ABSTRACT Nascent transcripts in permeabilized HeLa cells were elongated by ∼30-2,000 nucleotides Br-UTP or biotin-14-CTP, before incorporation sites immunolabelled either pre- post-embedding, and visualized light electron microscopy. Analogues concentrated ∼2,100 (range 2,000-2,700) discrete attached to a nucleoskeleton surrounded chromatin. A typical site contained cluster (diameter 71 nm) of at least 4, probably about 20, engaged polymerases, plus associated that partially overlapped zone...

Background Hypoxia in cancers results the upregulation of hypoxia inducible factor 1 (HIF-1) and a microRNA, hsa-miR-210 (miR-210) which is associated with poor prognosis. Methods Findings In human cancer cell lines tumours, we found that miR-210 targets mitochondrial iron sulfur scaffold protein ISCU, required for assembly iron-sulfur clusters, cofactors key enzymes involved Krebs cycle, electron transport, metabolism. Down regulation ISCU was major cause induction reactive oxygen species...

It is widely assumed that the vital processes of transcription and translation are spatially separated in eukaryotes no occurs nuclei. We localized sites by incubating permeabilized mammalian cells with [3H]lysine or lysyl-transfer RNA tagged biotin BODIPY; although most nascent polypeptides were cytoplasmic, some found discrete nuclear known as "factories." Some this also depends on concurrent polymerase II. This coupling simply explained if ribosomes translate transcripts those emerge from...

We analyzed the organization and function of mitochondrial DNA in a stable human cell line (ECV304, which is also known as T-24) containing mitochondria tagged with yellow fluorescent protein.

Using HeLa cells, we have developed methods to determine 1) the number of RNA polymerases that are active at any moment, 2) transcription sites, and 3) associated with one unit. To count engaged polymerases, cells were encapsulated in agarose, permeabilized, treated ribonuclease, now-truncated transcripts extended [ 32 P]uridine triphosphate; then, growing was calculated from total nucleotides incorporated average increment length transcripts. Approximately 15,000 elongated by polymerase I,...

Human embryonic stem cell (HESC) lines vary in their characteristics and behaviour not only because they are derived from genetically outbred populations, but also may undergo progressive adaptation upon long-term culture vitro . Such reflect selection of variants with altered propensity for survival retention an undifferentiated phenotype. Elucidating the mechanisms involved will be important understanding normal self-renewal commitment to differentiation validating safety HESC-based...

Genes on different chromosomes can be spatially associated in the nucleus several transcriptional and regulatory situations; however, functional significance of such associations remains unclear. Using human erythropoiesis as a model, we show that five cotranscribed genes, which are found four chromosomes, associate with each other at significant but variable frequencies. Those genes most frequently association lie decondensed stretches chromatin. By replacing mouse α-globin gene cluster...

The organization of genes within the nucleus may influence transcription. We have analyzed nuclear positioning coordinately regulated α- and β-globin show that gene-dense chromatin surrounding human α-globin is frequently decondensed, independent Against this background, we frequent juxtaposition active homologous loci occurs at speckles correlates with However, did not see increased colocalization signals, which would be expected direct physical interaction. same degree proximity does occur...

Populations of genetically identical eukaryotic cells show significant cell-to-cell variability in gene expression. However, we lack a good understanding the origins this variation. We have found marked average cellular rates transcription. also amount mitochondrial mass. undertook fusion studies that suggested transcription rate depends on small diffusible factors. Following this, vitro showed has sensitive dependence [ATP] but not concentration other nucleotide triphosphates (NTPs)....

Fractional killing is the main cause of tumour resistance to chemotherapy. This phenomenon observed even in genetically identical cancer cells homogeneous microenvironments. To understand this variable resistance, here we investigate individual responses TRAIL a clonal population HeLa using live-cell microscopy and computational modelling. We show that cellular mitochondrial content determines apoptotic fate modulates time death, with higher are more prone die. find all protein levels...

We present a study investigating the role of mitochondrial variability in generating noise eukaryotic cells. Noise cellular physiology plays an important many fundamental processes, including transcription, translation, stem cell differentiation and response to medication, but specific random influences that affect these processes have yet be clearly elucidated. Here we mechanism by which volume functionality, along with cycle dynamics, is linked transcription rate hence has profound effect...

Noise in gene expression is a main determinant of phenotypic variability. Increasing experimental evidence suggests that genome-wide cellular constraints largely contribute to the heterogeneity observed products. It still unclear, however, which global factors affect noise and what extent. Since eukaryotic an energy demanding process, differences budget each cell could determine differences. Here, we quantify contribution mitochondrial variability (a natural source ATP variation) expression....

