Characterization of the full length uracil-DNA glycosylase in the extreme thermophile Thermotoga maritima

A full length (192 amino acids) uracil-DNA glycosylase (TMUDG) has been expressed and purified from the extreme thermophile Thermotoga maritima. This protein is active up to 85 degrees C. The enzyme is product inhibited by abasic sites in DNA and weakly inhibited by uracil. TMUDG was originally cloned from an ORF which encoded a protein of 185 amino acids. This shorter protein was stable up to 70-75 degrees C and it seemed unusual that this enzyme had an optimal activity temperature below the growth temperature of the organism (80-90 degrees C). Following the publication of the complete genomic sequence of T. maritima, it was shown that the gene contains an additional seven amino acids (LYTREEL) at the N-terminal end of the protein. It is suggested that these seven residues are important in maintaining proper protein folding that results in increased temperature stability. We have also demonstrated that TMUDG can substitute for the Escherichia coli uracil-DNA glycosylase and initiate base excision repair using a closed circular DNA substrate containing a unique U:G base pair.     

Clinical outcome of a protocol to produce immunosuppression in rhesus monkeys (Macaca mulatta): application to infectious disease and gene therapy studies

Induced immunosuppression is required for a number of studies using rhesus monkeys (Macaca mulatta). This report describes the clinical outcome and safety of a dose-finding experiment that determined doses of cyclophosphamide and prednisone that could be used to induce a state of immunosuppression in rhesus monkeys. After determining the optimum dose of immunosuppressive agents, the protocol was then used on animals participating in infectious disease and gene therapy studies. Splenectomy was performed in some animals to increase the severity of immunosuppression. The onset, duration, and severity of lymphopenia and leukopenia were consistent in all animals. In most animals, physical examination findings and clinical serum chemistry profiles demonstrated only transient abnormalities. With proper clinical monitoring, combination treatment with cyclophosphamide and prednisone is an effective and safe method for inducing immunosuppression in rhesus monkeys.     

The Distribution of Microbial Communities in Anaerobic and Aerobic Zones of a Shallow Coastal Plain Aquifer

Abstract Randomly amplified polymorphic DNA (RAPD) fingerprinting was used to determine the genetic similarity of whole-community DNA extracts from unattached microorganisms in several groundwater wells. The study site was a shallow coastal plain aquifer on the Eastern Shore of Virginia that contains distinct regions of anaerobic and aerobic groundwater. Several wells in each region were sampled, and principal component and cluster analyses showed a clear separation of the microbial communities from the two chemical zones of the aquifer. Within these zones, there was no relationship between the genetic relatedness of a pair of communities and their spatial separation. Two additional sets of samples were taken at later times, and the same clear separation between communities in the different zones of the aquifer was observed. The specific relationships between wells within each zone changed over time, however, and the magnitude and direction of these changes corresponded to concurrent changes in the groundwater chemistry at each well. Together, these results suggest that local variation in groundwater chemistry can support genetically distinct microbial communities, and that the composition of the microbial communities can follow seasonal fluctuations in groundwater chemistry. Received: 25 May 1999; Accepted: 4 August 1999; Online Publication: 9 December 1999     

Chemolithoautotrophy in the marine,magnetotactic bacterial strains MV-1 and MV-2

Magnetite-producing magnetotactic bacteria collected from the oxic–anoxic transition zone of chemically stratified marine environments characterized by O₂/H₂S inverse double gradients, contained internal S-rich inclusions resembling elemental S globules, suggesting they oxidize reduced S compounds that could support autotrophy. Two strains of marine magnetotactic bacteria, MV-1 and MV-2, isolated from such sites grew in O₂-gradient media with H₂S or thiosulfate (S₂O₃^2–) as electron sources and O₂ as electron acceptor or anaerobically with S₂O₃^2– and N₂O as electron acceptor, with bicarbonate (HCO₃^–)/CO₂ as sole C source. Cells grown with H₂S contained S-rich inclusions. Cells oxidized S₂O₃^2– to sulfate (SO₄^2–). Both strains grew microaerobically with formate. Neither grew microaerobically with tetrathionate (S₄O₆^2–), methanol, or Fe²⁺ as FeS, or siderite (FeCO₃). Growth with S₂O₃^2– and radiolabeled ¹⁴C-HCO₃^– showed that cell C was derived from HCO₃^–/CO₂. Cell-free extracts showed ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity. Southern blot analyses indicated the presence of a form II RubisCO (cbbM) but no form I (cbbL) in both strains. cbbM and cbbQ, a putative post-translational activator of RubisCO, were identified in MV-1. MV-1 and MV-2 are thus chemolithoautotrophs that use the Calvin–Benson–Bassham pathway. cbbM was also identified in Magnetospirillum magnetotacticum. Thus, magnetotactic bacteria at the oxic–anoxic transition zone of chemically stratified aquatic environments are important in C cycling and primary productivity.     

Modelling the impact of antigen kinetics on T-cell activation and response

Cytotoxic T lymphocyte (CTL) responses are thought to be important for the control of many viral and other infections. Qualitative aspects of the CTL response, including the epitope specificity, affinity, and clonal composition, may affect the ability of T cells to mediate infection control. Although it is clear that the mode of introduction and the dose of antigen can affect these qualitative aspects of the response, little is understood of the mechanisms. We have developed an in silico model of the CTL response, which we use to study the impact of antigen dose, antigen kinetics and repeated antigen delivery on the response. The results suggest that recent observations on differences in response to killed antigen can be explained simply by differences in timing of T-cell activation. These findings may provide insight into how different vaccination strategies can quantitatively and qualitatively affect the outcome of the immune response.