A complete phylogeny of the whales, dolphins and even-toed hoofed mammals (Cetartiodactyla)

Despite the biological and economic importance of the Cetartiodactyla, the phylogeny of this clade remains controversial. Using the supertree approach of matrix representation with parsimony, we present the first phylogeny to include all 290 extant species of the Cetacea (whales and dolphins) and Artiodactyla (even-toed hoofed mammals). At the family-level, the supertree is fully resolved. For example, the relationships among the Ruminantia appear as (((Cervidae, Moschidae) Bovidae) (Giraffidae, Antilocapridae) Tragulidae). However, due to either lack of phylogenetic study or contradictory information, polytomies occur within the clades Sus, Muntiacus, Cervus, Delphinidae, Ziphiidae and Bovidae. Complete species-level phylogenies are necessary for both illustrating and analysing biological, geographical and ecological patterns in an evolutionary framework. The present species-level tree of the Cetartiodactyla provides the first opportunity to examine comparative hypotheses across entirely aquatic and terrestrial species within a single mammalian order.     

Multiple sclerosis: MHC associations and therapeutic implications

Multiple sclerosis (MS) is an autoimmune disease with an important genetic component. The strongest genetic association is with the major histocompatibility complex (MHC) region. Several MHC alleles predispose to the disease, the most prominent of which are certain alleles in the HLA-DR2 haplotype. Functional and structural studies have helped to explain the molecular basis of these associations. Although there is currently no curative treatment for MS, an increased understanding of the disease has aided the design of immunotherapies that act on the immune system more specifically than the longstanding drugs. Many of these therapies work at the antigen-specific level, disrupting the interaction between T-cell receptors and MHC molecules that leads to disease.     

Novel vacuolate sulfur bacteria from the Gulf of Mexico reproduce by reductive division in three dimensions

Large spherical sulfur bacteria, 180-375 microm in diameter, were found regularly and in abundance in surface sediments collected from hydrocarbon seeps (water depth 525-640 m) in the Gulf of Mexico. These bacteria were characterized by a thin 'shell' of sulfur globule-filled cytoplasm that surrounded a central vacuole (roughly 80% of biovolume) containing high concentrations of nitrate (average 460 mM). Approximately 800 base pairs of 16S rRNA gene sequence data, linked to this bacterium by fluorescent in situ hybridization, showed 99% identity with Thiomargarita namibiensis, previously described only from sediments collected off the coast of Namibia (Western Africa). Unlike T. namibiensis, where cells form a linear chain within a common sheath, the Gulf of Mexico strain occurred as single cells and clusters of two, four and eight cells, which were clearly the product of division in one to three planes. In sediment cores maintained at 4 degrees C, which undoubtedly experienced a diminishing flux of hydrogen sulfide over time, the Thiomargarita-like bacterium remained viable for up to 2 years. During that long period, each cell appeared to undergo (as judged by change in biovolume) one to three reductive divisions, perhaps as a dispersal strategy in the face of diminished availability of its putative electron donor.     

An encoded N-terminal extension results in low levels of heterologous protein production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

The tdk gene (encoding deoxythymidine kinase) of the gamma-proteobacterium Xenorhabdus nematophila has two potential translation start sites. The promoter-distal start site was predicted to be functional based on amino acid sequence alignment with closely related Tdk proteins. However, to experimentally determine if either of the two possible start codons allows production of a functional Tdk, we expressed the "long-form" (using the promoter-proximal start codon) and "short-form" (using the promoter-distal start codon) X. nematophila tdk genes from the T7 promoter of the pET-28a(+) vector. We assessed Tdk production and activity using a functional assay in an Escherichia coli tdk mutant, which, since it lacks functional Tdk, is able to grow in 5-fluorodeoxyuridine (FUdR)-containing medium.     

Classification of the extracellular fields produced by activated neural structures

Background  

Classifying the types of extracellular potentials recorded when neural structures are activated is an important component in understanding nerve pathophysiology. Varying definitions and approaches to understanding the factors that influence the potentials recorded during neural activity have made this issue complex.     

