Genome-wide high-resolution mapping and functional analysis of DNA methylation in arabidopsis.

Cytosine methylation is important for transposon silencing and epigenetic regulation of endogenous genes, although the extent to which this DNA modification functions to regulate the genome is still unknown. Here we report the first comprehensive DNA methylation map of an entire genome, at 35 base pair resolution, using the flowering plant Arabidopsis thaliana as a model. We find that pericentromeric heterochromatin, repetitive sequences, and regions producing small interfering RNAs are heavily methylated. Unexpectedly, over one-third of expressed genes contain methylation within transcribed regions, whereas only approximately 5% of genes show methylation within promoter regions. Interestingly, genes methylated in transcribed regions are highly expressed and constitutively active, whereas promoter-methylated genes show a greater degree of tissue-specific expression. Whole-genome tiling-array transcriptional profiling of DNA methyltransferase null mutants identified hundreds of genes and intergenic noncoding RNAs with altered expression levels, many of which may be epigenetically controlled by DNA methylation.

ApcD is necessary for efficient energy transfer from phycobilisomes to photosystem I and helps to prevent photoinhibition in the cyanobacterium Synechococcus sp PCC 7002

Phyrobilisomes (PBS) are the major light-harvesting, protein-pigment complexes in cyanobacteria and red algae. PBS absorb and transfer light energy to photosystem (PS) II as well as PS I, and the distribution of light energy from PBS to the two photosystems is regulated by light conditions through a mechanism known as state transitions. In this study the quantum efficiency of excitation energy transfer from PBS to PS I in the cyanobacterium Synechococcus sp. PCC 7002 was determined, and the results showed that energy transfer from PBS to PS I is extremely efficient. The results further demonstrated that energy transfer from PBS to PS I occurred directly and that efficient energy transfer was dependent upon the allophycocyanin-B alpha subunit, ApcD. In the absence of ApcD, cells were unable to perform state transitions and were trapped in state 1. Action spectra showed that light energy transfer from PBS to PS I was severely impaired in the absence of ApcD. An apcD mutant grew more slowly than the wild type in light preferentially absorbed by phyrobiliproteins and was more sensitive to high light intensity. On the other hand, a mutant lacking ApcF, which is required for efficient energy transfer from PBS to PS II, showed greater resistance to high light treatment. Therefore, state transitions in cyanobacteria have two roles: (1) they regulate light energy distribution between the two photosystems; and (2) they help to protect cells from the effects of light energy excess at high light intensities. (C) 2009 Elsevier B.V. All rights reserved.

Integration of double-fluorescence expression vectors into zebrafish genome for the selection of site-directed knockout/knockin

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7x10(-6). In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F-1 generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P-0 generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism.

Physiological and biochemical microcystin-RR toxicity to the Synechococcus elongatus

Freshwater Microcystis may form dense blooms in eutrophic lakes. It is known to produce a family of related cyclic hepatopeptides (microcystins, MC) that constitute a threat to aquatic ecosystems. Most toxicological studies of microcystins have focused on aquatic animals and plants, with few examining the possible effects of microcystins on phytoplankton. In this study we chose the unicellular Synechococcus elongatus (one of the most studied and geographically most widely distributed cyanobacteria in the picoplankton) as the test material and investigated the biological parameters: growth, pigment (chlorophyll-a, phycocyanin), photosynthetic activity, nitrate reductase activity, and protein and carbohydrate content. The results revealed that microcystin-RR concentrations above 100 mug (.) L-1 significantly inhibited the growth of Synechococcus elongatus. In addition, a change in color of the toxin-treated algae (chlorosis) was observed in the experiments. Furthermore, MC-RR markedly inhibited the synthesis of the pigments chlorophyll-a and phycocyanin. A drastic reduction in photochemical efficiency of PSII (F-v/F-m) was found after a 96-h incubation. Changes in protein and carbohydrate concentrations and in nitrate reductase activity also were observed during the exposure period. This study aimed to evaluate the mechanisms of microcystin toxicity on a cyanobacterium, according to the physiological and biochemical responses of Synechococcus elongatus to different doses of microcystin-RR. The ecological role of microcystins as an allelopathic substance also is discussed in the article. (C) 2004 Wiley Periodicals, Inc.

