Deterrence factors for copyright infringement online

The purpose of this study was to investigate deterrence factors for online file-sharing by analyzing different conditions that affect compliance with the law through survey of the students in a large university in Southern U.S. The findings show that certainty of punishment, stigma of the label, knowledge of the laws and consensus with the rule negatively correlated with both actual and likely future file-sharing activities of the user

Contribution of telomerase RNA retrotranscription to DNA double-strand break repair during mammalian genome evolution

A comparative analysis of two primate and two rodent genomes suggests that telomerase was utilized, in some instances, for the repair of DNA double-strand breaks during mammalian evolution

Gene amplification in human cells knocked down for RAD54

Background: In mammalian cells gene amplification is a common manifestation of genome instability promoted by DNA double-strand breaks (DSBs). The repair of DSBs mainly occurs through two mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR). We previously showed that defects in the repair of DSBs via NHEJ could increase the frequency of gene amplification. In this paper we explored whether a single or a combined defect in DSBs repair pathways can affect gene amplification. Results: We constructed human cell lines in which the expression of RAD54 and/or DNA-PKcs was constitutively knocked-down by RNA interference. We analyzed their radiosensitivity and their capacity to generate amplified DNA. Our results showed that both RAD54 and DNA-PKcs deficient cells are hypersensitive to γ-irradiation and generate methotrexate resistant colonies at a higher frequency compared to the proficient cell lines. In addition, the analysis of the cytogenetic organization of the amplicons revealed that isochromosome formation is a prevalent mechanism responsible for copy number increase in RAD54 defective cells. Conclusions: Defects in the DSBs repair mechanisms can influence the organization of amplified DNA. The high frequency of isochromosome formation in cells deficient for RAD54 suggests that homologous recombination proteins might play a role in preventing rearrangements at the centromeres

CENP-A binding domains and recombination patterns in horse spermatocytes

Centromeres exert an inhibitory effect on meiotic recombination, but the possible contribution of satellite DNA to this "centromere effect" is under debate. In the horse, satellite DNA is present at all centromeres with the exception of the one from chromosome 11. This organization of centromeres allowed us to investigate the role of satellite DNA on recombination suppression in horse spermatocytes at the stage of pachytene. To this aim we analysed the distribution of the MLH1 protein, marker of recombination foci, relative to CENP-A, marker of centromeric function. We demonstrated that the satellite-less centromere of chromosome 11 causes crossover suppression, similarly to satellite-based centromeres. These results suggest that the centromere effect does not depend on satellite DNA. During this analysis, we observed a peculiar phenomenon: while, as expected, the centromere of the majority of meiotic bivalent chromosomes was labelled with a single immunofluorescence centromeric signal, double-spotted or extended signals were also detected. Their number varied from 0 to 7 in different cells. This observation can be explained by positional variation of the centromeric domain on the two homologs and/or misalignment of pericentromeric satellite DNA arrays during homolog pairing confirming the great plasticity of equine centromeres

Uncoupling of Satellite DNA and Centromeric Function in the Genus Equus

In a previous study, we showed that centromere repositioning, that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement, has occurred frequently during the evolution of the genus Equus. In this work, the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus, E. asinus, E. grevyi, and E. burchelli highlighted two atypical features: 1) several centromeres, including the previously described evolutionary new centromeres (ENCs), seem to be devoid of satellite DNA, and 2) satellite repeats are often present at non-centromeric termini, probably corresponding to relics of ancestral now inactive centromeres. Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function. The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA. The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs

The telomeric transcriptome of Schizosaccharomyces pombe

Eukaryotic telomeres are transcribed into telomeric repeat-containing RNA (TERRA). Telomeric transcription has been documented in mammals, birds, zebra fish, plants and budding yeast. Here we show that the chromosome ends of Schizosaccharomyces pombe produce distinct RNA species. As with budding yeast and mammals, S. pombe contains G-rich TERRA molecules and subtelomeric RNA species transcribed in the opposite direction of TERRA (ARRET). Moreover, fission yeast chromosome ends produce two novel RNA species: C-rich telomeric repeat-containing transcripts (ARIA) and subtelomeric transcripts complementary to ARRET (αARRET). RNA polymerase II (RNAPII) associates with pombe chromosome ends in vivo and the telomeric factor Rap1 negatively regulates this association, as well as the cellular accumulation of RNA emanating from chromosome ends. We also show that the RNAPII subunit Rpb7 and the non-canonical poly(A) polymerases Cid12 and Cid14 are involved in the regulation of TERRA, ARIA, ARRET and αARRET transcripts. We confirm the evolutionary conservation of telomere transcription, and reveal intriguing similarities and differences in the composition and regulation of telomeric transcripts among model organisms

Non-coding telomeric and subtelomeric transcripts are differentially regulated by telomeric and heterochromatin assembly factors in fission yeast

