A portable single-sided magnetic-resonance sensor for the grading of liver steatosis and fibrosis.

Low-cost non-invasive diagnostic tools for staging the progression of non-alcoholic chronic liver failure from fatty liver disease to steatohepatitis are unavailable. Here, we describe the development and performance of a portable single-sided magnetic-resonance sensor for grading liver steatosis and fibrosis using diffusion-weighted multicomponent T2 relaxometry. In a diet-induced mouse model of non-alcoholic fatty liver disease, the sensor achieved overall accuracies of 92% (Cohen's kappa, κ = 0.

DNA ligase 4 stabilizes the ribosomal DNA array upon fork collapse at the replication fork barrier.

DNA double-strand breaks (DSB) were shown to occur at the replication fork barrier in the ribosomal DNA of Saccharomyces cerevisiae using 2D-gel electrophoresis. Their origin, nature and magnitude, however, have remained elusive. We quantified these DSBs and show that a surprising 14% of replicating ribosomal DNA molecules are broken at the replication fork barrier in replicating wild-type cells.

Chromatin structure of ribosomal genes in Chironomus thummi (Diptera: Chironomidae): tissue specificity and behaviour under drug treatment.

In eukaryotes the ribosomal gene population shows two different states in terms of chromatin structure. One subset is organized as nucleosomes (silent copies) while the other has a non-nucleosomal configuration (active copies). Insect cells are not the exception and this bimodal distribution of ribosomal chromatin also occurs in salivary gland cells, and cells of other larval tissues, of the midge Chironomus thummi.

Methods to study replication fork collapse in budding yeast.

Replication of the eukaryotic genome is a difficult task, as cells must coordinate chromosome replication with chromatin remodeling, DNA recombination, DNA repair, transcription, cell cycle progression, and sister chromatid cohesion. Yet, DNA replication is a potentially genotoxic process, particularly when replication forks encounter a bulge in the template: forks under these conditions may stall and restart or even break down leading to fork collapse. It is now clear that fork collapse stimulates chromosomal rearrangements and therefore represents a potential source of DNA damage.

Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions.

DNA replication forks pause in front of lesions on the template, eventually leading to cytotoxic chromosomal rearrangements. The in vivo structure of damaged eukaryotic replication intermediates has been so far elusive. Combining electron microscopy (EM) and two-dimensional (2D) gel electrophoresis, we found that UV-irradiated S.

Exo1 processes stalled replication forks and counteracts fork reversal in checkpoint-defective cells.

The replication checkpoint coordinates the cell cycle with DNA replication and recombination, preventing genome instability and cancer. The budding yeast Rad53 checkpoint kinase stabilizes stalled forks and replisome-fork complexes, thus preventing the accumulation of ss-DNA regions and reversed forks at collapsed forks. We searched for factors involved in the processing of stalled forks in HU-treated rad53 cells.

rDNA enhancer affects replication initiation and mitotic recombination: Fob1 mediates nucleolytic processing independently of replication.

To investigate the influence of the ribosomal DNA enhancer on initiation of replication and recombination at the ribosomal array, we used yeast S. cerevisiae strains with adjacent, tagged rRNA genes. We found that the enhancer is an absolute requirement for replication fork barrier function, while it only modulates initiation of replication.

Rad52-independent accumulation of joint circular minichromosomes during S phase in Saccharomyces cerevisiae.

We investigated the formation of X-shaped molecules consisting of joint circular minichromosomes (joint molecules) in Saccharomyces cerevisiae by two-dimensional neutral/neutral gel electrophoresis of psoralen-cross-linked DNA. The appearance of joint molecules was found to be replication dependent. The joint molecules had physical properties reminiscent of Holliday junctions or hemicatenanes, as monitored by strand displacement, branch migration, and nuclease digestion.

Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects.

Checkpoint-mediated control of replicating chromosomes is essential for preventing cancer. In yeast, Rad53 kinase protects stalled replication forks from pathological rearrangements. To characterize the mechanisms controlling fork integrity, we analyzed replication intermediates formed in response to replication blocks using electron microscopy.

Supercoiling, knotting and replication fork reversal in partially replicated plasmids.

To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18. The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy. Replication forks stop at the Ter-TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.

Nucleosome positioning at the replication fork.

In order to determine the time required for nucleosomes assembled on the daughter strands of replication forks to assume favoured positions with respect to DNA sequence, psoralen cross-linked replication intermediates purified from preparative two-dimensional agarose gels were analysed by exonuclease digestion or primer extension. Analysis of sites of psoralen intercalation revealed that nucleosomes in the yeast Saccharomyces cerevisiae rDNA intergenic spacer are positioned shortly after passage of the replication machinery. Therefore, both the 'old' randomly segregated nucleosomes as well as the 'new' assembled histone octamers rapidly position themselves (within seconds) on the newly replicated DNA strands, suggesting that the positioning of nucleosomes is an initial step in the chromatin maturation process.

Architecture of the replication fork stalled at the 3' end of yeast ribosomal genes.

Every unit of the rRNA gene cluster of Saccharomyces cerevisiae contains a unique site, termed the replication fork barrier (RFB), where progressing replication forks are stalled in a polar manner. In this work, we determined the positions of the nascent strands at the RFB at nucleotide resolution. Within an HpaI-HindIII fragment essential for the RFB, a major and two closely spaced minor arrest sites were found.

Replication of yeast rDNA initiates downstream of transcriptionally active genes.

