Structure and function of repetitive sequence elements associated with a highly polymorphic domain of the Neisseria meningitidis PilQ protein

Secretins are a large family of proteins associated with membrane translocation of macromolecular complexes, and a subset of this family, termed PilQ proteins, is required for type IV pilus biogenesis. We analysed the status of PilQ expression in Neisseria meningitidis (Mc) and found that PilQ^? mutants were non-piliated and deficient in the expression of pilus-associated phenotypes. Sequence analysis of the 5′ portion of the pilQ ORF of the serogroup B Mc strain 44/76 showed the presence of seven copies of a repetitive sequence element, in contrast to the situation in N. gonorrhoeae (Gc) strains, which carry either two or three copies of the repeat. The derived amino acid sequence of the consensus nucleotide repeat was an octapeptide PAKQQAAA, designated as the small basic repeat (SBR). This gene segment was studied in more detail in a collection of 52 Mc strains of diverse origin by screening for variability in the size of the PCR-generated DNA fragments spanning the SBRs. These strains were found to harbour from four to seven copies of the repetitive element. No association between the number of copies and the serogroup, geographic origin or multilocus genotype of the strains was evident. The presence of polymorphic repeat elements in Mc PilQ is unprecedented within the secretin family. To address the potential function of the repeat containing domain, Mc strains were constructed so as to express chimeric PilQ molecules in which the number of SBR repeats was increased or in which the repeat containing domain was replaced in toto by the corresponding region of the Pseudomonas aeruginosa (Pa) PilQ protein. Although the strain expressing PilQ with an increased number of SBRs was identical to the parent strain in pilus phenotypes, a strain expressing PilQ with the equivalent Pa domain had an eightfold reduction in pilus expression level. The findings suggest that the repeat containing domain of PilQ influences Mc pilus expression quantitatively but not qualitatively.     

The number of Na:K pump sites on red blood cells from HK and LK lambs

Lambs of known genotype with respect to the locus determining cation composition of red cells were obtained by selective matings. Numbers of K⁺ pump sites per cell were determined on HK and LK lambs 10–20 days postnatal by simultaneously determining [³H]ouabain binding and inhibition of active K⁺ transport. Red cells from HK lambs were indistinguishable from adult HK cells with regard to the K⁺ pump flux and number of pump sites. Cells from genetically LK lambs had pump fluxes and numbers of pump sites intermediate between those from adult HK and LK sheep. The results suggest that the change in cation composition and in the K⁺ pump during the first 60 days in genetically LK lambs can be correlated with a reduced number of K⁺ pump sites.     

The extent of linkage disequilibrium in four populations with distinct demographic histories

The design and feasibility of whole-genome-association studies are critically dependent on the extent of linkage disequilibrium (LD) between markers. Although there has been extensive theoretical discussion of this, few empirical data exist. The authors have determined the extent of LD among 38 biallelic markers with minor allele frequencies >.1, since these are most comparable to the common disease-susceptibility polymorphisms that association studies aim to detect. The markers come from three chromosomal regions-1,335 kb on chromosome 13q12-13, 380 kb on chromosome 19q13.2, and 120 kb on chromosome 22q13.3-which have been extensively mapped. These markers were examined in approximately 1,600 individuals from four populations, all of European origin but with different demographic histories; Afrikaners, Ashkenazim, Finns, and East Anglian British. There are few differences, either in allele frequencies or in LD, among the populations studied. A similar inverse relationship was found between LD and distance in each genomic region and in each population. Mean D' is.68 for marker pairs <5 kb apart and is.24 for pairs separated by 10-20 kb, and the level of LD is not different from that seen in unlinked marker pairs separated by >500 kb. However, only 50% of marker pairs at distances <5 kb display sufficient LD (delta>.3) to be useful in association studies. Results of the present study, if representative of the whole genome, suggest that a whole-genome scan searching for common disease-susceptibility alleles would require markers spaced < or = 5 kb apart.     

Effect of Rainbow Trout Growth Hormone Complementary DNA on Body Shape,Carcass Yield,and Carcass Composition of F1 and F2 Transgenic Common Carp (Cyprinus carpio

The effect of rainbow trout growth hormone complementary DNA on body shape, dress-out yield, and body composition were assessed in the F1 and F2 generations of transgenic common carp (Cyprinus carpio). All measurements were compared with those for nontransgenic full-sibling common carp in their respective families, and the fish were communally evaluated in earthen ponds. The body weight and length were highly correlated (P <0.01) in both genotypes in all the families. Head morphometrics were negatively correlated (P <0.05) to weight and length of the fish. Various head, body, and caudal traits grew disproportionately faster in transgenic fish in both generations. The altered body shape of transgenic fish resulted in improved dressing percentage in the F2 generation. The carcass composition of transgenic muscle had a lower percentage of (P <0.01) moisture and lipids and higher (P <0.01) percentage of protein in both generations. Six of the 18 amino acids analyzed in F1 transgenic common carp muscle were higher F1 (P <0.05) than the control genotype; however, amino acid ratios were minimally changed. Also, the fatty acid profiles of both genotypes were minimally altered. Higher histidine and lysine ratios in the diet are recommended for maximum growth and health of transgenic common carp in intensive culture systems on the basis of essential amino acid ratios.     

