Characterization of the upp gene encoding uracil phosphoribosyltransferase of Escherichia coli K12

Abstract

The upp gene coding for uracil phosphoribosyltransferase was subcloned on a 5‐kb EcoRI restriction fragment along with the purMN operon. By a combination of complementation, deletion and minicell analyses, the upp gene was located adjacent to and divergently transcribed from the purMN operon. All three gene products could be identified in minicell extracts. The cloned upp gene shows an elevated expression upon uracil starvation. The nucleotide sequence and transcription start of the gene were determined. The sequence yields an open reading frame of 624 nucleotides encoding a protein of 22.5 kDa which is in agreement with the previously determined subunit Mr of the purified enzyme. A putative 5‐phosphoribosyl‐α‐1‐diphosphate (PRPP) binding site has been identified which is similar to the PRPP binding site of the yeast uracil phosphoribosyltransferase.

Potential therapeutic applications of recombinant, invasive E. coli

“…This vector contains a conditional promoter that is active upon L-arabinose addition to the culture medium. A PCR product containing the upp gene (encoding the UPRT protein, see nucleotide sequence) 38 was cloned from the genome of BM2710 E. coli (primers: 5 0 -ctcgagaagatcgtggaagtcaaacac-3 0 and 5 0 -aagcttttatttcgtaccaaagattttgtc-3 0 ). The XhoI and a HindIII sites flanking the PCR product were used to clone the upp gene into the prokaryotic expression vector pBAD/HisA, in-frame with an N-terminal leader peptide.…”

Modified vaccinia virus Ankara as a vector for suicide gene therapy

“…In Escherichia coli and Saccharomyces cerevisiae 5-FU is converted to 5-FUMP by uracil phosphoribosyltransferase (UPRTase) 11,12 and it has been reported that adenovirus-mediated transduction of the E. coli upp gene that encodes UPRTase markedly sensitized tumor cells to 5-FU in vitro and in vivo. 13 We had previously reported the efficacy of a new suicide gene derived from a fusion of the S. cerevisiae CDase (FCY1) and UPRTase genes (FUR1).…”

Complete sequence of the upp gene encoding uracil phosphoribosyltransferase from Mycobacterium bovis BCG

“…UPRTase functiolls as the key enzyme in the salvage pathway of uracil in many microorganisms (10)(11)(12). The genes encoding UPRTase (upp) have been cloned and sequenced frorn Escherichia coli (13), Streptococcus salivarius (14), Lactococcus lactis (11), Bacillus subtilis (12), Haemophilus influenzae (15), Mycoplasma genitalium (16), and Saccharomyces cerevisiae (17). Here we report the nucleotide sequence of the putative upp gene of M. bovis BCG and compare the dedvccd amino acid (aa) sequence with the corresponding UPRTases from other microorganisms.…”

“…2). One is the C-temainal region (frora pro-173 to arg-207 in the BCG enzyme) which may correspond to a uracil binding site (11,13). Another region (from leu-ll6 or asp-122 to ser-137 in BCG) has been aligned with the consensus sequence of PRPP binding sites based on 22 sequences (13) …”