✦ LIBER ✦

Cloning and characterization of the prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli

✍ Scribed by Hove-Jensen, Biarne

Publisher: Springer
Year: 1985
Tongue: English
Weight: 865 KB
Volume: 201
Category: Article
ISSN: 0026-8925
DOI: 10.1007/bf00425670

No coin nor oath required. For personal study only.

✦ Synopsis

The gene, prs, encoding phosphoribosylpyrophosphate (PRPP) synthetase of Escherichia coli was isolated from a library of E. coli genes cloned in the bacteriophage 2D69 vector. A strain with a temperature-lethal defect in PRPP synthetase, (prs-2), was used as the host and cloning was performed by lysogenic complementation. The prs gene resided on a 5.6 kilobase-pair (kbp) DNA fragment generated by hydrolysis with restriction endonuclease BamHI. The nearby gene pth, encoding peptidyl-tRNA hydrolase, was also on this fragment. Subcloning of the fragment in the multi-copy plasmid pBR322 and subsequent deletion of parts of the insert resulted in a 1.7 kbp DNA fragment containing the entire prs gene. Bacterial strains harbouring prs-bearing plasmids showed up to 50-fold increased PRPP synthetase activity. The PRPP synthetase subunit was identified by analysis of plasmid-harbouring minicells and the subunit molecular mass established as 33,000 daltons. Analysis, by the minicell procedure, of plasmids with deletions extending into the prs gene established the direction of transcription as counterclockwise. A putative leader sequence of approximately 400 bp preceded the coding sequence. By deletion analysis and by cloning fragments of this leader sequence in a galK expression vector it was found to contain the prs promoter as well as a potential transcription termination site.

📜 SIMILAR VOLUMES

Cloning and orientation of the gene enco

Cloning and orientation of the gene encoding aminopeptidase N in Escherichia coli

✍ Bally, Marc ;Murgier, Maryse ;Lazdunski, Andrée 📂 Article 📅 1984 🏛 Springer 🌐 English ⚖ 450 KB

The pepN gene, that encodes aminopeptidase N in Escherichia coli, has been cloned into the multicopy plasmid pBR322. Expression of the cloned pepN gene results in overproduction of the enzyme. The restriction map of the 6.7 Kb insert was established and the gene was further localized by analysis of

Cloning and characterization of the xyl

Cloning and characterization of the xyl genes from Escherichia coli

✍ Rosenfeld, Stuart A. ;Stevis, Panayiotis E. ;Ho, Nancy W. Y. 📂 Article 📅 1984 🏛 Springer 🌐 English ⚖ 772 KB

Specific xylose utilization mutants of Escherichia coli were isolated that had altered xylose isomerase ( xylA ), xylulokinase ( xylB ), and regulatory ( xylR ) or transport ( xylT ) activities. We screened the Clarke and Carbon E. coli gene bank and one clone, pLC10 -15, was found to complement the

Molecular cloning and characterization o

Molecular cloning and characterization of the alkB gene of Escherichia coli

✍ Kataoka, Hiroko ;Sekiguchi, Mutsuo 📂 Article 📅 1985 🏛 Springer 🌐 English ⚖ 825 KB

Using methods of in vitro recombination we constructed hybrid plasmids that can suppress the increased methylmethane sulfonate sensitivity caused by alkB mutation. Since the cloned DNA fragment was mapped at 47 rain on the Escherichia coli K12 genetic map, an area where the alkB gene is located, we

Cloning of Escherichia coli genes encodi

Cloning of Escherichia coli genes encoding 3-methyladenine DNA glycosylases I and II

✍ Clarke, Neil D. ;Kvaal, Marit ;Seeberg, Erling 📂 Article 📅 1984 🏛 Springer 🌐 English ⚖ 507 KB

We have constructed two recombinant plasmids which harbour functions involved in DNA repair of alkylation damage in Escherichia coli. One plasmid carries the tag+ gene encoding 3-methyladenine DNA glycosylase I while the other carries alkA+ encoding 3-methyladenine DNA glycosylase II. The plasmids w

The cloning of the rorB gene of Escheric

The cloning of the rorB gene of Escherichia coli

✍ Debenham, Paul G. ;Webb, Michael B. T. ;Law, John 📂 Article 📅 1988 🏛 Springer 🌐 English ⚖ 457 KB

A segment of the Escherichia coli genome which complements the ionising radiation sensitivity of the rorB mutation was cloned into pBR322. This DNA segment also complements the mitomycin C sensitivity of the rorB mutation. The gene was subcloned until defined in a fragment of 1.05 kb. Only one gene

Cloning of the tyrocidine synthetase 1 g

Cloning of the tyrocidine synthetase 1 gene from Bacillus brevis and its expression in Escherichia coli

✍ Marahiel, Mohamed A. ;Krause, Michael ;Skarpeid, Hans-Jacob 📂 Article 📅 1985 🏛 Springer 🌐 English ⚖ 907 KB

The entire structural gene for tyrocidine synthetase 1 from Bacillus brevis ATCC 8185 has been cloned and expressed in Escherichia coli. Transformed E. coli cells were screened for their ability to produce tyrocidine synthetase 1 by in situ immunoassay using antibodies against gramicidin S synthetas