Polymerization and nucleic acid-binding properties of human L1 ORF1 protein

The L1 (LINE 1) retrotransposable element encodes two proteins, ORF1p and ORF2p. ORF2p is the L1 replicase, but the role of ORF1p is unknown. Mouse ORF1p, a coiled-coil-mediated trimer of ∼42-kDa monomers, binds nucleic acids and has nucleic acid chaperone activity. We purified human L1 ORF1p expressed in insect cells and made two findings that significantly advance our knowledge of the protein. First, in the absence of nucleic acids, the protein polymerizes under the very conditions (0.05 M NaCl) that are optimal for high (∼1 nM)-affinity nucleic acid binding. The non-coiled-coil C-terminal half mediates formation of the polymer, an active conformer that is instantly resolved to trimers, or multimers thereof, by nucleic acid. Second, the protein has a biphasic effect on mismatched double-stranded DNA, a proxy chaperone substrate. It protects the duplex from dissociation at 37°C before eventually melting it when largely polymeric. Therefore, polymerization of ORF1p seemingly affects its interaction with nucleic acids. Additionally, polymerization of ORF1p at its translation site could explain the heretofore-inexplicable phenomenon of cis preference—the favored retrotransposition of the actively translated L1 transcript, which is essential for L1 survival

Plant and Aphid Partners of Poleroviruses: Role in Virus Transmission by Aphids?

Comité de lecture : trueConférence invitée : falseDate de début de l'événement : 2011-07-11Date de fin de l'évenement : 2011-07-14Date de validation : Tue Aug 13 15:11:30 CEST 2013Diffusion de la pièce jointe : Publique, PubliqueIdentifiant : 200587Langue du titre : engNombre de consultation de la notice : 77Nombre de téléchargements de la pièce jointe : 8Pays de l'événement : BRAPublic visé : ScientifiqueType de communication avec actes : Présentation oraleType d'événement : SymposiumPoleroviruses are phloem limited viruses strictly transmitted by aphids in a circulative and non propagative manner. Virions are acquired by aphids when ingesting sap from infected plants. Virus particles cross the gut epithelium and the accessory salivary gland cells before being released, together with saliva, into the plant during a subsequent feed. This highly specific transcytosis mechanism relies on the presence of virus receptors on the surface of the aphid cells. We developed several approaches to identify virus partners in the plant and in the aphid to analyse their role in virus transmission by the vector. By screening different aphid cDNA libraries using a yeast two hybrid system, only few candidates were able to bind virus structural proteins. Among them, we found two nuclear proteins (GAR1 and ALY) which may not be the true virusreceptors but could be considered as virus-sensors. An Ephrin receptor-like protein was also found to interact with the viral proteins. Involvement of these candidates in virus transport through the aphid needs to be analyzed by developing in the insect RNAi-based techniques. These experiments are in progress. We also looked for plant virus-partners and identified several phloem proteins able to bind purified virions in vitro. We showed that these proteins could stimulate virus transmission by aphids when added together with purified virus to the aphid diet (Bencharki et al. 2010, M.P.M.I., 23: 799). By developing a yeast two hybrid system using a phloem specific cDNA library, we identified five additional proteins able to bind viral proteins. Among them, we found ALY proteins already identified as aphid virus-partners suggesting that orthologous plant and aphid proteins could be implicated in the virus cycle. So far, a direct implication of these proteins in aphid transmission has not been observed and experiments are on going to analyze their functions

In Vitro Acquisition of Specific Small Interfering RNAs Inhibits the Expression of Some Target Genes in the Plant Ectoparasite Xiphinema index

Xiphinema index is an important plant parasitic nematode that induces direct damages and specifically transmits the Grapevine fanleaf virus, which is particularly harmful for grapevines. Genomic resources of this nematode species are still limited and no functional gene validation technology is available. RNA interference (RNAi) is a powerful technology to study gene function and here we describe the application of RNAi on several genes in X. index. Soaking the nematodes for 48 h in a suspension containing specific small interfering RNAs resulted in a partial inhibition of the accumulation of some targeted mRNA. However, low reproducible silencing efficiency was observed which could arise from X. index silencing pathway deficiencies. Indeed, essential accustomed proteins for these pathways were not found in the X. index proteome predicted from transcriptomic data. The most reproducible silencing effect was obtained when targeting the piccolo gene potentially involved in endo-exocytosis of synaptic molecules. This represents the first report of gene silencing in a nematode belonging to the Longidoridae family

