Cloning of Escherichia coli uridine phosphorylase gene: localization of structural and regulatory regions in the cloned fragment and identification of the protein product
Brikun, I.A.; Mironov, A.S.; Masiliunaĭte, R.V.; Sukhodolets, V.V.
Molekuliarnaia Genetika Mikrobiologiia i Virusologiia 6: 7-11
1990
ISSN/ISBN: 0208-0613 PMID: 2172808 Document Number: 365528
Plasmid pUD5 carrying Escherichia coli udp gene was mutagenized with the Tn5 to determine the direction of udp gene transcription. Three independent Tn5 insertions into the plasmid borne udp gene were obtained (udp::Tn5-1, udp::Tn5-4 and udp::Tn5-5). These insertions cause disappearance of the uridine phosphorylase activity, though the ability of the plasmids to derepress transcription of chromosomal udp gene as a consequence of "titration" of cytoplasmic repressor CytR by operator regions of plasmid udp copies is retained. All the three Tn5 insertions were physically mapped within the 0.6 kb PstI-HindII fragment and transferred into the bacterial chromosome of Escherichia coli recBCsbcB strain. Integrated into the chromosomal udp gene the Tn 5 insertions were genetically mapped (by P1 transduction), with regard to the udp7 point mutation and two markers metE and zif9::Tn10 which flank the udp gene. The position of Tn5 on the constructed genetic map (metE-udp::Tn5-5 - udp::Tn5-1 - udp7 - udp::Tn5-4 - zif9::Tn10) coincides with the positions of Tn5 insertions on the pUD5. This allows one to predict the direction of transcription of the cloned udp gene from udp::Tn5-5 towards udp::Tn5-4, considering the known fact (Alkhimova et al, 1981) that the udp promoter is located in the vicinity of metE gene.