While telomere repeat-containing non-coding RNA has been identified in a variety of eukaryotes, its biological role is not yet clear. We have identified telomeric transcripts in fission yeast, a model system that combines precise genetic manipulability with telomeres remarkably similar to those of human. Like human and budding yeast, fission yeast harbours a population of telomeric RNA molecules containing G-rich telomeric repeats transcribed from the subtelomere to the telomere. In addition, we detect substantial levels of C-rich telomeric RNA whose appearance is independent of the RNA-dependent RNA polymerase, suggesting that the telomere repeats themselves serve as promoter sites; multiple distinct subtelomeric RNAs are also present. The regulation of these transcripts depends on the telomere-associated proteins Taz1 and Rap1, as deletion of taz1+ or rap1+ leads to increased levels of both telomere repeat-containing and subtelomeric transcripts. In contrast, loss of the heterochromatin proteins Swi6 or Clr4 or the telomerase regulator Rif1 results in elevated subtelomeric RNA levels while telomere-repeat containing transcript levels remain repressed. Coupled with the large body of knowledge surrounding the functions of telomeric and heterochromatin factors in fission yeast, these in vivo analyses suggest testable models for the roles of TERRA in telomere function

Early-life telomere dynamics differ between the sexes and predict growth in the barn swallow (Hirundo rustica)

Telomeres are conserved DNA-protein structures at the termini of eukaryotic chromosomes which contribute to maintenance of genome integrity, and their shortening leads to cell senescence, with negative consequences for organismal functions. Because telomere erosion is influenced by extrinsic and endogenous factors, telomere dynamics may provide a mechanistic basis for evolutionary and physiological trade-offs. Yet, knowledge of fundamental aspects of telomere biology under natural selection regimes, including sex- and context-dependent variation in early-life, and the covariation between telomere dynamics and growth, is scant. In this study of barn swallows (Hirundo rustica) we investigated the sex-dependent telomere erosion during nestling period, and the covariation between relative telomere length and body and plumage growth. Finally, we tested whether any covariation between growth traits and relative telomere length depends on the social environment, as influenced by sibling sex ratio. Relative telomere length declined on average over the period of nestling maximal growth rate (between 7 and 16 days of age) and differently covaried with initial relative telomere length in either sex. The frequency distribution of changes in relative telomere length was bimodal, with most nestlings decreasing and some increasing relative telomere length, but none of the offspring traits predicted the a posteriori identified group to which individual nestlings belonged. Tail and wing length increased with relative telomere length, but more steeply in males than females, and this relationship held both at the within- and among-broods levels. Moreover, the increase in plumage phenotypic values was steeper when the sex ratio of an individual's siblings was female-biased. Our study provides evidence for telomere shortening during early life according to subtly different dynamics in either sex. Furthermore, it shows that the positive covariation between growth and relative telomere length depends on sex as well as social environment, in terms of sibling sex ratio

Telomerase Efficiently Elongates Highly Transcribing Telomeres in Human Cancer Cells

RNA polymerase II transcribes the physical ends of linear eukaryotic chromosomes into a variety of long non-coding RNA molecules including telomeric repeat-containing RNA (TERRA). Since TERRA discovery, advances have been made in the characterization of TERRA biogenesis and regulation; on the contrary its associated functions remain elusive. Most of the biological roles so far proposed for TERRA are indeed based on in vitro experiments carried out using short TERRA-like RNA oligonucleotides. In particular, it has been suggested that TERRA inhibits telomerase activity. We have exploited two alternative cellular systems to test whether TERRA and/or telomere transcription influence telomerase-mediated telomere elongation in human cancer cells. In cells lacking the two DNA methyltransferases DNMT1 and DNMT3b, TERRA transcription and steady-state levels are greatly increased while telomerase is able to elongate telomeres normally. Similarly, telomerase can efficiently elongate transgenic inducible telomeres whose transcription has been experimentally augmented. Our data challenge the current hypothesis that TERRA functions as a general inhibitor of telomerase and suggest that telomere length homeostasis is maintained independently of TERRA and telomere transcription

Polymorphic organization of constitutive heterochromatin in Equus asinus (2n = 62) chromosome 1

In the karyotype of Equus asinus (domestic donkey, 2n = 62), non-centromeric heterochromatic bands have been described in subcentromeric and telomeric positions. In particular, chromosome 1 is characterised by heterochromatic bands in the proximal region of the long arm and in the short arm; it has been shown that these regions are polymorphic in size. Here we investigated the variation in the intensity and distribution of fluorescence signals observed on donkey chromosome 1 after in situ hybridization with two DNA probes containing fragments from the two major equine satellite DNA families. Our results show that, in Equus asinus chromosome 1, the amount and distribution of large clusters of satellite DNA can define at least nine polymorphic variants of the constitutive heterochromatin that cannot be detected by C-banding alone