The pMTL nic− cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing

Chambers, Stephen P.; Prior, Sue E.; Barstow, David A.; Minton, Nigel P.

doi:10.1016/0378-1119(88)90606-3

Cited by 429 publications

(220 citation statements)

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“…The PCR product was cloned in pMTL25 (Chambers et al, 1988) in the NcoI-Sall sites to form plasmid pM25EC. Plasmid pM25EC was digested and the NcoI-Bglll fragment was cloned in the expression vector pQE-60 (Qiagen GmbH, Dusseldorf, Germany) carrying a 6×His affinity tag at the 3' site of the expression box.…”

Section: Preparation Of Polyclonal Antibodies Against E Coil Branchimentioning

confidence: 99%

Expression of Escherichia coli branching enzyme in tubers of amylose‐free transgenic potato leads to an increased branching degree of the amylopectin

et al. 1996

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SummaryIn order to increase the branching degree of potato tuber starch, the gene encoding branching enzyme (glgB) of Escherichia coil was expressed in the amylose-free potato mutant. The E. coil glgB was cloned in the binary vector pBIN19 under the transcriptional control of the potato Granule Bound Starch Synthase (GBSS) promoter and transitpeptide sequence. The E. coil glgB was cloned behind the two N-terminal amino acids of the GBSS mature protein, creating a chimeric protein. Transgenic plants were obtained which expressed the E. coil branching enzyme as was shown by the presence of mRNA and protein in the tubers. Correctly processed protein was found both in the soluble and starch granule bound protein fraction. Analysis of the starch showed an increase in the branching degree (DE) of up to 25% more branchpoints. The increase in the number of branchpoints was due to the presence of more short chains, with a degree of polymerization (DP) of 16 glucose-residues or less in the amylopectin. Changes in other characteristics of the starch, such as average chain length (CL) and ;~ax, indicated a more branched structure for starch of transformed plants as well.

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Section: Preparation Of Polyclonal Antibodies Against E Coil Branchimentioning

confidence: 99%

Expression of Escherichia coli branching enzyme in tubers of amylose‐free transgenic potato leads to an increased branching degree of the amylopectin

et al. 1996

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“…From this vector (pORI) the gene encoding the replication initiation protein RepA was removed. Therefore, pORI is unable to replicate in any Km , Tc , pKVB2 (Kiel et al 1987) carrying the repA gene of pWV01 Law et al (1995) pUC19E Ap , Em , pUC19 (Yanisch-Perron et al 1985) carrying the Em of pE194 (Horinouchi and Weisblum 1982) in the SmaI restriction site Laboratory collection pTC2 Ap , Tc , pMTL25 (Chambers et al 1988) carrying the Tc of pLS1 (Lacks et al 1986) Buist et al (1995) bacterial strain unless RepA is provided in trans. The construction of integration vectors from pORI plasmids is possible by the availability of Escherichia coli, Bacillus subtilis and¸actococcus lactis helper strains that produce the replication protein from a chromosomally incorporated copy of repA (Leenhouts et al 1991a;Law et al 1995;Leenhouts, unpublished data).…”

Section: Introductionmentioning

confidence: 99%

A general system for generating unlabelled gene replacements in bacterial chromosomes

et al. 1996

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A general system for generating unlabelled gene replacements in bacterial chromosomes Leenhouts, K.; Buist, Girbe; Bolhuis, A.; Berge, A. ten; Kiel, Jan; Mierau, I.; Dabrowska, M.; Venema, G.; Kok, Jan IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document VersionPublisher's PDF, also known as Version of record Publication date: 1996Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Leenhouts, K., Buist, G., Bolhuis, A., Berge, A. T., Kiel, J., Mierau, I., ... Kok, J. (1996). A general system for generating unlabelled gene replacements in bacterial chromosomes. Molecular and General Genetics MGG, 253(1-2), 217-224. DOI: 10.1007/s004380050315 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Abstract A general system is described that facilitates gene replacements such that the recombinant strains are not labelled with antibiotic resistance genes. The method is based on the conditional replication of derivatives of the lactococcal plasmid pWV01, which lacks the repA gene encoding the replication initiation protein. Replacement vectors can be constructed in and isolated from gram-positive and gram-negative helper strains that provide RepA in trans. Cointegrate formation of the integration vectors with the chromosome of the target strain is selected by antibiotic resistance. Resolution of the cointegrate structure is identified in the second step of the procedure by the loss of the lacZ reporter gene present in the delivery vector. The second recombination event results either in gene replacement or in restoration of the original copy of the gene. As no antibiotic resistance marker is present in the genome of the mutant the system can be used to introduce multiple mutations in one strain. A feasibility study was performed using¸actococcus lactis and Bacillus subtilis as model organisms. The results indicate that the method should be applicable to any non-essential gene in numerous bacterial species.

