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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 13 December 2011, Vol.108(50), pp.20130-5
    Description: The fixation of atmospheric N(2) by cyanobacteria is a major source of nitrogen in the biosphere. In Nostocales, such as Anabaena, this process is spatially separated from oxygenic photosynthesis and occurs in heterocysts. Upon nitrogen step-down, these specialized cells differentiate from vegetative cells in a process controlled by two major regulators: NtcA and HetR. However, the regulon controlled by these two factors is only partially defined, and several aspects of the differentiation process have remained enigmatic. Using differential RNA-seq, we experimentally define a genome-wide map of 〉10,000 transcriptional start sites (TSS) of Anabaena sp. PCC7120, a model organism for the study of prokaryotic cell differentiation and N(2) fixation. By analyzing the adaptation to nitrogen stress, our global TSS map provides insight into the dynamic changes that modify the transcriptional organization at a critical step of the differentiation process. We identify 〉900 TSS with minimum fold change in response to nitrogen deficiency of eight. From these TSS, at least 209 were under control of HetR, whereas at least 158 other TSS were potentially directly controlled by NtcA. Our analysis of the promoters activated during the switch to N(2) fixation adds hundreds of protein-coding genes and noncoding transcripts to the list of potentially involved factors. These data experimentally define the NtcA regulon and the DIF(+) motif, a palindrome at or close to position -35 that seems essential for heterocyst-specific expression of certain genes.
    Keywords: Transcription Initiation Site ; Anabaena -- Cytology ; Nitrogen -- Pharmacology ; Stress, Physiological -- Drug Effects
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 2011, Vol.108(5), pp.2124-2129
    Description: There has been an increasing interest in cyanobacteria because these photosynthetic organisms convert solar energy into biomass and because of their potential for the production of biofuels. However, the exploitation of cyanobacteria for bioengineering requires knowledge of their transcriptional organization. Using differential RNA sequencing, we have established a genome-wide map of 3,527 transcriptional start sites (TSS) of the model organism Synechocystis sp. PCC6803. One-third of all TSS were located upstream of an annotated gene; another third were on the reverse complementary strand of 866 genes, suggesting massive antisense transcription. Orphan TSS located in intergenic regions led us to predict 314 noncoding RNAs (ncRNAs). Complementary microarray-based RNA profiling verified a high number of noncoding transcripts and identified strong ncRNA regulations. Thus, ~64% of all TSS give rise to antisense or ncRNAs in a genome that is to 87% protein coding. Our data enhance the information on promoters by a factor of 40, suggest the existence of additional small peptide-encoding mRNAs, and provide corrected 5' annotations for many genes of this cyanobacterium. The global TSS map will facilitate the use of Synechocystis sp. PCC6803 as a model organism for further research on photosynthesis and energy research. ; Includes references ; p. 2124-2129.
    Keywords: Transcription (Genetics) -- Physiological Aspects ; Cyanobacteria -- Genetic Aspects ; Genetic Regulation -- Research ; Rna Polymerases -- Properties;
    ISSN: 0027-8424
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  • 3
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 01 February 2011, Vol.108(5), pp.2124-9
    Description: There has been an increasing interest in cyanobacteria because these photosynthetic organisms convert solar energy into biomass and because of their potential for the production of biofuels. However, the exploitation of cyanobacteria for bioengineering requires knowledge of their transcriptional organization. Using differential RNA sequencing, we have established a genome-wide map of 3,527 transcriptional start sites (TSS) of the model organism Synechocystis sp. PCC6803. One-third of all TSS were located upstream of an annotated gene; another third were on the reverse complementary strand of 866 genes, suggesting massive antisense transcription. Orphan TSS located in intergenic regions led us to predict 314 noncoding RNAs (ncRNAs). Complementary microarray-based RNA profiling verified a high number of noncoding transcripts and identified strong ncRNA regulations. Thus, ∼64% of all TSS give rise to antisense or ncRNAs in a genome that is to 87% protein coding. Our data enhance the information on promoters by a factor of 40, suggest the existence of additional small peptide-encoding mRNAs, and provide corrected 5' annotations for many genes of this cyanobacterium. The global TSS map will facilitate the use of Synechocystis sp. PCC6803 as a model organism for further research on photosynthesis and energy research.
