Active translocon complexes labeled with GFP–Dad1 diffuse slowly as large polysome arrays in the endoplasmic reticulum

In the ER, the translocon complex (TC) functions in the translocation and cotranslational modification of proteins made on membrane-bound ribosomes. The oligosaccharyltransferase (OST) complex is associated with the TC, and performs the cotranslational N-glycosylation of nascent polypeptide chains. Here we use a GFP-tagged subunit of the OST complex (GFP–Dad1) that rescues the temperature-sensitive (ts) phenotype of tsBN7 cells, where Dad1 is degraded and N-glycosylation is inhibited, to study the lateral mobility of the TC by FRAP. GFP–Dad1 that is functionally incorporated into TCs diffuses extremely slow, exhibiting an effective diffusion constant (Deff) about seven times lower than that of GFP-tagged ER membrane proteins unhindered in their lateral mobility. Termination of protein synthesis significantly increases the lateral mobility of GFP–Dad1 in the ER membranes, but to a level that is still lower than that of free GFP–Dad1. This suggests that GFP–Dad1 as part of the OST remains associated with inactive TCs. Our findings that TCs assembled into membrane-bound polysomes diffuse slowly within the ER have mechanistic implications for the segregation of the ER into smooth and rough domains

RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications

The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV) RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA) residues at its termini (LNA/DNA gapmer) by inserting 8-oxo-2'-deoxyguanosine (8-oxo-dG) residues into the central DNA region. Obtained compounds, designed with the aim to analyze the effects of 8-oxo-dG modifications on the antisense oligonucleotides, displayed a unique set of properties. Compared to conventional LNA/DNA gapmers, the melting temperatures of the duplexes formed by modified LNA/DNA gapmers and DNA or RNA targets were reduced by approximately 1.6-3.3 degrees C per modification. Comparative transfection studies showed that small interfering RNA was the most potent HCV RNA replication inhibitor (effective concentration 50 (EC50) : 0.13 nM), whereas isosequential standard and modified LNA/DNA gapmers were approximately 50-fold less efficient (EC50 : 5.5 and 7.1 nM, respectively). However, the presence of 8-oxo-dG residues led to a more complete suppression of HCV replication in transfected cells. These modifications did not affect the efficiency of RNase H cleavage of antisense oligonucleotide: RNA duplexes but did alter specificity, triggering the appearance of multiple cleavage products. Moreover, the incorporation of 8-oxo-dG residues increased the stability of antisense oligonucleotides of different configurations in human serum.Peer reviewe

[Mathematical assessment of BAC-based interspecies hybridization data in the process of genomic mapping in the white-throated sparrow as an avian behavioral model] Математическая оценка данных межвидовой БАК-гибридизации в процессе геномного картирования у белошейной зонотрихии как модели поведения птиц

The white-throated sparrow (Zonotrichia albicollis) known for its morphological, behavioral and chromosomal polymorphisms represents a quite new model system to study genomic mechanisms underlying variable behavioral repertoire interwoven with population biology, reproduction and adaptation in this species. It was previously shown that these polymorphisms could be due to chromosomal rearrangements (inversions) on sparrow chromosome 2 (ZAL2) that is characterized by a heterogeneity in two distinct morphs, tan (ZAL2/ZAL2) and white (ZAL2/ZAL2m). To construct a comparative genomic map of ZAL2 and other chromosomes, we used a sparrow genomic BAC library, CHORI-264. Following a cross-species overgo hybridization approach, we screened the library and developed a first-generation BAC-based comparative physical map using the chicken and zebra finch reference genomes. The map includes 640 BAC-gene assignments for 77 loci and serves for further refining the genomic regions and identifying candidate genes that are affected by rearrangements and contribute to the observed behavioral polymorphisms. Mathematical assessment of the BAC-based hybridization data was undertaken to show evolutionary relationships of avian genomes. В настоящей работе представлена математическая оценка межвидовой БАК-гибридизации в процессе геномного картирования у белошейной зонотрихии – вида воробьев, рассматриваемого в качестве удобной модели поведения птиц

[Determination of intestinal microbiocenoses of chickens of the Hisex breed by the T-RFLP method in ontogenesis] Определение микробиоценозов кишечника кур породы «Хайсекс» методом T-RFLP в онтогенезе

