Global landscape of replicative DNA polymerase usage in the human genome

The division of labour between DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequence (Pu-seq) in HCT116 cells and mapped Polε and Polα usage genome wide. The polymerase usage profiles show Polε synthesises the leading strand and Polα contributes mainly to lagging strand synthesis. Combing the Polε and Polα profiles, we accurately predict the genome-wide pattern of fork directionality, zones of replication initiation and termination. We confirm that transcriptional activity shapes the patterns of initiation and termination and, by separately analysing the effect of transcription on both co-directional and converging forks, demonstrate that coupled DNA synthesis of leading and lagging strands in both co-directional and convergent forks is compromised by transcription. Polymerase uncoupling is particularly evident in the vicinity of large genes, including the two most unstable common fragile sites, FRA3B and FRA3D, thus linking transcription-induced polymerase uncoupling to chromosomal instability.

Large Intronic Deletion of the Fragile Site Gene PRKN Dramatically Lowers Its Fragility Without Impacting Gene Expression

Common chromosomal fragile sites (CFSs) are genomic regions prone to form breaks and gaps on metaphase chromosomes during conditions of replication stress. Moreover, CFSs are hotspots for deletions and amplifications in cancer genomes. Fragility at CFSs is caused by transcription of extremely large genes, which contributes to replication problems. These extremely large genes do not encode large proteins, but the extreme sizes of the genes originate from vast introns. Intriguingly, the intron sizes of extremely large genes are conserved between mammals and birds. Here, we have used reverse genetics to address the function and significance of the largest intron in the extremely large gene PRKN, which is highly fragile in our model system. Specifically, we have introduced an 80-kilobase deletion in intron 7 of PRKN. We find that gene expression of PRKN is largely unaffected by this intronic deletion. Strikingly, the intronic deletion, which leads to a 12% reduction of the overall size of the PRKN gene body, results in an almost twofold reduction of the PRKN fragility. Our results stress that while the large intron clearly contributes to the fragility of PRKN, it does not play an important role for PRKN expression. Taken together, our findings further add to the mystery concerning conservation of the seemingly non-functional but troublesome large introns in PRKN.

DNA molecular combing-based replication fork directionality profiling

The replication strategy of metazoan genomes is still unclear, mainly because definitive maps of replication origins are missing. High-throughput methods are based on population average and thus may exclusively identify efficient initiation sites, whereas inefficient origins go undetected. Single-molecule analyses of specific loci can detect both common and rare initiation events along the targeted regions. However, these usually concentrate on positioning individual events, which only gives an overview of the replication dynamics. Here, we computed the replication fork directionality (RFD) profiles of two large genes in different transcriptional states in chicken DT40 cells, namely untranscribed and transcribed DMD and CCSER1 expressed at WT levels or overexpressed, by aggregating hundreds of oriented replication tracks detected on individual DNA fibres stretched by molecular combing. These profiles reconstituted RFD domains composed of zones of initiation flanking a zone of termination originally observed in mammalian genomes and were highly consistent with independent population-averaging profiles generated by Okazaki fragment sequencing. Importantly, we demonstrate that inefficient origins do not appear as detectable RFD shifts, explaining why dispersed initiation has remained invisible to population-based assays. Our method can both generate quantitative profiles and identify discrete events, thereby constituting a comprehensive approach to study metazoan genome replication.

Autoregulation dependent and independent mechanisms are responsible for the systemic control of nodule formation by the plant N demand

In legumes interacting with rhizobia the formation of symbiotic organs responsible for the acquisition of atmospheric nitrogen is depending of the plant nitrogen (N) demand. We discriminated between local and systemic impact of nitrogen on nodule formation using Medicago truncatula plants cultivated in split-root systems. We obtained evidence of the control of nodule formation by whole plant systemic N-satisfaction signaling but obtained little evidence of a local control by mineral nitrogen. We characterized the impact of systemic N signaling on the root transcriptome reprogramming associated to nodule formation. We identified, large genes clusters displaying common expression profiles in response to systemic N signaling enriched in particular fonctions required during these biological processes. We found evidence of a strong effect of SUNN in the control by systemic N signaling of many genes involved in the early interaction with rhizobium as well as organogenesis supporting a role of autoregulation pathway in systemic N signaling. However, we also found evidence that major SUNN independent systemic N signaling controls were maintained in the mutant. This study shed light on the unexpected high complexity of the control of nodule formation by systemic N signaling, that probably involves multiple pathways.

