment, we also manipulated larval diet and male competitive environment in the first generation. We found separate and interactive effects of parental and grand-parental ages at breeding on descendants’ mortality rate and life span in both matrilines and patrilines. These breeding age effects were not modulated by grand-parental larval diet quality or competitive environment. Our findings suggest that variation in maternal and paternal ages at breeding could contribute substantially to intrapopulation variation in mortality and longevity.
... Other types of noncoding RNAs could also be involved. Flies maintain chromosome length through retrotranscription [84], which requires complex and specific chromatin structures [85]. Retrotransposon proliferation can promote mutagenesis [86]. ...
Individuals within populations vary enormously in mortality risk and longevity, but the causes of this variation remain poorly understood. A potentially important and phylogenetically widespread source of such variation is maternal age at breeding, which typically has negative effects on offspring longevity. Here, we show that paternal age can affect offspring longevity as strongly as maternal age does, and that breeding age effects can interact over two generations in both matrilines and patrilines. We manipulated maternal and paternal ages at breeding over two generations in the neriid fly Telostylinus angusticollis. To determine whether breeding age effects can be modulated by the environment, we also manipulated larval diet and male competitive environment in the first generation. We found separate and interactive effects of parental and grandparental ages at breeding on descendants’ mortality rate and lifespan in both matrilines and patrilines. These breeding age effects were not modulated by grandparental larval diet quality or competitive environment. Our findings suggest that variation in maternal and paternal ages at breeding could contribute substantially to intra-population variation in mortality and longevity.
... -HeT-A encodes its own 5'UTR, Gag, and 3'UTR(Pardue and DeBaryshe 2003), yet its 220 phylogenetic position reveals that this single-domain element is effectively a TAHRE element 221 missing an RT domain. Henceforth, we refer to this single lineage as TAHRE. ...
In virtually all eukaryotes, telomerase counteracts chromosome erosion by adding repetitive sequence to terminal ends. Drosophila melanogaster instead relies on specialized retrotransposons that insert preferentially at telomeres. This exchange of goods between host and mobile element - wherein the mobile element provides an essential genome service and the host provides a hospitable niche for mobile element propagation - has been called a genomic symbiosis. However, these telomere-specialized, jockey family elements may actually evolve to selfishly over-replicate in the genomes that they ostensibly serve. Under this intra-genomic conflict model, we expect rapid diversification of telomere-specialized retrotransposon lineages and possibly, the breakdown of this tenuous relationship. Here we report data consistent with both predictions. Searching the raw reads of the 15-million-year-old melanogaster species group, we generated de novo jockey retrotransposon consensus sequences and used phylogenetic tree-building to delineate four distinct telomere-associated lineages. Recurrent gains, losses, and replacements account for this striking retrotransposon lineage diversity. Moreover, an ancestrally telomere-specialized element has escaped, residing now throughout the genome of D. rhopaloa . In D. biarmipes , telomere-specialized elements have disappeared completely. De novo assembly of long-reads and cytogenetics confirmed this species-specific collapse of retrotransposon-dependent telomere elongation. Instead, telomere-restricted satellite DNA and DNA transposon fragments occupy its terminal ends. We infer that D. biarmipes relies instead on a recombination-based mechanism conserved from yeast to flies to humans. Combined with previous reports of adaptive evolution at host proteins that regulate telomere length, telomere-associated retrotransposon diversification and disappearance offer compelling evidence that intra-genomic conflict shapes Drosophila telomere evolution.
... Telomerase was not found in the Drosophila genome [6]. A unique feature of Drosophila telomeres is that they are composed of LINE (long interspersed nuclear element) retrotransposons; namely, HeT-A, TART and TAHRE, among which HeT-A is the most abundant [7]. Despite the different nature of telomeric repeats between Drosophila and species encoding telomerase, the basic mechanisms of telomere maintenance are similar [8]. ...
The study of the telomeric complex in oogenesis and early development is important for understanding the mechanisms which maintain genome integrity. Telomeric transcripts are the key components of the telomeric complex and are essential for regulation of telomere function. We study the biogenesis of transcripts generated by the major Drosophila telomere repeat HeT-A in oogenesis and early development with disrupted telomeric repeat silencing. In wild type ovaries, HeT-A expression is downregulated by the Piwi-interacting RNAs (piRNAs). By repressing piRNA pathway, we show that overexpressed HeT-A transcripts interact with their product, RNA-binding protein Gag-HeT-A, forming ribonucleoprotein particles (RNPs) during oogenesis and early embryonic development. Moreover, during early stages of oogenesis, in the nuclei of dividing cystoblasts, HeT-A RNP form spherical structures, which supposedly represent the retrotransposition complexes participating in telomere elongation. During the later stages of oogenesis, abundant HeT-A RNP are detected in the cytoplasm and nuclei of the nurse cells, as well as in the cytoplasm of the oocyte. Further on, we demonstrate that HeT-A products co-localize with the transporter protein Egalitarian (Egl) both in wild type ovaries and upon piRNA loss. This finding suggests a role of Egl in the transportation of the HeT-A RNP to the oocyte using a dynein motor. Following germline piRNA depletion, abundant maternal HeT-A RNP interacts with Egl resulting in ectopic accumulation of Egl close to the centrosomes during the syncytial stage of embryogenesis. Given the essential role of Egl in the proper localization of numerous patterning mRNAs, we suggest that its abnormal localization likely leads to impaired embryonic axis specification typical for piRNA pathway mutants.
... It is in fact agreed upon today that the evolution of various fundamental cellular mechanisms in many species originally arose through TE domestication. A classic example for the evolutionary significance of TE domestication is found in Drosophila, where telomeres are maintained not by telomerases but by two domesticated, non-LTR Jockey LINE-like retrotransposons, HeT-A and TART (Pardue & DeBaryshe, 2003). HeT-A and TART actively and in tandem add their long repeats at terminal regions of SCHRADER AND SCHMITZ | 3 chromosomes to compensate for the loss of terminal nucleotides during DNA replication. ...
The growing knowledge about the influence of transposable elements (TEs) on (1) long‐term genome and transcriptome evolution, (2) genomic, transcriptomic and epigenetic variation within populations, and (3) patterns of somatic genetic differences in individuals continues to spur the interest of evolutionary biologists in the role of TEs in adaptive evolution. As TEs can trigger a broad range of molecular variation in a population with potentially severe fitness and phenotypic consequences for individuals, different mechanisms evolved to keep TE activity in check, allowing for a dynamic interplay between the host, its TEs and the environment in evolution.
Here, we review evidence for adaptive phenotypic changes associated with TEs and the basic molecular mechanisms by which the underlying genetic changes arise: (1) domestication, (2) exaptation, (3) host gene regulation, (4) TE‐mediated formation of intron‐less gene copies – so‐called retrogenes, and (5) overall increased genome plasticity. Furthermore, we review and discuss how the stress‐dependent incapacitation of defense mechanisms against the activity of TEs might facilitate adaptive responses to environmental challenges, and how such mechanisms might be particularly relevant in species frequently facing novel environments, such as invasive, pathogenic, or parasitic species.
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... The telomeres of D. melanogaster are maintained by transpositions of specialized telomeric retrotransposons, while the telomerase gene has likely been lost in an ancestor of Diptera [21]. The non-LTR HeT-A, TART, and TAHRE retroelements are organized in tandem head-totail telomeric arrays, with HeT-A being the most abundant [22][23][24]. Telomere-associated sequences (TASs) consist of complex satellite-like repeats and are located proximally to retrotransposon arrays. Analysis of the ovarian small RNA-seq data revealed abundant piRNAs corresponding to both genomic strands of telomeric retrotransposons and TAS [10]; thus, telomeric piRNA clusters can be formally related to the dual-strand type. ...
Background:
Telomeric small RNAs related to PIWI-interacting RNAs (piRNAs) have been described in various eukaryotes; however, their role in germline-specific telomere function remains poorly understood. Using a Drosophila model, we performed an in-depth study of the biogenesis of telomeric piRNAs and their function in telomere homeostasis in the germline.
Results:
To fully characterize telomeric piRNA clusters, we integrated the data obtained from analysis of endogenous telomeric repeats, as well as transgenes inserted into different telomeric and subtelomeric regions. The small RNA-seq data from strains carrying telomeric transgenes demonstrated that all transgenes belong to a class of dual-strand piRNA clusters; however, their capacity to produce piRNAs varies significantly. Rhino, a paralog of heterochromatic protein 1 (HP1) expressed exclusively in the germline, is associated with all telomeric transgenes, but its enrichment correlates with the abundance of transgenic piRNAs. It is likely that this heterogeneity is determined by the sequence peculiarities of telomeric retrotransposons. In contrast to the heterochromatic non-telomeric germline piRNA clusters, piRNA loss leads to a dramatic decrease in HP1, Rhino, and trimethylated histone H3 lysine 9 in telomeric regions. Therefore, the presence of piRNAs is required for the maintenance of telomere chromatin in the germline. Moreover, piRNA loss causes telomere translocation from the nuclear periphery toward the nuclear interior but does not affect telomere end capping. Analysis of the telomere-associated sequences (TASs) chromatin revealed strong tissue specificity. In the germline, TASs are enriched with HP1 and Rhino, in contrast to somatic tissues, where they are repressed by Polycomb group proteins.
Conclusions:
piRNAs play an essential role in the assembly of telomeric chromatin, as well as in nuclear telomere positioning in the germline. Telomeric arrays and TASs belong to a unique type of Rhino-dependent piRNA clusters with transcripts that serve simultaneously as piRNA precursors and as their only targets. Telomeric chromatin is highly sensitive to piRNA loss, implying the existence of a novel developmental checkpoint that depends on telomere integrity in the germline.
