Molecular evolution of the Escherichia coli chromosome. VI. Two regions of high effective recombination

Two 6- to 8-min regions, centered respectively near 45 min (O-antigen region) and 99 min (restriction-modification region) on the Escherichia coli chromosome, display unusually high variability among 11 otherwise very similar strains. This variation, revealed by restriction fragment length polymorphism (RFLP) and nucleotide sequence comparisons, appears to be due to a great local increase in the retention frequency of recombinant replacements. We infer a two-step mechanism. The first step is the acquisition of a small stretch of DNA from a phylogenetically distant source. The second is the successful retransmission of the imported DNA, together with flanking native DNA, to other strains of E. coli. Each cell containing the newly transferred DNA has a very high selective advantage until it reaches a high frequency and (in the O-antigen case) is recognized by the new host's immune system. A high selective advantage increases the probability of retention greatly; the effective recombination rate is the product of the basic recombination rate and the probability of retention. Nearby nucleotide sequences clockwise from the O-antigen (rfb) region are correlated with specific O antigens, confirming local hitchhiking. Comparable selection involving imported restriction endonuclease genes is proposed for the region near 99 min.     

The VirB4 family of proposed traffic nucleoside triphosphatases: common motifs in plasmid RP4 TrbE are essential for conjugation and phage adsorption

Proteins of the VirB4 family are encoded by conjugative plasmids and by type IV secretion systems, which specify macromolecule export machineries related to conjugation systems. The central feature of VirB4 proteins is a nucleotide binding site. In this study, we asked whether members of the VirB4 protein family have similarities in their primary structures and whether these proteins hydrolyze nucleotides. A multiple-sequence alignment of 19 members of the VirB4 protein family revealed striking overall similarities. We defined four common motifs and one conserved domain. One member of this protein family, TrbE of plasmid RP4, was genetically characterized by site-directed mutagenesis. Most mutations in trbE resulted in complete loss of its activities, which eliminated pilus production, propagation of plasmid-specific phages, and DNA transfer ability in Escherichia coli. Biochemical studies of a soluble derivative of RP4 TrbE and of the full-length homologous protein R388 TrwK revealed that the purified forms of these members of the VirB4 protein family do not hydrolyze ATP or GTP and behave as monomers in solution.     

Human Mps1 kinase is required for the spindle assembly checkpoint but not for centrosome duplication

Budding yeast Mps1p kinase has been implicated in both the duplication of microtubule-organizing centers and the spindle assembly checkpoint. Here we show that hMps1, the human homolog of yeast Mps1p, is a cell cycle-regulated kinase with maximal activity during M phase. hMps1 localizes to kinetochores and its activity and phosphorylation state increase upon activation of the mitotic checkpoint. By antibody microinjection and siRNA, we demonstrate that hMps1 is required for human cells to undergo checkpoint arrest in response to microtubule depolymerization. In contrast, centrosome (re-)duplication as well as cell division occur in the absence of hMps1. We conclude that hMps1 is required for the spindle assembly checkpoint but not for centrosome duplication.     

Starch biosynthesis and intermediary metabolism in maize kernels. Quantitative analysis of metabolite flux by nuclear magnetic resonance

The seeds of cereals represent an important sink for metabolites during the accumulation of storage products, and seeds are an essential component of human and animal nutrition. Understanding the metabolic interconversions (networks) underpinning storage product formation could provide the foundation for effective metabolic engineering of these primary nutritional sources. In this paper, we describe the use of retrobiosynthetic nuclear magnetic resonance analysis to establish the metabolic history of the glucose (Glc) units of starch in maize (Zea mays) kernels. Maize kernel cultures were grown with [U-(13)C(6)]Glc, [U-(13)C(12)]sucrose, or [1,2-(13)C(2)]acetate as supplements. After 19 d, starch was hydrolyzed, and the isotopomer composition of the resulting Glc was determined by quantitative nuclear magnetic resonance analysis. [1,2-(13)C(2)]Acetate was not incorporated into starch. [U-(13)C(6)]Glc or [U-(13)C(12)]sucrose gave similar labeling patterns of polysaccharide Glc units, which were dominated by [1,2,3-(13)C(3)]- and [4,5,6-(13)C(3)]-isotopomers, whereas the [U-(13)C(6)]-, [3,4,5,6-(13)C(4)]-, [1,2-(13)C(2)]-, [5,6-(13)C(2)], [3-(13)C(1)], and [4-(13)C(1)]-isotopomers were present at lower levels. These isotopomer compositions indicate that there is extensive recycling of Glc before its incorporation into starch, via the enzymes of glycolytic, glucogenic, and pentose phosphate pathways. The relatively high abundance of the [5,6-(13)C(2)]-isotopomer can be explained by the joint operation of glycolysis/glucogenesis and the pentose phosphate pathway.     

