A transducing bacteriophage λ carrying the structural gene for elongation factor Ts

Friesen, James D.; Parker, Jack; Watson, Robert J.; Bendiak, Dirk S.; Reeh, Solvejg; Pedersen, Steen; Fiil, Niels P.

doi:10.1007/bf00268549

Cited by 27 publications

(11 citation statements)

References 17 publications

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“…A strain which is partially diploid for the 4 min region, however, does not produce altered sigma subunit. Thus, the phenomenon that we observe is different from that of Friesen et al (31). If the phenomenon we observe is due to a structural gene for sigma protein and if a structural gene for sigma exists at 4 min on the E. coli map, then that structural gene is either deleted from our F' plasmid or is not expressed in Salmonella.…”

Section: Resultscontrasting

confidence: 96%

“…This treatment did not modify the electrophoretic mobility of the labeled sigma protein. Friesen et al (31) have reported that a transducing phage carrying the gene for E. coli protein synthesis elongation factor Ts (4 min on the E. coli map) also codes for the synthesis of a sigma-like protein. Thus, it is possible that the E. coli carries more than one structural gene for sigma protein.…”

Section: Resultsmentioning

confidence: 99%

“…One such phage should cause synthesis of sigma subunit in cells pretreated with ultraviolet light (31,32) or in transcription-translation system in vitro (32).…”

Section: Resultsmentioning

confidence: 99%

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A gene from Escherichia coli affecting the sigma subunit of RNA polymerase.

Harris¹,

Martinez²,

Calendar³

1977

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

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The RNA polymerase sigma subunits of Escherichia coli K, E. coli C, and Salmonella typhimurium can be resolved by electrophoresis. Using this technique, we have analyzed Salmonella strains carrying F' plasmids from E. coli K in order to map the gene for the sigma factor. Partial diploid analyses show the location of the sigma gene at 62-66 min on the E. coli genetic map. This gene is cotransducible with toWC and dnaG, at 66 min. The properties of homologous components of the transcription and translation apparatuses often vary between strains of enteric bacteria. Such natural differences provide a convenient tool for genetic mapping of these components when mutants are not available. In order to make use of such differences, investigators individually transfer small regions of the chromosome from one bacterial strain to another strain. The recipients are then screened for the desired phenotype, by using an appropriate assay. With this technique, it has been possible to map some of the genes coding for ribosomal proteins (1) as well as the gene coding for transcription termination factor rho (2). Until now, however, no genetic mapping has been reported for the RNA polymerase (RNA nucleotidyltransferase, nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) sigma subunit, which determines the selectivity of transcription in vitro (3). We have observed electrophoretic differences between the RNA polymerase sigma subunits of Escherichia coli strain K-12 (K), E. coli strain C, and Salmonella typhimurium, and have used these differences to map an E. coli K gene which causes synthesis of the sigma subunit characteristic of E. coli strain K.MATERIALS AND METHODS Chemicals and Buffers. Buffers and DNA-cellulose were prepared as described by Burgess and Jendrisak (4 Bacterial Strains. The bacterial strains used are described in Table 1. New merodiploid strains were constructed by transferring F' plasmids from E. coli K merodiploid strains into various Salmonella typhimurium and E. coli C strains, each containing nutritional mutations such that the presence of the F' plasmid would allow growth on minimal glucose medium. Transfer was accomplished by either of two methods. Method 1: a minimal glucose plate was spread with 0.1 ml (107-108 colony-forming units) of an overnight culture of the donor strain and 0.05 ml of the recipient strain, followed by incubation at 370 for 48 hr (13). Method 2: 0.5 ml samples of each overnight culture were inoculated into flasks containing 10 ml of minimal glucose medium supplemented with the nutritional requirements of each strain. These cultures were shaken at 370 until the optical density of the suspension was doubled. One milliliter of the recipient strain, 2 ml of the donor strain and 3 ml of LB broth were then added to a 250 ml Erlenmeyer flask and shaken very slowly at 370 for 60-90 min. The mating mixtures were centrifuged, concentrated to 10% of the initial volume, and 0.2 ml aliquots were spread on minimal glucose plates, followed by incubation at 370 for 48 hr. C...

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Section: Resultscontrasting

confidence: 96%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

A gene from Escherichia coli affecting the sigma subunit of RNA polymerase.

