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      Transcription Profiling of the Model Cyanobacterium Synechococcus sp. Strain PCC 7002 by Next-Gen (SOLiD™) Sequencing of cDNA

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          Abstract

          The genome of the unicellular, euryhaline cyanobacterium Synechococcus sp. PCC 7002 encodes about 3200 proteins. Transcripts were detected for nearly all annotated open reading frames by a global transcriptomic analysis by Next-Generation (SOLiD™) sequencing of cDNA. In the cDNA samples sequenced, ∼90% of the mapped sequences were derived from the 16S and 23S ribosomal RNAs and ∼10% of the sequences were derived from mRNAs. In cells grown photoautotrophically under standard conditions [38°C, 1% (v/v) CO 2 in air, 250 μmol photons m −2 s −1], the highest transcript levels (up to 2% of the total mRNA for the most abundantly transcribed genes; e.g., cpcAB, psbA, psaA) were generally derived from genes encoding structural components of the photosynthetic apparatus. High-light exposure for 1 h caused changes in transcript levels for genes encoding proteins of the photosynthetic apparatus, Type-1 NADH dehydrogenase complex and ATP synthase, whereas dark incubation for 1 h resulted in a global decrease in transcript levels for photosynthesis-related genes and an increase in transcript levels for genes involved in carbohydrate degradation. Transcript levels for pyruvate kinase and the pyruvate dehydrogenase complex decreased sharply in cells incubated in the dark. Under dark anoxic (fermentative) conditions, transcript changes indicated a global decrease in transcripts for respiratory proteins and suggested that cells employ an alternative phosphoenolpyruvate degradation pathway via phosphoenolpyruvate synthase ( ppsA) and the pyruvate:ferredoxin oxidoreductase ( nifJ). Finally, the data suggested that an apparent operon involved in tetrapyrrole biosynthesis and fatty acid desaturation, acsF2–ho2–hemN2–desF, may be regulated by oxygen concentration.

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          Reliability and reproducibility issues in DNA microarray measurements.

          DNA microarrays enable researchers to monitor the expression of thousands of genes simultaneously. However, the current technology has several limitations. Here we discuss problems related to the sensitivity, accuracy, specificity and reproducibility of microarray results. The existing data suggest that for relatively abundant transcripts the existence and direction (but not the magnitude) of expression changes can be reliably detected. However, accurate measurements of absolute expression levels and the reliable detection of low abundance genes are difficult to achieve. The main problems seem to be the sub-optimal design or choice of probes and some incorrect probe annotations. Well-designed data-analysis approaches can rectify some of these problems.
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            Protein-based organelles in bacteria: carboxysomes and related microcompartments.

            Many bacteria contain intracellular microcompartments with outer shells that are composed of thousands of protein subunits and interiors that are filled with functionally related enzymes. These microcompartments serve as organelles by sequestering specific metabolic pathways in bacterial cells. The carboxysome, a prototypical bacterial microcompartment that is found in cyanobacteria and some chemoautotrophs, encapsulates ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase, and thereby enhances carbon fixation by elevating the levels of CO2 in the vicinity of RuBisCO. Evolutionarily related, but functionally distinct, microcompartments are present in diverse bacteria. Although bacterial microcompartments were first observed more than 40 years ago, a detailed understanding of how they function is only now beginning to emerge.
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              Microarray and cDNA sequence analysis of transcription during nerve-dependent limb regeneration

              Background Microarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. Innervated (NR) and denervated (DL) forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration. Results Considerable similarity was observed between NR and DL transcriptional programs at 5 and 14 days post amputation (dpa). Genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. Thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. The majority of the transcriptional differences between NR and DL limbs were correlated with blastema formation; cell numbers increased in NR limbs after 5 dpa and this yielded distinct transcriptional signatures of cell proliferation in NR limbs at 14 dpa. These transcriptional signatures were not observed in DL limbs. Instead, gene expression changes within DL limbs suggest more diverse and protracted wound-healing responses. 454 cDNA sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. Assembly of new 454 cDNA sequences with existing expressed sequence tag (EST) contigs from the Ambystoma EST database more than doubled (3935 to 9411) the number of non-redundant human-A. mexicanum orthologous sequences. Conclusion Many new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.
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                Author and article information

                Journal
                Front Microbiol
                Front. Microbio.
                Frontiers in Microbiology
                Frontiers Research Foundation
                1664-302X
                27 January 2011
                07 March 2011
                2011
                : 2
                : 41
                Affiliations
                [1] 1simpleDepartment of Biochemistry and Molecular Biology, The Pennsylvania State University University Park, PA, USA
                Author notes

                Edited by: Thomas E. Hanson, University of Delaware, USA

                Reviewed by: Martin Hagemann, University Rostock, Germany; Jack Meeks, University of California, USA

                *Correspondence: Donald A. Bryant, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, S-235 Frear Building, University Park, PA 16802, USA. e-mail: dab14@ 123456psu.edu

                This article was submitted to Frontiers in Microbial Physiology and Metabolism, a specialty of Frontiers in Microbiology.

                Article
                10.3389/fmicb.2011.00041
                3133671
                21779275
                415ca84b-1f13-40dc-848c-fd8dbb0926fc
                Copyright © 2011 Ludwig and Bryant.

                This is an open-access article subject to an exclusive license agreement between the authors and Frontiers Media SA, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are credited.

                History
                : 07 January 2011
                : 22 February 2011
                Page count
                Figures: 8, Tables: 4, Equations: 0, References: 81, Pages: 23, Words: 16810
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                transcription profiling,cyanobacteria,synechococcus 7002,fermentation,cdna sequencing

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