    Keywords: Transcription, Genetic ; Synechocystis -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 4
    In: New Phytologist, July 2009, Vol.183(1), pp.224-236
    Description: •  Dual targeting of proteins to more than one subcellular localization has been found in animals, in fungi and in plants. In the latter, ambiguous N‐terminal targeting signals have been described that result in the protein being located in both mitochondria and plastids. We have developed ambiguous targeting predictor (ATP), a machine‐learning implementation that classifies such ambiguous targeting signals. •  Ambiguous targeting predictor is based on a support vector machine implementation that makes use of 12 different amino acid features. Prediction results were validated using fluorescent protein fusion. •  Both in silico and in vivo evaluations demonstrate that ambiguous targeting predictor is useful for predicting dual targeting to mitochondria and plastids. Proteins that are targeted to both organelles by tandemly arrayed signals (so‐called twin targeting) can be predicted by both ambiguous targeting predictor and a combination of single targeting prediction tools. Comparison of ambiguous targeting predictor with previous experimental approaches, as well as in silico approaches, shows good congruence. •  Based on the prediction results, land plant genomes are expected to encode, on average, 〉 400 proteins that are located in mitochondria and plastids. Ambiguous targeting predictor is helpful for functional genome annotation and can be used as a tool to further our understanding about dual protein targeting and its evolution.
    Keywords: Ambiguous Targeting ; Chloroplast ; Genome Annotation ; Intracellular Sorting ; Mitochondrion
    ISSN: 0028-646X
    E-ISSN: 1469-8137
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  • 5
    Language: English
    In: Microbiology (Reading, England), November 2014, Vol.160(Pt 11), pp.2538-50
    Description: In contrast to Synechococcus elongatus PCC 7942, few data exist on the timing mechanism of the widely used cyanobacterium Synechocystis sp. PCC 6803. The standard kaiAB1C1 operon present in this organism was shown to encode a functional KaiC protein that interacted with KaiA, similar to the S. elongatus PCC 7942 clock. Inactivation of this operon in Synechocystis sp. PCC 6803 resulted in a mutant with a strong growth defect when grown under light-dark cycles, which was even more pronounced when glucose was added to the growth medium. In addition, mutants showed a bleaching phenotype. No effects were detected in mutant cells grown under constant light. Microarray experiments performed with cells grown for 1 day under a light-dark cycle revealed many differentially regulated genes with known functions in the ΔkaiABC mutant in comparison with the WT. We identified the genes encoding the cyanobacterial phytochrome Cph1 and the light-repressed protein LrtA as well as several hypothetical ORFs with a complete inverse behaviour in the light cycle. These transcripts showed a stronger accumulation in the light but a weaker accumulation in the dark in ΔkaiABC cells in comparison with the WT. In general, we found a considerable overlap with microarray data obtained for hik31 and sigE mutants. These genes are known to be important regulators of cell metabolism in the dark. Strikingly, deletion of the ΔkaiABC operon led to a much stronger phenotype under light-dark cycles in Synechocystis sp. PCC 6803 than in Synechococcus sp. PCC 7942.
    Keywords: Gene Deletion ; Multigene Family ; Bacterial Proteins -- Genetics ; Synechocystis -- Growth & Development
    ISSN: 13500872
    E-ISSN: 1465-2080
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  • 6
    In: The ISME Journal, 2014
    Description: Prochlorococcus is a genus of abundant and ecologically important marine cyanobacteria. Here, we present a comprehensive comparison of the structure and composition of the transcriptomes of two Prochlorococcus strains, which, despite their similarities, have adapted their gene pool to specific environmental constraints. We present genome-wide maps of transcriptional start sites (TSS) for both organisms, which are representatives of the two most diverse clades within the two major ecotypes adapted to high- and low-light conditions, respectively. Our data suggest antisense transcription for three-quarters of all genes, which is substantially more than that observed in other bacteria. We discovered hundreds of TSS within genes, most notably within 16 of the 29 prochlorosin genes, in strain MIT9313. A direct comparison revealed very little conservation in the location of TSS and the nature of non-coding transcripts between both strains. We detected extremely short 5' untranslated regions with a median length of only 27 and 29 nt for MED4 and MIT9313, respectively, and for 8% of all protein-coding genes the median distance to the start codon is only 10 nt or even shorter. These findings and the absence of an obvious Shine-Dalgarno motif suggest that leaderless translation and ribosomal protein S1-dependent translation constitute alternative mechanisms for translation initiation in Prochlorococcus. We conclude that genome-wide antisense transcription is a major component of the transcriptional output from these relatively small genomes and that a hitherto unrecognized high degree of complexity and variability of gene expression exists in their transcriptional architecture.
    Keywords: Transcriptome ; Prochlorococcus -- Genetics;
    ISSN: 1751-7362
    E-ISSN: 17517370
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  • 7
    In: International Journal of Cancer, 15 October 2019, Vol.145(8), pp.2292-2303
    Description: This prospective trial aimed to investigate whether tumor‐specific and mutations can be detected and quantified in circulating tumor (ct)DNA in patients with active GIST, and whether detection indicates disease activity. We included 25 patients with active disease and or mutations detected in tissue.... What's new? For Gastrointestinal Stromal Tumors (GIST), early detection of relapsed or progressive disease is mandatory to provide adequate surgical procedures or switch medication in case of drug resistance. However, until now monitoring of GIST patients has been restricted to imaging with technical limits in sensitivity...
