Radmila Capkova Frydrychova

Repeated Losses of TTAGG Telomere Repeats in Evolution of Beetles (Coleoptera)

We studied the occurrence of (TTAGG)n telomere repeats in 12 species of beetles, representing main lineages of the Coleoptera phylogenetic tree, by Southern hybridization and fluorescence in situ hybridization (FISH). In contrast to other insect orders, beetles were heterogeneous with respect to the occurrence of TTAGG repeats. In addition, the presence or absence of (TTAGG)n motif was irrespective of phylogenetic relationships. In the suborder Polyphaga, six species displayed positive hybridization signals. These were Silpha obscura, Agrilus viridis, Ampedus sanguineus, Stegobium paniceum, Oryzaephilus surinamensis, and Leptinotarsa decemlineata. Whereas negative signals were obtained in three polyphagan species, Geotrupes stercorarius, Thanasimus formicarius, and Sitophilus granarius. In the suborder Adephaga, the TTAGG sequence was present in one species, Graphoderus cinereus, and absent in two species, Orectochilus villosus and Pterostichus oblongopunctatus. We concluded that the telomerase-dependent (TTAGG)n motif had been repeatedly lost in different phylogenetic branches of Coleoptera and probably replaced with another mechanism of telomere elongation. This had to happen at least 5–6 times. The results suggest a predisposition or a backup mechanism of telomere maintenance in the genome of beetles that enabled them to make frequent evolutionary changes in the telomere composition.

Neo-sex chromosomes and adaptive potential in tortricid pests

Changes in genome architecture often have a significant effect on ecological specialization and speciation. This effect may be further enhanced by involvement of sex chromosomes playing a disproportionate role in reproductive isolation. We have physically mapped the Z chromosome of the major pome fruit pest, the codling moth, Cydia pomonella (Tortricidae), and show that it arose by fusion between an ancestral Z chromosome and an autosome corresponding to chromosome 15 in the Bombyx mori reference genome. We further show that the fusion originated in a common ancestor of the main tortricid subfamilies, Olethreutinae and Tortricinae, comprising almost 700 pest species worldwide. The Z–autosome fusion brought two major genes conferring insecticide resistance and clusters of genes involved in detoxification of plant secondary metabolites under sex-linked inheritance. We suggest that this fusion significantly increased the adaptive potential of tortricid moths and thus contributed to their radiation and subsequent speciation.

Karyotype, sex chromatin and sex chromosome differen- tiation in the carob moth, Ectomyelois ceratoniae (Lepi- doptera: Pyralidae)

Abstract This paper reports results of the first cytogenetic investigation carried out in the carob moth, Ectomyelois ceratoniae Zeller, the serious polyphagous pest of both stored products and field crops in the Mediterranean basin and Near East regions. Preparations of mitotically dividing spermatogonia and oogonia revealed that the carob moth karyotype consists of 2n = 62 chromosomes. As typical for Lepidoptera, the chromosomes were of a holokinetic type, only slightly differed in their sizes, and displayed no morphological structures including primary constrictions (the centromeres) that could enable us their identification. Metaphase I spermatocytes showed 31 bivalents. Accordingly, 31 chromosome elements were observed in metaphase II spermatocytes. Hence the haploid chromosome number is n = 31. In pachytene complements, two NOR bivalents were observed. In highly polyploid nuclei of the Malpighian tubule cells and silk glands, females showed a heterochromatin body, the so-called sex chromatin or W chromatin, that was absent in male nuclei. This indicated that the carob moth possesses a WZ/ZZ sex chromosome system. However, we failed to identify the sex chromosome bivalent WZ in pachytene oocytes. In order to differentiate the sex chromosomes, we employed genomic in situ hybridization (GISH) and comparative genomic hybridization (CGH). GISH detected the W chromosome by strong binding of the Cy3-labelled, female-derived DNA probe. With CGH, both the Cy3-labelled female-derived probe and Fluor-X labelled male-derived probe evenly bound to the W. This suggested that the W is composed predominantly of repetitive DNA sequences occurring scattered in other chromosomes but accumulated in the W chromosome.

HP1 is distributed within distinct chromatin domains at Drosophila telomeres.