... In Drosophila, though transposons threaten the genome, they are also essential for the integrity of both centromeres and telomeres (Pardue and DeBaryshe 2003;Wong and Choo 2004). In most other species, telomeres are composed of simple repeats and are maintained by telomerase, but, in Drosophila, telomeres consist of three non-LTR transposons, namely HeT-A, TART, and TAHRE DeBaryshe 2003, 2011;Abad et al. 2004). ...
Gametogenesis represents the most dramatic cellular differentiation pathways in both female and male flies. At the genome level, meiosis ensures that diploid germ cells become haploid gametes. At the epigenome level, extensive changes are required to turn on and shut off gene expression in a precise spatiotemporally controlled manner. Research applying conventional molecular genetics and cell biology, in combination with rapidly advancing genomic tools have helped us to investigate (1) how germ cells maintain lineage specificity throughout their adult reproductive lifetime; (2) what molecular mechanisms ensure proper oogenesis and spermatogenesis, as well as protect genome integrity of the germline; (3) how signaling pathways contribute to germline-soma communication; and (4) if such communication is important. In this chapter, we highlight recent discoveries that have improved our understanding of these questions. On the other hand, restarting a new life cycle upon fertilization is a unique challenge faced by gametes, raising questions that involve intergenerational and transgenerational epigenetic inheritance. Therefore, we also discuss new developments that link changes during gametogenesis to early embryonic development-a rapidly growing field that promises to bring more understanding to some fundamental questions regarding metazoan development.
... TERT assembles with the integral RNA template within TER to repeatedly synthesize tandem repeats of DNA at the telomeric end of the chromosome. Although the general mechanism of chromosomal end maintenance by telomerase remains fairly conserved throughout the eukaryotic lineage from flagellated protozoan to multicellular eukaryotes, it seems that other organisms like Drosophila have lost TERT and TER and instead evolved reterotransposon based mechanism to maintain their chromosomal termini [8]. ...
Replicative capacity of a cell is strongly correlated with telomere length regulation. Aberrant
lengthening or reduction in the length of telomeres can lead to health anomalies, such as cancer
or premature aging. Telomerase is a master regulator for maintaining replicative potential in most
eukaryotic cells. It does so by controlling telomere length at chromosome ends. Akin to cancer
cells, most single-cell eukaryotic pathogens are highly proliferative and require persistent telomerase
activity to maintain constant length of telomere and propagation within their host. Although
telomerase is key to unlimited cellular proliferation in both cases, not much was known about the role
of telomerase in human parasites (malaria, Trypanosoma, etc.) until recently. Since telomerase
regulation is mediated via its own structural components, interactions with catalytic reverse
transcriptase and several factors that can recruit and assemble telomerase to telomeres in a cell
cycle-dependent manner, we compare and discuss here recent findings in telomerase biology in cancer,
aging and parasitic diseases to give a broader perspective of telomerase function in human diseases.
... They are localized in the peri-centromeric regions (5S nrDNA) and nucleolar organizer regions (45S nrDNA) of most plant species, including Brassica 17-19 . Growing evidence supports the importance of TRs in genome function and evolution [20][21][22][23][24][25] . ...
Decoding complete genome sequences is prerequisite for comprehensive genomics studies. However, the currently available reference genome sequences of Brassica rapa (A genome), B. oleracea (C) and B. napus (AC) cover 391, 540, and 850 Mbp and represent 80.6, 85.7, and 75.2% of the estimated genome size, respectively, while remained are hidden or unassembled due to highly repetitive nature of these genome components. Here, we performed the first comprehensive genome-wide analysis using low-coverage whole-genome sequences to explore the hidden genome components based on characterization of major repeat families in the B. rapa and B. oleracea genomes. Our analysis revealed 10 major repeats (MRs) including a new family comprising about 18.8, 10.8, and 11.5% of the A, C and AC genomes, respectively. Nevertheless, these 10 MRs represented less than 0.7% of each assembled reference genome. Genomic survey and molecular cytogenetic analyses validates our insilico analysis and also pointed to diversity, differential distribution, and evolutionary dynamics in the three Brassica species. Overall, our work elucidates hidden portions of three Brassica genomes, thus providing a resource for understanding the complete genome structures. Furthermore, we observed that asymmetrical accumulation of the major repeats might be a cause of diversification between the A and C genomes.
... There are three telomere-specific retroelements found in D. melanogaster, HeTA, TAHRE and TART (collectively abbreviated HTT) that are present in multiple copies on each chromosome end ( Capkova Frydrychova et al., 2009;Mason et al., 2008). Transcription of telomeric retroelements, which is a key step in telomere elongation in Drosophila, has been found to correlate with cell proliferation ( Danilevskaya et al., 1997;Pardue and DeBaryshe, 2003;Walter and Biessmann, 2004). Telomere length is maintained through the interplay between telomere extension mechanisms and telomere shortening events. ...
It has been proposed that oxidative stress, elicited by high levels of reactive oxygen species, accelerates telomere shortening by erosion of telomeric DNA repeats. While most eukaryotes counteract telomere shortening by telomerase-driven addition of these repeats, telomeric loss in Drosophila is compensated by retrotransposition of the telomeric retroelements HeT-A, TART and TAHRE to chromosome ends. In this study we tested the effect of chronic exposure of flies to non-/sub-lethal doses of paraquat, which is a redox cycling compound widely used to induce oxidative stress in various experimental paradigms including telomere length analyses. Indeed, chronic paraquat exposure for five generations resulted in elevated transcriptional activity of both telomeric and non-telomeric transposable elements, and extended telomeric length in the tested fly lines. We propose that low oxidative stress leads to increased telomere length within Drosophila populations. For a mechanistic understanding of the observed phenomenon we discuss two scenarios: adaption, acting through a direct stimulation of telomere extension, or positive selection favoring individuals with longer telomeres within the population.
... Like in dipterans, we failed to identify TERT in the bdelloid rotifer Adineta vaga, suggesting the loss of TERT from this group of long-term asexual species [99,100]. In the absence of TERT and canonical telomere repeats, Drosophila melanogaster uses a retrotransposon-based telomere maintenance in which telomere-specific retrotransposons are reverse transcribed onto chromosome ends [101]. Three non-Long Terminal Repeat (LTR) retrotransposons, HeT-A [102,103], TART [104,105] and TAHRE [106] are specifically used for telomere maintenance in this species. ...
Background
Most animals employ telomerase, which consists of a catalytic subunit known as the telomerase reverse transcriptase (TERT) and an RNA template, to maintain telomere ends. Given the importance of TERT and telomere biology in core metazoan life history traits, like ageing and the control of somatic cell proliferation, we hypothesised that TERT would have patterns of sequence and regulatory evolution reflecting the diverse life histories across the Animal Kingdom. ResultsWe performed a complete investigation of the evolutionary history of TERT across animals. We show that although TERT is almost ubiquitous across Metazoa, it has undergone substantial sequence evolution within canonical motifs. Beyond the known canonical motifs, we also identify and compare regions that are highly variable between lineages, but show conservation within phyla. Recent data have highlighted the importance of alternative splice forms of TERT in non-canonical functions and although animals may share some conserved introns, we find that the selection of exons for alternative splicing appears to be highly variable, and regulation by alternative splicing appears to be a very dynamic feature of TERT evolution. We show that even within a closely related group of triclad flatworms, where alternative splicing of TERT was previously correlated with reproductive strategy, we observe highly diverse splicing patterns. Conclusions
Our work establishes that the evolutionary history and structural evolution of TERT involves previously unappreciated levels of change and the emergence of lineage specific motifs. The sequence conservation we describe within phyla suggests that these new motifs likely serve essential biological functions of TERT, which along with changes in splicing, underpin diverse functions of TERT important for animal life histories.
... The telomeres of Drosophila are also protected by TART and HeT TEs. 5 TEs are also known to regulate genes in many instances. 6 With the advancement of better technology, TEs have proved their role and importance in the genome, being involved in genetic recombination, chromosomal rearrangements, gene regulation, and mutations and having the ability to be transformed into new genes. ...
Transposable elements (TEs) have shown a great significance in regulatory elements research, being responsible for different types of cancers. They are divided into three classes on the basis of their mode of transposition, structural properties, and their homology with DNA sequence. In evaluating their role in cancers and other pathologies, researchers have found extensive evidence of their involvement. TEs can also be used as genetic markers for cancers and help in identifying potential therapeutic targets. There have been advancements in the management of hepatocellular carcinoma using TEs as regulatory components involved in the cancer’s pathogenesis.
... Given the differences in telomere lengths, overall lifespan, susceptibility to cancer, and other telomere-associated properties in the two species, this is perhaps not surprising. It may be one of many functional consequences of the accelerated evolution of subtelomeric genome regions, where a wide variety of lineage-specific and speciesspecific functionalities have arisen[302,303,348,349]. ...