Protein--protein interactions in the eubacterial replisome

Replication of genomic DNA is a universal process that proceeds in distinct stages, from initiation to elongation and finally to termination. Each stage involves multiple stable or transient interactions between protein subunits with functions that are more or less conserved in all organisms. In Escherichia coli, initiation of bidirectional replication at the origin (oriC) occurs through the concerted actions of the DnaA replication initiator protein, the hexameric DnaB helicase, the DnaC?helicase loading partner and the DnaG primase, leading to establishment of two replication forks. Elongation of RNA primers at each fork proceeds simultaneously on both strands by actions of the multimeric replicase, DNA polymerase III holoenzyme. The fork that arrives first in the terminus region is halted by its encounter with a correctly-oriented complex of the Tus replication terminator protein bound at one of several Ter sites, where it is trapped until the other fork arrives. We summarize current understanding of interactions among the various proteins that act in the different stages of replication of the chromosome of E. coli, and make some comparisons with the analogous proteins in Bacillus subtilis and the coliphages T4 and T7.     

A phylogeny of bacterial RNA nucleotidyltransferases: Bacillus halodurans contains two tRNA nucleotidyltransferases

We have analyzed the distribution of RNA nucleotidyltransferases from the family that includes poly(A) polymerases (PAP) and tRNA nucleotidyltransferases (TNT) in 43 bacterial species. Genes of several bacterial species encode only one member of the nucleotidyltransferase superfamily (NTSF), and if that protein functions as a TNT, those organisms may not contain a poly(A) polymerase I like that of Escherichia coli. The genomes of several of the species examined encode more than one member of the nucleotidyltransferase superfamily. The function of some of those proteins is known, but in most cases no biochemical activity has been assigned to the NTSF. The NTSF protein sequences were used to construct an unrooted phylogenetic tree. To learn more about the function of the NTSFs in species whose genomes encode more than one, we have examined Bacillus halodurans. We have demonstrated that B. halodurans adds poly(A) tails to the 3' ends of RNAs in vivo. We have shown that the genes for both of the NTSFs encoded by the B. halodurans genome are transcribed in vivo. We have cloned, overexpressed, and purified the two NTSFs and have shown that neither functions as poly(A) polymerase in vitro. Rather, the two proteins function as tRNA nucleotidyltransferases, and our data suggest that, like some of the deep branching bacterial species previously studied by others, B. halodurans possesses separate CC- and A-adding tRNA nucleotidyltransferases. These observations raise the interesting question of the identity of the enzyme responsible for RNA polyadenylation in Bacillus.     

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (IspC) from Mycobacterium tuberculosis: towards understanding mycobacterial resistance to fosmidomycin

1-Deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the mevalonate-independent isopentenyl diphosphate biosynthetic pathway and is a potential drug target in some pathogenic bacteria. The antibiotic fosmidomycin has been shown to inhibit IspC in a number of organisms and is active against most gram-negative bacteria but not gram positives, including Mycobacterium tuberculosis, even though the mevalonate-independent pathway is the sole isopentenyl diphosphate biosynthetic pathway in this organism. Therefore, the enzymatic properties of recombinant IspC from M. tuberculosis were characterized. Rv2870c from M. tuberculosis converts 1-deoxy-d-xylulose 5-phosphate to 2-C-methyl-d-erythritol 4-phosphate in the presence of NADPH. The enzymatic activity is dependent on the presence of Mg(2+) ions and exhibits optimal activity between pH 7.5 and 7.9; the K(m) for 1-deoxyxylulose 5-phosphate was calculated to be 47.1 microM, and the K(m) for NADPH was 29.7 microM. The specificity constant of Rv2780c in the forward direction is 1.5 x 10(6) M(-1) min(-1), and the reaction is inhibited by fosmidomycin, with a 50% inhibitory concentration of 310 nM. In addition, Rv2870c complements an inactivated chromosomal copy of IspC in Salmonella enterica, and the complemented strain is sensitive to fosmidomycin. Thus, M. tuberculosis resistance to fosmidomycin is not due to intrinsic properties of Rv2870c, and the enzyme appears to be a valid drug target in this pathogen.