Downregulation of the Escherichia coli guaB promoter by FIS

The Escherichia coli guaB promoter (PguaB) regulates transcription of two genes, guaB and guaA, that are required for the synthesis of guanosine 5′-monophosphate (GMP), a precursor for the synthesis of guanine nucleoside triphosphates. Transcription from PguaB increases as a function of increasing cellular growth rate, and this is referred to as growth rate-dependent control (GRDC). Here we investigated the role of the factor for inversion stimulation (FIS) in the regulation of this promoter. The results showed that there are three binding sites for FIS centred near positions −11, +8 and +29 relative to the guaB transcription start site. Binding of FIS to these sites results in repression of PguaB in vitro but not in vivo. Deletion of the fis gene results in increased PguaB activity in vivo, but GRDC of PguaB is maintained

Regulation of the gua operon of Escherichia coli by the DnaA protein

The guaBA operon of E.coli determines the production of the two enzymes required to convert hypoxanthine to guanine at the nucleotide level during nucleotide biosynthesis. Two DnaA boxes, binding sites for the DnaA replication-initiating DnaA protein, are present in the gua operon. One box (with a 8/9 fit to the consensus sequence DnaA box TTATACCAACA) overlaps the gua promoter, the other box (matching the consensus sequence) lies 200 base pairs within the guaB coding sequence. Regulation of the guaBA operon by DnaA protein was studied using strains carrying chromosomal gua-lacZ fusions. In these strains β-galactosidase acts as a reporter enzyme for transcription initiation at guaP. When the intracellular levels of DnaA were increased (by induction of a multicopy plasmid carrying the dnaA gene fused to the tac promoter) transcription from the gua promoter was repressed. Reducing the intracellular level of DnaA, either by sequestration with a multicopy oriC plasmid or by placing a temperature-sensitive dnaA mutant at the restrictive temperature, resulted in increased transcription from guaP. Repression of guaP by DnaA was dependent on the presence of both DnaA boxes in the gua-lacZ fusion; constructs containing only the box at guaP were unaffected by DnaA. The DnaA protein was purified to near homogeneity from an overproducing strain and supported DNA synthesis in vitro in a complementation assay. Using the techniques of filter-binding and gel retardation, the purified DnaA protein was found to bind restriction fragments from the gua operon. Footprinting demonstrated that DnaA protein binds specifically at the consensus sequence DnaA box within guaB. This box may therefore function as a transcriptional terminator. A computer search of nucleotide sequences in the EMBL data library (release 30, 1992) for the E.coli chromosome revealed 48 sites conforming to the consensus sequence for the DnaA box. These sites are located non-randomly on the chromosome map with a bias towards the origin of replication (oriC). A model is proposed whereby genes containing DnaA boxes (including guaB) comprise a DnaA regulon. Transcriptional activity of these genes may be regulated by the cellular concentration of the DnaA protein.</p

Regulation of the divergentguaBAandxseApromoters of Escherichia coliby the cyclic AMP receptor protein

The gua promoter (guaP) of Escherichia coli resembles those for ribosomal RNA (rrn) operons and lies in a close back-to-back arrangement with the promoter for xseA (xseP). Transcription from guaP is subject to stringent control and growth-rate-dependent regulation, and to repression by DnaA and PurR. In addition, transcription from guaP is regulated by the cyclic AMP receptor protein (CRP). Plasmid-borne promoter fusions to the receptor gene for chloramphenicol acetyl transferase were used to assess the role of CRP in controlling transcription from guaP and xseP following a downshift of cultures from rich into minimal medium. CRP is required to activate guaBA transcription and repress xseA transcription following downshift. Bandshift assays with a DNA fragment carrying the divergent promoters revealed specific binding of CRP. We propose that CRP, binding to a near-consensus site centred at -117.5, activates transcription from guaP and obstructs transcription from the xseA promoter

Detection of minimal residual cancer to investigate why oral tumors recur despite seemingly adequate treatment

Improvements in surgery and radiotherapy techniques have led to only a modest increase in the 5-year survival rate for patients with head and neck cancer. This is because the pattern of clinical disease Is changing, such that locoregional recurrence now accounts for fewer treatment failures, but more patients develop a second primary cancer or distant metastatic disease, In this study, we have used the p53 phage plaque assay, immunocytochemistry, and mutational analysis to assess the contribution of minimal residual cancer and genetic aberrations in clinically normal upper aerodigestive tract mucosa to treatment failure. Eighteen consecutive patients with oral tumors, with conventional clear margins, have been followed for a minimum of 36 months, Molecular assessment identified tumor-positive surgical margins for 6 of 11 assessable patients and additional tumor-positive lymph nodes for three cases. Disseminated malignant cells were detected in the hematopoietic cell compartment for six cases, and one patient had molecular evidence of field cancerization, Locoregional recurrence developed in five patients with tumors harboring a p53 gene mutation; four of these were associated with tumor-positive surgical margins, and one was associated with molecular evidence of field cancerization. Radiotherapy to the primary site did not prevent development of local recurrence when the residual tumor harbored ap53 gene mutation, Three of six cases with a tumor-positive bone marrow aspirate developed distant metastases, These findings reveal that molecular and immunocytochemical detection of minimal residual cancer and field cancerization can help identify patients who may develop locoregional or distant recurrence and justify further studies to evaluate the contribution of these remaining malignant cells to treatment failure