The three-dimensional (3D) organization of the chromosomal fiber in the human interphase nucleus is an important but poorly understood aspect of gene regulation. Here we quantitatively analyze and compare the 3D structures of two types of genomic domains as defined by the human transcriptome map. While ridges are gene dense and show high expression levels, antiridges, on the other hand, are gene poor and carry genes that are expressed at low levels. We show that ridges are in general less condensed, more irregularly shaped, and located more closely to the nuclear center than antiridges. Six human cell lines that display different gene expression patterns and karyotypes share these structural parameters of chromatin. This shows that the chromatin structures of these two types of genomic domains are largely independent of tissue-specific variations in gene expression and differentiation state. Moreover, we show that there is remarkably little intermingling of chromatin from different parts of the same chromosome in a chromosome territory, neither from adjacent nor from distant parts. This suggests that the chromosomal fiber has a compact structure that sterically suppresses intermingling. Together, our results reveal novel general aspects of 3D chromosome architecture that are related to genome structure and function.
We show that double strand breaks (DSBs) induced in chromatin of low as well as high density by exposure of human cells to gamma-rays are repaired in low-density chromatin. Extensive chromatin decondensation manifested in the vicinity of DSBs by decreased intensity of chromatin labelling, increased H4K5 acetylation, and decreased H3K9 dimethylation was observed already 15 min after irradiation. Only slight movement of sporadic DSB loci for short distances was noticed in living cells associated with chromatin decondensation around DSBs. This frequently resulted in their protrusion into the low-density chromatin domains. In these regions, the clustering (contact or fusion) of DSB foci was seen in vivo, and in situ after cell fixation. The majority of these clustered foci were repaired within 240 min, but some of them persisted in the nucleus for several days after irradiation, indicating damage that is not easily repaired. We propose that the repair of DSB in clustered foci might lead to misjoining of ends and, consequently, to exchange aberrations. On the other hand, the foci that persist for several days without being repaired could lead instead to cell death.
Cyanobacteria are among the most important primary producers on the Earth. However, the evolutionary forces driving cyanobacterial species diversity remain largely enigmatic due to both their distinction from macro-organisms and an undersampling of sequenced genomes. Thus, we present a new genome of a Synechococcus-like cyanobacterium from a novel evolutionary lineage. Further, we analyse all existing 16S rRNA sequences and genomes of Synechococcus-like cyanobacteria. Chronograms showed extremely polyphyletic relationships in Synechococcus, which has not been observed in any other cyanobacteria. Moreover, most Synechococcus lineages bifurcated after the Great Oxidation Event, including the most abundant marine picoplankton lineage. Quantification of horizontal gene transfer among 70 cyanobacterial genomes revealed significant differences among studied genomes. Horizontal gene transfer levels were not correlated with ecology, genome size or phenotype, but were correlated with the age of divergence. All findings were synthetized into a novel model of cyanobacterial evolution, characterized by serial convergence of the features, that is multicellularity and ecology.
Filamentous epipelic cyanobacteria were isolated from ponds and lakes in the Czech Republic, Austria and Italy. Morphological and genetic variation of 20 isolated strains within the genera Geitlerinema, Microcoleus and Phormidium were studied. Partial sequences of the 16S rRNA gene were used for phylogenetic analyses, and secondary structure of the 16S-23S ITS region was used to additionally define clades. Morphological and molecular were congruent, and we were able to identify the majority of strains correctly to species on the basis of morphological features. Overall diversity and morphological/genetic variation of epipelic species is not as high as described from other benthic habitats, possibly due to the relative microhabitat uniformity of lake/pond bottom sediments. The M. vaginatus clade is well defined by an 11 bp insert in 16S rRNA gene (bp 423-433) and populations from different ecological conditions differ in secondary structure in the 16S-23S ITS regions, particularly in Box-B helices. Ph. autumnale and the genus Geitlerinema appear to be polyphyletic as presently defined.
Abstract:The study of genome size variation in microalgae lags behind that of comparable research in higher plants and seaweeds. This situation is essentially caused by: (1) difficulties in obtaining sufficient biomass for experiments; (2) problems with protoplast isolation due to cell-wall heterogeneity and complexity; and (3) the absence of suitable standards for routine measurements. We propose a multi-step protocol that leads to the quantification of DNA content in desmids using flow cytometry. We present detailed culture conditions, the minimal biomass necessary for three repetitive measurements, a method to isolate protoplasts and selection of suitable standards. Our protocol, which is mainly based on studies with higher plants and commercially available enzyme mixtures, is useful in Streptophyta, especially members of the Zygnematophyceae, because of their close phylogenetic relationship to higher plants, in particular the similarity of their cell wall organization. Moreover, the suggested protocol also works for some Chlorophyta (Chloroidium ellipsoideum, Tetraselmis subcordiformis) and Heterokontophyta (Tribonema vulgare). We suggest and characterize a new standard for flow cytometry of microalgae (Micrasterias pinnatifida). Modification of the enzyme mixture is probably necessary for microalgae whose cell walls are surrounded by a mucilaginous envelope (Planktosphaeria), those that contain alganan (Chlorella), monads with a pellicle or chlamys (Euglena, Chlamydomonas). While we did not anticipate any success with diatoms (Pinnularia), because of their silica frustules, the enzyme mixture also failed for some other green microalgae (Xanthidium, Kentrosphaera, Stigeoclonium, Trentepohlia and Pseudendoclonium).
This work is focused on the function of the microtubule and actin networks in plasmid DNA transport during liposomal transfection. We observed strong binding of plasmid DNA-lipid complexes (lipoplexes) to both networks and directional long-range motion of these lipoplexes along the microtubules. Disruption of either of these networks led to the cessation of plasmid transport to the nucleus, a decreased mobility of plasmids, and accumulation of plasmid DNA in large aggregates at the cell periphery. Our findings show an indispensable but different role of both types of cytoskeleton, actin and microtubular, in the processes of gene delivery.
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This paper reports on the structural rearrangement of satellite DNA type I repeats and heterochromatin during the dedifferentiation and cell cycling of mesophyll protoplasts of cucumber (Cucumis sativus). These repeats were localized in the telomeric heterochromatin of cucumber chromosomes and in the chromocenters of interphase nuclei. The dramatic reduction of heterochromatin involves decondensation of subtelomeric repeats in freshly isolated protoplasts; however, there are not a great many remarkable changes in the expression profile. In spite of that, reformation of the chromocenters, occurring 48 h after protoplast isolation, is accompanied by recondensation of satellite DNA type I; however, only partial reassembly of these repeats was revealed. In this study, FISH and a flow cytometry assay show a correlation between the partial chromocenter and the repeats reassembly, and with the reentry of cultivated protoplasts into the cell cycle and first cell division. After that, divided cells displayed a higher variability in the expression profile than did leaves' mesophyll cells and protoplasts.
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