Uncovering metabolic pathways relevant to phenotypic traits of microbial genomes

Identifying the biochemical basis of microbial phenotypes is a main objective of comparative genomics. Here we present a novel method using multivariate machine learning techniques for comparing automatically derived metabolic reconstructions of sequenced genomes on a large scale. Applying our method to 266 genomes directly led to testable hypotheses such as the link between the potential of microorganisms to cause periodontal disease and their ability to degrade histidine, a link also supported by clinical studies.     

Elevated atmospheric CO2 concentration enhances salinity tolerance in Aster tripolium L.

Our study aimed at investigating the influence of elevated atmospheric CO₂ concentration on the salinity tolerance of the cash crop halophyte Aster tripolium L., thereby focussing on protein expression and enzyme activities. The plants were grown in hydroponics using a nutrient solution with or without addition of NaCl (75% seawater salinity), under ambient (380 ppm) and elevated (520 ppm) CO₂. Under ambient CO₂ concentration enhanced expressions and activities of the antioxidant enzymes superoxide dismutase, ascorbate peroxidase, and glutathione-S-transferase in the salt-treatments were recorded as a reaction to oxidative stress. Elevated CO₂ led to significantly higher enzyme expressions and activities in the salt-treatments, so that reactive oxygen species could be detoxified more effectively. Furthermore, the expression of a protective heat shock protein (class 20) increased under salinity and was even further enhanced under elevated CO₂ concentration. Additional energy had to be provided for the mechanisms mentioned above, which was indicated by the increased expression of a β ATPase subunit and higher v-, p- and f-ATPase activities under salinity. The higher ATPase expression and activities also enable a more efficient ion transport and compartmentation for the maintenance of ion homeostasis. We conclude that elevated CO₂ concentration is able to improve the survival of A. tripolium under salinity because more energy is provided for the synthesis and enhanced activity of enzymes and proteins which enable a more efficient ROS detoxification and ion compartmentation/transport.     

Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns

As part of the exploratory sequencing program Génolevures, visual scrutinisation and bioinformatic tools were used to detect spliceosomal introns in seven hemiascomycetous yeast species. A total of 153 putative novel introns were identified. Introns are rare in yeast nuclear genes (<5% have an intron), mainly located at the 5′ end of ORFs, and not highly conserved in sequence. They all share a clear non-random vocabulary: conserved splice sites and conserved nucleotide contexts around splice sites. Homologues of metazoan snRNAs and putative homologues of SR splicing factors were identified, confirming that the spliceosomal machinery is highly conserved in eukaryotes. Several introns’ features were tested as possible markers for phylogenetic analysis. We found that intron sizes vary widely within each genome, and according to the phylogenetic position of the yeast species. The evolutionary origin of spliceosomal introns was examined by analysing the degree of conservation of intron positions in homologous yeast genes. Most introns appeared to exist in the last common ancestor of present day yeast species, and then to have been differentially lost during speciation. However, in some cases, it is difficult to exclude a possible sliding event affecting a pre-existing intron or a gain of a novel intron. Taken together, our results indicate that the origin of spliceosomal introns is complex within a given genome, and that present day introns may have resulted from a dynamic flux between intron conservation, intron loss and intron gain during the evolution of hemiascomycetous yeasts.     

Identification of transgenic mice by direct PCR analysis of lysates of epithelial cells obtained from the inner surface of the rectum

Conventional screening protocols for transgene integration in mice employ tail tips or blood samples as sources to obtain genomic DNA preparations. We have developed a simple alternative non-surgical method. Epithelial cells are scraped off the inner surface of the rectum with a sterile plastic inoculation loop and are lysed with Kawasaki buffer. The lysate can be directly examined in a polymerase chain reaction (PCR) analysis without any need for further DNA purification. This procedure causes minimal harm and stress to the animals and repeated samples can be obtained as often as necessary. This technique has been used successfully to identify transgenic mice from a number of different lines. The method allows quick screening of numerous animals and contributes to a reduction of the number of surgical biopsies required     

Is It Always Unethical to Use a Placebo in a Clinical Trial?

Background to the debate: Placebos are used in trials to conceal whether a treatment is being given or not and hence to control for the psychosomatic effects of offering treatment. Placebo-controlled trials are controversial. Critics of such trials argue that if a proven effective therapy exists, a placebo should not be used. But proponents argue that placebo trials are still crucial to prove the efficacy and safety of many treatments.     

Bacteriophage lambda display of complex cDNA libraries: a new approach to functional genomics

We describe the construction and characterization of two lambda surface displayed cDNA expression libraries derived from human brain and mouse embryo. cDNA inserts were obtained by tagged random-priming elongation of commercially available cDNA libraries and cloned into a novel lambda vector at the 3' end of the D capsid protein gene, which produced highly complex repertoires (1x10(8) and 2x10(7) phage). These libraries were affinity selected with a monoclonal antibody against the neural specific factor GAP-43 and with polyclonal antibodies that recognize the EMX1 and EMX2 homeoproteins. In both cases rapid identification of specific clones was achieved, which demonstrates the great potential of the lambda display system for generating affinity selectable cDNA libraries from complex genomes.     

