Protein composition of interband regions in polytene and cell line chromosomes of Drosophila melanogaster

<p>Abstract</p> <p>Background</p> <p>Despite many efforts, little is known about distribution and interactions of chromatin proteins which contribute to the specificity of chromomeric organization of interphase chromosomes. To address this issue, we used publicly available datasets from several recent Drosophila genome-wide mapping and annotation projects, in particular, those from modENCODE project, and compared molecular organization of 13 interband regions which were accurately mapped previously.</p> <p>Results</p> <p>Here we demonstrate that in interphase chromosomes of <it>Drosophila </it>cell lines, the interband regions are enriched for a specific set of proteins generally characteristic of the "open" chromatin (RNA polymerase II, CHRIZ (CHRO), BEAF-32, BRE1, dMI-2, GAF, NURF301, WDS and TRX). These regions also display reduced nucleosome density, histone H1 depletion and pronounced enrichment for ORC2, a pre-replication complex component. Within the 13 interband regions analyzed, most were around 3-4 kb long, particularly those where many of said protein features were present. We estimate there are about 3500 regions with similar properties in chromosomes of <it>D. melanogaster </it>cell lines, which fits quite well the number of cytologically observed interbands in salivary gland polytene chromosomes.</p> <p>Conclusions</p> <p>Our observations suggest strikingly similar organization of interband chromatin in polytene chromosomes and in chromosomes from cell lines thereby reflecting the existence of a universal principle of interphase chromosome organization.</p

Paucity and preferential suppression of transgenes in late replication domains of the D. melanogaster genome

<p>Abstract</p> <p>Background</p> <p>Eukaryotic genomes are organized in extended domains with distinct features intimately linking genome structure, replication pattern and chromatin state. Recently we identified a set of long late replicating euchromatic regions that are underreplicated in salivary gland polytene chromosomes of <it>D. melanogaster</it>.</p> <p>Results</p> <p>Here we demonstrate that these underreplicated regions (URs) have a low density of <it>P</it>-<it>element </it>and <it>piggyBac </it>insertions compared to the genome average or neighboring regions. In contrast, <it>Minos</it>-based transposons show no paucity in URs but have a strong bias to testis-specific genes. We estimated the suppression level in 2,852 stocks carrying a single <it>P</it>-<it>element </it>by analysis of eye color determined by the mini-<it>white </it>marker gene and demonstrate that the proportion of suppressed transgenes in URs is more than three times higher than in the flanking regions or the genomic average. The suppressed transgenes reside in intergenic, genic or promoter regions of the annotated genes. We speculate that the low insertion frequency of <it>P-elemen</it>ts and <it>piggyBac</it>s in URs partially results from suppression of transgenes that potentially could prevent identification of transgenes due to complete suppression of the marker gene. In a similar manner, the proportion of suppressed transgenes is higher in loci replicating late or very late in Kc cells and these loci have a lower density of <it>P-elements </it>and <it>piggyBac </it>insertions. In transgenes with two marker genes suppression of mini-<it>white </it>gene in eye coincides with suppression of <it>yellow </it>gene in bristles.</p> <p>Conclusions</p> <p>Our results suggest that the late replication domains have a high inactivation potential apparently linked to the silenced or closed chromatin state in these regions, and that such inactivation potential is largely maintained in different tissues.</p

Identical Functional Organization of Nonpolytene and Polytene Chromosomes in Drosophila melanogaster

Salivary gland polytene chromosomes demonstrate banding pattern, genetic meaning of which is an enigma for decades. Till now it is not known how to mark the band/interband borders on physical map of DNA and structures of polytene chromosomes are not characterized in molecular and genetic terms. It is not known either similar banding pattern exists in chromosomes of regular diploid mitotically dividing nonpolytene cells. Using the newly developed approach permitting to identify the interband material and localization data of interband-specific proteins from modENCODE and other genome-wide projects, we identify physical limits of bands and interbands in small cytological region 9F13-10B3 of the X chromosome in D. melanogaster, as well as characterize their general molecular features. Our results suggests that the polytene and interphase cell line chromosomes have practically the same patterns of bands and interbands reflecting, probably, the basic principle of interphase chromosome organization. Two types of bands have been described in chromosomes, early and late-replicating, which differ in many aspects of their protein and genetic content. As appeared, origin recognition complexes are located almost totally in the interbands of chromosomes

Effect of electron irradiation on vortex dynamics in YBa_2Cu_3O_{7-x} single crystals

We report on drastic change of vortex dynamics with increase of quenched disorder: for rather weak disorder we found a single vortex creep regime, which we attribute to a Bragg-glass phase, while for enhanced disorder we found an increase of both the depinning current and activation energy with magnetic field, which we attribute to entangled vortex phase. We also found that introduction of additional defects always increases the depinning current, but it increases activation energy only for elastic vortex creep, while it decreases activation energy for plastic vortex creep.Comment: 4 pages, 3 figures, submited to Phys. Rev.