Telomeric regions in Drosophila are composed of three subdomains. A chromosome cap distinguishes the chromosome end from a DNA double-strand break; an array of retrotransposons, HeT-A, TART, and TAHRE (HTT), maintains telomere length by targeted transposition to chromosome ends; and telomere-associated sequence (TAS), which consists of a mosaic of complex repeated sequences, has been identified as a source of gene silencing. Heterochromatin protein 1 (HP1) and HP1-ORC-associated protein (HOAP) are major protein components of the telomere cap in Drosophila and are required for telomere stability. Besides the chromosome cap, HP1 is also localized along the HTT array and in TAS. Mutants for Su(var)205, the gene encoding HP1, have decreased the HP1 level in the HTT array and increased transcription of individual HeT-A elements. This suggests that HP1 levels directly affect HeT-A activity along the HTT array, although they have little or no effect on transcription of a white reporter gene in the HTT. Chromatin immunoprecipitation to identify other heterochromatic proteins indicates that TAS and the HTT array may be distinct from either heterochromatin or euchromatin.

Transcriptional Activity of the Telomeric Retrotransposon HeT-A in Drosophila melanogaster Is Stimulated as a Consequence of Subterminal Deficiencies at Homologous and Nonhomologous Telomeres

ABSTRACT Drosophila melanogaster telomeres have two DNA domains: a terminal array of retrotransposons and a subterminal repetitive telomere-associated sequence (TAS), a source of telomere position effect (TPE). We reported previously that deletion of the 2L TAS array leads to dominant suppression of TPE by stimulating in trans expression of a telomeric transgene. Here, we compared the transcript activities of a w transgene inserted between the retrotransposon and TAS arrays at the 2L telomere in genotypes with different lengths of the 2L TAS. In contrast to individuals bearing a wild-type 2L homologue, flies with a TAS deficiency showed a significant increase in the level of telomeric w transcript during development, especially in pupae. Moreover, we identified a read-through w transcript initiated from a retrotransposon promoter in the terminal array. Read-through transcript levels also significantly increased with the presence of a 2L TAS deficiency in trans, indicating a stimulating force of the TAS deficiency on retrotransposon promoter activity. The read-through transcript contributes to total w transcript, although most w transcript originates at the w promoter. While silencing of transgenes in nonhomologous telomeres is suppressed by 2L TAS deficiencies, suggesting a global effect, the overall level of HeT-A transcripts is not increased under similar conditions.

Effect of low doses of herbicide paraquat on antioxidant defense in Drosophila.

Despite a high toxicity, paraquat is one of the most widely used herbicides in the world. Our study evaluated the effect of paraquat exposure on antioxidant response and locomotion activity in Drosophila melanogaster. We examined the enzymatic activity of superoxide dismutase (SOD) and catalase, and the transcript levels of both enzymes. Flies were exposed to a wide range of paraquat concentrations (0.25 μM to 25 mM) for 12 h. SOD, at both transcript and enzymatic levels, revealed a biphasic dose-response curve with the peak at 2.5 μM paraquat. A similar dose-response curve was observed at transcript levels of catalase. Males revealed higher susceptibility to paraquat exposure, displaying higher lethality, increased levels of SOD activity, and increased peroxide levels than in females. We found that the exposure of females to 2.5 μM paraquat leads to an increase in locomotion activity. Because susceptibility to paraquat was enhanced by mating, the study supports the hypothesis of elevation of stress sensitivity as a physiological cost of reproduction.

Distribution of TTAGG-specific telomerase activity in insects

In most eukaryotes, telomeres consist of tandem arrays of a short repetitive DNA sequence. Insect telomeres are generally constituted by a (TTAGG)n repeat motif. Usually, telomeres are maintained by telomerase, a specialized reverse transcriptase that adds this sequence to chromosome ends. We examined telomerase activity in 15 species across Insecta. Telomerase activity was revealed in Isoptera, Blattaria, Lepidoptera, Hymenoptera, Trichoptera, Coleoptera, and Sternorrhyncha. In contrast, we were not able to detect telomerase activity in Orthoptera, Zygentoma, and Phasmida. Because we found telomerase activity in phylogenetically distant species, we conclude that a distribution pattern of (TTAGG)n sequence in Insecta is generally consistent with that of telomerase activity. Thus, the TTAGG-telomerase system is functional across the Insecta. Using real-time quantitative telomeric repeat amplification protocol (RTQ-TRAP) system, we quantified telomerase activity in different developmental stages and different tissues of a cockroach, Periplaneta americana. We show that telomerase is upregulated in young instars and gradually declines during development. In adults, it is most active in testes and ovaries. Thus, the telomerase activity of hemimetabolous insects seems to be associated with cell proliferation and organismal development.