A model for the hepatitis C virus envelope glycoprotein E2

Several experimental studies on hepatitis C virus (HCV) have suggested the envelope glycoprotein E2 as a key antigen for an effective vaccine against the virus. Knowledge of its structure, therefore, would present a significant step forward in the fight against this disease. This paper reports the application of fold recognition methods in order to produce a model of the HCV E2 protein. Such investigation highlighted the envelope protein E of Tick Borne Encephalitis virus as a possible template for building a model of HCV E2. Mapping of experimental data onto the model allowed the prediction of a composite interaction site between E2 and its proposed cellular receptor CD81, as well as a heparin binding domain. In addition, experimental evidence is provided to show that CD81 recognition by E2 is isolate or strain specific and possibly mediated by the second hypervariable region (HVR2) of E2. Finally, the studies have also allowed a rough model for the quaternary structure of the envelope glycoproteins E1 and E2 complex to be proposed. Proteins 2000;40:355-366.     

Characterization of the hepatitis C virus NS2/3 processing reaction by using a purified precursor protein

The NS2-NS3 region of the hepatitis C virus polyprotein encodes a proteolytic activity that is required for processing of the NS2/3 junction. Membrane association of NS2 and the autocatalytic nature of the NS2/3 processing event have so far constituted hurdles to the detailed investigation of this reaction. We now report the first biochemical characterization of the self-processing activity of a purified NS2/3 precursor. Using multiple sequence alignments, we were able to define a minimal domain, devoid of membrane-anchoring sequences, which was still capable of performing the processing reaction. This truncated protein was efficiently expressed and processed in Escherichia coli. The processing reaction could be significantly suppressed by growth in minimal medium in the absence of added zinc ions, leading to the accumulation of an unprocessed precursor protein in inclusion bodies. This protein was purified to homogeneity, refolded, and shown to undergo processing at the authentic NS2/NS3 cleavage site with rates comparable to those observed using an in vitro-translated full-length NS2/3 precursor. Size-exclusion chromatography and a dependence of the processing rate on the concentration of truncated NS2/3 suggested a functional multimerization of the precursor protein. However, we were unable to observe trans cleavage activity between cleavage-site mutants and active-site mutants. Furthermore, the cleavage reaction of the wild-type protein was not inhibited by addition of a mutant that was unable to undergo self-processing. Site-directed mutagenesis data and the independence of the processing rate from the nature of the added metal ion argue in favor of NS2/3 being a cysteine protease having Cys993 and His952 as a catalytic dyad. We conclude that a purified protein can efficiently reproduce processing at the NS2/3 site in the absence of additional cofactors.     

Nitric oxide reacts with the single-electron reduced active site of cytochrome c oxidase

The reduction kinetics of the mutants K354M and D124N of the Paracoccus denitrificans cytochrome oxidase (heme aa(3)) by ruthenium hexamine was investigated by stopped-flow spectrophotometry in the absence/presence of NO. Quick heme a reduction precedes the biphasic heme a(3) reduction, which is extremely slow in the K354M mutant (k(1) = 0.09 +/- 0.01 s(-1); k(2) = 0.005 +/- 0.001 s(-1)) but much faster in the D124N aa(3) (k(1) = 21 +/- 6 s(-1); k(2) = 2.2 +/- 0.5 s(-1)). NO causes a very large increase (>100-fold) in the rate constant of heme a(3) reduction in the K354M mutant but only a approximately 5-fold increase in the D124N mutant. The K354M enzyme reacts rapidly with O(2) when fully reduced but is essentially inactive in turnover; thus, it was proposed that impaired reduction of the active site is the cause of activity loss. Since at saturating [NO], heme a(3) reduction is approximately 100-fold faster than the extremely low turnover rate, we conclude that, contrary to O(2), NO can react not only with the two-electron but also with the single-electron reduced active site. This mechanism would account for the efficient inhibition of cytochrome oxidase activity by NO in the wild-type enzyme, both from P. denitrificans and from beef heart. Results also suggest that the H(+)-conducting K pathway, but not the D pathway, controls the kinetics of the single-electron reduction of the active site.     

Transgenic mouse models for studying the functions of insulin-like growth factor-binding proteins.

The insulin-like growth factor-binding proteins (IGFBPs) comprise a family of six related peptides that interact with high affinity with IGFs. IGFBPs compete with IGF receptors for IGF binding, and as a consequence of this competition they can affect cell growth. In addition, IGF-independent regulatory mechanisms of IGFBPs have been described. Despite their common property to interact with IGFs every IGFBP is expressed in a tightly regulated time- and tissue-specific manner suggesting that each protein may have its own distinct functions. Several transgenic mouse models overexpressing IGFBP-1, -2, -3, or -4 were developed in the past few years. Brain abnormalities were a common feature of IGFBP-1 transgenic models. Individual strains showed alterations in glucose homeostasis, reproductive performance, and a reduction of somatic growth as the most prominent phenotypes. The latter was also the main effect observed in IGFBP-2 transgenic mice. The overexpression of IGFBP-3 under the control of an ubiquitous promoter resulted in selective organomegaly, whereas mammary gland-targeted expression of this protein caused an altered involution after pregnancy in this organ. Tissue-specific overexpression of IGFBP-4 resulted in hypoplasia and reduced weight of smooth muscle-rich tissues such as bladder, aorta, and stomach. This review summarizes the current knowledge about the actions of IGFBPs in vivo based on the presently established transgenic mice.