FANCD2 modulates the mitochondrial stress response to prevent common fragile site instability

Common fragile sites (CFSs) are genomic regions frequently involved in cancer-associated rearrangements. Most CFSs lie within large genes, and their instability involves transcription- and replication-dependent mechanisms. Here, we uncover a role for the mitochondrial stress response pathway in the regulation of CFS stability in human cells. We show that FANCD2, a master regulator of CFS stability, dampens the activation of the mitochondrial stress response and prevents mitochondrial dysfunction. Genetic or pharmacological activation of mitochondrial stress signaling induces CFS gene expression and concomitant relocalization to CFSs of FANCD2. FANCD2 attenuates CFS gene transcription and promotes CFS gene stability. Mechanistically, we demonstrate that the mitochondrial stress-dependent induction of CFS genes is mediated by ubiquitin-like protein 5 (UBL5), and that a UBL5-FANCD2 dependent axis regulates the mitochondrial UPR in human cells. We propose that FANCD2 coordinates nuclear and mitochondrial activities to prevent genome instability.

Analysis on Overweight and Inadequate Physical Activity by Nutritional Behaviors such as Obesity

We study all the review article related to Obesity. The Obesity is the disease in which imbalance between the calorie intake and its use. Obesity has touched widespread levels in established as well as in developing countries. It progressively existence experiential by altering the lifestyle of the family. The mechanism of Obesity is the unusual calorie convert into fat in adipose tissue. Adipose tissue consists of an adipose cell and unusual calorie accumulate into the adipose cell they swell increase their Weight and increase the bodyweight of the body. Universally in an investigation of 199 nations, 1.46 billion grown-ups are assessed to actuality overweight, and 502 million are assessed as existence stout. Individuals are commonly viewed as fat when their weight file (BMI) an estimation developed by segmentation an individual load by the rectangular of the separate tallness is more than 30 Kg/m2, by the reference range 25-30 kg/m2 considered as overweight. Weight Index (BMI) is commonplace uses to clarify the overweight and heftiness in the study of disease transmission contemplates. In any case, BMI has low affectability and there is bigger entomb singular changes in the percent muscle versus fat for some random BMI esteem, somewhat trait to age sex and nationality. Causes of Obesity, both genes and environmental factor participate in Obesity. However, by the large, genes are now thought to set only the stage and provide the background, against which the decisive effects and provide effects are eventually driven by the natural and conduct factors.

Simultaneous Genome Sequencing of Prosthecochloris ethylica and Desulfuromonas acetoxidans within a Syntrophic Mixture Reveals Unique Pili and Protein Interactions

Strains of Chloropseudomonas ethylica, 2-K, N2, and N3 are known to be composed of a syntrophic mixture of a green sulfur bacterium and a sulfur-reducing colorless component. Upon sequence analysis, the green sulfur photosynthetic bacterial component of strain N3 was dominant and was readily sequenced, but the less abundant sulfur-reducing bacterial component was apparent only when analyzed by metagenomic binning. Whole-genome comparison showed that the green bacterium belonged to the genus Prosthecochloris and apparently was a species for which there was no genome sequence on file. For comparison, we also sequenced the genome of Prosthecochloris sp. DSM 1685, which had previously been isolated from the 2-K mixture in pure culture and have shown that all three Prosthecochloris genomes belong to a new species, which we propose to be named Prosthecochloris ethylica comb. nov. Whole genomes were also sequenced for the isolated Desulfuromonas strains DSM 1675 (from strain 2-K) and DSM 1676 (from strain N2) and shown to be nearly identical to the genome found in the N3 mixture. The genome of the green sulfur bacterium contains large genes for agglutination proteins, similar to the ones proposed to be involved in larger photosynthetic consortia of Chlorochromatium aggregatum. In addition, we also identified several unique “tight adhesion (tad)” pili genes that are presumably involved in the formation of cell–cell interactions. The colorless component, on the other hand, contained a unique large multiheme cytochrome C and unique genes for e-pili (geopilin) formation, genetically clustered with a conserved ferredoxin gene, which are all expected to play an electron transfer role in the closed sulfur cycle in the syntrophic mixture. The findings from the simultaneous genome sequencing of the components of Cp. ethylica have implications for the phenomenon of direct interspecies interactions and coupled electron transfer in photosynthetic symbionts. The mechanisms for such interactions appear to be more common in the environment than originally anticipated.