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  1. Article ; Online: Plastid Gene Transcription: An Update on Promoters and RNA Polymerases.

    Ortelt, Jennifer / Link, Gerhard

    Methods in molecular biology (Clifton, N.J.)

    2021  Volume 2317, Page(s) 49–76

    Abstract: Chloroplasts, the sites of photosynthesis and sources of reducing power, are at the core of the success story that sets apart autotrophic plants from most other living organisms. Along with their fellow organelles (e.g., amylo-, chromo-, etio-, and ... ...

    Abstract Chloroplasts, the sites of photosynthesis and sources of reducing power, are at the core of the success story that sets apart autotrophic plants from most other living organisms. Along with their fellow organelles (e.g., amylo-, chromo-, etio-, and leucoplasts), they form a group of intracellular biosynthetic machines collectively known as plastids. These plant cell constituents have their own genome (plastome), their own (70S) ribosomes, and complete enzymatic equipment covering the full range from DNA replication via transcription and RNA processive modification to translation. Plastid RNA synthesis (gene transcription) involves the collaborative activity of two distinct types of RNA polymerases that differ in their phylogenetic origin as well as their architecture and mode of function. The existence of multiple plastid RNA polymerases is reflected by distinctive sets of regulatory DNA elements and protein factors. This complexity of the plastid transcription apparatus thus provides ample room for regulatory effects at many levels within and beyond transcription. Research in this field offers insight into the various ways in which plastid genes, both singly and groupwise, can be regulated according to the needs of the entire cell. Furthermore, it opens up strategies that allow to alter these processes in order to optimize the expression of desired gene products.
    MeSH term(s) DNA-Binding Proteins/genetics ; DNA-Binding Proteins/metabolism ; DNA-Directed RNA Polymerases/genetics ; DNA-Directed RNA Polymerases/metabolism ; Gene Expression Regulation, Plant ; Plant Proteins/genetics ; Plant Proteins/metabolism ; Plastids/genetics ; Plastids/metabolism ; Promoter Regions, Genetic ; Transcription, Genetic
    Chemical Substances DNA-Binding Proteins ; Plant Proteins ; DNA-Directed RNA Polymerases (EC 2.7.7.6)
    Language English
    Publishing date 2021-05-24
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-0716-1472-3_2
    Database MEDical Literature Analysis and Retrieval System OnLINE

    Full text online

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  2. Article: Simplified Point-of-Care Full SARS-CoV-2 Genome Sequencing Using Nanopore Technology

    Pembaur, Anton / Sallard, Erwan / Weil, Patrick Philipp / Ortelt, Jennifer / Ahmad-Nejad, Parviz / Postberg, Jan

    Microorganisms. 2021 Dec. 16, v. 9, no. 12

    2021  

    Abstract: The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment of a global decentralized surveillance system to recognize local outbreaks and the emergence of novel variants of concern. Among available deep-sequencing technologies, ... ...

    Abstract The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment of a global decentralized surveillance system to recognize local outbreaks and the emergence of novel variants of concern. Among available deep-sequencing technologies, nanopore-sequencing could be an important cornerstone, as it is mobile, scalable, and cost-effective. Therefore, streamlined nanopore-sequencing protocols need to be developed and optimized for SARS-CoV-2 variants identification. We adapted and simplified existing workflows using the ‘midnight’ 1200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost the entire SARS-CoV-2 genome. Subsequently, we applied Oxford Nanopore Rapid Barcoding and the portable MinION Mk1C sequencer combined with the interARTIC bioinformatics pipeline. We tested a simplified and less time-consuming workflow using SARS-CoV-2-positive specimens from clinical routine and identified the CT value as a useful pre-analytical parameter, which may help to decrease sequencing failures rates. Complete pipeline duration was approx. 7 h for one specimen and approx. 11 h for 12 multiplexed barcoded specimens. The adapted protocol contains fewer processing steps and can be completely conducted within one working day. Diagnostic CT values deduced from qPCR standardization experiments can act as principal criteria for specimen selection. As a guideline, SARS-CoV-2 genome copy numbers lower than 4 × 10⁶ were associated with a coverage threshold below 20-fold and incompletely assembled SARS-CoV-2 genomes. Thus, based on the described thermocycler/chemistry combination, we recommend CT values of ~26 or lower to achieve full and high-quality SARS-CoV-2 (+)RNA genome coverage.
    Keywords RNA ; Severe acute respiratory syndrome coronavirus 2 ; barcoding ; bioinformatics ; chemistry ; cost effectiveness ; genome ; guidelines ; high-throughput nucleotide sequencing ; monitoring ; nanopores ; pandemic ; point-of-care systems
    Language English
    Dates of publication 2021-1216
    Publishing place Multidisciplinary Digital Publishing Institute
    Document type Article
    ZDB-ID 2720891-6
    ISSN 2076-2607
    ISSN 2076-2607
    DOI 10.3390/microorganisms9122598
    Database NAL-Catalogue (AGRICOLA)

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  3. Article: Simplified Point-of-Care Full SARS-CoV-2 Genome Sequencing Using Nanopore Technology.