Expression of human epidermal growth factor gene in cyanobacteria

The human epidermal growth factor (hEGF) is a small single-chain polypeptide of 53 amino acid residues. It can stimulate the proliferation of many cell types, mainly those of epidermal and epithelial tissues both in vivo and in vitro. A vector pRL-hEGF was constructed using plasmids pRL-489 and pUC-hEGF. The synthetic hEGF gene was recombined into the downstream of strong promoter psbA in plasmids pRL-489. Then, the vector was introduced into Synechococcus sp. PCC 7002 and Anabaena sp. PCC 7120 by triparental conjugative transfer. The transformation was confirmed by PCR amplification. The pRL-hEGF is thought to be retained as a plasmid form in the transgenic Anabaena sp. PCC 7120, since it can be recovered. However, it has been integrated into the chromosome of Synechococcus sp. PCC 7002 as there is no duplication origin in the pRL-hEGF in this cyanobacterium. and plasmid cannot be isolated from the Synechococcus sp. PCC 7002 either. The radioimmunoassay (RIA) proved that the hEGF gene has been expressed as the protein existed in these two strains of transgenic cyanobacteria, and the hEGF protein in Anabaena sp. PCC 7002 could be secreted into the medium.

Lineage-specific domain fusion in the evolution of purine nucleotide cyclases in cyanobacteria

Cyclic nucleotides (both cAMP and cGMP) play extremely important roles in cyanobacteria, such as regulating heterocyst formation, respiration, or gliding. Catalyzing the formation of cAMP and cGMP from ATP and GTP is a group of functionally important enzymes named adenylate cyclases and guanylate cyclases, respectively. To understand their evolutionary patterns, in this study, we presented a systematic analysis of all the cyclases in cyanobacterial genomes. We found that different cyanobacteria had various numbers of cyclases in view of their remarkable diversities in genome size and physiology. Most of these cyclases exhibited distinct domain architectures, which implies the versatile functions of cyanobacterial cyclases. Mapping the whole set of cyclase domain architectures from diverse prokaryotic organisms to their phylogenetic tree and detailed phylogenetic analysis of cyclase catalytic domains revealed that lineage-specific domain recruitment appeared to be the most prevailing pattern contributing to the great variability of cyanobacterial cyclase domain architectures. However, other scenarios, such as gene duplication, also occurred during the evolution of cyanobacterial cyclases. Sequence divergence seemed to contribute to the origin of putative guanylate cyclases which were found only in cyanobacteria. In conclusion, the comprehensive survey of cyclases in cyanobacteria provides novel insight into their potential evolutionary mechanisms and further functional implications.

Spatial variations of size-fractionated chlorophyll, cyanobacteria and heterotrophic bacteria in the Central and Western Pacific

Geographic and vertical variations of size-fractionated (0.2-1 mu m, 1-10 mu m, and >10 mu m) Chlorophyll a (Chl.a) concentration, cyanobacteria abundance and heterotrophic bacteria abundance were investigated at 13 stations from 4 degrees S, 160 degrees W to 30 degrees N, 140 degrees E in November 1993. The results indicated a geographic distribution pattern of these parameters with instances of high values occurring in the equatorial region and offshore areas, and with instance of low values occurring in the oligotrophic regions where nutrients were almost undetectable. Cyanobacteria showed the highest geographic variation (ranging from 27x10(3) to 16,582x10(3) cell l(-1)), followed by Chl.a (ranging from 0.048 to 0.178 mu g l(-1)), and heterotrophic bacteria (ranging from 2.84x10(3) to 6.50 x 10(5) cell l(-1)). Positive correlations were observed between nutrients and Chl.a abundance. Correspondences of cyanobacteria and heterotrophic bacteria abundances to nutrients were less significant than that of Chl.a. The total Chl.a was accounted for 1.0-30.9%, 35.9-53.7%, and 28.1-57.3% by the >10 mu m, 1-10 mu m and 0.2-1 mu m fractions respectively. Correlation between size-fractionated Chl.a and nutrients suggest that the larger the cell size, the more nutrient-dependent growth and production of the organism. The ratio of pheophytin to chlorophyll implys that more than half of the > 10 mu m and about one third of the 1-10 mu m pigment-containing particles in the oligotrophic region were non-living fragments, while most of the 1-10 mu m fraction was living cells. In the depth profiles, cyanobacteria were distributed mainly in the surface layer, whereas heterotrophic bacteria were abundant from surface to below the euphotic zone. Chl.a peaked at the surface layer (0-20 m) in the equatorial area and at the nitracline (75-100 m) in the oligotrophic regions. Cyanobacteria were not the principle component of the picoplankton. The carbon biomass ratio of heterotroph to phytoplankton was greater than 1 in the eutrophic area and lower than 1 in oligotrophic waters.