The telomere is a specialized simple sequence repeat found at the end of all linear chromosomes. It acts as a substrate for telomere binding factors that in coordination with other interacting elements form what is known as the shelterin complex to protect the end of the chromosome from the DNA damage repair machinery. The telomere shortens with each cell division, and once critically short is no longer able to perform this role. Short dysfunctional telomeres result in cellular senescence, apoptosis, or genome instability. Telomere length is regulated by many factors including cis-acting elements in the proximal sequence which is known as the subtelomere. The Riethman lab played a pivotal role in generating the reference sequence of the subtelomere in both the human and mouse genomes, providing an essential resource for this work. Short high throughput sequencing (HTS) reads generated from the simple repeat containing telomere or the segmental duplication rich subtelomere cannot be aligned to a reference genome uniquely. They are filtered and excluded from many HTS analysis methods. A ChIP-Seq analysis pipeline was developed to incorporate these multimapping reads to study DNA-protein interactions in the subtelomere. This pipeline was employed to search for factors regulating the expression TERRA, an essential long non-coding RNA, and to better characterize their transcription start sites. ChIP-seq analysis in the human subtelomere found colocalization of CTCF and Cohesin directly adjacent to the telomere and throughout the subtelomere specific repeats. Follow up functional studies showed this binding regulated TERRA transcription at these sites. Extending these analyses in the mouse genome showed very different patterns of CTCF and cohesin binding, with no evidence of binding at apparent sites of TERRA transcription. Mouse subtelomere sequence analysis showed the co-occurence of two repeats at sites of putative TERRA expression, MurSatRep1 and MMSAT4, one of which was previously shown to be expressed in lincRNAs. The Telomere Analysis from SEquencing Reads(TASER) pipeline was developed to capture telomere information from HTS data sets and used to investigate telomere changes that occur in prostate cancer. TASER analysis of 53 paired prostate tumor and normal samples revealed an overall decrease in telomere length in tumor samples relative to matched paired normal tissue, especially sequence containing the exact canonical telomere repeat. Multimapping reads contain important information, that when used properly, help elucidate understanding of telomere biology, cancer biology, and genome regulation and stability.
The taxonomy of the three species of the genus Secale is discussed followed by a description of their morphology, and account of their distribution, cytogenetics, and evolutionary aspects. The relationships of these species to one another and to other Triticineae, as well as processes that have led to the domestication of Secale cereale , are reviewed too.
Long INterspersed Element-1 (LINE-1 or L1) is the only autonomously active transposable element in the human genome. The vast majority of L1s are inactive, but a small number (~80-100 per human genome) retain the ability to mobilize by a ‘copy and paste’ mechanism called retrotransposition. L1 encodes two proteins (ORF1p and ORF2p) required for retrotransposition. ORF2p is a 150kDa protein that has endonuclease (EN) and reverse transcriptase (RT) activities that are responsible for initiating L1 integration by a mechanism termed target-site primed reverse transcription (TPRT). During canonical TPRT, the L1 EN makes a single-strand endonucleolytic nick at a double-stranded genomic DNA target sequence (typically 5’-TTTT/A-3’ and variants of that sequence), to expose a 3’-hydroxyl group that is used as a primer by the L1 RT to reverse transcribe L1 messenger RNA. Different types of transposable elements (TEs) have evolved convergent strategies to target genomic ‘safe havens,’ where TE insertions are predicted to have relatively minimal effects on host fitness and gene expression. Whether L1 integrates into specific genomic regions requires elucidation. In this thesis, I have examined L1 integration preferences in four human cell lines that are proxies for in vivo cell types known to accommodate endogenous de novo L1 retrotransposition events. By combining cultured cell, molecular biological, the Pacific Bioscience sequencing platform, and computational approaches, I characterized 65,079 de novo engineered human L1 integration sites. I compared our L1 insertion dataset to a weighted random model, which assumes that L1 integration preferences are mediated solely by the presence of a degenerate L1 EN consensus cleavage site in the human genome. The data suggest that gene content, transcriptional activity, strand bias, epigenetic environment, and DNA replication status have minimal effects on L1 integration. Thus, L1 EN is the principal determinant of L1 integration. In contrast to canonical EN-dependent L1 retrotransposition, previous studies indicated that L1s could also integrate at sites of DNA damage, including dysfunctional telomeres, by an endonuclease-independent (ENi) mechanism in certain cultured cell lines that contain mutations in genes that render the non-homologous end-joining (NHEJ) pathway of DNA repair and p53 inactive. Here, we explored whether the disruption of other DNA repair pathways influence ENi L1 integration. We observed ENi retrotransposition in certain tissue culture cell lines containing defects in the Fanconi anemia (FA) DNA repair pathway. Since defects in the FA pathway can lead to the accumulation of inter-strand DNA crosslinks that, if left unrepaired, can interfere with DNA replication, we hypothesized that lesions arising at stalled DNA replication forks may provide substrates for enhanced ENi retrotransposition. Indeed, the examination of L1 EN mutant integration sites in FANCD2-deficient cells, suggests that a 3’-hydroxyl group present at Okazaki fragments and/or double-strand DNA breaks generated at collapsed DNA replication forks might be used as a primer to initiate ENi L1 retrotransposition. In sum, our results suggest that ENi L1 retrotransposition may represent an ancestral mobilization mechanism used by LINE-like retrotransposons prior to the acquisition of an endonuclease domain. Under this scenario, LINE-like elements were reliant upon genomic features (e.g., sites of genomic DNA damage, replication forks, and, less frequently, dysfunctional telomeres) to initiate TPRT in the absence of an endonuclease. Indeed, we posit that the acquisition of an endonuclease domain allowed L1 to autonomously insert throughout the genome and, as originally implied by its name, become an interspersed retrotransposon.
Telomeres are protective structures at the ends of eukaryotic chromosomes, and disruption of their nucleoprotein composition usually results in genome instability and cell death. Telomeric DNA sequences have generally been found to be exceptionally conserved in evolution, and the most common pattern of telomeric sequences across eukaryotes is (TxAyGz)n maintained by telomerase. However, telomerase-added DNA repeats in some insect taxa frequently vary, show unusual features, and can even be absent. It has been speculated about factors that might allow frequent changes in telomere composition in Insecta. Coleoptera (beetles) is the largest of all insect orders and based on previously available data, it seemed that the telomeric sequence of beetles varies to a great extent. We performed an extensive mapping of the (TTAGG)n sequence, the ancestral telomeric sequence in Insects, across the main branches of Coleoptera. Our study indicates that the (TTAGG)n sequence has been repeatedly or completely lost in more than half of the tested beetle superfamilies. Although the exact telomeric motif in most of the (TTAGG)n-negative beetles is unknown, we found that the (TTAGG)n sequence has been replaced by two alternative telomeric motifs, the (TCAGG)n and (TTAGGG)n, in at least three superfamilies of Coleoptera. The diversity of the telomeric motifs was positively related to the species richness of taxa, regardless of the age of the taxa. The presence/absence of the (TTAGG)n sequence highly varied within the Curculionoidea, Chrysomeloidea, and Staphylinoidea, which are the three most diverse superfamilies within Metazoa. Our data supports the hypothesis that telomere dysfunctions can initiate rapid genomic changes that lead to reproductive isolation and speciation.
It is suggested to read the definitions of Telomere, Telomerase and Subtelomere in the section Terminology before this chapter.
While the medicinal importance of Panax ginseng has long been established, its molecular cytogenetic data have not been available until the recent decade. Slow growth, low seed yield, long seed dormancy, sensitivity to light and high temperature, and low mitotic index are just some of the biological factors that hinder speedy progress in P. ginseng cytogenetics. Past reports were simple chromosome counting using chromogenic dyes, but the development of fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) technologies have sparked a renewed interest in P. ginseng cytogenetics in the context of genomics. Different classes of repetitive elements (REs) including transposable elements (TEs) and tandem repeats (TRs) representing about 80% of the genome have been identified from whole-genome screening of NGS reads and the draft P. ginseng genome assembly. Chromosomal mapping of these repetitive elements through FISH was instrumental in establishing a high-resolution P. ginseng karyotype (2n = 4x = 48), in elucidating its allopolyploid nuclear genome, and in providing insights into its genome history. Chromosome data have contributed valuable information in understanding the P. ginseng genome structure and continued integration of new innovative approaches like CRISPR/dCas9 chromosome tagging and chromosome flow sorting will continue to highlight the importance of cytogenetic works in P. ginseng. In this chapter, we review the history, challenges, achievements, and future directions of P. ginseng cytogenetics in the post-genomics era.
The release of the Brassica oleracea draft genome sequence opens numerous opportunities to understand its genome structure and evolution. A 515-Mb (82% of the total genome) high-quality draft assembly was made up of 56% repetitive elements (REs). Although the contribution of REs to genome structures, organization and evolution is relatively poorly understood, advances in bioinformatics have enabled genome-wide quantification and downstream analyses of REs in plant genomes. This chapter provides an overview of the classification, abundance, and genomic organization of the major types of REs that make up the main repeat component in the B. oleracea genome. Eight elements namely, 5S and 45S nrDNA, centromeric and subtelomeric tandem repeats (CentBo1, CentBo2, BoSTRa/b, and BoSTRc), a centromeric retrotransposon (BoCRB), and a Ty1/copia LTR retrotransposon (BoCopia-1) were classified into this repeat component. Whole-genome shotgun (WGS) mapping and molecular cytogenetic analyses provided an in-depth view of the abundance and distribution of these repeats both in the in silico generated draft assembly and mitotic metaphase chromosomes. The information not only validates the abundance of repeat elements in draft genomes, but also provide an avenue for understanding overall genome structure.
Retrotransposons are mobile genetics elements, which form almost half of our genome. Only the L1HS subfamily of the Long Interspersed Element-1 class (LINE-1 or L1) has retained the ability to jump autonomously in humans. Their mobilization in the germline – but also in some somatic tissues – contributes to human genetic diversity and to diseases, such as cancer. L1 reactivation can be directly mutagenic by disrupting genes or regulatory sequences. In addition, L1 sequences themselves contain many regulatory cis-elements. Thus, L1 insertions near a gene or within intronic sequences can also produce more subtle genic alterations. To explore L1-mediated genic alterations in a genome-wide manner, we have developed a dedicated RNA-seq analysis software able to identify L1 chimeric or antisense transcripts and to annotate these novel isoforms with their associated alternative splicing events. During the course of this work, it appeared that understanding the link between L1HS insertion polymorphisms and phenotype or disease requires a comprehensive view of the different L1HS copies present in a given individual or sample. To provide a comprehensive summary of L1HS insertion polymorphisms identified in healthy or pathological human samples and published in peer-reviewed journals, we developed euL1db, the European database of L1HS retrotransposon insertions in humans. This work will help understanding the overall impact of L1 insertions on gene expression, at a genome-wide scale.