Experience in genetic testing of hypertrophic cardiomyopathy using nanopore DNA sequencing

Aim. To investigate the application of the Oxford Nanopore Technologies’ third generation sequencing for the genetic testing of hypertrophic cardiomyopathy.Material and methods. The study involved 12 patients with hypertrophic cardiomyopathy aged 18 to 67 years (women, 9; men, 3). Using the PCR barcoding amplicons (SQK-LSK109) protocol, DNA libraries were created which contained long-range PCR fragments of the MYH7, MYBPC3, TNNT2, TNNI3 and TPM1 genes. The sequencing was performed using the MinION system by Oxford Nanopore Technologies (UK). Bioinformatic algorithms for data analysis included Guppy v.5.0.7, Nanopolish and Clairvoyante. The identified genetic variants were confirmed by Sanger sequencing.Results. Data on the complete sequence of the five major sarcomeric genes for hypertrophic cardiomyopathy were obtained. We found eight potentially disease-causing sequence variants in MYH7, MYBPC3 and TNNT2 genes by monomolecular sequencing. However, only three mutations p.Arg243Cys, p.Tyr609Asn, p.Arg870His in the MYH7 gene, and one mutation p.Lys985Asn in the MYBPC3 were confirmed by Sanger sequencing. Cascade screening of pathogenic variant p.Arg870His in the MYH7 gene was performed. We found one asymptomatic carrier.Conclusion. It appears that monomolecular sequencing technology is a feasible approach to identify mutations in patients with hypertrophic cardiomyopathy. Although improvement in accuracy of DNA sequencing, as well as optimization and simplification of bioinformatic algorithms for identification of the genetic variants are needed

Late Replication Domains in Polytene and Non-Polytene Cells of Drosophila melanogaster

In D. melanogaster polytene chromosomes, intercalary heterochromatin (IH) appears as large dense bands scattered in euchromatin and comprises clusters of repressed genes. IH displays distinctly low gene density, indicative of their particular regulation. Genes embedded in IH replicate late in the S phase and become underreplicated. We asked whether localization and organization of these late-replicating domains is conserved in a distinct cell type. Using published comprehensive genome-wide chromatin annotation datasets (modENCODE and others), we compared IH organization in salivary gland cells and in a Kc cell line. We first established the borders of 60 IH regions on a molecular map, these regions containing underreplicated material and encompassing ∼12% of Drosophila genome. We showed that in Kc cells repressed chromatin constituted 97% of the sequences that corresponded to IH bands. This chromatin is depleted for ORC-2 binding and largely replicates late. Differences in replication timing between the cell types analyzed are local and affect only sub-regions but never whole IH bands. As a rule such differentially replicating sub-regions display open chromatin organization, which apparently results from cell-type specific gene expression of underlying genes. We conclude that repressed chromatin organization of IH is generally conserved in polytene and non-polytene cells. Yet, IH domains do not function as transcription- and replication-regulatory units, because differences in transcription and replication between cell types are not domain-wide, rather they are restricted to small “islands” embedded in these domains. IH regions can thus be defined as a special class of domains with low gene density, which have narrow temporal expression patterns, and so displaying relatively conserved organization

Frequently asked questions about chlorophyll fluorescence, the sequel

[EN] Using chlorophyll (Chl) a fluorescence many aspects of the photosynthetic apparatus can be studied, both in vitro and, noninvasively, in vivo. Complementary techniques can help to interpret changes in the Chl a fluorescence kinetics. Kalaji et al. (Photosynth Res 122: 121-158, 2014a) addressed several questions about instruments, methods and applications based on Chl a fluorescence. Here, additionalChl a fluorescence-related topics are discussed again in a question and answer format. Examples are the effect of connectivity on photochemical quenching, the correction of F-V/F-M values for PSI fluorescence, the energy partitioning concept, the interpretation of the complementary area, probing the donor side of PSII, the assignment of bands of 77 K fluorescence emission spectra to fluorescence emitters, the relationship between prompt and delayed fluorescence, potential problems when sampling tree canopies, the use of fluorescence parameters in QTL studies, the use of Chl a fluorescence in biosensor applications and the application of neural network approaches for the analysis of fluorescence measurements. The answers draw on knowledge fromdifferent Chl a fluorescence analysis domains, yielding in several cases new insights.Kalaji, H.; Schansker, G.; Brestic, M.; Bussotti, F.; Calatayud, A.; Ferroni, L.; Goltsev, V.... (2017). Frequently asked questions about chlorophyll fluorescence, the sequel. 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Co-composting: An Opportunity to Produce Compost with Designated Tailor-Made Properties

AbstractCo-composting is a technique that allows the aerobic degradation of organic waste mixtures, primarily aiming at obtaining compost that can be used as fertiliser or soil amendment. As compared to the typical composting activity, the main difference is not merely the use of more than one feedstock to start and sustain the biodegradation process, but also the possibility of combining various kinds of waste to obtain 'tailored' products with designed properties, or to reclaim and valorise natural resources, such as degraded soils or polluted soils and sediments. Set up of appropriate co-composting protocols can be a way to optimise the management of waste produced by different sectors of agriculture and industry and also from human settlements. Different formulations can not only optimise the biodegradation process through the adjustment of nutrient ratios, but also lead to the formation of products with innovative properties. Moreover, co-composting can be a technique of choice for the reclamation of soils degraded by intensive agriculture or contaminated soils and sediments. In fact, an appropriate mix of organic waste and soils can restore the soil structure and induce fertility in nutrient-depleted soils, and also remediate polluted soils and sediments through degradation of organic pollutants and stabilisation of heavy metals. While the selection of different mixes of organic waste may lead to the design of composts with specific properties and the potential valorisation of selected waste materials, there are still several factors that hamper the development of co-composting platforms, mainly insufficient knowledge of some chemical and microbiological processes, but also some legislative aspects. This chapter illustrates the progress achieved in co-composting technology worldwide, some key legislative aspects related to the co-composting process, the main scientific and technical aspects that deserve research attention to further develop co-composting technology, and successful applications of co-composting for the reclamation of soils and sediments, allowing their use for cultivation or as growing media in plant nurseries. A specific case study of the production of fertile plant-growing media from sediment co-composting with green waste is also illustrated