Crowdfunding the Azolla fern genome project: a grassroots approach.

Much of science progresses within the tight boundaries of what is often seen as a "black box". Though familiar to funding agencies, researchers and the academic journals they publish in, it is an entity that outsiders rarely get to peek into. Crowdfunding is a novel means that allows the public to participate in, as well as to support and witness advancements in science. Here we describe our recent crowdfunding efforts to sequence the Azolla genome, a little fern with massive green potential. Crowdfunding is a worthy platform not only for obtaining seed money for exploratory research, but also for engaging directly with the general public as a rewarding form of outreach.

Characterisation of Genome-Wide PLZF/RARA Target Genes

The PLZF/RARA fusion protein generated by the t(11;17)(q23;q21) translocation in acute promyelocytic leukaemia (APL) is believed to act as an oncogenic transcriptional regulator recruiting epigenetic factors to genes important for its transforming potential. However, molecular mechanisms associated with PLZF/RARA-dependent leukaemogenesis still remain unclear. We searched for specific PLZF/RARA target genes by ChIP-on-chip in the haematopoietic cell line U937 conditionally expressing PLZF/RARA. By comparing bound regions found in U937 cells expressing endogenous PLZF with PLZF/RARA-induced U937 cells, we isolated specific PLZF/RARA target gene promoters. We next analysed gene expression profiles of our identified target genes in PLZF/RARA APL patients and analysed DNA sequences and epigenetic modification at PLZF/RARA binding sites. We identify 413 specific PLZF/RARA target genes including a number encoding transcription factors involved in the regulation of haematopoiesis. Among these genes, 22 were significantly down regulated in primary PLZF/RARA APL cells. In addition, repressed PLZF/RARA target genes were associated with increased levels of H3K27me3 and decreased levels of H3K9K14ac. Finally, sequence analysis of PLZF/RARA bound sequences reveals the presence of both consensus and degenerated RAREs as well as enrichment for tissue-specific transcription factor motifs, highlighting the complexity of targeting fusion protein to chromatin. Our study suggests that PLZF/RARA directly targets genes important for haematopoietic development and supports the notion that PLZF/RARA acts mainly as an epigenetic regulator of its direct target genes.

Photo-dynamic biocidal action of methylene blue and hydrogen peroxide on the cyanobacterium Synechococcus leopoliensis under visible light irradiation

Biofilm growth on stone surfaces is a significant contributing factor to stone biodeterioration. Current market based biocides are hazardous to the environment and to public health. We have investigated the photo-dynamic effect of methylene blue (MB) in the presence of hydrogen peroxide (H₂O₂) on the destruction of the cyanobacterium Synechococcus leopoliensis (S. leopoliensis) under irradiation with visible light. Data presented in this paper illustrate that illumination of S. leopoliensis in the presence of a photosensitiser (MB) and H₂O₂ results in the decomposition of both the cyanobacterium and the photosensitiser. The presence of MB and H₂O₂ affects the viability of the photosensitiser and the cyanobacterium with the fluorescence of both decreasing by 80% over the irradiation time investigated. The photo-dynamic effect was observed under aerobic and anaerobic conditions indicating that oxygen was not necessary for the process. This novel combination could be effective for the remediation of biofilm colonised stone surfaces

The mechanism of the regulation of energy distribution between photosystems 1 and 2 in the cyanobacterium Synechococcus sp. strain PCC 7002 /