Telomerase deficiency in human somatic cells results in telomere erosion and senescence. Small RNAs that target telomeres have been observed in diverse organisms but their functions are not well characterized. We define an endogenous small RNA pathway in Caenorhabditis elegans that promotes heterochromatin formation at telomeres via Dicer, the perinuclear Argonaute protein WAGO-1 and the nuclear Argonaute protein HRDE-1. Loss of telomerase induces biogenesis of siRNAs that target the telomeric lncRNA TERRA, whereas loss of both telomerase and small RNA-mediated telomeric silencing induces TERRA expression, DNA damage, and an accelerated sterility phenotype. These phenotypes can be rescued by exogenous telomeric siRNAs or by loss of the DNA damage response protein EXO-1. Thus, endogenous siRNAs interact with TERRA to promote heterochromatin formation in a manner that is critical for the stability of naturally eroding telomeres. We propose that small RNA-mediated genome silencing could be broadly relevant to regulation of proliferative aging.
Most animals employ telomerase, which consists of a catalytic subunit known as the telomerase reverse transcriptase (TERT) and an RNA template, to maintain telomere ends. Given the importance of TERT and the apparent importance of telomere biology in core metazoan life history traits like ageing and the control of somatic cell proliferation, we hypothesised that TERT would have patterns of sequence and regulatory evolution reflecting adaptations to diverse evolutionary and life histories across the Animal Kingdom. To test this, we performed a complete investigation of the evolutionary history of TERT across animals. We show that although TERT is almost ubiquitous across Metazoa, it has undergone substantial sequence evolution in canonical motifs. Beyond the known canonical motifs, we also identify and compare regions that are highly variable between lineages, but for which conservation exists within phyla. Recent data have highlighted the importance of alternate splice forms of TERT in non-canonical functions in some animals. Although animals may share some conserved introns, we find that the selection of exons for alternative splicing appears to be highly variable, and regulation by alternative splicing appears to be a very dynamic feature of TERT evolution. We show that even within a closely related group of triclad flatworms, where alternative splicing of TERT was previously correlated with reproductive strategy, we observe highly diverse alternative splicing patterns. Our work establishes that the evolutionary history and structural evolution of TERT involves previously unappreciated levels of change, supporting the view that this core eukaryotic protein has adapted to the requirements of diverse animal life histories.
Telomere length maintenance is essential for most eukaryotes to ensure genome stability and integrity. A non-long terminal repeat (LTR) retrotransposon, SART1Bm, targets telomeric repeats (TTAGG)n of the silkworm Bombyx mori and is presumably involved in telomere length maintenance. However, how many telomeric repeats are required for its retrotransposition and how reverse transcription is initiated at the target site are not well understood. Here, using an ex vivo and trans-in vivo recombinant baculovirus retrotransposition system, we demonstrated that SART1Bm requires at least three (TTAGG) telomeric repeats and a longer poly(A) tail for its accurate retrotransposition. We found that SART1Bm retrotransposed only in the third (TTAGG) tract of three repeats and that the A residue of the (TTAGG) unit was essential for its retrotransposition. Interestingly, SART1Bm also retrotransposed into telomeric repeats of other species, such as human (TTAGGG)n repeats, albeit with low retrotransposition efficiency. We further showed that the reverse transcription of SART1Bm occurred inaccurately at the internal site of the 3′ untranslated region (UTR) when using a short poly(A) tail but at the accurate site when using a longer poly(A) tail. These findings promote our understanding of the general mechanisms of site-specific retrotransposition and aid the development of a site-specific gene knock-in tool.
Background
The evolutionary dynamics of transposable elements (TEs) vary across the tree of life and even between closely related species with similar ecologies. In Drosophila , most of the focus on TE dynamics has been completed in Drosophila melanogaster and the overall pattern indicates that TEs show an excess of low frequency insertions, consistent with their frequent turn over and high fitness cost in the genome. Outside of D. melanogaster , insertions in the species Drosophila algonquin , suggests that this situation may not be universal, even within Drosophila . Here we test whether the pattern observed in D. melanogaster is similar across five Drosophila species that share a common ancestor more than fifty million years ago.
Results
For the most part, TE family and order insertion frequency patterns are broadly conserved between species, supporting the idea that TEs have invaded species recently, are mostly costly and dynamics are conserved in orthologous regions of the host genome
Conclusions
Most TEs retain similar activities and fitness costs across the Drosophila phylogeny, suggesting little evidence of drift in the dynamics of TEs across the phylogeny, and that most TEs have invaded species recently.
Repetitive elements (REs) play major roles in genome organization, size, and evolution, but are often underrepresented in genome assemblies. The recent genome assembly of the allotetraploid Brassica napus genome revealed that 48% of the genome comprised REs, including transposons and tandem repeats. In the present work, we show the overall quantity and comparative analyses of major repeat families in both the assembled and unassembled portions of the reference B. napus genome. We surveyed the abundance, distribution, diversity, and dynamics of ten major REs in the B. napus genome, which represented less than 1% of the total 1130 Mb B. napus genome in the current assembly. However, in silico mapping of raw whole-genome sequence reads from nine B. napus accessions revealed about 11% of the genome as represented by these ten repeat families. Comparative analyses of these major repeats showed their evolutionary dynamics in the B. rapa (Ar), B. oleracea (Co), and B. napus (AnCn) genomes as well as a considerable inter- and intraspecies repeat diversity among different B. napus accessions. Cytogenetic mapping of these major repeats showed their genomic abundance and distribution, with some families having a conserved subgenomic distribution pattern in the B. napus genome. Finally, the impact of genetic changes to REs and their corresponding epigenetic readjustments during B. napus evolution are also discussed in this chapter.
The “selfish DNA” theory postulates that transposable elements (TEs) are intragenomic parasites, and that natural selection against deleterious effects associated with their presence is the main force preventing their genomic spread in natural populations (Orgel and Crick 1980).
The Tc1 mariner element is widely distributed among organisms and have been already described in different species of fish. The genus Ancistrus (Kner, 1854) has 68 nominal species and is part of an interesting taxonomic and cytogenetic group, as well as presenting a variation of chromosome number, ranging from 2n=34 to 54 chromosomes, and the existence of simple and multiple sex chromosome system and the occurrence of chromosomal polymorphisms involving chromosomes that carry the nucleolus organizer region. In this study, a repetitive element by restriction enzyme, from Ancistrus sp.1 "Flecha" was isolated, which showed similarity with a transposable element Tc1-mariner. Its chromosomal location is distributed in heterochromatic regions and along the chromosomal arms of all specimens covered in this study, confirming the pattern dispersed of this element found in other studies carried out with other species. Thus, this result reinforces the hypothesis that the sequence AnDraI is really a dispersed element isolated. As this isolated sequence showed the same pattern in all species which have different sex chromosomes systems, including in all sex chromosomes, we could know that it is not involved in sex chromosome differentiation.
The telomere is a nucleoprotein complex at the ends of linear chromosomes that protects them from fusion and degradation. The telomere consists of telomeric DNA, a protective protein complex and telomeric RNA. Biogenesis of telomeric transcripts in development is still far from being understood. Drosophila telomeres are elongated by a transposition of specialized telomeric retrotransposons that encode proteins. Using transgenic constructs encoding tagged telomeric protein, we found that transcripts of Drosophila telomeric element HeT-A bind Gag-HeT-A protein encoded by these transcripts. Maternal HeT-A transcripts and Gag-HeT-A form ribonucleoprotein granules around centrosomes, centers of microtubule organization, during blastoderm formation, upon disruption of telomere silencing during oogenesis. The specific localization of HeT-A RNA is dependent on microtubules since disruption of microtubules caused delocalization of HeT-A transcripts. This transgenic system is a valuable model for the study of telomeric RNA biogenesis.
Drosophila melanogaster is the only invertebrate that contains endogenous retroviruses, which are called errantiviruses. Two domesticated genes, Grp and Iris, which originate from errantivirus gag and env, respectively, have been found in the D. melanogaster genome. The functions performed by the genes in Drosophila are still unclear. To identify the functions of domesticated gag and env in the D. melanogaster genome, expression of Iris and Grp was studied in strains differing by the presence or absence of the functional gypsy errantivirus. In addition, the expression levels were measured after injection of gram-positive and gram-negative bacteria, which activate different immune response pathways, and exposure to various abiotic stress factors. The presence of functional D. melanogaster retrovirus gypsy was found to increase the Grp expression level in somatic tissues of the carcass, while exerting no effect on the Iris expression level. Activation of the immune response in D. melanogaster by bacteria Bacillus cereus increased the Grp expression level and did not affect Iris expression. As for the effects of abiotic stress factors (oxidative stress, starvation, and heat and cold stress), the Grp expression level increased in response to starvation in D. melanogaster females, and the Iris expression level was downregulated in heat shock and oxidative stress. Based on the findings, Grp was assumed to play a direct role in the immune response in D. melanogaster; Iris is not involved in immune responses, but and apparently performs a cell function that is inhibited in stress.
Loss of telomeric DNA during cell proliferation may play a role in ageing and cancer. Since telomeres permit complete replication of eukaryotic chromosomes and protect their ends from recombination, we have measured telomere length, telomerase activity and chromosome rearrangements in human cells before and after transformation with SV40 or Ad5. In all mortal populations, telomeres shortened by approximately 65 bp/generation during the lifespan of the cultures. When transformed cells reached crisis, the length of the telomeric TTAGGG repeats was only approximately 1.5 kbp and many dicentric chromosomes were observed. In immortal cells, telomere length and frequency of dicentric chromosomes stabilized after crisis. Telomerase activity was not detectable in control or extended lifespan populations but was present in immortal populations. These results suggest that chromosomes with short (TTAGGG)n tracts are recombinogenic, critically shortened telomeres may be incompatible with cell proliferation and stabilization of telomere length by telomerase may be required for immortalization.
