Constructing Recombinant <scp>DNA</scp> Molecules by PCR

Elion, Elaine A.; Marina, Pablo; Lu, Yu

doi:10.1002/0471142727.mb0317s78

Cited by 23 publications

(26 citation statements)

References 15 publications

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“…DNA manipulations were performed using standard techniques (4,14). Restriction endonucleases were purchased from Fermentas.…”

Section: Methodsmentioning

confidence: 99%

“…Acs was radiolabeled using Pat protein acetyltransferase (37) and [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]acetylCoA in a final volume of 1 ml. Excess [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]acetyl-CoA was removed by buffer exchange; NAD ϩ was added to the Acs Ac reaction mixture to a final concentration of 1 mM, and deacetylation reactions were initiated by the addition of CobB L or CobB S . Samples were periodically removed from the reaction mixture and quenched with SDS-PAGE loading buffer.…”

Section: Methodsmentioning

confidence: 99%

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Biologically Active Isoforms of CobB Sirtuin Deacetylase in Salmonella enterica and Erwinia amylovora

Tucker

Escalante‐Semerena

2010

J Bacteriol

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Sirtuins are NAD؉ -dependent protein deacylases that are conserved in all domains of life and are involved in diverse cellular processes, including control of gene expression and central metabolism. Eukaryotic sirtuins have N-terminal extensions that have been linked to protein multimerization and cellular localization. Here the first evidence of sirtuin isoforms in bacteria is reported. The enterobacterium Salmonella enterica synthesizes two isoforms of CobB sirtuin, a shorter 236-amino-acid isoform (here CobB S ) and a longer 273-amino-acid isoform (here CobB L ). The N-terminal 37-amino-acid extension of CobB L is amphipathic, containing 18 basic amino acids (12 of which are Arg) and 13 hydrophobic ones; both isoforms were active in vivo and in vitro. Northern blot and transcription start site analyses revealed that cobB is primarily expressed as two monocistronic cobB mRNAs from two transcription start sites, one of which was mapped within the neighboring ycfX gene and the other of which was located within cobB. Additionally, a low-abundance ycfX-cobB bicistronic mRNA was observed which could encode up to three proteins (YcfX, CobB L , and CobB S ). CobB L isoforms are common within the family Enterobacteriaceae, but species of the genus Erwinia (including the plant pathogen Erwinia amylovora) encode only the CobB L isoform. The CobB L isoform from E. amylovora restored growth of as S. enterica cobB mutant strain on low acetate.

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“…DNA manipulations were performed using standard techniques (4,14). Restriction endonucleases were purchased from Fermentas.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Biologically Active Isoforms of CobB Sirtuin Deacetylase in Salmonella enterica and Erwinia amylovora

Tucker

Escalante‐Semerena

2010

J Bacteriol

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“…To produce larger sequences that will encode for repetitive polypeptides of high Mw, these chemically synthesised short DNA sequences must be assembled. There are different methods to gather the small sequences such as concatenation of oligonucleotides [54], recursive directional ligation (RDL) [55], and mutagenesis or amplification of existing gene segments using polymerase chain reaction (PCR) [56]. However, it is not within the scope of this review to discuss in detail all the methods, please check [54][55][56] for further details.…”

Section: Recombinant Dna Expression Of Proteinsmentioning

confidence: 99%

Peptide-Based Polymer Therapeutics

2014

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Abstract:Polypeptides are envisaged to achieve a major impact on a number of different relevant areas such as biomedicine and biotechnology. Acquired knowledge and the increasing interest on amino acids, peptides and proteins is establishing a large panel of these biopolymers whose physical, chemical and biological properties are ruled by their controlled sequences and composition. Polymer therapeutics has helped to establish these polypeptide-based constructs as polymeric nanomedicines for different applications, such as disease treatment and diagnostics. Herein, we provide an overview of the advantages of these systems and the main methodologies for their synthesis, highlighting the different polypeptide architectures and the current research towards clinical applications.

