The Alpha Project: a model system for systems biology research.

One goal of systems biology is to understand how genome-encoded parts interact to produce quantitative phenotypes. The Alpha Project is a medium-scale, interdisciplinary systems biology effort that aims to achieve this goal by understanding fundamental quantitative behaviours of a prototypic signal transduction pathway, the yeast pheromone response system from Saccharomyces cerevisiae. The Alpha Project distinguishes itself from many other systems biology projects by studying a tightly bounded and well-characterised system that is easily modified by genetic means, and by focusing on deep understanding of a discrete number of important and accessible quantitative behaviours. During the project, the authors have developed tools to measure the appropriate data and develop models at appropriate levels of detail to study a number of these quantitative behaviours. The authors have also developed transportable experimental tools and conceptual frameworks for understanding other signalling systems. In particular, the authors have begun to interpret system behaviours and their underlying molecular mechanisms through the lens of information transmission, a principal function of signalling systems. The Alpha Project demonstrates that interdisciplinary studies that identify key quantitative behaviours and measure important quantities, in the context of well-articulated abstractions of system function and appropriate analytical frameworks, can lead to deeper biological understanding. The authors' experience may provide a productive template for systems biology investigations of other cellular systems.

Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid.

The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti contains three replicons: pSymA, pSymB, and the chromosome. We report here the complete 1,354,226-nt sequence of pSymA. In addition to a large fraction of the genes known to be specifically involved in symbiosis, pSymA contains genes likely to be involved in nitrogen and carbon metabolism, transport, stress, and resistance responses, and other functions that give S. meliloti an advantage in its specialized niche.

Reduction of adenosine-5'-phosphosulfate instead of 3'-phosphoadenosine-5'-phosphosulfate in cysteine biosynthesis by Rhizobium meliloti and other members of the family Rhizobiaceae.

We have cloned and sequenced three genes from Rhizobium meliloti (Sinorhizobium meliloti) that are involved in sulfate activation for cysteine biosynthesis. Two of the genes display homology to the Escherichia coli cysDN genes, which code for an ATP sulfurylase (EC 2.7.7.4). The third gene has homology to the E. coli cysH gene, a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase (EC 1.8.99.4), but has greater homology to a set of genes found in Arabidopsis thaliana that encode an adenosine-5'-phosphosulfate (APS) reductase. In order to determine the specificity of the R. meliloti reductase, the R. meliloti cysH homolog was histidine tagged and purified, and its specificity was assayed in vitro. Like the A. thaliana reductases, the histidine-tagged R. meliloti cysH gene product appears to favor APS over PAPS as a substrate, with a Km for APS of 3 to 4 microM but a Km for PAPS of >100 microM. In order to determine whether this preference for APS is unique to R. meliloti among members of the family Rhizobiaceae or is more widespread, cell extracts from R. leguminosarum, Rhizobium sp. strain NGR234, Rhizobium fredii (Sinorhizobium fredii), and Agrobacterium tumefaciens were assayed for APS or PAPS reductase activity. Cell extracts from all four species also preferentially reduce APS over PAPS.

Characterization of human adenovirus proteinase activity in disrupted virus particles.

Virus-coded proteinases are attractive targets for antiviral therapy; however, lack of quick, sensitive, quantitative, and selective assays for enzyme activity makes it difficult to characterize these proteinases and to screen large numbers of potential inhibitors. Here we describe new substrates for the adenovirus proteinase, fluorogenic Rhodamine-based substrates containing tetrapeptides corresponding to sequences cleaved in adenovirus precursor proteins. Proteinase activity in as few as 10(9) disrupted virions could be quantitatively detected in a 30-min assay. With the substrate (Leu-Arg-Gly-Gly-NH)2-Rhodamine, the Km was 1.4 microM and the Vmax was 3.24 pmol substrate hydrolyzed/sec/pmol virus. Enzyme activity was stimulated by dithiothreitol and inhibited by several serine-specific as well as cysteine-specific proteinase inhibitors. In a thiol protection experiment, the virion enzyme was shown to have a cysteine residue with an unusually low pKa, a pKa similar to that of the active-site nucleophile of the cysteine proteinase papain. The curve of Vmax as a function of pH is unlike the curve from papain and implied that there are at least three ionizable groups whose protonation state can affect catalysis - one with a pKa of 6.2, another with a pKa of 7.2, and a third with a pKa of 8.3.

Contributions of supercoiling to Tn3 resolvase and phage Mu Gin site-specific recombination.

Members of the resolvase/invertase family of site-specific recombinases require supercoiled substrates containing two recombination sites. To dissect the roles of supercoiling in recombination by the Tn3 and gamma delta resolvases and the phage Mu Gin invertase, we used substrates that provided some but not all of the topological features of the standard substrate. We divided the Tn3 resolvase reaction into two stages, synapsis and postsynapsis. Using structural and functional topological analyses, we verified that the resolvase synaptic complexes with nicked catenanes were recombination intermediates. The requirement for supercoiling was even less stringent for the gamma delta resolvase, which recombined nicked catenanes about half as well as it did supercoiled substrates. Gin recombination of catenanes occurred even if the recombinational enhancer was on a nicked ring, as long as both crossover sites were on a supercoiled ring. Therefore, supercoiling is required at the Gin crossover sites but not at the enhancer. We conclude that solely conformational effects of supercoiling are required for resolvase synapsis and the function of the Gin enhancer, but that a torsional effect, probably double helix unwinding, is needed for Tn3 resolvase postsynapsis and at the Gin recombination sites.