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“…The 1·0 kbp EcoRI fragment from pAZR-1 was ligated into the EcoRI site of pUR19 and designated pAZR-3 and the 1·2 kbp ScaI-HindIII fragment was inserted between the SmaI and HindIII sites within the pUR19 polylinker and termed pAZR-12. In order to construct the final plasmid used in this study, pAZR-17, the 2·0 kbp XhoI-SphI fragment of pAZR-1 was ligated into the XhoI and SphI sites of the bacterial vector pMTL20 (Chambers et al, 1988) and the insert then isolated as a HindIII/SmaI fragment and again cloned into the HindIII and SmaI sites of pUR19. The chromosomal insert DNA contained within these four plasmids (pAZR-1, pAZR-3, pAZR-12 and pAZR-17) is shown diagrammatically in Figure 2A.…”

Section: Media Strains and Growth Conditionsmentioning

confidence: 99%

“…In addition to pAZR-1 the following subclones in pMTL23 (Chambers et al, 1988) were used as templates for nucleotide sequencing: 1·0 kbp EcoRI, 0·5 kbp EcoRI, 0·4 kbp EcoRI, 0·3 kbp EcoRI, 0·45 kbp EcoRI-HindIII, 2·0 kbp EcoRVHindIII, 1·2 kbp ScaI-HindIII. Independent clones were sequenced on both strands using the Taq Dye DeoxyTM Terminator Cycle Sequencing Kit (Applied Biosystems) and samples analysed on an Applied Biosystems Model 373A DNA Sequencing System.…”

Section: Media Strains and Growth Conditionsmentioning

confidence: 99%

“…The 0·35 kb XhoI-EcoRV fragment from pAZR-1 was ligated into the XhoI and SmaI polylinker sites of pMTL23 and termed pSD-1. The 1·0 kbp ScaI/ SphI fragment of pAZR-1 was then cloned into the SmaI and SphI polylinker sites of pMTL22 (Chambers et al, 1988) to create the plasmid pSD-2. The 1·78 kbp HindIII ura4 + gene fragment from vector pREP42 (Basi et al, 1993) was cloned into the HindIII site of pSD-1 (this clone was designated pSD-3).…”

Section: Media Strains and Growth Conditionsmentioning

confidence: 99%

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Isolation of a Schizosaccharomyces pombe Gene Which in High Copy Confers Resistance to the Nucleoside Analogue 5‐Azacytidine

Platt

Price

1997

Yeast

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Treatment of Schizosaccharomyces pombe with the C5 DNA methyltransferase (C5Mtase) inhibitor 5-azacytidine (5-azaC) has previously been shown to induce G2 checkpoint-dependent cell cycle arrest. S. pombe strains defective in both the checkpoint control pathways and in DNA repair processes are sensitive to 5-azaC. Here we describe the isolation of azr1 + , as a multi-copy suppressor of the 5-azaC sensitivity of G2 checkpoint and DNA repair-deficient strains. azr1 + encodes a putative 25 kDa protein with limited homology to a Saccharomyces cerevisiae open reading frame of unknown function. The azr1 + gene is not essential and the null mutant shows no alteration in either DNA repair or checkpoint properties. We also report the sequence of the putative fission yeast cytidine deaminase gene, designated pcd1 + , which lies immediately adjacent to azr1 + but which plays only a moderate role in suppression of 5-azaC sensitivity. These data have been deposited with EMBL nucleotide sequence database, Accession Number X98329.

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The pMTL nic− cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing

Cited by 429 publications

References 21 publications

Expression of Escherichia coli branching enzyme in tubers of amylose‐free transgenic potato leads to an increased branching degree of the amylopectin

Expression of Escherichia coli branching enzyme in tubers of amylose‐free transgenic potato leads to an increased branching degree of the amylopectin

A general system for generating unlabelled gene replacements in bacterial chromosomes

Isolation of a Schizosaccharomyces pombe Gene Which in High Copy Confers Resistance to the Nucleoside Analogue 5‐Azacytidine

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