A simple modification to the luminometric methylation assay to control for the effects of DNA fragmentation.

Variations in DNA methylation have been implicated in a number of disorders. Changes in global DNA methylation levels have long been associated with various types of cancer. One of the recently described methods for determining global DNA methylation levels is the LUminometric Methylation Assay (LUMA), which utilizes methylation sensitive and insensitive restriction endonucleases and pyrosequencing technology for quantification.

Inter- and intra-specific pan-genomes of Borrelia burgdorferi sensu lato: genome stability and adaptive radiation.

Background: Lyme disease is caused by spirochete bacteria from the Borrelia burgdorferi sensu lato (B. burgdorferi s.l.

Heterologous expression and purification of Arabidopsis thaliana VIM1 protein: in vitro evidence for its inability to recognize hydroxymethylcytosine, a rare base in Arabidopsis DNA.

The discovery of 5-hydroxymethyl-cytosine (5hmC) in mammalian cells prompted us to look for this base in the DNA of Arabidopsis thaliana (thale cress), and to ask how well the Arabidopsis Variant in Methylation 1 (VIM1) protein, an essential factor in maintaining 5-cytosine methylation (5mC) homeostasis and epigenetic silencing in this plant, recognizes this novel base. We found that the DNA of Arabidopsis' leaves and flowers contain low levels of 5hmC. We also cloned and expressed in Escherichia coli full-length VIM1 protein, the archetypal member of the five Arabidopsis VIM gene family.

Genome stability of Lyme disease spirochetes: comparative genomics of Borrelia burgdorferi plasmids.

Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B.

Whole-genome sequences of Borrelia bissettii, Borrelia valaisiana, and Borrelia spielmanii.

It has been known for decades that human Lyme disease is caused by the three spirochete species Borrelia burgdorferi, Borrelia afzelii, and Borrelia garinii. Recently, Borrelia valaisiana, Borrelia spielmanii, and Borrelia bissettii have been associated with Lyme disease. We report the complete genome sequences of B.

Distinct p53 genomic binding patterns in normal and cancer-derived human cells.

We report here genome-wide analysis of the tumor suppressor p53 binding sites in normal human cells. 743 high-confidence ChIP-seq peaks representing putative genomic binding sites were identified in normal IMR90 fibroblasts using a reference chromatin sample. More than 40% were located within 2 kb of a transcription start site (TSS), a distribution similar to that documented for individually studied, functional p53 binding sites and, to date, not observed by previous p53 genome-wide studies.

Whole-genome sequences of two Borrelia afzelii and two Borrelia garinii Lyme disease agent isolates.

Human Lyme disease is commonly caused by several species of spirochetes in the Borrelia genus. In Eurasia these species are largely Borrelia afzelii, B. garinii, B.

Enzyme activity in dialkyl phosphate ionic liquids.

The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariella volvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate.

A new approach to a Lyme disease vaccine.

A single recombinant outer surface protein A (OspA) antigen designed to contain protective elements from 2 different OspA serotypes (1 and 2) is able to induce antibody responses that protect mice against infection with either Borrelia burgdorferi sensu stricto (OspA serotype-1) or Borrelia afzelii (OspA serotype-2). Protection against infection with B burgdorferi ss strain ZS7 was demonstrated in a needle-challenge model. Protection against B.

Whole genome sequence of an unusual Borrelia burgdorferi sensu lato isolate.

Human Lyme disease is caused by a number of related Borrelia burgdorferi sensu lato species. We report here the complete genome sequence of Borrelia sp. isolate SV1 from Finland.

Whole-genome sequences of thirteen isolates of Borrelia burgdorferi.

Borrelia burgdorferi is a causative agent of Lyme disease in North America and Eurasia. The first complete genome sequence of B. burgdorferi strain 31, available for more than a decade, has assisted research on the pathogenesis of Lyme disease.

Differential binding of Escherichia coli McrA protein to DNA sequences that contain the dinucleotide m5CpG.

The Escherichia coli McrA protein, a putative C(5)-methylcytosine/C(5)-hydroxyl methylcytosine-specific nuclease, binds DNA with symmetrically methylated HpaII sequences (Cm5CGG), but its precise recognition sequence remains undefined. To determine McrA's binding specificity, we cloned and expressed recombinant McrA with a C-terminal StrepII tag (rMcrA-S) to facilitate protein purification and affinity capture of human DNA fragments with m5C residues. Sequence analysis of a subset of these fragments and electrophoretic mobility shift assays with model methylated and unmethylated oligonucleotides suggest that N(Y > R) m5CGR is the canonical binding site for rMcrA-S.