In this paper, we examine first the steadiness of the rate of evolutionary change in a larval hemolymph protein, LHP, in numerousDrosophila species. We estimated amino acid sequence divergence from immunological distances measured with the quantitative microcomplement fixation technique. Using tests not depending on knowledge of absolute times of divergence, we estimated the variance of the rate of evolutionary change to be at least 4 times as large as that for a process resembling radioactive decay. Thus, the rate of evolution of this protein is as uniform as that of vertebrate proteins. Our analysis indicates no acceleration of protein evolution in the lineages leading to Hawaiian drosophilines. Second, we give an explicit description of a procedure for calculating the absolute value of the mean rate of evolutionary change in this protein. This procedure is suggested for general use in calculating absolute rates of molecular evolution. The mean rate of evolution of LHP is about 1.2 immunological distance units per million years, which probably coreesponds to a unit evolutionary period of 4 million years; LHP thus evolves at a rate comparable to that of mammalian hemoglobins. Finally, we utilize the calibrated rate of LHP evolution to derive a time scale of evolution in the Drosophilidae and higher Diptera.
Unlabelled:
We have developed a new software package, Molecular Evolutionary Genetics Analysis version 2 (MEGA2), for exploring and analyzing aligned DNA or protein sequences from an evolutionary perspective. MEGA2 vastly extends the capabilities of MEGA version 1 by: (1) facilitating analyses of large datasets; (2) enabling creation and analyses of groups of sequences; (3) enabling specification of domains and genes; (4) expanding the repertoire of statistical methods for molecular evolutionary studies; and (5) adding new modules for visual representation of input data and output results on the Microsoft Windows platform.
Availability:
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Contact:
s.kumar@asu.edu
HeT-A was the first transposable element shown to have a bona fide role in chromosome structure, maintenance of telomeres in Drosophila melanogaster. HeT-A has hallmarks of non-long-terminal-repeat (non-LTR) retrotransposable elements but also has several unique features. We have now isolated HeT-A elements from Drosophila yakuba, showing that the retrotransposon mechanism of telomere maintenance predates the separation of D. melanogaster and D. yakuba (5-15 million years ago). HeT-A elements from the two species show significant sequence divergence, yet unusual features seen in HeT-Amel are conserved in HeT-Ayak. In both species, HeT-A elements are found in head-to-tail tandem arrays in telomeric heterochromatin. In both species, nearly half of the HeT-A sequence is noncoding and shows a distinctive imperfect repeat pattern of A-rich segments. Neither element encodes reverse transcriptase. The HeT-Amel promoter appears to be intermediate between the promoters of non-LTR and of LTR retrotransposons. The HeT-Ayak promoter shows similar features. HeT-Amel has a frameshift within the coding region. HeT-Ayak does not require a frameshift but shows conservation of the polypeptide sequence of the frameshifted product of D. melanogaster.
HeT-A elements are a new family of transposable elements in Drosophila that are found exclusively in telomeric regions and in the pericentric heterochromatin. Transposition of these elements onto broken chromosome ends has been implicated in chromosome healing. To monitor the fate of HeT-A elements that had attached to broken ends of the X chromosome, we examined individual X chromosomes from a defined population over a period of 17 generations. The ends of the X chromosomes with new HeT-A additions receded at the same rate as the broken ends before the HeT-A elements attached. In addition, some chromosomes, approximately 1% per generation, had acquired new HeT-A sequences of an average of 6 kb at their ends with oligo(A) tails at the junctions. Thus, the rate of addition of new material per generation matches the observed rate of terminal loss (70-75 bp) caused by incomplete replication at the end of the DNA molecule. One such recently transposed HeT-A element which is at least 12 kb in length has been examined in detail. It contains a single open reading frame of 2.8 kb which codes for a gag-like protein.
Loss of telomeric DNA during cell proliferation may play a role in ageing and cancer. Since telomeres permit complete replication of eukaryotic chromosomes and protect their ends from recombination, we have measured telomere length, telomerase activity and chromosome rearrangements in human cells before and after transformation with SV40 or Ad5. In all mortal populations, telomeres shortened by approximately 65 bp/generation during the lifespan of the cultures. When transformed cells reached crisis, the length of the telomeric TTAGGG repeats was only approximately 1.5 kbp and many dicentric chromosomes were observed. In immortal cells, telomere length and frequency of dicentric chromosomes stabilized after crisis. Telomerase activity was not detectable in control or extended lifespan populations but was present in immortal populations. These results suggest that chromosomes with short (TTAGGG)n tracts are recombinogenic, critically shortened telomeres may be incompatible with cell proliferation and stabilization of telomere length by telomerase may be required for immortalization.
Drosophila melanogaster F elements are mobile, oligo(A)-terminated DNA sequences that likely propagate by the retrotranscription of RNA intermediates. Plasmids bearing DNA segments from the left-hand region of a full-length F element fused to the CAT gene were used as templates for transient expression assays in Drosophila Schneider II cultured cells. Protein and RNA analyses led to the identification of two promoters, Fin and Fout, that transcribe in opposite orientations. The Fin promoter drives the synthesis of transcripts that initiate around residue +6 and are directed toward the element. Fin, that probably controls the formation of F transposition RNA intermediates and gene products, is internal to the transcribed region. Sequences important for accumulation of Fin transcripts are included within the +1 to +30 interval; an additional regulatory element may coincide with a heptamer located downstream of this region also found in the 5' end regions of F-like Drosophila retrotransposons. Analysis of the template activity of 3' deletion derivatives indicates that the level of accumulation of Fin RNA is also dependent upon the presence of sequences located within the +175 to +218 interval. The Fout promoter drives transcription in the opposite orientation with respect to Fin. Fout transcripts initiate at nearby sites within the +92 to +102 interval. Sequences downstream of these multiple RNA start sites are not required for the activity of the Fout promoter. Deletions knocking out the Fin promoter do not impair Fout transcription; conversely, initiation at the Fin promoter still takes place in templates that lack the Fout promoter. At a low level, both promoters are active in cultured cells.
A constructed human LINE-1 (L1Hs) element containing intact 5' and 3' untranslatable regions and an in-frame fusion between
the L1Hs open reading frame 1 and the bacterial lacZ gene (p1LZ) was found to promote the expression of beta-galactosidase
in a variety of transiently transfected cell types in tissue culture. Full-length RNA was detected in the transfected cells.
Most of the RNA transcripts initiated at or near the beginning of the L1Hs segment. Sequences within the L1Hs segment of p1LZ
were sufficient for expression of the reporter gene; however, modulation of the transcriptional regulatory region by upstream
sequences was not ruled out. Deletion analysis revealed that the sequences most critical for transcription were located within
the first 100 bp of L1Hs. Other sequences within the first 668 bp of L1Hs also contributed to overall expression. Expression
of p1LZ was high in human teratocarcinoma cells and low in all other cell types. This pattern of cell-type-specific expression
matches the known pattern of endogenous L1Hs transcription in cultured cells.
The process of polytenization plays a crucial role in Drosophila development, and most of the larval tissues are polytene. By analyzing the pattern of DNA replication in embryos pulse-labeled with BrdU, we show that many larval tissues undergo a transition to begin becoming polytene late in embryogenesis. Our results demonstrate that in these larval tissues polyteny results from a modified cell cycle, the endo cell cycle, in which there is only an S (synthesis) phase and a G (gap) phase. A key regulator of the mitotic cell cycle, the product of the string gene (the Drosophila homologue of cdc25), is not required for the endo cell cycle. The developmental regulation of the endo cell cycle is striking in that tissue-specific domains undergo polytene DNA replication in a dynamic pattern at defined times in embryogenesis. During subsequent rounds of the endo cell cycle in late embryogenesis and first instar larval development, the domains are subdivided and the temporal control is not as rigid. The length of the G phase varies among different tissues. By quantifying DNA content, we show that during the early polytene S phases the genome is not fully duplicated.
We have recovered terminal chromosome deletions of the X chromosome of Drosophila [Df(1)RT; RT = receding tips] that break in various positions of the yellow gene (y) region and delete all distal DNA sequences. Terminal DNA fragments are heterogeneous in length. Molecular cloning and sequencing of the terminal DNA fragments revealed that the broken ends of the deleted chromosomes do not carry any telomeric DNA sequences, yet the broken chromatids do not fuse to one another. Moreover, we confirmed by sequence analysis of 49 independently cloned terminal DNA fragments from two RT lines collected at different times that they lose DNA sequences from their distal ends at a rate of 70-75 base pairs per fly generation. We calculate that the rate of loss from these ends is consistent with the removal of an octanucleotide RNA primer at each round of DNA replication in the germ line.
The terminus of a DNA helix has been called its Achilles' heel. Thus to prevent possible incomplete replication and instability of the termini of linear DNA, eukaryotic chromosomes end in characteristic repetitive DNA sequences within specialized structures called telomeres. In immortal cells, loss of telomeric DNA due to degradation or incomplete replication is apparently balanced by telomere elongation, which may involve de novo synthesis of additional repeats by novel DNA polymerase called telomerase. Such a polymerase has been recently detected in HeLa cells. It has been proposed that the finite doubling capacity of normal mammalian cells is due to a loss of telomeric DNA and eventual deletion of essential sequences. In yeast, the est1 mutation causes gradual loss of telomeric DNA and eventual cell death mimicking senescence in higher eukaryotic cells. Here, we show that the amount and length of telomeric DNA in human fibroblasts does in fact decrease as a function of serial passage during ageing in vitro and possibly in vivo. It is not known whether this loss of DNA has a causal role in senescence.