Cyanobacteria are able to regulate the distribution of absorbed light energy between photo systems 1 and 2 in response to light conditions. The mechanism of this regulation (the state transition) was investigated in the marine cyanobacterium Synechococcus sp. strain PCC 7002. Three cell types were used: the wild type, psaL mutant (deletion of a photo system 1 subunit thought to be involved in photo system 1 trimerization) and the apcD mutant (a deletion of a phycobilisome subunit thought to be responsible for energy transfer to photo system 1). Evidence from 77K fluorescence emission spectroscopy, room temperature fluorescence and absorption cross-section measurements were used to determine a model of energy distribution from the phycobilisome and chlorophyll antennas in state 1 and state 2. The data confirm that in state 1 the phycobilisome is primarily attached to PS2. In state 2, a portion of the phycobilisome absorbed light energy is redistributed to photo system 1. This energy is directly transferred to photo system 1 by one of the phycobilisome terminal emitters, the product of the apcD gene, rather than via the photo system 2 chlorophyll antenna by spillover (energy transfer between the photo system 2 and photo system 1 chlorophyll antenna). The data also show that energy absorbed by the photo system 2 chlorophyll antenna is redistributed to photo system 1 in state 2. This could occur in one of two ways; by spillover or in a way analogous to higher plants where a segment of the chlorophyll antenna is dissociated from photo system 2 and becomes part of the photo system 1 antenna. The presence of energy transfer between neighbouring photo system 2 antennae was determined at both the phycobilisome and chlorophyll level, in states 1 and 2. Increases in antenna absorption cross-section with increasing reaction center closure showed that there is energy transfer (connectivity) between photosystem 2 antennas. No significant difference was shown in the amount of connectivity under these four conditions.

The mysterious presence of a 5-methylcytosine oxidase in the Drosophila genome: Possible explanations

5-methylcytosine is an important epigenetic modification involved in gene control in vertebrates and many other complex living organisms. Its presence in Drosophila has been a matter of debate and recent bisulfite sequencing studies of early-stage fly embryos have concluded that the genome of Drosophila is essentially unmethylated. However, as we outline here, the Drosophila genome harbors a well-conserved homolog of the TET protein family. The mammalian orthologs TET1/2/3 are known to convert 5-methylcytosine into 5-hydroxymethylcytosine. We discuss several possible explanations for these seemingly contradictory findings. One possibility is that the 2 modified cytosine bases are generated in Drosophila only at certain developmental stages and in a cell type-specific manner during neurogenesis. Alternatively, Drosophila Tet and its mammalian homologs may carry out catalytic activity-independent functions, and the possibility that these proteins may oxidize 5-methylcytosine in RNA created by the methyltransferase Dnmt2 should also be strongly considered.

Genome-wide mapping of 5-hydroxymethylcytosine in three rice cultivars reveals its preferential localization in transcriptionally silent transposable element genes

5-Hydroxymethylcytosine (5hmC), a modified form of cytosine that is considered the sixth nucleobase in DNA, has been detected in mammals and is believed to play an important role in gene regulation. In this study, 5hmC modification was detected in rice by employing a dot-blot assay, and its levels was further quantified in DNA from different rice tissues using liquid chromatography-multistage mass spectrometry (LC-MS/MS/MS). The results showed large intertissue variation in 5hmC levels. The genome-wide profiles of 5hmC modification in three different rice cultivars were also obtained using a sensitive chemical labelling followed by a next-generation sequencing method. Thousands of 5hmC peaks were identified, and a comparison of the distributions of 5hmC among different rice cultivars revealed the specificity and conservation of 5hmC modification. The identified 5hmC peaks were significantly enriched in heterochromatin regions,and mainly located in transposable element (TE) genes, especially around retrotransposons. The correlation analysis of 5hmC and gene expression data revealed a close association between 5hmC and silent TEs. These findings provide a resource for plant DNA 5hmC epigenetic studies and expand our knowledge of 5hmC modification.

Non-referenced genome assembly from epigenomic short-read data

Current computational methods used to analyze changes in DNA methylation and chromatin modification rely on sequenced genomes. Here we describe a pipeline for the detection of these changes from short-read sequence data that does not require a reference genome. Open source software packages were used for sequence assembly, alignment, and measurement of differential enrichment. The method was evaluated by comparing results with reference-based results showing a strong correlation between chromatin modification and gene expression. We then used our de novo sequence assembly to build the DNA methylation profile for the non-referenced Psammomys obesus genome. The pipeline described uses open source software for fast annotation and visualization of unreferenced genomic regions from short-read data.