Various helicases and single-stranded DNA (ssDNA) binding proteins are known to destabilize G-quadruplex (GQ) structures, which otherwise result in genomic instability. Bulk biochemical studies have shown that Bloom helicase (BLM) unfolds both intermolecular and intramolecular GQ in the presence of ATP. Using single molecule FRET, we show that binding of RecQ-core of BLM (will be referred to as BLM) to ssDNA in the vicinity of an intramolecular GQ leads to destabilization and unfolding of the GQ in the absence of ATP. We show that the efficiency of BLM-mediated GQ unfolding correlates with the binding stability of BLM to ssDNA overhang, as modulated by the nucleotide state, ionic conditions, overhang length and overhang directionality. In particular, we observed enhanced GQ unfolding by BLM in the presence of non-hydrolysable ATP analogs, which has implications for the underlying mechanism. We also show that increasing GQ stability, via shorter loops or higher ionic strength, reduces BLM-mediated GQ unfolding. Finally, we show that while WRN has similar activity as BLM, RecQ and RECQ5 helicases do not unfold GQ in the absence of ATP at physiological ionic strength. In summary, our study points to a novel and potentially very common mechanism of GQ destabilization mediated by proteins binding to the vicinity of these structures.

• MeSH
• adenosindifosfát metabolismus   MeSH
• adenosintrifosfát analogy a deriváty   metabolismus   MeSH
• G-kvadruplexy * MeSH
• helikasy RecQ chemie   metabolismus   MeSH
• jednovláknová DNA metabolismus   MeSH
• lidé MeSH
• telomery chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• adenosindifosfát MeSH
• adenosine 5'-O-(3-thiotriphosphate) MeSH Prohlížeč
• adenosintrifosfát MeSH
• Bloom syndrome protein MeSH Prohlížeč
• helikasy RecQ MeSH
• jednovláknová DNA MeSH

At the end of the growth phase, mouse antral follicle oocytes acquire full developmental competence. In the mouse, this event is marked by the transition from the so-called non-surrounded nucleolus (NSN) chromatin configuration into the transcriptionally quiescent surrounded nucleolus (SN) configuration, which is named after a prominent perinucleolar condensed chromatin ring. However, the SN chromatin configuration alone is not sufficient for determining the developmental competence of the SN oocyte. There are additional nuclear and cytoplamic factors involved, while a little is known about the changes occurring in the cytoplasm during the NSN/SN transition. Here, we report functional analysis of maternal ELAVL2 an AU-rich element binding protein. Elavl2 gene encodes an oocyte-specific protein isoform (denoted ELAVL2°), which acts as a translational repressor. ELAVL2° is abundant in fully grown NSN oocytes, is ablated during the NSN/SN transition and remains low during the oocyte-to-embryo transition (OET). ELAVL2° overexpression during meiotic maturation causes errors in chromosome segregation, indicating the significance of naturally reduced ELAVL2° levels in SN oocytes. On the other hand, during oocyte growth, prematurely reduced Elavl2 expression results in lower yields of fully grown and meiotically matured oocytes, suggesting that Elavl2 is necessary for proper oocyte maturation. Moreover, Elavl2 knockdown showed stimulating effects on translation in fully grown oocytes. We propose that ELAVL2 has an ambivalent role in oocytes: it functions as a pleiotropic translational repressor in efficient production of fully grown oocytes, while its disposal during the NSN/SN transition contributes to the acquisition of full developmental competence.