The inferred amino acid sequences of 10 specific gene products from nine retroviruses were aligned by computer, all evolutionary distances between them calculated, and evolutionary trees constructed. Not unexpectedly, the various gene products are changing at different rates, the reverse transcriptase being the least and the envelope proteins the most different from one retrovirus to another. For the most part, trees based on the retroviral enzyme sequences are congruent, indicating that extensive genetic recombination has not been a major factor in the evolution of the central part of the genome. In the case of envelope protein sequences, however, the sequences clearly exhibit evidence of multiple cross-over events between quite distantly related retroviruses. A composite phylogenetic tree was constructed from the four retroviral enzyme sequences, and a number of important historical happenings were interpreted in the light of the time scale it affords.
As is the case for some other RNA viruses, the amino acid sequences of retroviral proteins change at an astonishing rate. For example, the proteases of the human immunodeficiency virus (HIV) and the visna lentivirus with which it is often compared are as different as the proteases of fungi and mammals, and those of the human type I leukemia virus are as different from HIV or visna as are the proteins of humans and bacteria. That the sequences of retrovirus proteins can be recognized as sharing common ancestry with non-retroviral proteins implies that the vastly accelerated change has begun only recently or occurs very sporadically. Only a scheme whereby exogenous retroviruses exist as short-lived bursts upon a backdrop of germline-encoded endogenous viruses is consistent with the sequence data. Retroviruses are related to many other reverse transcriptase-bearing entities present in the genomes of eukaryotes. They also have proteins that are homologous with those of some plant and animal DNA viruses, and their reverse transcriptase is recognizably similar to sequences found in the introns of some fungal mitochondria. Computer alignment of all these sequences allows an overall phylogeny to be constructed that chronicles the history of events leading to infectious retroviruses.
We have isolated a 5.7-kbp dispersed moderately repeated DNA sequence (TOC1) from the mutant OEE1 gene of the Chlamydomonas reinhardtii strain FUD44. The copy number (2 to over 30) and genomic locations of TOC1 elements vary widely in different C. reinhardtii strains. Our standard laboratory photosynthetic strain exhibits a high degree of TOC1 instability during short periods of mitotic growth. TOC1 appears to be a retrotransposon: it contains LTRs and an oligonucleotide stretch that corresponds to a conserved pentapeptide of reverse transcriptase. TOC1 is an unusual retrotransposon: it is not flanked by a target site duplication in the OEE1 gene, the left end of TOC1 only contains a fraction of the LTR the remainder of which is present at its right end and TOC1 does not start with a 5' TG and end with a 3' CA. In most cases, TOC1 excision leaves behind a complete solo LTR sequence (577 bp) and in one case a deleted solo LTR sequence (191 bp). Solo LTR sequences form a separate family of repeated sequences in most of the strains tested.
An x-ray-dependent mutator on chromosome 3 of Drosophila melanogaster is described that specifically increases the recovery of deletions for chromosomal tip regions. Such deficiencies can be induced on any chromosome. More centromere proximal mutations, as assayed by the sex-linked recessive lethal test, are not increased over the wild-type control. As far as can be determined by genetic, cytological, and molecular assays, the deletions extend to the very end of the chromosome involved. In addition, the frequency of these deletions is directly proportional to x-ray dose, suggesting that they are one-break rearrangements. It is proposed that the mutator is blocked in a major pathway for the repair of DNA double-strand breaks, and that a minor repair pathway is responsible for the addition of new telomeres under these conditions.
To investigate the precise structure of eucaryotic primer RNA made in vivo, short DNA chains isolated from nuclei of Drosophila
melanogaster embryos were analyzed. Post-labeling of 5' ends of short DNA chains with polynucleotide kinase and [gamma-32P]ATP
revealed that 7% of the DNA fragments were covalently linked with mono- to octaribonucleotide primers at their 5' ends. Octaribonucleotides,
the major component (ca. 30%), formed the cap structure in the reaction with vaccinia guanylyltransferase and [alpha-32P]GTP,
indicating that they were the intact primer RNA with tri- (or di-) phosphate termini, and the shorter ribooligomers were degradation
intermediates. The intact primers started with purine (A/G ratio, 4:1), and the starting few ribonucleotide residues were
rich in A.
Heterochromatin protein 1 (HP1) was initially discovered as a protein that is associated with the heterochromatin at the chromocenter of polytene chromosomes in Drosophila larval salivary glands. In this paper we investigate the localization of heterochromatin protein 1 in the diploid nuclei of Drosophila embryos. We focus on its association with the interphase heterochromatin in fixed embryos before and during cycle 14, the developmental time at which heterochromatin becomes most conspicuous, and also follow its localization during mitosis. The GAGA transcription factor was recently shown to be localized at sequences within α-heterochromatin in pre-cycle 14 embryos, and an antibody against this protein serves as a convenient marker for these sequences.
We find an enrichment of heterochromatin protein 1 in the intensely DAPI-staining regions near the apical surface of nuclear cycle 10 embryos. At this stage GAGA factor is localized into punctate structures in this same region. This enrichment for HP1 is markedly increased during nuclear cycle 14.
Surprisingly, whereas GAGA factor retains its association with the heterochromatin throughout the cell cycle, a significant fraction of HP1 is dispersed throughout the spindle around the segregating chromosomes during mitosis. This dispersed pool of heterochromatin protein 1 was observed during mitosis in both early and late Drosophila embryos and in an analysis of a bacterially produced 6× histidine-heterochromatin protein 1 fusion protein injected into living Drosophila embryos. When Drosophila tissue culture cells were prepared by a method which removes soluble protein and avoids fixation of the mitotic chromosomes, an enrichment for heterochromatin protein 1 in the heterochromatin of the chromosomes was discovered also.
A family of 340-bp tandem telomere-associated DNA repeats is present in 50- to 200-kb blocks in seven of the eight paired
chromosome ends in Chironomus pallidivittatus. It consists of four main subfamilies, differing from each other by small clusters
of mutations. This differentiation may reflect different functional roles for the repeats. Here we find that one subfamily,
D3, is consistently localized most peripherally and extends close to the ends of the chromosomes, as shown by its sensitivity
to the exonuclease Bal 31. The amounts of D3 are highly variable between individuals. The repeat characteristic for D3 forms
a segment with pronounced dyad symmetry, which in single-strand form would give rise to a hairpin. Evidence from an interspecies
comparison suggests that a similar structure is the result of selective forces. Another subfamily, M1, is present more proximally
in a subgroup of telomeres characterized by a special kind of repeat variability. Thus, a complex block with three kinds of
subfamilies may occupy different M1 telomeres depending on the stock of animals. We conclude that subfamilies are differentially
distributed between and within telomeres and are likely to serve different functions.
The Dictyostelium discoideum NC4 genome harbors approximately 150 individual copies of a retrotransposable element called the Dictyostelium repetitive element (DRE). This element contains nonidentical terminal repeats (TRs) consisting of conserved building blocks A and B in the left TR and B and C in the right TR. Seven different-sized classes of RNA transcripts from these elements were resolved by Northern (RNA) blot analysis, but their combined abundance was very low. When D. discoideum cells were grown in the presence of the respiratory chain blocker antimycin A, steady-state concentrations of these RNA species increased 10- to 20-fold. The D. discoideum genome contains two DRE subtypes, the full-length 5.7-kb DREa and the internally deleted 2.4-kb DREb. Both subtypes are transcribed, as confirmed by analysis of cloned cDNA. Primary transcripts from the sense strand originate at nucleotide +1 and terminate at two dominant sites, located 21 or 28 nucleotides upstream from the 3' end of the elements. The activity of a reasonably strong polymerase II promoter in the 5'-terminal A module is slightly upregulated by the tRNA gene located 50 +/- 4 nucleotides upstream and drastically reduced by the adjacent B module of the DRE. Transcripts from the opposite DNA strand (complementary-sense transcripts) were also detected, directed by an internally located polymerase II promoter residing within the C module. This latter transcription was initiated at multiple sites within the oligo(dA12) stretch which terminates DREs.
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In dipteran insects the most distal telomere-associated DNA known to exist consists of long, complex tandem repeats. We have classified the 340-bp tandemly arranged repeats in Chironomus pallidivittatus. The repeats are distributed in a small number of subfamilies. One type of the repeat has the character of a master unit from which other main units can be derived usually by simple changes. The derived subfamilies contain segments that are degenerate versions of the corresponding segment in the master sequence. Such segments can also occur together in one and the same repeat unit in different combinations. There is a complete absence of subfamily-specific base variants in regions lying outside of the degenerate segments. Homogenization takes place between DNA sequences that are often smaller than a whole repeat unit. The mosaic structure of the repeat arrays suggests that gene conversion is an important force in the generation and maintenance of this family of repeats.
In Drosophila, chromosome ends (telomeres) are composed of telomere-specific transposable elements (the retroposons HeT-A and TART). These elements are a bona fide part of the cellular machinery yet have many of the hallmarks of retrotransposable elements and retroviruses, raising the possibility that parasitic transposable elements and viruses might have evolved from mechanisms that the cell uses to maintain its chromosomes. It is striking that Drosophila, the model organism for many discoveries in genetics, development and molecular biology (including the classical concept of telomeres), should prove to have chromosome ends different from the generally accepted model. Studies of these telomere-specific retrotransposable elements raise questions about conventional wisdom concerning not only telomeres, but also transposable elements and heterochromatin.
We investigated the fate of dicentric chromosomes in the mitotic divisions of Drosophila melanogaster. We constructed chromosomes that were not required for viability and that carried P elements with inverted repeats of the target sites (FRTs) for the FLP site-specific recombinase. FLP-mediated unequal sister-chromatid exchange between inverted FRTs produced dicentric chromosomes at a high rate. The fate of the dicentric chromosome was evaluated in the mitotic cells of the male germline. We found that dicentric chromosomes break in mitosis, and the broken fragments can be transmitted. Some of these chromosome fragments exhibit dominant semilethality. Nonlethal fragments were broken at many sites along the chromosome, but the semilethal fragments were all broken near the original site of sister-chromatid fusion, and retained P element sequences near their termini. We discuss the implications of the recovery and behavior of broken chromosomes for checkpoints that detect double-strand break damage and the functions of telomeres in Drosophila.