    Pembaur, Anton / Sallard, Erwan / Weil, Patrick Philipp / Ortelt, Jennifer / Ahmad-Nejad, Parviz / Postberg, Jan

    Microorganisms

    2021  Volume 9, Issue 12

    Abstract: The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment of a global decentralized surveillance system to recognize local outbreaks and the emergence of novel variants of concern. Among available deep-sequencing technologies, ... ...

    Abstract The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment of a global decentralized surveillance system to recognize local outbreaks and the emergence of novel variants of concern. Among available deep-sequencing technologies, nanopore-sequencing could be an important cornerstone, as it is mobile, scalable, and cost-effective. Therefore, streamlined nanopore-sequencing protocols need to be developed and optimized for SARS-CoV-2 variants identification. We adapted and simplified existing workflows using the 'midnight' 1200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost the entire SARS-CoV-2 genome. Subsequently, we applied Oxford Nanopore Rapid Barcoding and the portable MinION Mk1C sequencer combined with the interARTIC bioinformatics pipeline. We tested a simplified and less time-consuming workflow using SARS-CoV-2-positive specimens from clinical routine and identified the CT value as a useful pre-analytical parameter, which may help to decrease sequencing failures rates. Complete pipeline duration was approx. 7 h for one specimen and approx. 11 h for 12 multiplexed barcoded specimens. The adapted protocol contains fewer processing steps and can be completely conducted within one working day. Diagnostic CT values deduced from qPCR standardization experiments can act as principal criteria for specimen selection. As a guideline, SARS-CoV-2 genome copy numbers lower than 4 × 10
    Language English
    Publishing date 2021-12-16
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2720891-6
    ISSN 2076-2607
    ISSN 2076-2607
    DOI 10.3390/microorganisms9122598
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Plastid gene transcription: promoters and RNA polymerases.

    Ortelt, Jennifer / Link, Gerhard

    Methods in molecular biology (Clifton, N.J.)

    2014  Volume 1132, Page(s) 47–72

    Abstract: Chloroplasts, the sites of photosynthesis and sources of reducing power, are at the core of the success story that sets apart autotrophic plants from most other living organisms. Along with their fellow organelles (e.g., amylo-, chromo-, etio-, and ... ...

    Abstract Chloroplasts, the sites of photosynthesis and sources of reducing power, are at the core of the success story that sets apart autotrophic plants from most other living organisms. Along with their fellow organelles (e.g., amylo-, chromo-, etio-, and leucoplasts), they form a group of intracellular biosynthetic machines collectively known as plastids. These plant cell constituents have their own genome (plastome), their own (70S) ribosomes, and complete enzymatic equipment covering the full range from DNA replication via transcription and RNA processive modification to translation. Plastid RNA synthesis (gene transcription) involves the collaborative activity of two distinct types of RNA polymerases that differ in their phylogenetic origin as well as their architecture and mode of function. The existence of multiple plastid RNA polymerases is reflected by distinctive sets of regulatory DNA elements and protein factors. This complexity of the plastid transcription apparatus thus provides ample room for regulatory effects at many levels within and beyond transcription. Research in this field offers insight into the various ways in which plastid genes, both singly and groupwise, can be regulated according to the needs of the entire cell. Furthermore, it opens up strategies that allow to alter these processes in order to optimize the expression of desired gene products.
    MeSH term(s) Arabidopsis/genetics ; Arabidopsis Proteins/genetics ; Chloroplast Proteins/genetics ; Chloroplasts/genetics ; DNA, Plant ; DNA-Binding Proteins ; DNA-Directed RNA Polymerases/genetics ; Escherichia coli Proteins/genetics ; Gene Expression Regulation, Plant/genetics ; Genome, Chloroplast ; Mitochondrial Proteins/genetics ; Promoter Regions, Genetic ; Sigma Factor/genetics ; Nicotiana/genetics ; Transcription, Genetic
    Chemical Substances Arabidopsis Proteins ; Chloroplast Proteins ; DNA, Plant ; DNA-Binding Proteins ; Escherichia coli Proteins ; Mitochondrial Proteins ; SIG6 protein, Arabidopsis ; Sig5 protein, Arabidopsis ; Sigma Factor ; rpoB protein, E coli ; DNA-Directed RNA Polymerases (EC 2.7.7.6) ; RNA polymerase alpha subunit (EC 2.7.7.6) ; RpoTmp protein, Arabidopsis (EC 2.7.7.6) ; RpoTp protein, Arabidopsis (EC 2.7.7.6)
    Language English
    Publishing date 2014-03-05
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-62703-995-6_3
    Database MEDical Literature Analysis and Retrieval System OnLINE