Conceptual data modelling for bioinformatics

Current research in the biosciences depends heavily on the effective exploitation of huge amounts of data. These are in disparate formats, remotely dispersed, and based on the different vocabularies of various disciplines. Furthermore, data are often stored or distributed using formats that leave implicit many important features relating to the structure and semantics of the data. Conceptual data modelling involves the development of implementation-independent models that capture and make explicit the principal structural properties of data. Entities such as a biopolymer or a reaction, and their relations, eg catalyses, can be formalised using a conceptual data model. Conceptual models are implementation-independent and can be transformed in systematic ways for implementation using different platforms, eg traditional database management systems. This paper describes the basics of the most widely used conceptual modelling notations, the ER (entity-relationship) model and the class diagrams of the UML (unified modelling language), and illustrates their use through several examples from bioinformatics. In particular, models are presented for protein structures and motifs, and for genomic sequences.     

NMR spectroscopic and theoretical study of the complexation of the inhibitor allosamidin in the binding pocket of the plant chitinase hevamine

Based on NMR spectroscopic information about the allosamidin-hevamine complex, ab initio MO calculations of the ring current effect of the aromatic moieties of Trp255, Tyr183 and Tyr6 of hevamine were carried out to investigate the role of these amino acid residues in binding interactions with allosamidin in solution. In addition, the intermolecular steric compression effect on the 13C chemical shifts of the allosamizoline carbon atoms and the hydrogen bonding to Glu127 was identified. It can be inferred that the binding forces are strongest in the allosamizoline moiety of allosamidin.     

Mitochondrial respiration at low levels of oxygen and cytochrome c

In the intracellular microenvironment of active muscle tissue, high rates of respiration are maintained at near-limiting oxygen concentrations. The respiration of isolated heart mitochondria is a hyperbolic function of oxygen concentration and half-maximal rates were obtained at 0.4 and 0.7 microM O(2) with substrates for the respiratory chain (succinate) and cytochrome c oxidase [N,N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)+ascorbate] respectively at 30 degrees C and with maximum ADP stimulation (State 3). The respiratory response of cytochrome c-depleted mitoplasts to external cytochrome c was biphasic with TMPD, but showed a monophasic hyperbolic function with succinate. Half-maximal stimulation of respiration was obtained at 0.4 microM cytochrome c, which was nearly identical to the high-affinity K(')(m) for cytochrome c of cytochrome c oxidase supplied with TMPD. The capacity of cytochrome c oxidase in the presence of TMPD was 2-fold higher than the capacity of the respiratory chain with succinate, measured at environmental normoxic levels. This apparent excess capacity, however, is significantly decreased under physiological intracellular oxygen conditions and declines steeply under hypoxic conditions. Similarly, the excess capacity of cytochrome c oxidase declines with progressive cytochrome c depletion. The flux control coefficient of cytochrome c oxidase, therefore, increases as a function of substrate limitation of oxygen and cytochrome c, which suggests a direct functional role for the apparent excess capacity of cytochrome c oxidase in hypoxia and under conditions of intracellular accumulation of cytochrome c after its release from mitochondria.     

Biphasic oxygen kinetics of cellular respiration and linear oxygen dependence of antimycin A inhibited oxygen consumption

Oxygen kinetics in fibroblasts was biphasic. This was quantitatively explained by a major mitochondrial hyperbolic component in the low-oxygen range and a linear increase of rotenone-and antimycin A-inhibited oxygen consumption in the high-oxygen range. This suggests an increased production of reactive oxygen species and oxidative stress at elevated, air-level oxygen concentrations. The high oxygen affinity of mitochondrial respiration provides the basis for the maintenance of a high aerobic scope at physiological low-oxygen levels, whereas further pronounced depression of oxygen pressure induces energetic stress under hypoxia.