Bacteriophage T4 DNA replication protein 41. Cloning of the gene and purification of the expressed protein

The bacteriophage T4 primase, composed of the T4 proteins 41 and 61, synthesizes pentaribonucleotides used to prime DNA synthesis on single-stranded DNA in vitro. 41 protein is also a DNA helicase that opens DNA in the same direction as the growing replication fork. Previously, Mattson et al. (Mattson, T., Van Houwe, G., Bolle, A., Selzer, G., and Epstein, R. (1977) Mol. Gen. Genet. 154, 319-326) located part of gene 41 on a 3400-base pair EcoRI fragment of T4 DNA (map units 24.3 to 21.15). In this paper, we report the cloning of T4 DNA representing map units 24.3 to 20.06 in a multicopy plasmid vector. Extracts of cells containing this plasmid complement gene 41- extracts in a DNA synthesis assay, indicating that this region contains all the information necessary for the expression of active 41 protein. We located gene 41 more precisely between T4 map units 22.01 to 20.06 since our cloning of this region downstream of the strong lambda promoter PL results in the production of active 41 protein at a level 100-fold greater than after T4 infection. We have purified 133 mg of homogeneous 41 protein from 27 g of these cells. Like the 41 protein from T4 infected cells, the purified 41 protein in conjunction with the T4 gene 61 priming protein catalyzes primer formation (assayed by RNA primer-dependent DNA synthesis with T4 polymerase, the genes 44/62 and 45 polymerase accessory proteins, and the gene 32 helix-destabilizing protein) and is a helicase whose activity is stimulated by T4 61 protein.     

Green Tea Polyphenols Precondition against Cell Death Induced by Oxygen-Glucose Deprivation via Stimulation of Laminin Receptor,Generation of Reactive Oxygen Species,and Activation of Protein Kinase C?

As the development of synthetic drugs for the prevention of stroke has proven challenging, utilization of natural products capable of preconditioning neuronal cells against ischemia-induced cell death would be a highly useful complementary approach. In this study using an oxygen-glucose deprivation and reoxygenation (OGD/R) model in PC12 cells, we show that 2-day pretreatment with green tea polyphenols (GTPP) and their active ingredient, epigallocatechin-3-gallate (EGCG), protects cells from subsequent OGD/R-induced cell death. A synergistic interaction was observed between GTPP constituents, with unfractionated GTPP more potently preconditioning cells than EGCG. GTPP-induced preconditioning required the 67-kDa laminin receptor (67LR), to which EGCG binds with high affinity. 67LR also mediated the generation of reactive oxygen species (ROS) via activation of NADPH oxidase. An exogenous ROS-generating system bypassed 67LR to induce preconditioning, suggesting that sublethal levels of ROS are indeed an important mediator in GTPP-induced preconditioning. This role for ROS was further supported by the fact that antioxidants blocked GTPP-induced preconditioning. Additionally, ROS induced an activation and translocation of protein kinase C (PKC), particularly PKCϵ from the cytosol to the membrane/mitochondria, which was also blocked by antioxidants. The crucial role of PKC in GTPP-induced preconditioning was supported by use of its specific inhibitors. Preconditioning was increased by conditional overexpression of PKCϵ and decreased by its knock-out with siRNA. Collectively, these results suggest that GTPP stimulates 67LR and thereby induces NADPH oxidase-dependent generation of ROS, which in turn induces activation of PKC, particularly prosurvival isoenzyme PKCϵ, resulting in preconditioning against cell death induced by OGD/R.     

Mutational analysis of the T4 gp59 helicase loader reveals its sites for interaction with helicase, single-stranded binding protein, and DNA

Efficient DNA replication involves coordinated interactions among DNA polymerase, multiple factors, and the DNA. From bacteriophage T4 to eukaryotes, these factors include a helicase to unwind the DNA ahead of the replication fork, a single-stranded binding protein (SSB) to bind to the ssDNA on the lagging strand, and a helicase loader that associates with the fork, helicase, and SSB. The previously reported structure of the helicase loader in the T4 system, gene product (gp)59, has revealed an N-terminal domain, which shares structural homology with the high mobility group (HMG) proteins from eukaryotic organisms. Modeling of this structure with fork DNA has suggested that the HMG-like domain could bind to the duplex DNA ahead of the fork, whereas the C-terminal portion of gp59 would provide the docking sites for helicase (T4 gp41), SSB (T4 gp32), and the ssDNA fork arms. To test this model, we have used random and targeted mutagenesis to generate mutations throughout gp59. We have assayed the ability of the mutant proteins to bind to fork, primed fork, and ssDNAs, to interact with SSB, to stimulate helicase activity, and to function in leading and lagging strand DNA synthesis. Our results provide strong biochemical support for the role of the N-terminal gp59 HMG motif in fork binding and the interaction of the C-terminal portion of gp59 with helicase and SSB. Our results also suggest that processive replication may involve the switching of gp59 between its interactions with helicase and SSB.     