Организаторы: Сибирское отделение Российской академии наук; Институт химической биологии и фундаментальной медицины Лаптев Г.Ю., Ильина Л.А., Никонов И.Н., Кочиш И.И., Романов М.Н., Смоленский В.И., Панин А.Н., Йылдырым Е.А., Новикова Н.И., Филиппова В.А., Дубровин А.В. Цель работы: выявление структуры и таксономического состава микроорганизмов слепых отростков ЖКТ кур породы «Хайсекс» в онтогенезе с применением комплекса молекулярно-генетических методов. Исследования состава бактериального сообщества слепых отростков кишечника 40-, 155- и 315-суточных кур-несушек (по 3 из каждой группы) проводили молекулярно-генетическими методами (Т-RFLP и ПЦР в реальном времени). У исследуемой птицы в онтогенезе происходило развитие микробного сообщества ЖКТ, изменение содержания и появление новых микроорганизмов. Отмечено, что спектр выявляемых бактерий был выше у 40- и 155-суточной птицы (221 11 и 258 9 филотипов соответственно) по сравнению с 315-суточными курами-несушками (178 8 филотипов). Также у 315-суточной птицы выявлено наименьшее содержание неидентифицированных филотипов. В слепых отростках ЖКТ взрослой птицы выявлено изменение доминирующих таксономических групп микроорганизмов - более высокая доля бактерий кислот-утилизирующих бактерий класса Negativicutes, целлюлозолитиков класса Clostridia. Обратная тенденция наблюдалась в отношении бактерий классов Bifidobacteriales, Bacillales. Большее содержание лактобактерий порядка Lactobacillales наблюдалась у 315-суточных кур-несушек (33,15 1,05%) по сравнению с 40- (5,13 0,23%) и 155-суточной (24,58 0,86%) птицей. Разнообразие и количество бактерий в слепых отростков ЖКТ, которых традиционно относят к возбудителям различных заболеваний птицы, из родов Enterobacter, Pantoea, Listeria, Acinetobacter, Mycoplasma, семейств Campylobacteraceae, Pasteurellaceae, филума Fusobacteria увеличивается с возрастом птицы. Таким образом, в ходе молекулярно-генетических исследований был определен видовой состав микробиоценозов слепых отростков ЖКТ кур яичной породы «Хайсекс» в онтогенезе. Исследования выполнены при поддержке Министерства образования и науки Российской Федерации, Договор № 14.W03.31.0013 от 20.02.2017 г

RNA-sõltuva RNA polümeraasi aktiivsuse kaudu on selgroogsete rakud võimelised tuvastama pluss ahelaga RNA viiruste nakkuse

