14 research outputs found
The structure of M.EcoKI Type I DNA methyltransferase with a DNA mimic antirestriction protein
Type-I DNA restriction–modification (R/M) systems are important agents in limiting the transmission of mobile genetic elements responsible for spreading bacterial resistance to antibiotics. EcoKI, a Type I R/M enzyme from Escherichia coli, acts by methylation- and sequence-specific recognition, leading to either methylation of DNA or translocation and cutting at a random site, often hundreds of base pairs away. Consisting of one specificity subunit, two modification subunits, and two DNA translocase/endonuclease subunits, EcoKI is inhibited by the T7 phage antirestriction protein ocr, a DNA mimic. We present a 3D density map generated by negative-stain electron microscopy and single particle analysis of the central core of the restriction complex, the M.EcoKI M2S1 methyltransferase, bound to ocr. We also present complete atomic models of M.EcoKI in complex with ocr and its cognate DNA giving a clear picture of the overall clamp-like operation of the enzyme. The model is consistent with a large body of experimental data on EcoKI published over 40 years
Impact of target site distribution for Type I restriction enzymes on the evolution of methicillin-resistant Staphylococcus aureus (MRSA) populations.
A limited number of Methicillin-resistant Staphylococcus aureus (MRSA) clones are responsible for MRSA infections worldwide, and those of different lineages carry unique Type I restriction-modification (RM) variants. We have identified the specific DNA sequence targets for the dominant MRSA lineages CC1, CC5, CC8 and ST239. We experimentally demonstrate that this RM system is sufficient to block horizontal gene transfer between clinically important MRSA, confirming the bioinformatic evidence that each lineage is evolving independently. Target sites are distributed randomly in S. aureus genomes, except in a set of large conjugative plasmids encoding resistance genes that show evidence of spreading between two successful MRSA lineages. This analysis of the identification and distribution of target sites explains evolutionary patterns in a pathogenic bacterium. We show that a lack of specific target sites enables plasmids to evade the Type I RM system thereby contributing to the evolution of increasingly resistant community and hospital MRSA
Seasonal fluctuations in birth weight and neonatal limb length; Does prenatal vitamin D influence neonatal size and shape?
Background: Birth weight is known to fluctuate with season of birth, however, there is little information about seasonal variation in neonatal anthropometric measures
Global loss of Bmal1 expression alters adipose tissue hormones, gene expression and glucose metabolism
Extent: 11p.The close relationship between circadian rhythm disruption and poor metabolic status is becoming increasingly evident, but role of adipokines is poorly understood. Here we investigated adipocyte function and the metabolic status of mice with a global loss of the core clock gene Bmal1 fed either a normal or a high fat diet (22% by weight). Bmal1 null mice aged 2 months were killed across 24 hours and plasma adiponectin and leptin, and adipose tissue expression of Adipoq, Lep, Retn and Nampt mRNA measured. Glucose, insulin and pyruvate tolerance tests were conducted and the expression of liver glycolytic and gluconeogenic enzyme mRNA determined. Bmal1 null mice displayed a pattern of increased plasma adiponectin and plasma leptin concentrations on both control and high fat diets. Bmal1 null male and female mice displayed increased adiposity (1.8 fold and 2.3 fold respectively) on the normal diet, but the high fat diet did not exaggerate these differences. Despite normal glucose and insulin tolerance, Bmal1 null mice had increased production of glucose from pyruvate, implying increased liver gluconeogenesis. The Bmal1 null mice had arrhythmic clock gene expression in epigonadal fat and liver, and loss of rhythmic transcription of a range of metabolic genes. Furthermore, the expression of epigonadal fat Adipoq, Retn, Nampt, AdipoR1 and AdipoR2 and liver Pfkfb3 mRNA were down-regulated. These results show for the first time that global loss of Bmal1, and the consequent arrhythmicity, results in compensatory changes in adipokines involved in the cellular control of glucose metabolism.David John Kennaway, Tamara Jayne Varcoe, Athena Voultsios, Michael James Bode
The structure of the KlcA and ArdB proteins reveals a novel fold and antirestriction activity against Type I DNA restriction systems in vivo but not in vitro
Plasmids, conjugative transposons and phage frequently encode anti-restriction proteins to enhance their chances of entering a new bacterial host that is highly likely to contain a Type I DNA restriction and modification (RM) system. The RM system usually destroys the invading DNA. Some of the anti-restriction proteins are DNA mimics and bind to the RM enzyme to prevent it binding to DNA. In this article, we characterize ArdB anti-restriction proteins and their close homologues, the KlcA proteins from a range of mobile genetic elements; including an ArdB encoded on a pathogenicity island from uropathogenic Escherichia coli and a KlcA from an IncP-1b plasmid, pBP136 isolated from Bordetella pertussis. We show that all the ArdB and KlcA act as anti-restriction proteins and inhibit the four main families of Type I RM systems in vivo, but fail to block the restriction endonuclease activity of the archetypal Type I RM enzyme, EcoKI, in vitro indicating that the action of ArdB is indirect and very different from that of the DNA mimics. We also present the structure determined by NMR spectroscopy of the pBP136 KlcA protein. The structure shows a novel protein fold and it is clearly not a DNA structural mimic
Pineal gland function during the reproductive cycle : a multispecies study
v, 175 leaves : photos., graphs, tables ; 31 cm.David John KennawayThesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 197
Plasma metabolites, insulin, and adipokines in 8 week old wild-type and <i>Bmal1</i> null mice.
<p>Plasma glucose (a), free fatty acids (b), insulin (c), adiponectin (d) and leptin (e) levels. Data are the mean ± s.e.m. for <i>n = </i>4 mice of each genotype at each time point, wild-type mice (open circles) and <i>Bmal1</i> null mice (closed circles). The accompanying histograms represent the estimated marginal means ± s.e.m. of the individual gene expression as calculated from the ANOVA. The shaded areas represent the period of darkness. The symbol * indicates that there was a significant difference (<i>P</i><0.05) between the genotypes.</p
The relative gene expression across 24 h of clock and liver enzyme genes in the liver of male wild-type and <i>Bmal1</i> null mice fed a normal rodent diet.
<p>(a) <i>Bmal1</i>, (b) <i>Per2</i>, (c) <i>Pfkfb3</i>, (d) <i>Pck</i>, (e) <i>Fbp1</i>, (f) <i>G6pc,</i> (g) <i>Gck</i> and (h) <i>Adipor2.</i> The data are the relative expression for each gene compared to <i>Actin</i> mRNA (mean ± s.e.m., n = 4 for each genotype), wild-type mice (open circles) and <i>Bmal1</i> null mice (closed circles). The highest expression of each gene for wild-type mice was set at one. The apparent absence of an SEM bar indicates that it is obscured by the symbol. The shaded areas represent the period of darkness.</p
The blood glucose response to the intra peritoneal administration of pyruvate (2 mg/g) to male and female wild-type and <i>Bmal1</i> null mice fed a normal rodent diet.
<p>The mean ± s.e.m. plasma glucose levels are shown for male (a) and female (c) wild-type (open circles) and <i>Bmal1</i> null (closed circles) mice. The mean area under the curve (± SEM) of the plasma glucose profiles up to 120 min post injection (b, d).</p