Molecular systematics and origin of sociality in mongooses (Herpestidae, Carnivora)

The Herpestidae are small terrestrial carnivores comprising 18 African and Asian genera, currently split into two subfamilies, the Herpestinae and the Galidiinae. The aim of this work was to resolve intra-familial relationships and to test the origin of sociality in the group. For this purpose we analysed sequences of the complete cytochrome b gene for 18 species of Herpestidae. The results showed that the mongooses were split into three clades: (1) the Malagasy taxa (Galidiinae and Cryptoprocta), (2) the true social mongooses and (3) the solitary mongooses, each group being also supported by morphological and chromosomal data. Our results suggested unexpected phylogenetic relationships: (1) the genus Cynictis is included in the solitary mongoose clade, (2) the genera Liberiictis and Mungos are sister-group, and (3) the genus Herpestes is polyphyletic. We examined the evolution of the sociality in mongooses by combining behavioural traits with the cytochrome b data. Some of the behavioural traits provided good synapomorphies for characterizing the social species clade, showing the potential benefit of using such characters in phylogeny. The mapping of ecological and behavioural features resulted in hypothesizing solitary behavior and life in forest as the conditions at the base of the mongoose clade.     

Oxidation alters the architecture of the phenylalanyl-tRNA synthetase editing domain to confer hyperaccuracy

High fidelity during protein synthesis is accomplished by aminoacyl-tRNA synthetases (aaRSs). These enzymes ligate an amino acid to a cognate tRNA and have proofreading and editing capabilities that ensure high fidelity. Phenylalanyl-tRNA synthetase (PheRS) preferentially ligates a phenylalanine to a tRNA^Phe over the chemically similar tyrosine, which differs from phenylalanine by a single hydroxyl group. In bacteria that undergo exposure to oxidative stress such as Salmonella enterica serovar Typhimurium, tyrosine isomer levels increase due to phenylalanine oxidation. Several residues are oxidized in PheRS and contribute to hyperactive editing, including against mischarged Tyr-tRNA^Phe, despite these oxidized residues not being directly implicated in PheRS activity. Here, we solve a 3.6 Å cryo-electron microscopy structure of oxidized S. Typhimurium PheRS. We find that oxidation results in widespread structural rearrangements in the β-subunit editing domain and enlargement of its editing domain. Oxidization also enlarges the phenylalanyl-adenylate binding pocket but to a lesser extent. Together, these changes likely explain why oxidation leads to hyperaccurate editing and decreased misincorporation of tyrosine. Taken together, these results help increase our understanding of the survival of S. Typhimurium during human infection.     

Cobalamin-dependent methionine synthase from Escherichia coli B: electron paramagnetic resonance spectra of the inactive form and the active methylated form of the enzyme

Cobalamin-dependent methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) has been isolated from Escherichia coli B in homogeneous form. The enzyme is isolated in an inactive form with the visible absorbance properties of cob(II)alamin. The inactive enzyme exhibits an electron paramagnetic resonance (EPR) spectrum at 38 K that is characteristic of cob(II)alamin at acid pH, where the protonated dimethylbenzimidazole substituent is not coordinated with the cobalt nucleus (base-off cobalamin). An additional, variable component of the EPR spectrum of the inactive enzyme has the characteristics of a cob(III)alamin-superoxide complex. Previous work by others [Taylor, R.T., & Weissbach, H. (1969) Arch. Biochem. Biophys. 129, 745-766. Fujii, K., & Huennekens, F.M. (1979) in Biochemical Aspects of Nutrition (Yagi, K., Ed.) pp 173-183, Japan Scientific Societies, Tokyo] has demonstrated that the enzyme can be activated by reductive methylation using adenosylmethionine as the methyl donor. We present data indicating that the conversion of inactive to methylated enzyme is correlated with the disappearance of the EPR spectrum as expected for the conversion of paramagnetic cob(II)alamin to diamagnetic methylcobalamin. When the methyl group is transferred from the methylated enzyme to homocysteine under aerobic conditions, cob(II)alamin/cob(III)alamin-superoxide enzyme is regenerated as indicated by the return of the visible absorbance properties of the initially isolated enzyme and partial return of the EPR spectrum. Our enzyme preparations contain copper in approximately 1:1 stoichiometry with cobalt as determined by atomic absorption spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Doc Toxin Is a Kinase That Inactivates Elongation Factor Tu

The Doc toxin from bacteriophage P1 (of the phd-doc toxin-antitoxin system) has served as a model for the family of Doc toxins, many of which are harbored in the genomes of pathogens. We have shown previously that the mode of action of this toxin is distinct from the majority derived from toxin-antitoxin systems: it does not cleave RNA; in fact P1 Doc expression leads to mRNA stabilization. However, the molecular triggers that lead to translation arrest are not understood. The presence of a Fic domain, albeit slightly altered in length and at the catalytic site, provided a clue to the mechanism of P1 Doc action, as most proteins with this conserved domain inactivate GTPases through addition of an adenylyl group (also referred to as AMPylation). We demonstrated that P1 Doc added a single phosphate group to the essential translation elongation factor and GTPase, elongation factor (EF)-Tu. The phosphorylation site was at a highly conserved threonine, Thr-382, which was blocked when EF-Tu was treated with the antibiotic kirromycin. Therefore, we have established that Fic domain proteins can function as kinases. This distinct enzymatic activity exhibited by P1 Doc also solves the mystery of the degenerate Fic motif unique to the Doc family of toxins. Moreover, we have established that all characterized Fic domain proteins, even those that phosphorylate, target pivotal GTPases for inactivation through a post-translational modification at a single functionally critical acceptor site.