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“…L-ARABINOSE METABOLISM IN C. GLUTAMICUM 3421 (10). E. coli JM109 transformants were selected on the basis of their chloramphenicol resistance.…”

Section: Vol 75 2009mentioning

confidence: 99%

Identification and Functional Analysis of the Gene Cluster for l -Arabinose Utilization in Corynebacterium glutamicum

Kawaguchi

Sasaki

Vertès

et al. 2009

Appl Environ Microbiol

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Corynebacterium glutamicum ATCC 31831 grew on L-arabinose as the sole carbon source at a specific growth rate that was twice that on D-glucose. The gene cluster responsible for L-arabinose utilization comprised a six-cistron transcriptional unit with a total length of 7.8 kb. Three L-arabinose-catabolizing genes, araA (encoding L-arabinose isomerase), araB (L-ribulokinase), and araD (L-ribulose-5-phosphate 4-epimerase), comprised the araBDA operon, upstream of which three other genes, araR (LacI-type transcriptional regulator), araE (L-arabinose transporter), and galM (putative aldose 1-epimerase), were present in the opposite direction. Inactivation of the araA, araB, or araD gene eliminated growth on L-arabinose, and each of the gene products was functionally homologous to its Escherichia coli counterpart. Moreover, compared to the wild-type strain, an araE disruptant exhibited a >80% decrease in the growth rate at a lower concentration of Larabinose (3.6 g liter Corynebacterium glutamicum is a gram-positive soil microorganism able to secrete large amounts of glutamic acid under suitable conditions (32, 57). Consequently, this organism been used for a long time for industrial production of amino acids and nucleotides, among other primary metabolites (17). Moreover, its potential as a commodity chemical producer is the focus of increasing research efforts (21, 39). Commonly, C. glutamicum is able to grow on various carbon sources, such as hexoses, sugar alcohols, and organic acids (9,29,30). In a future where pentose sugar-rich lignocellulosics should play an important role in global renewable energy, growth of C. glutamicum on pentose sugar-based media is vital to its continued relevance as a producer of commodity chemicals. This has not been possible owing to the lack of essential pentose sugar assimilation pathways in C. glutamicum (4). This contrasts with the characteristics of two major industrial workhorse bacteria, the gram-negative organism Escherichia coli and the grampositive organism Bacillus subtilis, which can grow on L-arabinose as the sole carbon source using the same metabolic pathway. L-Arabinose uptake by E. coli and B. subtilis occurs via two distinct systems involving both a low-affinity H ϩ symporter (encoded by araE) and a high-affinity ATP-dependent transport system (araFGH) (5,18,45). After entering the cell, L-arabinose is sequentially converted to L-ribulose, L-ribulose 5-phosphate, and D-xylulose 5-phosphate by the action of Larabinose isomerase (encoded by araA), L-ribulokinase (araB), and L-ribulose-5-phosphate 4-epimerase (araD), respectively (Fig. 1). D-Xylulose 5-phosphate is eventually metabolized through the reductive pentose phosphate pathway (12). No similar genes for L-arabinose metabolism have been identified in any of the corynebacterial genomes sequenced so far (3,24,38,52,60). Nevertheless, L-arabinose metabolism by C. glutamicum was previously achieved by expression of E. coli araA, araB, and araD genes (26). Although the resulting transformant was able to grow on L-ara...

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Constructing Recombinant DNA Molecules by PCR

Cited by 23 publications

References 15 publications

Biologically Active Isoforms of CobB Sirtuin Deacetylase in Salmonella enterica and Erwinia amylovora

Biologically Active Isoforms of CobB Sirtuin Deacetylase in Salmonella enterica and Erwinia amylovora

Peptide-Based Polymer Therapeutics

Identification and Functional Analysis of the Gene Cluster for l -Arabinose Utilization in Corynebacterium glutamicum

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