    Keywords: Liquid Biopsy ; Circulating Tumor Dna ; Gist ; Cancer Biomarker
    ISSN: 0020-7136
    E-ISSN: 1097-0215
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  • 8
    In: Molecular Systems Biology, 2009, Vol.5(1), pp.n/a-n/a
    Description: Information on the numbers and functions of naturally occurring antisense RNAs (asRNAs) in eubacteria has thus far remained incomplete. Here, we screened the model cyanobacterium sp. PCC 6803 for asRNAs using four different methods. In the final data set, the number of known noncoding RNAs rose from 6 earlier identified to 60 and of asRNAs from 1 to 73 (28 were verified using at least three methods). Among these, there are many asRNAs to housekeeping, regulatory or metabolic genes, as well as to genes encoding electron transport proteins. Transferring cultures to high light, carbon‐limited conditions or darkness influenced the expression levels of several asRNAs, suggesting their functional relevance. Examples include the asRNA to , which accumulates in a light‐dependent manner and may be required for processing the L11 r‐operon and the SyR7 noncoding RNA, which is antisense to the 5′ UTR, possibly modulating murein biosynthesis. Extrapolated to the whole genome, ∼10% of all genes in are influenced by asRNAs. Thus, chromosomally encoded asRNAs may have an important function in eubacterial regulatory networks. In addition to regulatory proteins, bacteria, as well as eukaryotes, possess a significant number of regulatory RNAs. In bacteria, the majority of regulatory RNAs appears to be encoded at genomic locations far away from their target genes and exhibit only partial base complementarity to their mRNA targets. However, a small number of regulatory RNAs is transcribed from the reverse complementary strand of an annotated gene and hence these exhibit full or partial overlaps with their potential targets (‐encoded regulatory RNAs, asRNAs). It was known early on that such asRNAs control phage development and plasmid replication in bacteria (Wagner and Simons, 1994), yet recent work has much more advanced on ‐encoded regulatory RNAs, leaving information on the numbers, functions and systemic relevance of chromosomally encoded asRNAs behind. There are three main technical problems in dealing with antisense transcription in bacteria: (i) the general lack of robust algorithms to predict them; (ii) the high risk of measuring experimental artifacts generated during cDNA synthesis in microarray analyses (Perocchi , 2007); (iii) a low level of transcription reported to occur virtually throughout the entire genome (Selinger , 2000), making it difficult to differentiate asRNAs with a regulatory function from transcriptional noise. Here, we have tried to overcome all three obstacles by (i) rigorously interrogating all predictions made in a computational approach using tiled microarrays. To overcome the problem of unintended second strand synthesis (ii) we labeled RNA samples directly before their hybridization on the microarray and finally (iii) we focused predominantly on very highly expressed asRNAs. A tiling microarray was developed, covering all genes and intergenic regions for which a terminator, and thus a candidate asRNA or ncRNA, was computationally predicted. The arrays were hybridized in quadruplicates with pooled RNA from nine different conditions, to detect also those transcripts, which are only induced under specific conditions. As a positive control, the asRNA IsrR (Duehring , 2006) was detected as one contiguous segment of the array (Figure 1). In the 20 kb genomic region, that also gives rise to the IsrR/ transcript pair, two further asRNAs were detected. The affected genes (as_sll1586 and as_ndhH) code for an unknown protein and NADH dehydrogenase subunit 7, respectively (Figure 1). 432 of 646 transcripts above the expression threshold of +1.0 corresponded to mono‐, di‐, and polycistronic mRNAs, whereas 60 originated from intergenic regions and were considered ncRNAs and 73 at least partially overlap sense transcripts and therefore were designated asRNAs. Earlier mathematical modeling of sRNA‐based gene regulation suggested a particular niche for regulatory RNA in allowing cells to transition quickly yet reliably between distinct states, consistent with the widespread appearance of bacterial sRNAs in stress regulatory networks (Mehta , 2008). To derive functional and quantitative data in an efficient way, we constructed a second microarray for measuring changes in expression levels of mRNAs together with their cognate asRNAs and derived the expression ratio as a proxy for the possible impact of the putative riboregulator. In detail, we show that transfer of cultures to stress conditions, which are highly relevant for a photosynthetic organism, causes distinct and characteristic changes in this ratio. For six selected asRNA/mRNA pairs and for the SyR7 ncRNA, we confirmed the changes in expression levels further by Northern blot hybridization (Figure 6). The ncRNA SyR7 overlaps with the 5′ UTR of the gene over its full length. The level of SyR7 was more than 20 times higher than that of the mRNA under three different conditions. However, the SyR7/ ratio declined dramatically to ∼1 on a shift to HL (Figure 6A). The enzyme encoded by is required for murein biosynthesis. Therefore, we assume that the translation of