Telomere Maintenance in Organisms without Telomerase

Telomeres serve two vital functions to eukaryotes. They act as a protective chromosome cap to distinguish natural chromosome ends from double stranded DNA breaks and to avoid inappropriate fusions of telomeric sequences, and they maintain chromosome length by adding DNA to the ends of chromosomes. Telomeres thus balance the loss of terminal DNA due to the inability of the replication machinery to completely replicate linear DNA molecules (Olovnikov, 1973; Watson, 1972). In many cases the newly replicated chromosome ends are resected to allow for the formation of a t-loop that helps to hide the tip (Griffith et al., 1999; Wellinger et al., 1996). Most eukaryotes elongate chromosome ends with a special reverse transcriptase, telomerase, that carries a specific RNA template with telomeric sequence (Greider, 1996). The telomerase enzyme repeatedly adds copies of the short telomeric DNA sequence to the chromosome end. While there is strict conservation of telomeric sequence repeat in most species, the repeat unit has changed over evolutionary time. Holotrichous ciliates, e. g. Tetrahymena, use the sequence (TTGGGG)n (Blackburn & Gall, 1978), while hypotrichous ciliates, e. g. Oxytricha, use (TTTTGGGG)n (Klobutcher et al., 1981; Oka et al., 1980). The primary telomeric sequence in plants is (TTTAGGG)n (Richards & Ausubel, 1988; Zellinger & Riha, 2007), although the alga Chlamydomonas uses (TTTTAGGG)n. In the yeasts the telomeric sequence has the same general motif, but is not as tightly controlled. Saccharomyces for example uses (TG1-3)n (Shampay et al., 1984; Wang & Zakian, 1990), while Schizosaccharomyces has (TTACAG1-8)n (Matsumoto et al., 1987). The sequence found at the telomeres of most metazoans is (TTAGGG)n (Meyne et al., 1989; Traut et al., 2007), although arthropods use (TTAGG)n (Okazaki et al., 1993). Lack of the predominant telomeric sequence in a species does not, however, signify that telomerase-generated terminal sequences are missing. For example, the metazoan-type telomeric sequence is found in place of the plant sequence in Aloe species (Weiss & Scherthan, 2002). In order to establish that a telomeraseindependent, chromosome maintenance system exists it is also necessary to show a lack of a telomerase gene and telomerase activity, and to identify the nature of the DNA sequence at the chromosome termini. Establishing the negative is always difficult, and confirming that a specific sequence is at, not merely near, the chromosome tip is not trivial. Conversely, the presence of a

Telomeres: Their Structure and Maintenance

Telomeres are essential nucleoprotein structures at the ends of eukaryotic chromosomes. They play several essential roles preserving genome stability and function, including distinguish‐ ing chromosome ends from DNA double stranded breaks (DSBs) and maintenance of chromo‐ some length. Due to the inability of conventional DNA polymerases to replicate the very end of a chromosome, sometimes known as the end replication problem, chromosome ends short‐ en with every round of DNA replication. In the absence of special telomere maintenance mechanisms this telomere shortening leads to replicative senescence and apoptosis. Several telomere maintenance mechanisms have been identified; these are reflected in several known types of telomeres. In most eukaryotes telomeres comprise a tandem array of a short, 5-8 bp, well conserved repeat unit, and telomere length is maintained by telomerase, a specialized reverse transcriptase that carries its own RNA template and adds telomeric sequences onto chromosome ends [1]. Nevertheless, in some organisms the array of short telomeric se‐ quence motifs has been replaced with less conventional sequences, such as satellite sequen‐ ces or transposable elements. The telomeres of such organisms are maintained through homologous recombination or through transposition of the mobile elements [2,3]. These dif‐ ferent telomere types present distinct difficulties for chromosome end protection. Telomeres maintained by telomerase are protected by a proteinaceous telomere cap, termed shelterin, that recognizes chromosome ends in a DNA sequence specific manner, while telomeres with long terminal repeat units are protected by a cap, termed terminin, that binds to chromo‐ some ends independently of DNA sequence.

Chronic low-dose pro-oxidant treatment stimulates transcriptional activity of telomeric retroelements and increases telomere length in Drosophila.

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.