A model of reverse transcription has been devised by which the detailed architecture of ten molecular structures is predicted. The model includes a number of novel features for which experimental evidence is presented. First, growing minus DNA strand is copied from the viral RNA only up to a position about 150 nucleotides from the 5' end of the RNA. Second, plus-strand DNA, after being copied from approximately 600 nucleotides at the 5' end of the minus-strand DNA, then transcribes the first approximately 20 nucleotides of the tRNApro primer (which is covalently attaced to the 5' end of the minus DNA strand). The 3' ends of the minus and plus DNA probably form a hybrid through the homology conferred by the primer binding site sequences. Third, the minus and plus DNA strands are elongated in a continuous fashion resulting in a linear double-stranded DNA molecule containing a 600 nucleotide direct repeat at both ends. The most of the features of the model have experimental support, and it appears to provide a credible description of reverse transcription.
Eight terminally deleted Drosophila melanogaster chromosomes have now been found to be "healed." In each case, the healed chromosome end had acquired sequence from the HeT DNA family, a complex family of repeated sequences found only in telomeric and pericentric heterochromatin. The sequences were apparently added by transposition events involving no sequence homology. We now report that the sequences transposed in healing these chromosomes identify a novel transposable element, HeT-A, which makes up a subset of the HeT DNA family. Addition of HeT-A elements to broken chromosome ends appears to be polar. The proximal junction between each element and the broken chromosome end is an oligo(A) tract beginning 54 nucleotides downstream from a conserved AATAAA sequence on the strand running 5' to 3' from the chromosome end. The distal (telomeric) ends of HeT-A elements are variably truncated; however, we have not yet been able to determine the extreme distal sequence of a complete element. Our analysis covers approximately 2,600 nucleotides of the HeT-A element, beginning with the oligo(A) tract at one end. Sequence homology is strong (greater than 75% between all elements studied). Sequence may be conserved for DNA structure rather than for protein coding; even the most recently transposed HeT-A elements lack significant open reading frames in the region studied. Instead, the elements exhibit conserved short-range sequence repeats and periodic long-range variation in base composition. These conserved features suggest that HeT-A elements, although transposable elements, may have a structural role in telomere organization or maintenance.
We investigated whether single P element insertional mutagenesis could be used to analyze heterochromatin within the Drosophila minichromosome Dp1187. Forty-five insertions of the P[lacZ,rosy+] element onto Dp1187 (recovered among 7,825 transpositions) were highly clustered. None was recovered in centromeric heterochromatin, but 39 occurred about 40 kb from the distal telomere within a 4.7-kb hotspot containing tandem copies of a novel 1.8-kb repetitive DNA sequence. The DNA within and distal to this region lacked essential genes and displayed several other properties characteristic of heterochromatin. The rosy+ genes within the inserted transposons were inhibited by position-effect variegation, and the subtelomeric region was underrepresented in polytene salivary gland cells. These experiments demonstrated that P elements preferentially transpose into a small subset of heterochromatic sites, providing a versatile method for studying the structure and function of these chromosome regions. This approach revealed that a Drosophila chromosome contains a large region of subtelomeric heterochromatin with specific structural and genetic properties.
Stocks of D. melanogaster X chromosomes carrying terminal deletions (RT chromosomes) have been maintained for several years. Some of the chromosomes are slowly losing DNA from the broken ends (as expected if replication is incomplete) and show no telomere-associated DNA added to the receding ends. Two stocks carry chromosomes that have become "healed" and are no longer losing DNA. In both stocks the broken chromosome end has acquired a segment of HeT DNA, a family of complex repeats found only at telomeres and in pericentric heterochromatin. Although the HeT family is complex, the HeT sequence joined to the broken chromosome end is the same in both stocks. In contrast, the two chromosomes are broken in different places and have no detectable sequence similarity at the junction with the new DNA. Sequence analysis suggests that the new telomere sequences have been added by a specific mechanism that does not involve homologous recombination.
A nucleic acid binding protein (NBP) derived from the gag gene of retroviruses that is thought to interact with genomic RNA in virion cores, contains a highly conserved arrangement
of cysteine residues. A search of available nucleic acid and protein sequences has revealed that the motif CysX2CysX4HisX4Cys (NBPcys) is invarlent in all replication competent retroviruses, a Syrian hamster intracisternal A-particle gene, the
Drosophila retrotransposon copia and in cauliflower mosaic virus (CaMV). In each case, NBPcys is located in that part of the ‘gag-pol’ region Just preceding a conserved protease amino acid sequence. This is of special significance for CaMV as NBPcys is in
the coat protein gene (ORF IV) upstream of the putative reverse transcrlptase gene (ORF V) and demonstrates that the gag-pol arrangement of reverse transcribing elements is preserved in CaMV. Moreover, CaMV differs from all other known NBPcys-con-taining
elements in that it packages a DNA genome in virions.
Destabilization of a P element transposon inserted in the subtelomeric region induced a set of similar chromosomal rearrangements. These rearrangements appear to be terminal deletions with endpoints clustered at the centromere-distal end of the transposon. The terminally deleted chromosome progressively loses sequences from the broken end at a rate of approximately 50-100 bp per fly generation, suggesting that the replication of this end may be incomplete. In most cases, capping of the broken end by readdition of new sequences was not observed. Past failures to recover terminal deletions of Drosophila chromosomes following X-ray mutagenesis may have been due to a cell cycle arrest in response to unrepaired DNA damage rather than to an absolute requirement for the telomere.
The mobile element jockey is similar in structural organization and coding potential to the LINEs of various organisms. As demonstrated here, two polyadenylated jockey transcripts detected at different stages of Drosophila ontogenesis and in cell cultures have the same length as genomic copies of jockey and correspond to the strand containing ORFs. alpha-amanitin experiments indicate that jockey is transcribed by RNA polymerase II. Analysis of both expression of CAT constructions and initiation of transcription in jockey genomic and transfected copies has shown that jockey transcription is controlled by an internal promoter. Inward location of the promoter allows it to be preserved in the course of replication via reverse transcription and accounts for the distribution of jockey and probably other LINEs throughout the genome. This is the first case of an internal promoter described for RNA polymerase II. The comparison of sequences at the beginning of LINE elements in Drosophila allows one to detect possible core sequences.
Ring chromosomes that have been opened to give linear chromosomes offer an opportunity to study the DNA sequences associated with new chromosome ends. The Drosophila melanogaster chromosome C(1)A was originally a ring chromosome, consisting of two linked X chromosomes, and thus had no telomeres. This chromosome has spontaneously opened in polytene region 13, a region near the middle of the euchromatic arm of the X chromosome. The opening of the ring has produced two new telomeres on the C(1)A chromosome. Each of the new telomeres has acquired He-T DNA sequences. He-T DNA is a complex family of repeated sequences found in the telomeric and pericentric heterochromatin of D. melanogaster chromosomes. He-T DNA sequences are detected, at various levels, in the most distal band on the end of each polytene chromosome in all D. melanogaster stocks. To our knowledge, these sequences have never been detected within the euchromatic chromosomal regions in any stock. The strong correlation between He-T DNA sequences and telomeric regions suggests that He-T sequences may have a role in organizing or maintaining the ends of chromosomes. The association of He-T DNA with newly acquired telomeres in a formerly euchromatic region, polytene region 13, strengthens this correlation.
The extent to which transcriptional and posttranscriptional regulation contributes to the coupling of histone gene expression and DNA replication was examined during the cell cycle in synchronized HeLa S3 cells. Rates of transcription were determined in vitro in isolated nuclei. A 3-5-fold increase in cell cycle dependent histone gene transcription was observed in early S phase, prior to the peak of DNA synthesis. This result is consistent with a previous determination of histone mRNA synthesis in intact cells [Plumb, M., Stein, J., & Stein, G. (1983) Nucleic Acids Res. 11, 2391]. The transcription of these genes did not change appreciably after inhibition of DNA replication by hydroxyurea treatment, although Northern blot analysis indicated that cellular levels of histone mRNA decreased rapidly in the presence of the drug. Total cellular levels of histone mRNA closely parallel the rate of DNA synthesis as a function of cell cycle progression, reaching a maximal 20-fold increase as compared with non S phase levels. This DNA synthesis dependent accumulation of histone mRNA occurs predominantly in the cytoplasm and appears to be mediated primarily by control of histone mRNA stability. Changes in nuclear histone mRNA levels were less pronounced. These combined observations suggest that both transcriptional regulation and posttranscriptional regulation contribute toward control of the cell cycle dependent accumulation of histone mRNA during S phase, while the stability of histone mRNA throughout S phase and the selective turnover of histone mRNAs, either at the natural termination of S phase or following inhibition of DNA synthesis, are posttranscriptionally regulated.(ABSTRACT TRUNCATED AT 250 WORDS)
In this paper, we examine first the steadiness of the rate of evolutionary change in a larval hemolymph protein, LHP, in numerous Drosophila species. We estimated amino acid sequence divergence from immunological distances measured with the quantitative microcomplement fixation technique. Using tests not depending on knowledge of absolute times of divergence, we estimated the variance of the rate of evolutionary change to be at least 4 times as large as that for a process resembling radioactive decay. Thus, the rate of evolution of this protein is as uniform as that of vertebrate proteins. Our analysis indicates no acceleration of protein evolution in the lineages leading to Hawaiian drosophilines. Second, we give an explicit description of a procedure for calculating the absolute value of the mean rate of evolutionary change in this protein. This procedure is suggested for general use in calculating absolute rates of molecular evolution. The mean rate of evolution of LHP is about 1.2 immunological distance units per million years, which probably corresponds to a unit evolutionary period of 4 million years; LHP thus evolves at a rate comparable to that of mammalian hemoglobins. Finally, we utilize the calibrated rate of LHP evolution to derive a time scale of evolution in the Drosophilidae and higher Diptera.