is controlled by SyR7 and that under HL synthesis of MurF is required for accelerated cell wall biosynthesis. Similar characteristic changes were also obtained for the other asRNA/mRNA pairs studied in more detail (Figure 6B and C). These selected examples show that a multitude of asRNA functions and mechanisms appear possible. It is well established that asRNAs and their targets can form RNA–RNA duplexes, which are degraded by dsRNA‐specific RNases (Hernandez , 2005; Duehring , 2006; Darfeuille , 2007; Kawano , 2007; Fozo , 2008). Hence antisense transcription is a powerful natural tool in repressing gene expression. There is a growing number of examples which support the idea of bacterial asRNAs serving as novel types of transcriptional terminators such as the 427 nt asRNA RNAβ in (Stork , 2007). Another possible level of regulation is represented by asRNAs, which directly modulate transcriptional activity. There is strong evidence to suggest that divergently located promoters can interfere with each other (Prescott and Proudfoot, 2002), and the length of transcripts generated from the divergently located promoter (Sneppen , 2005) is one important factor for this interaction. Here, we observed ∼180 nt as the average ncRNA length, whereas the lengths of the asRNAs ranged from 65 nt to 700 nt, with many asRNAs longer than 300 nt, lending support to the idea that some of them may have a function in transcriptional interference. An example of the transcriptional interference mechanism is an 1000 nt long asRNA involved in the sulfur‐dependent expression of the operon in (Andre , 2008). Extrapolated to the whole genome, we estimated the total number of chromosomally encoded asRNAs in to be at least 300. Chromosomally encoded ‐asRNAs are much more frequent than originally thought and seem to outnumber intergenic ncRNAs. Antisense RNAs may affect 8–10% of all genes in , a number that lies within the range of asRNAs in eukaryotic genomes. It is very likely that chromosomally encoded asRNAs constitute an important component of another, not yet fully appreciated, level of gene regulation in bacteria. The presence of specific antisense transcripts (asRNAs) was scrutinized in Synechocystis sp. PCC 6803 by two different types of microarrays, a biocomputational prediction, Northern hybridizations and 5′ RACE experiments. Our results raise the number of strongly expressed asRNAs known from this model cyanobacterium from one previously described to 73 and of non‐coding RNAs transcribed from free‐standing genes from six to 60. The expression levels of several asRNAs were influenced upon transfer of cultures to high light, carbon limitation or darkness, suggesting their functional relevance. Extrapolated to the whole genome, ∼10% of all genes in Synechocystis are influenced by asRNAs. Thus, chromosomally encoded asRNAs may play a much more important role in eubacterial regulatory networks than previously expected.
    Keywords: Antisense Rna ; Cyanobacteria ; Microarray ; Noncoding Rna ; Synechocystis
    ISSN: 1744-4292
    E-ISSN: 1744-4292
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  • 9
    In: Environmental Microbiology, July 2019, Vol.21(7), pp.2544-2558
    Description: Nitrogen is frequently limiting microbial growth in the environment. As a response, many filamentous cyanobacteria differentiate heterocysts, cells devoted to N fixation. Heterocyst differentiation is under the control of the master regulator HetR. Through the characterization of the HetR‐dependent transcriptome in sp. PCC 7120, we identified the new candidate genes likely involved in heterocyst differentiation. According to their maximum induction, we defined E‐DIF (early in differentiation) and L‐DIF (late in differentiation) genes. Most of the genes known to be involved in the critical aspects of heterocyst differentiation or function were also classified into these groups, showing the validity of the approach. Using fusions to , we verified the heterocyst‐specific transcription of several of the found genes, antisense transcripts and potentially trans‐acting sRNAs. Through comparative sequence analysis of promoter regions, we noticed the prevalence of the previously described DIF1 motif and identified a second motif, called DIF2, in other promoters of the E‐DIF cluster. Both motifs are widely conserved in heterocystous cyanobacteria. We assigned as a third member, besides and , to the CnfR regulon. The elements identified here are of interest for understanding cell differentiation, engineering of biological nitrogen fixation or production of O‐sensitive molecules in cyanobacteria.
    Keywords: Transcription (Genetics) – Analysis ; Genetic Research – Analysis ; Cell Differentiation – Analysis ; Nitrogen Fixation – Analysis;
    ISSN: 1462-2912
    E-ISSN: 1462-2920
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  • 10
    Language: English
    In: Cell Reports, 01 November 2019, Vol.29(6), pp.1645-1659.e9
    Description: Molecular chaperones such as heat-shock proteins (HSPs) help in protein folding. Their function in the cytosol has been well studied. Notably, chaperones are also present in the nucleus, a compartment where proteins enter after completing de novo folding...
    Keywords: Biology
    ISSN: 2211-1247
    E-ISSN: 2211-1247
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