• Klíčová slova
• ARE, ELAVL2, NSN, SN, chromatin, oocyte,
• MeSH
• buněčné linie MeSH
• ELAV-like protein 2 genetika   metabolismus   MeSH
• genový knockdown MeSH
• lidé MeSH
• meióza fyziologie   MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• oocyty cytologie   metabolismus   MeSH
• ovariální folikul cytologie   metabolismus   MeSH
• protein - isoformy genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ELAV-like protein 2 MeSH
• Elavl2 protein, mouse MeSH Prohlížeč
• protein - isoformy MeSH

Reciprocal interactions between a tumor and its microenvironment control expansion of tumor cells. Here we show a specific type of interaction in which blasts of experimental leukemia destroy the bone marrow (BM) structures and kill stromal cells. The in vitro experiments showed that the cytotoxic agent released by leukemic cells is the fragmented DNA derived from their genome and occurring in nucleosome-like complexes. This DNA entered nuclei of BM or other cells and induced H2A.X phosphorylation at serine 139, similar to double-strand break-inducing agents. There was a correlation between large amounts of acquired DNA and death of recipient cells. Moreover, the DNA integrated into chromosomal DNA of recipient cells. Primary human acute myeloid leukemia cells also released fragmented DNA that penetrated the nuclei of other cells both in vitro and in vivo. We suggest that DNA fragments released from leukemic and also perhaps other types of tumor cells can activate DNA repair mechanisms or death in recipient cells of a tumor microenvironment, depending on the amount of the acquired DNA. This can impair DNA stability and viability of tumor stromal cells, undermine homeostatic capacity of tumor microenvironment and facilitate tumor progression.

• MeSH
• akutní myeloidní leukemie patologie   MeSH
• apoptóza MeSH
• buněčné jádro metabolismus   MeSH
• buňky stromatu fyziologie   MeSH
• DNA metabolismus   MeSH
• histony metabolismus   MeSH
• kostní dřeň patologie   MeSH
• kur domácí MeSH
• kuřecí embryo MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorové mikroprostředí * MeSH
• nádory kostní dřeně patologie   MeSH
• nukleozomy metabolismus   MeSH
• oprava DNA MeSH
• poškození DNA MeSH
• progrese nemoci MeSH
• transplantace nádorů MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• histony MeSH
• nukleozomy MeSH

Double-stranded RNA (dsRNA) is involved in different biological processes. At least three different pathways can respond to dsRNA in mammals. One of these pathways is RNA interference (RNAi) where long dsRNA induces sequence-specific degradation of transcripts carrying sequences complementary to dsRNA. Long dsRNA is also a potent trigger of the interferon pathway, a sequence-independent response that leads to global suppression of translation and global RNA degradation. In addition, dsRNA can be edited by adenosine deamination, which may result in nuclear retention and degradation of dsRNA or in alteration of RNA coding potential. Here, we provide a technical review summarizing different strategies of long dsRNA usage. While the review is largely focused on long dsRNA-induced RNAi in mammalian cells, it also provides helpful information on both the in vitro production and in vivo expression of dsRNAs. We present an overview of currently available vectors for dsRNA expression and provide the latest update on oocyte-specific transgenic RNAi approaches.

• MeSH
• buněčné linie MeSH
• dvouvláknová RNA biosyntéza   chemie   genetika   MeSH
• genetické inženýrství metody   MeSH
• genetické vektory genetika   MeSH
• lidé MeSH
• oocyty metabolismus   MeSH
• RNA interference MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• dvouvláknová RNA MeSH

Transient plasmid transfection is a common approach in studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We have found that the entire plasmid sequence is transcribed at different levels. Spurious transcription may have undesirable effects as some plasmids, when co-transfected, inhibited expression of luciferase reporters in a dose-dependent manner. In one case, we attributed this effect to a Kan/Neo resistance cassette, which generated a unique population of edited sense and antisense small RNAs. The unexpected complexity of expression from transiently transfected plasmids underscores the importance of appropriate experimental controls.

• MeSH
• antisense RNA genetika   MeSH
• buněčné linie MeSH
• lidé MeSH
• plazmidy genetika   MeSH
• průtoková cytometrie MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antisense RNA MeSH

The TCR signal transduction is initiated by the activation of Src-family kinases (SFK) which phosphorylate Immunoreceptor tyrosine-based activation motifs (ITAM) present in the intracellular parts of the T-cell receptor (TCR) signaling subunits. Numerous data suggest that after stimulation TCR interacts with membrane rafts and thus it gains access to SFK and other important molecules involved in signal transduction. However, the precise mechanism of this process is unclear. One of the key questions is how SFK access TCR and what is the importance of non-raft and membrane raft-associated SFK for the initiation and maintenance of the TCR signaling. To answer this question we targeted a negative regulator of SFK, C-terminal Src kinase (Csk) to membrane rafts, recently described "heavy rafts" or non-raft membrane. Our data show that only Csk targeted into "classical" raft but not to "heavy raft" or non-raft membrane effectively inhibits TCR signaling, demonstrating the critical role of membrane raft-associated SFK in this process.

