Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in Entamoeba histolytica

<p>Abstract</p> <p>Background</p> <p>In eukaryotic and prokaryotic cells, homologous recombination is an accurate mechanism to generate genetic diversity, and it is also used to repair DNA double strand-breaks. <it>RAD52 </it>epistasis group genes involved in recombinational DNA repair, including <it>mre11, rad50, nsb1/xrs2, rad51, rad51c/rad57, rad51b/rad55, rad51d, xrcc2, xrcc3, rad52, rad54, rad54b/rdh54 </it>and <it>rad59 </it>genes, have been studied in human and yeast cells. Notably, the RAD51 recombinase catalyses strand transfer between a broken DNA and its undamaged homologous strand, to allow damaged region repair. In protozoan parasites, homologous recombination generating antigenic variation and genomic rearrangements is responsible for virulence variation and drug resistance. However, in <it>Entamoeba histolytica </it>the protozoan parasite responsible for human amoebiasis, DNA repair and homologous recombination mechanisms are still unknown.</p> <p>Results</p> <p>In this paper, we initiated the study of the mechanism for DNA repair by homologous recombination in the primitive eukaryote <it>E. histolytica </it>using UV-C (150 J/m²) irradiated trophozoites. DNA double strand-breaks were evidenced in irradiated cells by TUNEL and comet assays and evaluation of the EhH2AX histone phosphorylation status. In <it>E. histolytica </it>genome, we identified genes homologous to yeast and human RAD52 epistasis group genes involved in DNA double strand-breaks repair by homologous recombination. Interestingly, the <it>E. histolytica </it>RAD52 epistasis group related genes were differentially expressed before and after UV-C treatment. Next, we focused on the characterization of the putative recombinase EhRAD51, which conserves the typical architecture of RECA/RAD51 proteins. Specific antibodies immunodetected EhRAD51 protein in both nuclear and cytoplasmic compartments. Moreover, after DNA damage, EhRAD51 was located as typical nuclear <it>foci</it>-like structures in <it>E. histolytica </it>trophozoites. Purified recombinant EhRAD51 exhibited DNA binding and pairing activities and exchanging reactions between homologous strands <it>in vitro</it>.</p> <p>Conclusion</p> <p><it>E. histolytica </it>genome contains most of the RAD52 epistasis group related genes, which were differentially expressed when DNA double strand-breaks were induced by UV-C irradiation. In response to DNA damage, EhRAD51 protein is overexpressed and relocalized in nuclear <it>foci</it>-like structures. Functional assays confirmed that EhRAD51 is a <it>bonafide </it>recombinase. These data provided the first insights about the potential roles of the <it>E. histolytica </it>RAD52 epistasis group genes and EhRAD51 protein function in DNA damage response of this ancient eukaryotic parasite.</p

Extreme genomic erosion after recurrent demographic bottlenecks in the highly endangered Iberian lynx

Background: Genomic studies of endangered species provide insights into their evolution and demographic history, reveal patterns of genomic erosion that might limit their viability, and offer tools for their effective conservation. The Iberian lynx (Lynx pardinus) is the most endangered felid and a unique example of a species on the brink of extinction. Results: We generate the first annotated draft of the Iberian lynx genome and carry out genome-based analyses of lynx demography, evolution, and population genetics. We identify a series of severe population bottlenecks in the history of the Iberian lynx that predate its known demographic decline during the 20th century and have greatly impacted its genome evolution. We observe drastically reduced rates of weak-to-strong substitutions associated with GC-biased gene conversion and increased rates of fixation of transposable elements. We also find multiple signatures of genetic erosion in the two remnant Iberian lynx populations, including a high frequency of potentially deleterious variants and substitutions, as well as the lowest genome-wide genetic diversity reported so far in any species. Conclusions: The genomic features observed in the Iberian lynx genome may hamper short- and long-term viability through reduced fitness and adaptive potential. The knowledge and resources developed in this study will boost the research on felid evolution and conservation genomics and will benefit the ongoing conservation and management of this emblematic species

Targets of the Entamoeba histolytica Transcription Factor URE3-BP

The Entamoeba histolytica transcription factor Upstream Regulatory Element 3-Binding Protein (URE3-BP) is a calcium-responsive regulator of two E. histolytica virulence genes, hgl5 and fdx1. URE3-BP was previously identified by a yeast one-hybrid screen of E. histolytica proteins capable of binding to the sequence TATTCTATT (Upstream Regulatory Element 3 (URE3)) in the promoter regions of hgl5 and fdx1. In this work, precise definition of the consensus URE3 element was performed by electrophoretic mobility shift assays (EMSA) using base-substituted oligonucleotides, and the consensus motif validated using episomal reporter constructs. Transcriptome profiling of a strain induced to produce a dominant-positive URE3-BP was then used to identify additional genes regulated by URE3-BP. Fifty modulated transcripts were identified, and of these the EMSA defined motif T[atg]T[tc][cg]T[at][tgc][tg] was found in over half of the promoters (54% p<0.0001). Fifteen of the URE3-BP regulated genes were potential membrane proteins, suggesting that one function of URE3-BP is to remodel the surface of E. histolytica in response to a calcium signal. Induction of URE3-BP leads to an increase in tranwell migration, suggesting a possible role in the regulation of cellular motility

Supporting SIP-based End-to-End Data Distribution Service QoS in WANs

International audienceAssuring end-to-end QoS in enterprise distributed real-time and embedded (DRE) systems is hard due to the heterogeneity and transient behavior of communication networks, the lack of integrated mechanisms that schedule communication and computing resources holistically, and the scalability limits of IP multicast in wide-area networks (WANs). This paper makes three contributions to research on overcoming these problems in the context of enterprise DRE systems that use the OMG Data Distribution Service (DDS) quality-of- service (QoS)-enabled publish/subscribe (pub/sub) middleware over WANs. First, it codifies the limitations of conventional DDS implementations deployed over WANs. Second, it describes a middleware component called Proxy DDS that bridges multiple, isolated DDS domains deployed over WANs. Third, it describes the NetQSIP framework that combines multi-layer, standards-based technologies including the OMG-DDS, Session Initiation Protocol (SIP), and IP DiffServ to support end-to-end QoS in a WAN and shield pub/sub applications from tedious and error-prone details of network QoS mechanisms. The results of experiments using Proxy DDS and NetQSIP show how combining DDS with SIP in DiffServ networks significantly improves dynamic resource reservation in WANs and provides effective end-to-end QoS management