An improved positive selection plasmid vector constructed by ollgonucleotide mediated mutagenesis

Nilsson, Björn; Uhlén, Mathias; Josephson, Staffan; Gatenbeck, Sten; Philipson, Lennart

doi:10.1093/nar/11.22.8019

Cited by 158 publications

(68 citation statements)

References 22 publications

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“…Chromosomal DNA for the construction of the genomic library was obtained from Thermotoga maritima MSB8 (DSM 3109), as described before [9]. The genomic library was constructed in vector pUN121 [10] and introduced into Escherichia coli JM83 [11]. Recombinant DNA techniques and expression of the recombinant gene were also performed in E. coli JM83.…”

Section: Methodsmentioning

confidence: 99%

Thermotoga maritima maltosyltransferase, a novel type of maltodextrin glycosyltransferase acting on starch and malto‐oligosaccharides

Meissner¹,

Liebl²

1998

European Journal of Biochemistry

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A novel enzyme acting on starch and malto-oligosaccharides was identified and characterised. The non-hydrolytic enzyme, designated maltosyltransferase (MTase), of the hyperthermophilic bacterium Thermotoga maritima MSB8 disproportionates malto-oligosaccharides via glycosyl transfer reactions. The enzyme has a unique transfer specificity strictly confined to the transfer of maltosyl units. Incubation of MTase with starch or its constituents, i.e. amylose and amylopectin, led to the formation of a set of multiples of maltose (i.e. maltose, maltotetraose, maltohexaose etc.). Malto-oligosaccharides with a degree of polymerization (DP) X were disproportionated to products with a DP of X Ϯ2n (with Xу 3 and n ϭ 0,1, 2, ...). Maximum activity in a 10-min assay was recorded at pH 6.5 and 85Ϫ90°C. The enzyme displayed extraordinary resistance to thermal inactivation. For example, at 90, 85, and 70°C (pH 6.5, 0.34 mg ml Ϫ1 protein), MTase half-lives of about 2.5 h, 17 h, and 21 days, respectively, were recorded.The gene for MTase, designated mmtA, was isolated from a gene library of T. maritima strain MSB8. Analysis of the MTase primary structure as deduced from the nucleotide sequence of mmtA revealed that the enzyme is not closely related to known protein sequences. However, low-level local similarity between MTase and the A-amylase enzyme family (glycosyl hydrolase family 13) was detected, including conserved acidic residues essential for catalysis. Therefore, MTase should be assigned to this family. Based on detailed sequence analyses and comparison with amylolytic enzymes of known crystal structure we propose that MTase contains a (β/A) 8 -fold as the core supersecondary structure which is typical for the Aamylase family. On the other hand, MTase is unique in that it lacks several residues highly conserved throughout this family. Also, MTase possesses an extraordinarily large domain B (a domain typical for the A-amylase family, inserted between β-strand 3 and A-helix 3 of the (β/A) 8 -barrel fold).Keywords : glycosyl transfer; mmtA gene; nucleotide sequence; hyperthermophilic bacterium; thermostability.Enzymes from organisms inhabiting extremely hot environments represent fascinating objects for basic as well as applied research. On the one hand, the industry is interested in thermostable biocatalysts for biotechnological processes demanding elevated temperatures. On the other hand, considerable efforts have been made over the last years to understand how the proteins of extreme thermophiles maintain their structure and function at high temperatures (for a review, see [1]). We have focused our interests on the enzymes involved in carbohydrate breakdown and utilisation of the hyperthermophilic bacterium, Thermotoga maritima. This organism, which represents one of the deepest branches in the phylogenetic tree of the bacterial domain [2], is characterised by a growth range between 55°C and 90°C with an optimum at 80°C [3], making it one of the most thermophilic bacteria known to date. T. maritima MSB8 (DSM 3109), the typ...