• MeSH
• buněčná membrána metabolismus   MeSH
• C-terminální Src kinasa MeSH
• fosforylace MeSH
• imunoblotting MeSH
• kultivované buňky MeSH
• lidé MeSH
• membránové mikrodomény * MeSH
• protoonkogenní proteiny metabolismus   MeSH
• receptory antigenů T-buněk metabolismus   MeSH
• signální transdukce MeSH
• skupina kinas odvozených od src-genu MeSH
• tyrosinkinasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• C-terminální Src kinasa MeSH
• CSK protein, human MeSH Prohlížeč
• protoonkogenní proteiny MeSH
• receptory antigenů T-buněk MeSH
• skupina kinas odvozených od src-genu MeSH
• tyrosinkinasy MeSH

Small RNA molecules regulating gene expression received a status of omnipresent master regulators of eukaryotic lives with almost supernatural powers. Mammals hold at least three mechanisms employing small RNA molecules for regulating gene expression. One of these mechanisms, the microRNA (miRNA) pathway, involves currently over a thousand of genome-encoded different miRNAs that are claimed to extend their control over more than a half of a genome. Here, I discuss how and why mouse oocytes and early embryos ignore the regulatory power of miRNAs, adding another surprising feature to the field of small RNAs.

• MeSH
• embryo savčí metabolismus   MeSH
• mikro RNA genetika   MeSH
• myši MeSH
• oocyty metabolismus   MeSH
• RNA interference * MeSH
• RNA messenger skladovaná MeSH
• vývojová regulace genové exprese * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mikro RNA MeSH
• RNA messenger skladovaná MeSH

A tumor cell is formed when a critical amount of endogenous and/or exogenous tumorigenic stimuli is exceeded. We have shown that the transient presence of nontumorigenic stray cells in tissues of experimental animals that contain cells with a subcritical set of genetic mutations can act as a tumor-promoting stimulus. To induce somatic mutations in all chicken tissues, we have used the MAV-2 retroviral insertion system that almost exclusively generates nephroblastomas. MAV-2 mutagenized animals i.v. inoculated with nonmalignant cells developed early clonal lung tumors before nephroblastomas. Importantly, the injected cells did not become a component of resultant tumors. Lung tumors displayed specific mutational signature characterized by an insertion of MAV-2 provirus into the fyn-related kinase (frk) promoter that results in the overexpression of the frk gene. In contrast, plag1, foxP, and twist genes were most often mutagenized in nephroblastomas. Based on such observations, we propose the mechanism termed industasis, a promotion of fully malignant phenotype of incipient tumor cell by stray cells, and hypothesize that it might be the underlying cause of human multiple primary tumors.