The control of RNA synthesis from protein-coding genes is fundamental in determining the various cell types higher eukaryotes. activation these driven by promoter complexes, and performed an enzyme mega-complex-the polymerase II holoenzyme. These two complexes are components required to initiate gene expression generate primary transcripts that, after processing, yield mRNAs that pass cytoplasm where protein occurs. But although this pathway has been studied intensively, aspects metabolism...

Lineage specification and cellular maturation require coordinated regulation of gene expression programs. In large part, this is dependent on the activator repressor functions protein complexes associated with tissue-specific transcriptional regulators. study, we have used a proteomic approach to characterize multiprotein containing key hematopoietic regulator SCL in erythroid megakaryocytic cell lines. One novel SCL-interacting proteins identified both types corepressor ETO-2. Interaction...

Clinically successful hematopoietic cell transplantation is dependent on stem and progenitor cells. Here we identify the matricellular protein Nephroblastoma Overexpressed (Nov, CCN3) as being essential for their functional integrity. Nov expression restricted to primitive (CD34) compartments of umbilical vein cord blood, its knockdown in these cells by lentivirus-mediated RNA interference abrogates function vitro vivo. Conversely, forced addition recombinant both enhance and/or activity....

The homeostasis of the hematopoietic stem/progenitor cell pool relies on a fine-tuned balance between self-renewal, differentiation and proliferation. Recent studies have proposed that mitochondria regulate these processes. Although recent work has contributed to understanding role during stem differentiation, it remains unclear whether mitochondrial content/function affects human versus progenitor function. We found mass correlates strongly with membrane potential in CD34+ cells. We,...

Summary Human aging is associated with accumulation of cells that have undergone replicative senescence. The rare premature Werner's syndrome (WS) provides a phenocopy normal human and WS patient recapitulate the phenotype in culture as they rapidly lose ability to proliferate or replicate their DNA. loss functional WRN protein. Although biochemical properties protein, which possesses both helicase exonuclease activities, suggest an involvement DNA metabolism, its action not clear. Here, we...

ERK and p38 MAP kinases, acting through the downstream mitogen- stress-activated kinase 1/2 (MSK1/2), elicit histone H3 phosphorylation on a subfraction of nucleosomes – including those at Fos Jun concomitant with gene induction. S10 S28 tail have both been shown to be phospho-acceptors in vivo. Both phospho-epitopes appear similar time-courses occur tails that are highly sensitive TSA-induced hyperacetylation, similarities which might suggest MSK1/2 phosphorylates sites same tails. Indeed,...

The TNF receptor-associated protein 1 (TRAP1) is a mitochondrial HSP that has been related to drug resistance and protection from apoptosis in colorectal prostate cancer. Here, the effect of TRAP1 ablation on cell proliferation, survival, apoptosis, function was determined non-small lung cancer (NSCLC). In addition, prognostic value evaluated patients with NSCLC. These results demonstrate knockdown reduces growth clonogenic survival. Moreover, downregulation impairs functions such as ATP...

In eukaryotic nuclei, DNA is wrapped around a protein octamer composed of the core histones H2A, H2B, H3, and H4, forming nucleosomes as fundamental units chromatin. The modification deposition specific histone variants play key roles in chromatin function. this study, we established an vitro system based on permeabilized cells that allows assembly exchange situ. H2A each tagged with green fluorescent (GFP), are incorporated into euchromatin by independently replication, H3.1-GFP assembled...

The DNA-binding hemopoietic zinc finger transcription factor GATA1 promotes terminal megakaryocyte differentiation and restrains abnormal immature expansion.How coordinates these fundamental processes is unclear.Previous studies of synthetic naturally occurring mutant molecules demonstrate that interaction with the essential cofactor FOG-1 (via N-terminal finger) are required for gene expression in terminally differentiating megakaryocytes platelet production.Moreover, acquired mutations...

The cell nucleus is highly compartmentalized with well-defined domains, it not well understood how this nuclear order maintained. Many scientists are fascinated by the different set of structures observed in to attribute functions them. In distinguish functional compartments from non-functional aggregates, I believe important investigate biophysical nature organisation.The various can be divided broadly as chromatin or protein and/or RNA based, and they have very dynamic properties....

The recent outbreaks of Zika virus (ZIKV), its association with Guillain⁻Barré syndrome and fetal abnormalities, the lack approved vaccines antivirals, highlight importance developing countermeasures to combat ZIKV disease. In this respect, infectious clones constitute excellent tools accomplish these goals. However, flavivirus are often difficult work due toxicity some sequences in bacteria. To bypass problem, several alternative approaches have been applied for generation including, among...

Alcohol dehydrogenase (ADH), the major enzyme catalyzing biological oxidation of ethanol in mammals, includes four classes with very different capacities for oxidation. Class III ADH is present all tissues and well conserved throughout evolution. This has a low activity ethanol, specific glutathione-dependent formaldehyde, therefore formaldehyde (FALDH). Until now there have been few conflicting studies concerning its intracellular distribution, which important understanding role cell...