    Full text online

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  5. Article ; Online: Simplified point-of-care full SARS-CoV-2 genome sequencing using nanopore technology

    Pembaur, Anton / Sallard, Erwan / Weil, Patrick Philipp / Ortelt, Jennifer / Ahmad-Nejad, Parviz / Postberg, Jan

    medRxiv

    Abstract: Background: The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment a global decentralized surveillance and warning system to recognize local outbreaks and the emergence of novel variants-of-concern. Among the available deep- ... ...

    Abstract Background: The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment a global decentralized surveillance and warning system to recognize local outbreaks and the emergence of novel variants-of-concern. Among the available deep-sequencing technologies, nanopore-sequencing could be an important cornerstone, since it is mobile, scalable and acquisition investments are comparably low. Therefore, streamlined and efficient nanopore-sequencing protocols need to be developed and optimized for SARS-CoV-2 variants identification, in particular for smaller hospital laboratories with lower throughput. Results: We adapted and simplified existing workflows using the midnight 1,200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost all of the SARS-CoV-2 genome. Subsequently, we applied the Oxford Nanopore Rapid barcoding protocol and the portable MinION Mk1C sequencer in combination with the ARTIC bioinformatics pipeline. We tested the simplified and less time-consuming workflow on confirmed SARS-CoV-2-positive specimens from clinical routine and identified pre-analytical parameters, which may help to decrease the rate of sequencing failures. Duration of the complete pipeline was approx. 7 hrs for one specimen and approx. 11 hrs for 12 multiplexed barcoded specimens. Conclusions: The adapted protocol contains less processing steps. Diagnostic CT values are the principal criteria for specimen selection. Subsequent to diagnostic qRT-PCR, multiplex library preparation, quality controls, nanopore sequencing and the bioinformatic pipeline can be completely conducted within one working-day.
    Keywords covid19
    Language English
    Publishing date 2021-07-09
    Publisher Cold Spring Harbor Laboratory Press
    Document type Article ; Online
    DOI 10.1101/2021.07.08.21260171
    Database COVID19

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  6. Article: AtSIG6 and other members of the sigma gene family jointly but differentially determine plastid target gene expression in Arabidopsis thaliana.

    Bock, Sylvia / Ortelt, Jennifer / Link, Gerhard

    Frontiers in plant science

    2014  Volume 5, Page(s) 667

    Abstract: Plants contain a nuclear gene family for plastid sigma factors, i.e., proteins that associate with the "bacterial-type" organellar RNA polymerase and confer the ability for correct promoter binding and transcription initiation. Questions that are still ... ...

    Abstract Plants contain a nuclear gene family for plastid sigma factors, i.e., proteins that associate with the "bacterial-type" organellar RNA polymerase and confer the ability for correct promoter binding and transcription initiation. Questions that are still unresolved relate to the "division of labor" among members of the sigma family, both in terms of their range of target genes and their temporal and spatial activity during development. Clues to the in vivo role of individual sigma genes have mainly come from studies of sigma knockout lines. Despite its obvious strengths, however, this strategy does not necessarily trace-down causal relationships between mutant phenotype and a single sigma gene, if other family members act in a redundant and/or compensatory manner. We made efforts to reduce the complexity by genetic crosses of Arabidopsis single mutants (with focus on a chlorophyll-deficient sig6 line) to generate double knockout lines. The latter typically had a similar visible phenotype as the parental lines, but tended to be more strongly affected in the transcript patterns of both plastid and sigma genes. Because triple mutants were lethal under our growth conditions, we exploited a strategy of transformation of single and double mutants with RNAi constructs that contained sequences from the unconserved sigma region (UCR). These RNAi/knockout lines phenotypically resembled their parental lines, but were even more strongly affected in their plastid transcript patterns. Expression patterns of sigma genes revealed both similarities and differences compared to the parental lines, with transcripts at reduced or unchanged amounts and others that were found to be present in higher (perhaps compensatory) amounts. Together, our results reveal considerable flexibility of gene activity at the levels of both sigma and plastid gene expression. A (still viable) "basal state" seems to be reached, if 2-3 of the 6 Arabidopsis sigma genes are functionally compromised.
    Language English
    Publishing date 2014-11-25
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2711035-7
    ISSN 1664-462X
    ISSN 1664-462X
    DOI 10.3389/fpls.2014.00667
    Database MEDical Literature Analysis and Retrieval System OnLINE

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