Frequency of resistance to Bacillus thuringiensis toxin Cry1Ab in southern United States Corn Belt population of European corn borer (Lepidoptera: Crambidae)

The high-dose refuge resistance management strategy is the main approach used to delay resistance in targeted pests to Bacillus thuringiensis (Bt) toxins in transgenic crops. We used an F2 screen to test a critical assumption of the high-dose refuge strategy, which is that resistance allele (R) frequencies are initially rare (<10(-3)) in Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) from the southern Corn Belt. We expanded the methodological scope of the F2 screen so that both males and females may be used to initiate a screen and determined how the results from both sexes may be combined. In total, 62 female and 131 male O. nubilalis lines from Kansas and 39 female and four male lines from Texas were screened. No major resistance alleles were found and estimated R frequency for the southern Corn Belt was updated to between 0 and 0.0044 with 95% credibility. The experiment-wise detection probability was 98.7%. These results suggest the frequency of resistance alleles is low enough that the high-dose refuge resistance management strategy may be effective for delaying resistance evolution in O. nubilalis to Bt corn in the southern Corn Belt.     

A novel strategy for the identification of genomic islands by comparative analysis of the contents and contexts of tRNA sites in closely related bacteria

We devised software tools to systematically investigate the contents and contexts of bacterial tRNA and tmRNA genes, which are known insertion hotspots for genomic islands (GIs). The strategy, based on MAUVE-facilitated multigenome comparisons, was used to examine 87 Escherichia coli MG1655 tRNA and tmRNA genes and their orthologues in E.coli EDL933, E.coli CFT073 and Shigella flexneri Sf301. Our approach identified 49 GIs occupying ~1.7 Mb that mapped to 18 tRNA genes, missing 2 but identifying a further 30 GIs as compared with Islander [Y. Mantri and K. P. Williams (2004), Nucleic Acids Res., 32, D55–D58]. All these GIs had many strain-specific CDS, anomalous GC contents and/or significant dinucleotide biases, consistent with foreign origins. Our analysis demonstrated marked conservation of sequences flanking both empty tRNA sites and tRNA-associated GIs across all four genomes. Remarkably, there were only 2 upstream and 5 downstream deletions adjacent to the 328 loci investigated. In silico PCR analysis based on conserved flanking regions was also used to interrogate hotspots in another eight completely or partially sequenced E.coli and Shigella genomes. The tools developed are ideal for the analysis of other bacterial species and will lead to in silico and experimental discovery of new genomic islands.     

Effects of glycine substitutions on the structure and function of gramicidin a channels

Tryptophan residues often are found at the lipid-aqueous interface region of membrane-spanning proteins, including ion channels, where they are thought to be important determinants of protein structure and function. To better understand how Trp residues modulate the function of membrane-spanning channels, we have examined the effects of Trp replacements on the structure and function of gramicidin A channels. Analogues of gramicidin A in which the Trp residues at positions 9, 11, 13, and 15 were sequentially replaced with Gly were synthesized, and the three-dimensional structure of each analogue was determined using a combination of two-dimensional NMR techniques and distance geometry-simulated annealing structure calculations. Though Trp --> Gly substitutions destabilize the beta6.3-helical gA channel structure, it is possible to determine the structure of analogues with Trp --> Gly substitutions at positions 11, 13, and 15, but not for the analogue with the Trp --> Gly substitution at position 9. The Gly11-, Gly13-, and Gly15-gA analogues form channels that adopt a backbone fold identical to that of native gramicidin A, with only small changes in the side chain conformations of the unsubstituted residues. Single-channel current measurements show that the channel function and lifetime of the analogues are significantly affected by the Trp --> Gly replacements. The conductance variations appear to be caused by sequential removal of the Trp dipoles, which alter the ion-dipole interactions that modulate ion movement. The lifetime variations did not appear to follow a clear pattern.