Harris¹,

Martinez²,

Calendar³

1977

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

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“…4 (5 Mg). Sample 6: prepared as sample 3 except that the same quantity of anti-IF3 antiserum has been incubated with 3.3 ug of IF3 (the incubation was in buffer A without NH4Cl and with 0.2 Mg/ml of NaN3 for 1 hr at room temperature and overnight at 40 in a total volume of 25 Ml) before addition of the 3200 cpm of the low-speed supernatant.…”

Section: Isolation Of X Transducing Bacteriophagesmentioning

confidence: 99%

Specialized transducing phage for the initiation factor 3 gene in Escherichia coli.

Springer¹,

Graffe²,

Hennecke³

1977

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

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A previously isolated thermosensitive mutant [Springer, M., Graffe, M. & Grunberg-Manago, M. (1977) Mol. Gen. Genet. 151,[17][18][19][20][21][22][23][24][25][26] exhibits two defects in vitro, one in the initiation factor IF3 and the other in the L-phenylalanine: tRNAPh' ligase (EC 6.1.1.20). Specialized X transducing phages that transduced this mutant to thermoresistance were selected. In vitro studies showed that the transductants had a normal MF3 activity. One of these transducing phages was shown to code for a protein synthesized under the control of Escherichia coli promoters, which has the same molecular weight as IF3. This protein crossreacts specifically with MF3 antisera and comigrates with pure 1F3 in a two-dimensional gel system. In Escherichia coli genes for the translation apparatus are located at a number of sites which are scattered along the chromosome (1-3). Transducing X bacteriophages have proved to be very useful for unraveling the genetic organization of these sites (4-6). Until recently similar studies on the initiation of translation have been lacking and as a result this process has only been studied biochemically (7). A previous work described a thermosensitive mutant exhibiting two defects in vitro: a thermolabile initiation factor IF3 and a modified L-phenylalanine:tRNAPhe ligase (phenylalanyl-tRNA synthetase, EC 6.1.1.20) (8); these defects were shown to be located at 38 min on the new E. coli map (9). Moreover, stable heterodiploids (mutant chromosome/wild-type episome) were shown to have a thermoresistant phenotype (8). Therefore, thermoresistant transductants could be selected for, using specialized transducing phages that generally form merodiploids when integrated into the E. coli chromosome.The present work describes the isolation of a X bacteriophage that, when integrated in the mutant chromosome, transduces the strain to thermoresistance in vvo and reverses both defects in vitro. This phage is shown to code for a 22,000 molecular weight protein that is under the control of E. coli promoters. This protein is also shown to be specifically recognized by antiserum against IF3, and to comigrate with pure IF3 in a twodimensional gel system. MATERIAL AND METHODSE. coli and bacteriophage X strains are listed in Table 1 (Fig. 1, 1 (Fig. 1, 3) ,4Ci/Amol) per ml was added. After 35-min aeration at 430, the label was isotopically diluted with an equal volume of nonradioactive leucine at 5 mg/ml. The cells were then centrifuged 20 min at 20,000 X g and resuspended in 500 ,ul of the sonication buffer (Tris-HCI, pH 7.5, 50 mM/Mg acetate, 10 mM/ NH4C1, 1.5 M). Two 100-W sonications were performed at 0°A bbreviations: IF, initiation factor; Mr, molecular weight. 3970The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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“…Data for comprehensive studies may be obtained by summing values obtained from individual polypeptides to provide a global picture of cell regulation (e.g., protein amounts, synthesis, and degradation under a variety of growth conditions [12,15,19,20,27], regulatory domains of the rel gene [4,23,26,31] or of cyclic AMP [17,24]). Also being studied are single proteins (7,9,14,17,30,33,34), composition of cell compartments (1, 32), and gene structure and function (6,10,29,33).…”

mentioning

confidence: 99%

Protein identifications of O'Farrell two-dimensional gels: locations of 81 Escherichia coli proteins

Bloch¹,

Phillips²,

Neidhardt³

1980

J Bacteriol

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The two-dimensional gel electrophoresis method of P. H. O'Farrell readily resolves approximately 1,000 proteins from whole-cell homogenates. We have found that the location of most individual proteins is sufficiently reproducible and precise to permit different laboratories to exchange information about them. We present the location of 81 Escherichia coli structural proteins, binding proteins, enzymes, and factors, identified with the aid of purified proteins supplied to us by many investigators.

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A transducing bacteriophage λ carrying the structural gene for elongation factor Ts

Cited by 27 publications

References 17 publications

A gene from Escherichia coli affecting the sigma subunit of RNA polymerase.

A gene from Escherichia coli affecting the sigma subunit of RNA polymerase.

Specialized transducing phage for the initiation factor 3 gene in Escherichia coli.

Protein identifications of O'Farrell two-dimensional gels: locations of 81 Escherichia coli proteins

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