Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.     

Understanding substrate specificity in human and parasite phosphoribosyltransferases through calculation and experiment.

We present molecular dynamics (MD) simulations on two enzymes: a human hypoxanthine-guanine-phosphoribosyltransferase (HGPRTase) and its analogue in the protozoan parasite Tritrichomonas foetus. The parasite enzyme has an additional ability to process xanthine as a substrate, making it a hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) [Chin, M. S., and Wang, C. C. (1994) Mol. Biochem. Parasitol. 63 (2), 221-229 (1)]. X-ray crystal structures of both enzymes complexed to guanine monoribosyl phosphate (GMP) have been solved, and show only subtle differences in the two active sites [Eads et al. (1994) Cell 78 (2), 325-334 (2); Somoza et al. (1996) Biochemistry 35 (22), 7032-7040 (3)]. Most of the direct contacts with the base region of the substrate are made by the protein backbone, complicating the identification of residues significantly associated with xanthine recognition. Our calculations suggest that the broader specificity of the parasite enzyme is due to a significantly more flexible base-binding region, and rationalize the effect of two mutations, R155E and D163N, that alter substrate specificity [Munagala, N. R., and Wang, C. C. (1998) Biochemistry 37 (47), 16612-16619 (4)]. In addition, our simulations suggested a double mutant (D106E/D163N) that might rescue the D163N mutant. This double mutant was expressed and assayed, and its catalytic activity was confirmed. Our molecular dynamics trajectories were also used with a structure-based design program, Pictorial Representation Of Free Energy Changes (PROFEC), to suggest parasite-selective derivatives of GMP. Our calculations here successfully rationalize the parasite-selectivity of two novel inhibitors derived from the computer-aided design of Somoza et al. (5) and demonstrate the utility of PROFEC in the design of species-selective inhibitors.     

Immobilization of Burkholderia cepacia in polyurethane-based foams: embedding efficiency and effect on bacterial activity

Immobilization of the trichloroethylene-degrading bacterium Burkholderia cepacia was evaluated using hydrophilic polyurethane foam. The influence of several foam formulation parameters upon cell retention was examined. Surfactant type was a major determinant of retention; a lecithin-based compound retained more cells than pluronic- or silicone-based surfactants. Excessive amounts of surfactant led to increased washout of bacteria. Increasing the biomass concentration in the foam from 4.8 to 10.5% dry weight per wet weight of foam resulted in fewer cells being washed out. Embedding at reduced temperature did not significantly affect retention, while the use of a silane binding agent gave inconsistent results. The optimal formulation retained all but 0.2% of total embedded cells during passage of 2 L of water through columns containing 2 g of foam. All foam formulations tested reduced the culturability of embedded cells by several orders of magnitude, but O₂ consumption and CO₂ evolution rates of embedded cells were never less than 50% of those of free cells. Nutrient amendments stimulated an increase in cell volume and ribosomal activity in immobilized cells as indicated by hybridization studies using fluorescently labeled ribosomal probes. These results indicate that, although immobilized cells were mostly nonculturable, they were metabolically active and thus could be used for biodegradation of toxic compounds. Received 23 December 1996/ Accepted in revised form 13 March 1997     

Restriction-map variation associated with the G6PD polymorphism in natural populations of Drosophila melanogaster

Restriction-map variation was studied in 126 copies of the G6pd region in X chromosome lines of Drosophila melanogaster from North America, Europe, and Africa. Special attention was focused on the distribution of variation relative to the geographically variable polymorphism for two electrophoretic variants. Nucleotide heterozygosity as determined by eight six-cutter restriction enzymes for the 13-kb region is estimated, on the basis of the worldwide sample, to be 0.065%, which is the lowest value reported for any comparable region in the D. melanogaster genome. Significant linkage disequilibrium between electrophoretic alleles and restriction-site variation is observed for several sites. In contrast to published studies of other genetic regions, there are large insertions that reach significant frequencies and are found across considerable geographic distances. There is a clustering of this variation inside the first large intervening sequence of the G6PD gene.      

Molecular evolution of the MAGUK family in metazoan genomes

Background  

Development, differentiation and physiology of metazoans all depend on cell to cell communication and subsequent intracellular signal transduction. Often, these processes are orchestrated via sites of specialized cell-cell contact and involve receptors, adhesion molecules and scaffolding proteins. Several of these scaffolding proteins important for synaptic and cellular junctions belong to the large family of membrane-associated guanylate kinases (MAGUK). In order to elucidate the origin and the evolutionary history of the MAGUKs we investigated full-length cDNA, EST and genomic sequences of species in major phyla.