Interaction of the human cytomegalovirus uracil DNA glycosylase UL114 with the viral DNA polymerase catalytic subunit UL54

Interaction between human cytomegalovirus uracil DNA glycosylase (UL114) and the viral DNA polymerase accessory subunit (UL44) has been reported; however, no such association was found in proteomic studies of UL44-interacting proteins. Utilizing virus expressing FLAG-tagged UL114, nuclease-resistant association of UL44 and the DNA polymerase catalytic subunit UL54 with UL114 was observed by co-immunoprecipitation. Contrary to a previous report, we observed that UL114 was much less abundant than UL44. Interaction of UL114 with UL54, independent of the UL54 carboxyl terminus, but not with UL44 was detected in vitro. Our data are consistent with a direct UL114–UL54 interaction, and suggest that UL114 and UL54 act in concert during base excision repair of the viral genome

Synthetic Biology: Mapping the Scientific Landscape

This article uses data from Thomson Reuters Web of Science to map and analyse the scientific landscape for synthetic biology. The article draws on recent advances in data visualisation and analytics with the aim of informing upcoming international policy debates on the governance of synthetic biology by the Subsidiary Body on Scientific, Technical and Technological Advice (SBSTTA) of the United Nations Convention on Biological Diversity. We use mapping techniques to identify how synthetic biology can best be understood and the range of institutions, researchers and funding agencies involved. Debates under the Convention are likely to focus on a possible moratorium on the field release of synthetic organisms, cells or genomes. Based on the empirical evidence we propose that guidance could be provided to funding agencies to respect the letter and spirit of the Convention on Biological Diversity in making research investments. Building on the recommendations of the United States Presidential Commission for the Study of Bioethical Issues we demonstrate that it is possible to promote independent and transparent monitoring of developments in synthetic biology using modern information tools. In particular, public and policy understanding and engagement with synthetic biology can be enhanced through the use of online interactive tools. As a step forward in this process we make existing data on the scientific literature on synthetic biology available in an online interactive workbook so that researchers, policy makers and civil society can explore the data and draw conclusions for themselves

The nucleotide recognized by the Escherichia coli A restriction and modification enzyme.

The nucleotide recognition sequence for the restriction-modification enzyme of Escherichia coli A (EcoA) has been determined to be GAG-7N-GTCA. This sequence is fairly similar, but distinctly different from the two other type I restriction enzyme recognition sites known for E. coli B and E. coli K12, respectively. N6-adenosine methylation has been observed at nucleotide positions 2 and 12 within that sequence after modification by EcoA. As a reference point for mapping the single EcoA site in lambda, the position of lambda point mutation Oam29 has been determined also

Histidine residues near the N terminus of staphylococcal alpha-toxin as reporters of regions that are critical for oligomerization and pore formation.

Chemical modification of histidine residues in staphylococcal alpha-toxin leads to loss of functional activity. Site-directed mutants of the toxin in which each of the four histidine residues was replaced by several amino acids were therefore produced. The mutant proteins were purified and characterized. Exchange of H-259 or H-144 was sometimes tolerated without reduction in hemolytic activity. These histidine residues are thus not essential for toxin function. Exchange of H-35 and H-48, however, had marked effects. H-35 mutant toxins bound with high affinity to rabbit erythrocytes but displayed faulty oligomerization and were unable to form pores. H-48 mutant toxins also had severely impaired hemolytic activity due probably to faulty hexamerization. We interpret these results to indicate that the N-terminal domain of alpha-toxin in the region of H-35 and H-48 is involved in protomer-protomer interactions that underlie the hexamerization and pore-forming process

Functional map of human cytomegalovirus AD169 defined by global mutational analysis

Human cytomegalovirus has a complex double-stranded DNA genome of ≈240,000 bp that contains ≈150 ORFs likely to encode proteins, most of whose functions are not well understood. We have used an infectious bacterial artificial chromosome to introduce 413 defined insertion and substitution mutations into the human cytomegalovirus AD169 genome by random and site-directed transposon mutagenesis. Mutations were produced in all unique ORFs with a high probability of encoding proteins for which mutants have not been previously documented and in many previously characterized ORFs. The growth of selected mutants was assayed in cultured human fibroblasts, and we now recognize 41 essential, 88 nonessential, and 27 augmenting ORFs. Most essential and augmenting genes are located in the central region, and nonessential genes generally cluster near the ends of the viral genome