The rates of synthesis and turnover of histone mRNA in Drosophila embryos were determined by hybridization of in vivo and in vitro labeled embryonic RNA to Drosophila histone DNA of the recombinant plasmid cDm500. There is a large store of maternal histone mRNA, equivalent to at least 7 X 10(7) copies of each of the five classes of histone mRNA per embryo. Embryonic synthesis of histone mRNA begins at 90 min after oviposition, making the histone genes among the first to be transcribed by embryonic nuclei. Embryonic histone mRNA accumulates rapidly during the blastoderm and gastrula stages. The peak in the rate of histone mRNA synthesis per embryo coincides with the peak in the rate of DNA synthesis per embryo, which occurs at 6 hr after oviposition. After 6 hr, as the rate of DNA synthesis per embryo decreases, the rate of histone mRNA synthesis and the total mass of histone mRNA per embryo both drop sharply. The rate of histone mRNA synthesis per gene falls more than 60 fold in the first 13 hr after oviposition, from 1.3 -2.5 copies per gene-min at 2 hr to 0.02-0.03 copies per gene-min at 13 hr. From measurements of the mass of histone mRNA per embryo and of the rate of accumulation of newly synthesized histone mRNA at a number of stages of early embryogenesis we determined that the cytoplasmic half-life of histone mRNA decreases approximately 7 fold during early Drosophila development, from 2.3 hr at blastoderm to 20 min by the end of gastrulation. Thus the level of expression of histone genes in Drosophila development is controlled not only by the size of the maternal mRNA pool and changes in the rate of histone mRNA synthesis, but also by changes in the rate of histone mRNA turnover.
The general inhibition in transcriptional activity during mitosis abolishes the stress-inducible expression of the human hsp70 gene. Among the four transcription factors that bind to the human hsp70 promoter, the DNA-binding activities of three (C/EBP, GBP, and HSF1) were normal, while Sp1 showed reduced binding activity in mitotic cell extracts. In vivo footprinting and immunocytochemical analyses revealed that all of the sequence-specific transcription factors were displaced from promoter sequences as well as from bulk chromatin during mitosis. The correlation of transcription factor displacement with chromatin condensation suggests an involvement of chromatin structure in mitotic repression. However, retention of DNase I hypersensitivity suggests that the hsp70 promoter was not organized in a canonical nucleosome structure in mitotic chromatin. Displacement of transcription factors from mitotic chromosomes could present another window in the cell cycle for resetting transcriptional programs.
R2 is a non-LTR retrotransposable element that inserts at a specific site in the 28S rRNA genes of most insects. We have expressed the open reading frame of the R2 element from Bombyx mori, R2Bm, in E. coli and shown that it encodes both sequence-specific endonuclease and reverse transcriptase activities. The R2 protein makes a specific nick in one of the DNA strands at the insertion site and uses the 3' hydroxyl group exposed by this nick to prime reverse transcription of its RNA transcript. After reverse transcription, cleavage of the second DNA strand occurs. A similar mechanism of insertion may be used by other non-LTR retrotransposable elements as well as short interspersed nucleotide elements.
TART, a telomere-associated DNA element from Drosophila, is shown in this paper to have structural homology to LINE (long interspersed element)-like retrotransposons and to transpose to broken chromosome ends. TART DNA was detected by in situ hybridization in 7 of 10 independent additions of DNA to a chromosome end. We found evidence that a TART element had transposed to the chromosome end in each of two additions that were examined in detail. From the DNA sequence of a TART element that recently transposed, we infer that TART encodes two proteins having significant sequence similarity to the putative proteins of many LINEs. These results support the hypothesis that TART elements preferentially retrotranspose to the termini of chromosomes as part of the essential process by which Drosophila telomeres are maintained.
The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment
of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order
to downweight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are
varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific
gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather
than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced
gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new
program, CLUSTAL W which is freely available.
Four inherited neurodegenerative diseases are linked to abnormally expanded repeats of glutamine residues in the affected proteins. Molecular modeling followed by optical, electron, and x-ray diffraction studies of a synthetic poly(L-glutamine) shows that it forms beta-sheets strongly held together by hydrogen bonds. Glutamine repeats may function as polar zippers, for example, by joining specific transcription factors bound to separate DNA segments. Their extension may cause disease either by increased, nonspecific affinity between such factors or by gradual precipitation of the affected proteins in neurons.
Yeast strains were constructed in which a single telomere could be eliminated from the end of a dispensable chromosome. In wild-type cells, elimination of a telomere caused a RAD9-mediated cell cycle arrest, indicating that telomeres help cells to distinguish intact chromosomes from damaged DNA. However, many cells recovered from the arrest without repairing the damaged chromosome, replicating and segregating it for as many as ten cell divisions prior to its eventual loss. Telomere elimination caused a dramatic increase in loss of the chromosome in all strains examined, demonstrating that yeast telomeres are also essential for maintaining chromosome stability. Thus, in spite of checkpoint and DNA damage repair systems, many chromosomes that lose a telomere are themselves destined for loss.
We present the first isolation of the terminal DNA of an intact Drosophila telomere. It differs from those isolated from other eukaryotes by the lack of short tandem repeats at the terminus. The terminal 14.5 kb is composed of four tandem elements derived from two families of non-long terminal repeat retrotransposons and is subject to slow terminal loss. One of these transposon families, TART (telomere-associated retrotransposon), is described for the first time here. The other element, HeT-A, has previously been shown to transpose to broken chromosome ends. Our results provide key evidence that these elements also transpose to natural chromosome ends. We propose that the telomere-associated repetitive DNA is maintained by saltatory expansions, including terminal transpositions of specialized retrotransposons, which serve to balance terminal loss.
The I-R system of hybrid dysgenesis in Drosophila melanogaster is controlled by a long interspersed nuclear element-like retroposon,
the I factor. Transposition of the I factor occurs at a high frequency only in the ovaries of females produced by crossing
males of inducer strains that contain functional I factors with females of reactive strains that lack them. In this study,
the 5' untranslated region of the I factor was joined to the chloramphenicol acetyltransferase gene, and activity was assayed
in transfected D. melanogaster tissue culture cells and transformed flies. The results have identified a promoter that lies
within the first 186 pb of the I factor. Deletion analysis shows that nucleotides +1 to +40 are sufficient for high promoter
activity and accurate transcription initiation. This region contains sequences that are found in a class of RNA polymerase
II promoters that lack both a TATA box and CpG-rich motifs. In transformed flies, high levels of expression from nucleotides
+1 to +186 are confined to the ovaries of reactive females, suggesting that the promoter is involved in the tissue and cytotype
specificity of transposition.
The dipteran Chironomus tentans has complex tandemly repeated 350-bp DNA sequences at or near the chromosome ends. As in Drosophila
melanogaster, short simple repeats with cytosines and guanines in different strands have never been observed. We were therefore
interested in learning whether the Chironomus repeats could have evolved from simple sequence telomeric DNA, which might suggest
that they constitute a functional equivalent. We screened for repeat units with evolutionarily ancient features within the
tandem arrays and recovered two clones with a less-evolved structure. Sequence analysis reveals that the present-day 350-bp
unit probably evolved from a simpler 165-bp unit through the acquisition of transposed sequences. The 165-bp unit contains
DNA with a highly biased distribution of cytosine and guanine between the two strands, although with the ratios inverted in
two minor parts of the repeat. It is largely built up of short degenerate subrepeats for which most of the sequence can be
reconstructed. The consensus for the subrepeat sequence is similar to the simple telomeric repeat sequences of several kinds
of eukaryotes. We propose that the present-day unit has evolved from telomeric, simple sequence, asymmetric DNA from which
it has retained some original sequence features and possibly functions.
A pentanucleotide repetitive sequence, (TTAGG)n, has been isolated from a silkworm genomic library, using cross-hybridization
with a (TTNGGG)5 sequence, which is conserved among most eukaryotic telomeres. Both fluorescent in situ hybridization and
Bal 31 exonuclease experiments revealed major clusters of (TTAGG)n at the telomeres of all Bombyx chromosomes. To determine
the evolutionary origin of this sequence, two types of telomeric sequence, (TTAGG)5 and a hexanucleotide repetitive sequence,
(TTAGGG)4, which is conserved mainly among vertebrate and several invertebrate telomeres so far examined, were hybridized
to DNAs from a wide variety of eukaryotic species under highly stringent hybridization conditions. The (TTAGGG)5 oligonucleotide
hybridized to genomic DNAs from vertebrates and several nonvertebrate species, as has been reported so far, but not to any
DNAs from insects. On the other hand, the Bombyx type of telomere sequence, (TTAGG)n, hybridized to DNAs from 8 of 11 orders
of insect species tested but not to vertebrate DNAs, suggesting that this TTAGG repetitive sequence is conserved widely among
insects.
Yeast cells lacking a functional EST1 gene show progressive shortening of the terminal G1-3T telomeric repeats and a parallel increase in the frequency of cell death. Although the majority of the cells in an est1- culture die, a minor subpopulation survives the potentially lethal consequences of the est1 mutation. We show that these est1- survivors arise as a result of the amplification and acquisition of subtelomeric elements (and their deletion derivatives) by a large number of telomeres. Hence, even when the primary pathway for telomere replication is defective, an alternative backup pathway can restore telomere function and keep the cell alive.