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Section: Methodsmentioning

confidence: 99%

Thermotoga maritima maltosyltransferase, a novel type of maltodextrin glycosyltransferase acting on starch and malto‐oligosaccharides

Meissner¹,

Liebl²

1998

European Journal of Biochemistry

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“…Nuclei were isolated as described elsewhere (Brown et al, 1984) and mn-on transcription was carried out as previously described (Matrisian et al, 1985a). Filter immobilized cDNAs (2 jig) for rat stromelysin (Breathnach et al, 1987), a rat actin (Matrisian et al, 1985b), rat lactate dehydrogenase (Matrisian et al, 1985b) and plasmid UN121 (Nilsson et al, 1983) (Matrisian et al, 1986b) was changed to a PstI site by using a specific oligonucleotide linker. A PstI site was created at position +8 bp of the stromelysin gene by oligonucleotide directed site specific mutagenesis using methods described previously (Sanchez-Lopez et al, 1988).…”

Section: Methodsmentioning

confidence: 99%

Negative regulation of the rat stromelysin gene promoter by retinoic acid is mediated by an AP1 binding site.

et al. 1990

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Stromelysin is a member of the metalloproteinase family which plays an important role in extracellular matrix remodelling during many normal and disease processes. We show here that in polyomavirus‐transformed rat embryo fibroblast cells (PyT21), the transcription from the stromelysin gene is repressed by the vitamin A derivative retinoic acid (RA). Furthermore, expression vectors encoding the human RA receptors hRAR‐alpha, hRAR‐beta and hRAR‐gamma repress chloramphenicol acetyltransferase (CAT) expression from stromelysin promoter‐CAT gene expression vectors in RA‐treated PyT21 and human HeLa cells, as determined by transient transfection assays. Through mutation and deletion analysis, we show that the RA dependent repression is mediated by a 25 bp region from nucleotide positions ‐72 to ‐48 of the rat stromelysin 5′‐flanking DNA sequence. Further mutation analysis of this region indicates that the DNA sequence required for RA dependent repression colocalizes with an AP1 binding site which is essential for promoter activity. We show also that RA represses the transcriptional activity of a reporter gene containing a TPA responding AP1 binding site driving the HSV tk promoter. Thus the RAR‐RA complex appears to repress transcription of the stromelysin gene by blocking activation by positive regulatory factors. However, we found no evidence supporting the possibility that the RA dependent repression could be due to RAR binding to the AP1 binding site or to the AP1 components c‐fos and c‐jun.

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“…Chromosomal DNA library construction A library of plasmids containing E. coli chromosomal DNA inserts was constructed by adding E. coli MG1655 chromosomal DNA partially digested with Sau 3A to Bcl I-digested pNU121 (Nilsson et al, 1983). DNA inserted at this Bcl I site disrupts the cI gene and allows expression of the tetracycline resistance gene transcribed from the cI-regulated Pr promoter.…”

Section: Dna Preparations and Manipulationsmentioning

confidence: 99%

Destabilized inheritance of pSC101 and other Escherichia coli plasmids by DpiA, a novel two‐component system regulator

Ingmer

Miller

Cohen

1998

Molecular Microbiology

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SummaryWe identified a gene (dpiA , destabilizer of plasmid inheritance) which, when overexpressed in Escherichia coli, destabilizes the inheritance of pSC101 and other iteron-containing plasmids as disparate as mini-F and RK6 but not the inheritance of P1, RSF1010 and ColD. These effects of DpiA, which functions like an effector protein for a previously undescribed two-component signal transduction system, were reduced by mutations known to promote pSC101 replication and partitioning. dpiB, a gene encoding the putative histidine kinase of this two-component system, is located immediately 5Ј to dpiA and adjacent to a DpiA-induced target promoter that transcribes genes having homology to citrate lyase operon genes, citC, citD and citE, of Klebsiella pneumoniae. Disruption of dpiB reversed or reduced the effect of DpiA overproduction on pSC101 inheritance. A second DpiA target, the promoter for a gene (appY ) implicated in E. coli's response to anaerobiosis, is repressed by DpiA. A mutation in dpiA at a site commonly conserved and phosphor ylated in two-component system effector proteins abolished the effects of DpiA overproduction on pSC101 inheritance and negative regulation of appY expression. Our findings suggest a possible mechanism by which environmental and/or cellular stimuli may influence plasmid inheritance.

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An improved positive selection plasmid vector constructed by ollgonucleotide mediated mutagenesis

Cited by 158 publications

References 22 publications

Thermotoga maritima maltosyltransferase, a novel type of maltodextrin glycosyltransferase acting on starch and malto‐oligosaccharides

Thermotoga maritima maltosyltransferase, a novel type of maltodextrin glycosyltransferase acting on starch and malto‐oligosaccharides

Negative regulation of the rat stromelysin gene promoter by retinoic acid is mediated by an AP1 binding site.

Destabilized inheritance of pSC101 and other Escherichia coli plasmids by DpiA, a novel two‐component system regulator

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