First spectroscopy of 66^{66}Se and 65^{65}As: Investigating shape coexistence beyond the N = Z line

The experiment was performed at the National Superconducting Cyclotron Laboratory (NSCL), at Michigan State University (USA).We report on the first γ spectroscopy of 66Se and 65As from two-neutron removal at intermediate beam energies. The deduced excitation energies for the first-excited states in 66Se and 65As are compared to mean-field-based predictions within a collective Hamiltonian formalism using the Gogny D1S effective interaction and to state-of-the-art shell-model calculations restricted to the pf5/2 g9/2 valence space. The obtained Coulomb-energy differences for the first excited states in 66Se and 65As are discussed within the shell-model formalism to assess the shape-coexistence picture for both nuclei. Our results support a favored oblate ground-state deformation in 66Se and 65As. A shape transition for the ground state of even-odd As isotopes from oblate in 65As to prolate in 67,69,71As is suggested

The genome of the green anole lizard and a comparative analysis with birds and mammals

The evolution of the amniotic egg was one of the great evolutionary innovations in the history of life, freeing vertebrates from an obligatory connection to water and thus permitting the conquest of terrestrial environments1. Among amniotes, genome sequences are available for mammals2 and birds3–5, but not for non-avian reptiles. Here we report the genome sequence of the North American green anole lizard, Anolis carolinensis. We find that A. carolinensis microchromosomes are highly syntenic with chicken microchromosomes, yet do not exhibit the high GC and low repeat content that are characteristic of avian microchromosomes3. Also, A. carolinensis mobile elements are very young and diverse – more so than in any other sequenced amniote genome. This lizard genome’s GC content is also unusual in its homogeneity, unlike the regionally variable GC content found in mammals and birds6. We describe and assign sequence to the previously unknown A. carolinensis X chromosome. Comparative gene analysis shows that amniote egg proteins have evolved significantly more rapidly than other proteins. An anole phylogeny resolves basal branches to illuminate the history of their repeated adaptive radiations

Copy number variations in East-Asian population and their evolutionary and functional implications

Recent discovery of the copy number variation (CNV) in normal individuals has widened our understanding of genomic variation. However, most of the reported CNVs have been identified in Caucasians, which may not be directly applicable to people of different ethnicities. To profile CNV in East-Asian population, we screened CNVs in 3578 healthy, unrelated Korean individuals, using the Affymetrix Genome-Wide Human SNP array 5.0. We identified 144 207 CNVs using a pooled data set of 100 randomly chosen Korean females as a reference. The average number of CNVs per genome was 40.3, which is higher than that of CNVs previously reported using lower resolution platforms. The median size of CNVs was 18.9 kb (range 0.2–5406 kb). Copy number losses were 4.7 times more frequent than copy number gains. CNV regions (CNVRs) were defined by merging overlapping CNVs identified in two or more samples. In total, 4003 CNVRs were defined encompassing 241.9 Mb accounting for ∼8% of the human genome. A total of 2077 CNVRs (51.9%) were potentially novel. Known CNVRs were larger and more frequent than novel CNVRs. Sixteen percent of the CNVRs were observed in ≥1% of study subjects and 24% overlapped with the OMIM genes. A total of 476 (11.9%) CNVRs were associated with segmental duplications. CNVS/CNVRs identified in this study will be valuable resources for studying human genome diversity and its association with disease

Anti-cancer effects and mechanism of actions of aspirin analogues in the treatment of glioma cancer

INTRODUCTION: In the past 25 years only modest advancements in glioma treatment have been made, with patient prognosis and median survival time following diagnosis only increasing from 3 to 7 months. A substantial body of clinical and preclinical evidence has suggested a role for aspirin in the treatment of cancer with multiple mechanisms of action proposed including COX 2 inhibition, down regulation of EGFR expression, and NF-κB signaling affecting Bcl-2 expression. However, with serious side effects such as stroke and gastrointestinal bleeding, aspirin analogues with improved potency and side effect profiles are being developed. METHOD: Effects on cell viability following 24 hr incubation of four aspirin derivatives (PN508, 517, 526 and 529) were compared to cisplatin, aspirin and di-aspirin in four glioma cell lines (U87 MG, SVG P12, GOS – 3, and 1321N1), using the PrestoBlue assay, establishing IC50 and examining the time course of drug effects. RESULTS: All compounds were found to decrease cell viability in a concentration and time dependant manner. Significantly, the analogue PN517 (IC50 2mM) showed approximately a twofold increase in potency when compared to aspirin (3.7mM) and cisplatin (4.3mM) in U87 cells, with similar increased potency in SVG P12 cells. Other analogues demonstrated similar potency to aspirin and cisplatin. CONCLUSION: These results support the further development and characterization of novel NSAID derivatives for the treatment of glioma