• MeSH
• biologické modely MeSH
• buňky patologie   virologie   MeSH
• fyziologie virů MeSH
• invazivní růst nádoru MeSH
• inzerční mutageneze fyziologie   MeSH
• kultivované buňky MeSH
• kur domácí MeSH
• kuřecí embryo MeSH
• mnohočetné primární nádory etiologie   MeSH
• nádorová transformace buněk patologie   MeSH
• nádory ledvin patologie   virologie   MeSH
• nádory plic patologie   virologie   MeSH
• pohyb buněk fyziologie   MeSH
• proviry růst a vývoj   fyziologie   MeSH
• Wilmsův nádor patologie   virologie   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Gene deregulation is a frequent cause of malignant transformation. Alteration of the gene structure and/or expression leading to cellular transformation and tumor growth can be experimentally achieved by insertion of the retroviral genome into the host DNA. Retrovirus-containing host loci found repeatedly in clonal tumors are called common viral integration sites (cVIS). cVIS are located in genes or chromosomal regions whose alterations participate in cellular transformation. Here, we present the chicken model for the identification of oncogenes and tumor suppressor genes in solid tumors by mapping the cVIS. Using the combination of inverse PCR and long terminal repeat-rapid amplification of cDNA ends technique, we have analyzed 93 myeloblastosis-associated virus type 2-induced clonal nephroblastoma tumors in detail, and mapped >500 independent retroviral integration sites. Eighteen genomic loci were hit repeatedly and thus classified as cVIS, five of these genomic loci have previously been shown to be involved in malignant transformation of different human cell types. The expression levels of selected genes and their human orthologues have been assayed in chicken and selected human renal tumor samples, and their possible correlation with tumor development, has been suggested. We have found that genes associated with cVIS are frequently, but not in all cases, deregulated at the mRNA level as a result of proviral integration. Furthermore, the deregulation of their human orthologues has been observed in the samples of human pediatric renal tumors. Thus, the avian nephroblastoma is a valid source of cancer-associated genes. Moreover, the results bring deeper insight into the molecular background of tumorigenesis in distant species.

• MeSH
• DNA vazebné proteiny genetika   MeSH
• geny ras genetika   MeSH
• integrace viru genetika   MeSH
• koncové repetice MeSH
• kur domácí * MeSH
• kuřecí embryo MeSH
• lidé MeSH
• mapování chromozomů MeSH
• nádory ledvin genetika   virologie   MeSH
• nemoci drůbeže genetika   MeSH
• onkogenní proteiny genetika   MeSH
• onkogeny genetika   MeSH
• polymerázová řetězová reakce MeSH
• proviry genetika   MeSH
• ptačí proteiny genetika   MeSH
• transkripční faktor Twist genetika   MeSH
• tumor supresorové geny MeSH
• virus ptačí myeloblastózy genetika   MeSH
• Wilmsův nádor genetika   virologie   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• onkogenní proteiny MeSH
• ptačí proteiny MeSH
• transkripční faktor Twist MeSH
• Twist protein, Gallus gallus MeSH Prohlížeč

Video rate confocal laser scanning microscopy at the highest spatial and temporal resolution of backscattered light (BSL) imaging allowed for regular observation of fast intracellular motion (FIM) first revealed in living neoplastic cells. However, the absence of an objective evaluation has hampered further study of the mechanisms and biological significance of FIM. Particularly, a quantification of apparent differences in velocities that would complement and improve the current demonstration of FIM by color coding using the combination of red-green-blue (RGB) images had been missing. Standard methods of tracking or pattern recognition could not be applied because of the fuzzy nature of images of FIM. A search for a suitable method led to correlation analysis. It was calibrated on Brownian motion and a known type of motion, such as cell marginal ruffling, compared with FIM. Results approved its explanatory potential. Therefore, several crucial incidences of FIM could be analyzed. Apart from an argument against viewing FIM as a manifestation of simple Brownian motion, the correlation analysis of FIM in the adjacent peripheries of a rat fibroblast and a K4 rat sarcoma cell confirmed the notion of higher and uneven distribution of velocity of FIM in a tumor cell so far shown in color-coded images only. This result and other yet unpublished observations indicate that the velocity and topology of FIM can also contribute to a biological distinction between neoplastic and normal cells. Regular application of the correlation analysis should further expand the study of FIM for its mechanisms and predictive value. Such an approach should be thoroughly examined for a contribution to the knowledge of cancer cells.

• MeSH
• audiovizuální záznam MeSH
• experimentální sarkom MeSH
• fibroblasty cytologie   fyziologie   MeSH
• kalibrace MeSH
• konfokální mikroskopie metody   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky fyziologie   MeSH
• listy rostlin cytologie   fyziologie   MeSH
• nádorové buňky kultivované fyziologie   MeSH
• počítačové zpracování obrazu MeSH
• pohyb buněk fyziologie   MeSH
• zobrazování trojrozměrné MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH