No abstract

An isothermal signal amplification technique for specific DNA sequences, known as cycling probe technology (CPT), was performed within a microfluidic chip. The presence of DNA from methicillin-resistant Staphylococcus aureus was determined by signal amplification of a specific DNA sequence. The microfluidic device consisted of four channels intersecting to mix the sample and reagents within 55 s, as they were directed toward the reactor coil by electrokinetic pumping. The 160-nL CPT reactor occupied approximately 220 mm2. Gel-free capillary electrophoresis separation of the biotin- and fluorescein-labeled probe from the probe fragments was performed on-chip following the on-chip reaction. An off-chip CPT reaction, with on-chip separation gave a detection limit of 2 fM (0.03 amol) target DNA and an amplification factor of 85,000. Calibration curves, linear at <5% probe fragmentation, obeyed a power law relationship with an argument of 0.5 [target] at higher target DNA concentrations for both on-chip and off-chip CPT reaction and analysis. An amplification factor of 42,000 at 250 fM target (25,000 target molecules) was observed on-chip, but the reaction was approximately 4 times less sensitive than off-chip under the conditions used. Relative SD values for on-chip CPT were 0.8% for the peak migration times, 9% for the area of intact probe peak, and 8% for the fragment/probe peak area ratio.

The complete nucleotide sequence of the 71V-1658 strain of western equine encephalitis virus (WEE) was determined (minus 25 nucleotides from the 5h end). A 5h RACE reaction was used to sequence the 5h terminus from WEE strain CBA87. The deduced WEE genome was 11 508 nucleotides in length, excluding the 5h cap nucleotide and 3h poly(A) tail. The nucleotide composition was 28 % A, 25 % C, 25 % G and 22 % U. Comparison with partial WEE sequences of strain 5614 (nsP2-nsP3 of the nonstructural region) and strain BFS1703 (26S structural region) revealed comparatively little variation ; a total of 149 nucleotide differences in 8624 bases (1n7% divergence), of which only 28 % (42 nucleotides) altered the encoded amino acids. Comparison of deduced nsP1 and nsP4 amino acid sequences from WEE with the corresponding proteins from eastern equine encephalitis virus (EEE) yielded identities of 84n9 and 83n8 %, respectively. Previously uncharacterized stem-loop structures were identified in the nontranslated terminal regions. A cDNA clone of the 26S region encoding the structural polyprotein of WEE strain 71V-1658 was placed under the control of a cytomegalovirus promoter and transfected into tissue culture cells. The viral envelope proteins were functionally expressed in tissue culture, as determined by histochemical staining with monoclonal antibodies that recognize WEE antigens, thus, forming the initial step in the investigation of subunit vaccines to WEE.

The amino radicals (.AH) formed by the h°Co radiolysis of N,O-saturated 0.05 M solutions of ethylene diamine tetraacetate (EDTA) at pH 7 and 11.2 and of glycine at pH 11.2 brought about an efficient two-electron reduction of lumiflavin (FI). The spectra of the products were identical to those formed by photolysis of the same solutions and by reduction of the lumiflavin with .CO,-radicals. The products were reoxidised to flavin by oxygen. The quantum yield for flavin disappearance was 0.52 2 0.07 and 0.17 ? 0.01 in the presence of EDTA at pH 7 and 1 1.2 and 0.065 + 0.008 and 0.17 ? 0.01 for glycine at the same pHs, respectively.The overall two-electron reduction can be explained by the mechanism:The rate constants of reaction [4] were found by pulse radiolysis to be 1.8 ? 0.3 x 10' and 1.5 ? 0.3 X 10" M-' s-' for the radicals of glycine and EDTA at pH 7 and 3.6 * 0.3 x 1 0 H M S -I for glycine radicals at pH 11.2. The spectrum of .FIH formed by glycine radicals at pH 7 is similar to that produced by .CO,-, but there was some perturbation, which is apparently due to interaction with the amine. The radicals formed from the secondary amines piperazine and diethylamine at pH I 1.8 also effected reversible two-electron reduction. However, the radicals from glycine anhydride and the primary amine ethylamine yielded significant amounts of non-oxidisable products. The reaction mechanisms are discussed and effects of pH are considered. PARMINDER S . SURDHAR, DOUGLAS E. BADER et DAVID A. ARMSTRONG. Can. J. Chem. 63, 1357 (1985). Les radicaux arninks (.AH) issus de la radiolyse au "Co de solutions saturkes en N,O, contenant 0,05 M de tetraacktate d'kthylene diamine (TAED) at maintenues a des pH de 7 ou 11,2 ou contenant de la glycine a un pH 11,2, provoquent une rkduction deux Clectrons efficace de la lumiflavine (FI). Les spectres des produits sont identiques a ceux des produits obtenus par la photolyse des mCmes solutions et par la reduction de la lumiflavine par les radicaux .CO,-. L'oxygkne provoque une rkoxydation des produits en flavine. A des pH de 7 et 11,2 et en presence du TAED, les rendements quantiques pour la disparition de la flavine sont respectivement kgaux a 0,52 2 0,07 et de 0,17 ? 0,01 alors qu'en presence de glycine et aux mCmes pH ces rendements sont Cgaux a 0,065 2 0,008 et 0.17 ? 0,Ol.On peut ilustrer la reduction globale a deux Clectrons, par le mecanisme suivant:Faisant appel a la radiolyse pulsation, on a pu Ctablir que les constantes de vitesse de la reaction [4] sont Cgales a 1,8 2 0,3

ABSTRACTEnvironmental samples were collected from carcass sites during and after anthrax outbreaks in 2000 and 2001 in the bison (Bison bison) population within Wood Buffalo National Park and the Hook Lake Region north of Wood Buffalo National Park.Bacillus anthracisspores were isolated from these samples and confirmed using phenotypic characterization and real-time PCR. ConfirmedB. anthracisisolates were typed using multiple-locus variable-number tandem repeat analysis (MLVA15) and single-nucleotide-repeat analysis (SNRA).B. anthracisisolates split into two clades based on MLVA15, while SNRA allowed some isolates between carcass sites to be distinguished from each other. SNRA polymorphisms were also present within a single carcass site. Some isolates from different carcass sites having the same SNRA type had divergent MLVA types; this finding leads to questions about hierarchical typing methods and the robustness of the fine-scale typing ofBacillus anthracis.

An efficient synthesis of a fluorescent probe is described that can be specifically bound by the mannose binding FimH protein.

No abstract

Elongation factor Tu (EF-Tu), encoded by tuf genes, carries aminoacyl-tRNA to the ribosome during protein synthesis. Duplicated tuf genes (tufA and tufB), which are commonly found in enterobacterial species, usually coevolve via gene conversion and are very similar to one another. However, sequence analysis of tuf genes in our laboratory has revealed highly divergent copies in 72 strains spanning the genus Yersinia (representing 12 Yersinia species). The levels of intragenomic divergence between tufA and tufB sequences ranged from 8.3 to 16.2% for the genus Yersinia, which is significantly greater than the 0.0 to 3.6% divergence observed for other enterobacterial genera. We further explored tuf gene evolution in Yersinia and other Enterobacteriaceae by performing directed sequencing and phylogenetic analyses. Phylogenetic trees constructed using concatenated tufA and tufB sequences revealed a monophyletic genus Yersinia in the family Enterobacteriaceae. Moreover, Yersinia strains form clades within the genus that mostly correlate with their phenotypic and genetic classifications. These genetic analyses revealed an unusual divergence between Yersinia tufA and tufB sequences, a feature unique among sequenced Enterobacteriaceae and indicative of a genus-wide loss of gene conversion. Furthermore, they provided valuable phylogenetic information for possible reclassification and identification of Yersinia species.