In the yeast S. cerevisiae, ARS (autonomously replicating sequence) elements located in the intergenic spacers of the rRNA gene locus are infrequently activated as origins of replication. We analyzed the rARS activation with a combination of neutral/neutral (N/N) two-dimensional (2D) gel electrophoresis and either the intercalating drug psoralen, which in vivo specifically marks the transcribing gene copies, or the selective accessibility of restriction sites in transcriptionally active genes.

Interaction of Ap1, Ap2, and Sp1 with the regulatory regions of the human pro-alpha1(I) collagen gene.

In the pro-alpha1(I) collagen gene a number of cis-regulatory elements, which interact with a variety of trans-acting factors, are present in the promoter and first intron. We have undertaken a comprehensive study of Sp1, Ap1, and Ap2 binding in the region spanning -442 to +1697 nt. DNase I footprinting analysis revealed these factors bind with varying affinities to some of the potential sites: Sp1 binds to 16 of 34 potential sites, Ap2 binds to 22 of 40 potential binding sites, and Ap1 binds to its only potential site.

Mechanisms ensuring rapid and complete DNA replication despite random initiation in Xenopus early embryos.

Chromosome replication initiates without sequence specificity at average intervals of approximately 10 kb during the rapid cell cycles of early Xenopus embryos. If the distribution of origins were random, some inter-origin intervals would be too long to be fully replicated before the end of S phase. To investigate what ensures rapid completion of DNA replication, we have examined the replication intermediates of plasmids of various sizes (5.

Asymmetry of Dam remethylation on the leading and lagging arms of plasmid replicative intermediates.

In Escherichia coli, adenine methylation at the sequence GATC allows coupling of cellular processes to chromosome replication and the cell cycle. The transient presence of hemimethylated DNA after replication facilitates post-replicative mismatch repair, induces transcription of some genes and allows transposition of mobile elements. We were interested in estimating the half-life of hemimethylated DNA behind the replication fork in plasmid molecules and in determining whether Dam methyltransferase restores N6 adenine methylation simultaneously on both replicative arms.

Electron microscopic analysis reveals that replication factor C is sequestered by single-stranded DNA.

Replication factor C (RF-C) is a eukaryotic heteropentameric protein required for DNA replication and repair processes by loading proliferating cell nuclear antigen (PCNA) onto DNA in an ATP-dependent manner. Prior to loading PCNA, RF-C binds to DNA. This binding is thought to be restricted to a specific DNA structure, namely to a primer/template junction.

Transcriptional activity and chromatin structure of enhancer-deleted rRNA genes in Saccharomyces cerevisiae.

We used the psoralen gel retardation assay and Northern blot analysis in an in vivo yeast system to analyze effects of rDNA enhancer deletions on the chromatin structure and the transcription of tagged rDNA units. We found that upon deletion of a single enhancer element, transcription of the upstream and downstream rRNA gene was reduced by about 50%. Although removing both flanking enhancers of an rRNA gene led to a further reduction in transcription levels, a significant amount of transcriptional activity remained, either resulting from the influence of more distantly located enhancer elements or reflecting the basal activity of the polymerase I promoter within the nucleolus.

Formation of knots in partially replicated DNA molecules.

Bacterial plasmids with two origins of replication in convergent orientation are frequently knotted in vivo. The knots formed are localised within the newly replicated DNA regions. Here, we analyse DNA knots tied within replication bubbles of such plasmids, and observe that the knots formed show predominantly positive signs of crossings.

Geometry and physics of catenanes applied to the study of DNA replication.

The concept of ideal geometric configurations was recently applied to the classification and characterization of various knots. Different knots in their ideal form (i.e.

Histone acetylation facilitates RNA polymerase II transcription of the Drosophila hsp26 gene in chromatin.

A number of activators are known to increase transcription by RNA polymerase (pol) II through protein acetylation. While the physiological substrates for those acetylases are poorly defined, possible targets include general transcription factors, activator proteins and histones. Using a cell-free system to reconstitute chromatin with increased histone acetylation levels, we directly tested for a causal role of histone acetylation in transcription by RNA pol II.

In vivo mapping of nucleosomes using psoralen-DNA crosslinking and primer extension.

By the use of psoralen crosslinking and primer extension, a method was developed which allows the analysis of chromatin structure in vivo. Using a yeast minichromosome, >9 nucleosomes were mapped with a resolution of at least +/-30 bp.

Altered 3'-terminal RNA structure in phage Qbeta adapted to host factor-less Escherichia coli.

The RNA phage Qbeta requires for the replication of its genome an RNA binding protein called Qbeta host factor or Hfq protein. Our previous results suggested that this protein mediates the access of replicase to the 3'-end of the Qbeta plus strand RNA. Here we report the results of an evolutionary experiment in which phage Qbeta was adapted to an Escherichia coli Q13 host strain with an inactivated host factor (hfq) gene.

Chromatin structure and methylation of rat rRNA genes studied by formaldehyde fixation and psoralen cross-linking.

By using formaldehyde cross-linking of histones to DNA and gel retardation assays we show that formaldehyde fixation, similar to previously established psoralen photocross-linking, discriminates between nucleosome- packed (inactive) and nucleosome-free (active) fractions of ribosomal RNA genes. By both cross-linking techniques we were able to purify fragments from agarose gels, corresponding to coding, enhancer and promoter sequences of rRNA genes, which were further investigated with respect to DNA methylation. This approach allows us to analyse independently and in detail methylation patterns of active and inactive rRNA gene copies by the combination of Hpa II and Msp I restriction enzymes.