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Peaaegu iga siiani uuritud elusorganism võib olla liigispetsiifiliste RNA viiruste nakkust ja levikut võimaldavaks peremeesorganismiks. Nakatamisvõimeline viirusosakene ehk virion sisaldab valgulise või valke ja lipiide sisaldavad kesta poolt ümbritsetud nukleiinhappe kujul viiruse genoomi. RNA viiruste genoomi paljundamise eestvedajaks on viiruse genoomi poolt kodeeritav RNA-sõltuv RNA polümeraas (RdRp). Täpsemalt viib viiruse RNA genoomi replikatsiooni läbi replikaas: mitmest subühikust koosnev ensümaatiline kompleks, mis omab RdRp aktiivsusega tuumikkomponenti. Kõigepealt sünteesib RdRp viiruse RNA genoomi matriitsina kasutades sellele komplementaarse RNA ahela, mis hakkab järgnevalt omakorda toimima kui viiruse genoomi paljundamise matriits. RdRp toimib kui molekulaarne masin, mis kannab genoomses materjalis peituva geneetilise informatsiooni ühelt RNA molekulilt teisele. Kõik RNA viirused kodeerivad ise replikaasi RdRp komponenti, kuna nende peremeesrakud kas ei ole võimelised paljundama viiruste pikki RNA genoome (e.g. selgrootud loomad ja taimed) või neil puudub RdRp aktiivsusega ensüüm (e.g. selgroogsed loomad). Satelliitviiruste hulka kuuluv hepatiit delta viirus on teadaolevalt ainsana selle reegli suhtes erandlik. Replikaasikompleksi teised subühikud võivad sõltuvalt viirusest olla kodeeritud kas viiruse enda või peremeesraku poolt. Käesoleva doktoritöö raames võeti Flaviviridae sugukonna “kollaste” ja Togaviridae sugukonna “ümbristega” viiruste RdRp ensüümide aktiivsuse mõju analüüsimiseks ette teekond katseklaasist peremeesrakku. Kõigepealt viisime läbi hepatiit C viiruse (HCV) RdRp tuumikkomponendi struktuuri ja funktsiooni seose analüüsi erinevates molekulaarsetes keskkondades. Teiseks tuvastasime Semliki Forest viiruse (SFV) replikatsiooni uurides, et SFV replikaas ei ole võimeline paljundama mitte ainult viiruslikku päritolu nukleiinhappeid, vaid transkribeerib ka peremeesraku RNA matriitse. Viimasena mainitud SFV replikaasi RdRp aktiivsuse “kõrvalnäht” kutsub esile võimsate rakuliste viirusvastaste kaitsemehhanismide vallandumise. Sellele avastusele tuginedes arendasime uudse üldistatud mudeli, mis kirjeldab, kuidas selgroogsete organismide rakud võivad detekteerida SFV-ga sarnaseid RNA viiruseid ja kuidas viirused üritavad säärast avastamist vältida.Essentially every living organism studied thus far may serve as a host for the infection and propagation of species-specific RNA viruses. An infectious virus particle, i.e. a virion, carries the genome in the form of nucleic acid surrounded by a protein or protein-lipid coat. RNA virus genome replication is driven by the viral genome-encoded RNA-dependent RNA polymerase (RdRp). More specifically, viral RNA genome replication is performed by a replicase, a multisubunit enzyme complex that possesses a core component with RdRp activity. RdRp first utilizes a viral RNA genome template to catalyze the synthesis of complementary RNA, which subsequently serves as a template for the production of viral genomes. RdRp is a molecular machine that transfers the genetic information embedded in genetic material from one RNA molecule to another. All RNA viruses encode the RdRp component of the replicase because host cells either cannot replicate long RNA genomes of viruses (e.g., invertebrates and plants) or do not possess intrinsic RdRp (e.g., vertebrates). Hepatitis delta virus, a satellite virus, is an exception to this rule. In addition to RdRp, all of the other protein subunits required for replicase assembly are encoded by either the viral RNA genome or the host genome. This dissertation involved a journey from the test tube to the host cell to analyze the effects of the viral RdRp activities of “yellow” and “mantled” RNA viruses of Flaviviridae and Togaviridae families. First, we performed structure-function relationship analyses of the hepatitis C virus (HCV) core RdRp component in different molecular environments. Second, by conducting Semliki Forest virus (SFV) replication studies, we found that the SFV replicase not only replicates viral nucleic acids but also has the capacity to transcribe host cell RNA templates. The latter “side effect” of SFV replicase RdRp activity triggers a potent cellular antiviral response. This finding led to the development of a generalized novel model that describes how vertebrate host cells might detect RNA viruses similar to SFV and how viruses counteract this detection

Can vegetation provide indications of ancient lake shorelines after more than one hundred years? : a case study of Iskander-kul Lake, Tajikistan

Bioindication is a common approach to assess and evaluate environmental changes over both short or long periods of time. Here we attempt to highlight that vegetation can provide indications of the palaeoshoreline of Lake Iskander-kul, even after at least 150 years. It is an example of a dammed lake that was created by a huge mass rockfall as a result of a strong earthquake during the late Pleistocene. Applying the two way indicator species analysis (TWINSPAN) we found that the shrubby vegetation is the particular one that can still effectively thrive along the palaeoshoreline despite the lake downlift. Using the phi coefficient as a fidelity measure for certain vegetation type, we found, that Juniperus seravschanica, Lonicera seravschanica, Berberis integerrima, Seseli lehmannianum, Astragalus kabadianus and Ephedra intermedia have the highest indicator value for palaeoshoreline. Apart from the ancient palaeoshoreline, on the screes and slopes surrounding the lake, significantly different vegetation types developed on the screes and slopes dominated by steppe (Hyssopus servaschanica, Galium turkestanicum, Festuca valesiaca) and scree (Ferula foetidissima, Stipa drobovii or Ferula kuhistanica) vegetation types. Using the multivariate analysis (CCA) we show that the slope inclination and distance from the palaeoshorelines has considerable influence on species composition within the study plots