Transcript profiling of human platelets using microarray and serial analysis of gene expression (SAGE).

Platelets are anucleated cells that are generated from megakaryocytes via thrombopoiesis. They lack genomic DNA but have a pool of individual mRNA transcripts. Taken together, these mRNAs constitute a platelet transcriptome.

Rapid detection and identification of a pathogen's DNA using Phi29 DNA polymerase.

Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents.

Cloning, purification and initial characterization of E. coli McrA, a putative 5-methylcytosine-specific nuclease.

Expression strains of Escherichia coli BL21(DE3) overproducing the E. coli m(5)C McrA restriction protein were produced by cloning the mcrA coding sequence behind a T7 promoter. The recombinant mcrA minus BL21(DE3) host produces active McrA as evidenced by its acquired ability to selectively restrict the growth of T7 phage containing DNA methylated in vitro by HpaII methylase.

Characterization of clinically-attenuated Burkholderia mallei by whole genome sequencing: candidate strain for exclusion from Select Agent lists.

Background: Burkholderia mallei is an understudied biothreat agent responsible for glanders which can be lethal in humans and animals. Research with this pathogen has been hampered in part by constraints of Select Agent regulations for safety reasons. Whole genomic sequencing (WGS) is an apt approach to characterize newly discovered or poorly understood microbial pathogens.

A new binding motif for the transcriptional repressor REST uncovers large gene networks devoted to neuronal functions.

The repressor element 1 (RE1) silencing transcription factor (REST) helps preserve the identity of nervous tissue by silencing neuronal genes in non-neural tissues. Moreover, in an epithelial model of tumorigenesis, loss of REST function is associated with loss of adhesion, suggesting the aberrant expression of REST-controlled genes encoding this property. To date, no adhesion molecules under REST control have been identified.

Paired-end genomic signature tags: a method for the functional analysis of genomes and epigenomes.

Because paired-end genomic signature tags are sequenced-based, they have the potential to become an alternate tool to tiled microarray hybridization as a method for genome-wide localization of transcription factors and other sequence-specific DNA binding proteins. As outlined here the method also can be used for global analysis of DNA methylation. One advantage of this approach is the ability to easily switch between different genome types without having to fabricate a new microarray for each and every DNA type.

Characterization of a unique borreliacidal epitope on the outer surface protein C of Borrelia burgdorferi.

The outer surface protein C (OspC) of the Lyme disease agent, Borrelia burgdorferi, is an immunoprotective antigen in laboratory models of infection. However, to understand its protective effects, it is important to identify the key epitopes of this protein. We produced a borreliacidal anti-OspC monoclonal antibody specific to the B31 strain and identified its binding site.

Enzymatic activity of the SARS coronavirus main proteinase dimer.

The enzymatic activity of the SARS coronavirus main proteinase dimer was characterized by a sensitive, quantitative assay. The new, fluorogenic substrate, (Ala-Arg-Leu-Gln-NH)(2)-Rhodamine, contained a severe acute respiratory syndrome coronavirus (SARS CoV) main proteinase consensus cleavage sequence and Rhodamine 110, one of the most detectable compounds known, as the reporter group. The gene for the enzyme was cloned in the absence of purification tags, expressed in Escherichia coli and the enzyme purified.

Structure-based design of a second-generation Lyme disease vaccine based on a C-terminal fragment of Borrelia burgdorferi OspA.

Here, we describe a structure-based approach to reduce the size of an antigen protein for a subunit vaccine. Our method consists of (i) determining the three-dimensional structure of an antigen, (ii) identifying protective epitopes, (iii) generation of an antigen fragment that contains the protective epitope, and (iv) rational design to compensate for destabilization caused by truncation. Using this approach we have successfully developed a second-generation Lyme disease vaccine.

Proteomic analysis of Lyme disease: global protein comparison of three strains of Borrelia burgdorferi.

The Borrelia burgdorferi spirochete is the causative agent of Lyme disease, the most common tick-borne disease in the United States. It has been studied extensively to help understand its pathogenicity of infection and how it can persist in different mammalian hosts. We report the proteomic analysis of the archetype B.

Structural investigation of Borrelia burgdorferi OspB, a bactericidal Fab target.

Certain antibody Fab fragments directed against the C terminus of outer surface protein B (OspB), a major lipoprotein of the Lyme disease spirochete, Borrelia burgdorferi, have the unusual property of being bactericidal even in the absence of complement. We report here x-ray crystal structures of a C-terminal fragment of B. burgdorferi OspB, which spans residues 152-296, alone at 2.