DNA-dependent in vitro synthesis of enzymes of the galactose operon of Escherichia coli

Wetekam, Waldemar; Staack, Karin; Ehring, Ruth

doi:10.1007/bf00266928

Cited by 57 publications

(34 citation statements)

References 27 publications

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“…The "ribosomal wash" fluid was treated with ammonium sulfate at 0.7 saturation, the precipitated protein was dissolved in 4 ml of disruption buffer, and dialyzed (crude initiation factor fraction). The templates for gene expression were prepared as follows: Xdgal (36) DNA was prepared from purified phage as described (37). Xplac DNA carrying the UV5 promotor (38) was isolated accordingly (experiment shown in Table 3).…”

Section: Nf Action In Vitromentioning

confidence: 99%

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Nitrofurans, a group of synthetic antibiotics, with a new mode of action: discrimination of specific messenger RNA classes.

Herrlich¹,

Schweiger²

1976

Proc. Natl. Acad. Sci. U.S.A.

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Nitrofurans, a class of antibacterial drugs in, extensive use, interferes with gene expression in a highly specific manner. While in the low dose range (0.5-25 ;&g/ml), 5-nitro-2-furfurylidene-l-aminohydantoin has no effect on transcription, it inhibits specifically the expression of one class of genes in translation. The specific inhibition concerns the inducible genes. The inhibition of messenger RNA expression occurs at the initiation step. The action of nitrofurans, thus, indicates heterogenei in e population of mRNA molecules and in the translational machinery, and suggests the possibility of selective translational control.

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Section: Nf Action In Vitromentioning

confidence: 99%

“…For each system, the optimal Mg2+ concentration was determined and used in the experiments shown here. Enzyme assays were performed as described (28,37). Yields of enzymes synthesized in vitro were as described previously (21,28).…”

Section: Nf Action In Vitromentioning

confidence: 99%

Nitrofurans, a group of synthetic antibiotics, with a new mode of action: discrimination of specific messenger RNA classes.

Herrlich¹,

Schweiger²

1976

Proc. Natl. Acad. Sci. U.S.A.

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“…DNA-directed protein synthesis in E. coli cell-free extract The method of Zubdy et al [12] was used with modifications [22,231. Reaction mixtures were preincubated for 3 min at 37 "C before adding cell-free extract.…”

Section: Preparation Of R Meliloti Cellqree Extractmentioning

confidence: 99%

A cell‐free system from Rhizobium meliloti to study the specific expression of nodulation genes

Dusha

Schröder

Putnoky

et al. 1986

European Journal of Biochemistry

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An in vitro transcription-translation system was developed using cell-free extracts from the symbiotic nitrogenfixing bacterium Rhizobium meliloti strain 41. Conditions for preparation of the 30000 x g supernatant extract and for measurement of protein-synthesizing activity were determined and compared to the activity of an Eschericha coli cell-free system. Genes expressed in the free-living or in the symbiotic state were studied. The product of a recA-like gene (41-kDa protein) was synthesized both in R. meliloti and E. coli extracts, although less efficiently in the heterologous system. In agreement with earlier results obtained in E. coli minicells, three proteins (44, 28.5 and 23 kDa) were synthesized from a cloned 3.3 x 103-base DNA region carrying genes for nodulation (nod). However, differences in the transcription-translation of nod and host specificity (hsn) genes were observed when protein expression was compared in R. meliloti and E. coli cell-free extracts, and the possible explanations of these findings are discussed.Rhizobium meliloti, a member of the family Rhizobiaceae, is able to establish a symbiotic relationship with leguminous plants. As a result of the interaction of plant and bacterial genes, root nodules are formed which are able to fix atmospheric nitrogen.Bacterial genes required for nodule formation have been identified in R . meliloti. The nodulation genes, described so far, are located on a large plasmid and are closely linked to nif genes [I -51. Nodulation genes are arranged into two clusters: early nodulation functions, which are conserved in many Rhizobium species, form one cluster ('common' nod genes) [4,6, 71 were reported. To explain the lack or decreased level of protein synthesis from some heterologous DNA templates in the E. coli system, differences in sequence signals involved in transcription and translation processes were invoked. Recently we published the observation that SmR of transposon Tn5 is expressed in vivo in R. meliloti although it is not detectable in E. coli [20]. This finding also supports the idea that differences either on the transcriptional or translational level or both between the two species may exist. To circumvent these difficulties our aim was to establish an in vitro proteinsynthesizing system from R. meliloti, which would enable us to study specific expression of Rhizobium genes. MATERIALS AND METHODS Preparation of R. meliloti cellqree extractStrain AK631, a prototrophic, symbiotically effective derivative of R. meliloti 41 with compact colony morphology, was used for preparation of the 30000 x g supernatant (S-30) extract. Cells were grown with shaking at 170 rpm at 32°C in TY medium [21] to A600 = 0.7-1.0. Cultures were cooled rapidly in an ice/water bath and cells were collected by centrifugation at 6000 rpm for 20 min at 4°C. The bacterial pellet was washed twice with buffer A containing 20 mM Trislacetate (pH 8.2), 10 mM magnesium acetate, 40 mM potassium acetate, 1 mM dithiothreitol. Bacteria were stored at -20°C until used. For the prepara...

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“…Until now the following methods have been used for detection of specific gene products: (i) Identification of individual bands in electrophoretic patterns (see [4]); (ii) Immunoprecipitation with monospecific antibodies [5][6][7]; (iii) Assay for a specific function like enzyme activity [1,6,8] or toxicity in the case of bacteriocins [9,10]. Method (i) gives no information about the functional activity of the protein.…”

Section: Introductionmentioning

confidence: 99%

Detection by affinity methods of a plasmid‐coded enzyme (guanine phosphoribosyltransferase) in a coupled transcription/translation system from escherichia coli

et al. 1980

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DNA-dependent in vitro synthesis of enzymes of the galactose operon of Escherichia coli

Cited by 57 publications

References 27 publications

Nitrofurans, a group of synthetic antibiotics, with a new mode of action: discrimination of specific messenger RNA classes.

Nitrofurans, a group of synthetic antibiotics, with a new mode of action: discrimination of specific messenger RNA classes.

A cell‐free system from Rhizobium meliloti to study the specific expression of nodulation genes

Detection by affinity methods of a plasmid‐coded enzyme (guanine phosphoribosyltransferase) in a coupled transcription/translation system from escherichia coli

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