Transcriptional analysis of the HeT-A retrotransposon in mutant and wild type stocks reveals high sequence variability at Drosophila telomeres and other unusual features

<p>Abstract</p> <p>Background</p> <p>Telomere replication in Drosophila depends on the transposition of a domesticated retroelement, the <it>HeT-A </it>retrotransposon. The sequence of the <it>HeT-A </it>retrotransposon changes rapidly resulting in differentiated subfamilies. This pattern of sequence change contrasts with the essential function with which the <it>HeT-A </it>is entrusted and brings about questions concerning the extent of sequence variability, the telomere contribution of different subfamilies, and whether wild type and mutant Drosophila stocks show different <it>HeT-A </it>scenarios.</p> <p>Results</p> <p>A detailed study on the variability of <it>HeT-A </it>reveals that both the level of variability and the number of subfamilies are higher than previously reported. Comparisons between GIII, a strain with longer telomeres, and its parental strain Oregon-R indicate that both strains have the same set of <it>HeT-A </it>subfamilies. Finally, the presence of a highly conserved splicing pattern only in its antisense transcripts indicates a putative regulatory, functional or structural role for the <it>HeT-A </it>RNA. Interestingly, our results also suggest that most <it>HeT-A </it>copies are actively expressed regardless of which telomere and where in the telomere they are located.</p> <p>Conclusions</p> <p>Our study demonstrates how the <it>HeT-A </it>sequence changes much faster than previously reported resulting in at least nine different subfamilies most of which could actively contribute to telomere extension in Drosophila. Interestingly, the only significant difference observed between Oregon-R and GIII resides in the nature and proportion of the antisense transcripts, suggesting a possible mechanism that would in part explain the longer telomeres of the GIII stock.</p

Developing a community-based genetic nomenclature for anole lizards

Background: Comparative studies of amniotes have been hindered by a dearth of reptilian molecular sequences. With the genomic assembly of the green anole, Anolis carolinensis available, non-avian reptilian genes can now be compared to mammalian, avian, and amphibian homologs. Furthermore, with more than 350 extant species in the genus Anolis, anoles are an unparalleled example of tetrapod genetic diversity and divergence. As an important ecological, genetic and now genomic reference, it is imperative to develop a standardized Anolis gene nomenclature alongside associated vocabularies and other useful metrics. Results: Here we report the formation of the Anolis Gene Nomenclature Committee (AGNC) and propose a standardized evolutionary characterization code that will help researchers to define gene orthology and paralogy with tetrapod homologs, provide a system for naming novel genes in Anolis and other reptiles, furnish abbreviations to facilitate comparative studies among the Anolis species and related iguanid squamates, and classify the geographical origins of Anolis subpopulations. Conclusions: This report has been generated in close consultation with members of the Anolis and genomic research communities, and using public database resources including NCBI and Ensembl. Updates will continue to be regularly posted to new research community websites such as lizardbase. We anticipate that this standardized gene nomenclature will facilitate the accessibility of reptilian sequences for comparative studies among tetrapods and will further serve as a template for other communities in their sequencing and annotation initiatives.Organismic and Evolutionary BiologyOther Research Uni

Effects of L1-ORF2 fragments on green fluorescent protein gene expression

The retrotransposon known as long interspersed nuclear element-1 (L1) is 6 kb long, although most L1s in mammalian and other eukaryotic cells are truncated. L1 contains two open reading frames, ORF1 and ORF2, that code for an RNA-binding protein and a protein with endonuclease and reverse transcriptase activities, respectively. In this work, we examined the effects of full length L1-ORF2 and ORF2 fragments on green fluorescent protein gene (GFP) expression when inserted into the pEGFP-C1 vector downstream of GFP. All of the ORF2 fragments in sense orientation inhibited GFP expression more than when in antisense orientation, which suggests that small ORF2 fragments contribute to the distinct inhibitory effects of this ORF on gene expression. These results provide the first evidence that different 280-bp fragments have distinct effects on the termination of gene transcription, and that when inserted in the antisense direction, fragment 280-9 (the 3' end fragment of ORF2) induces premature termination of transcription that is consistent with the effect of ORF2