17 research outputs found
Investigations on the occurrence of Plasmodium knowlesi in travellers returning from the endemic areas of simian malaria
Malaria remains an important public health issue all over the world. Among 5 Plasmodium species invasive to humans, Plasmodium knowlesi has been identified most recently. It is sometimes difficult to differentiate this species from P. malariae with the use of microscopic examination. However, P. knowlesi infection may be associated with rapidly increasing parasitaemia and severe clinical course with the risk of death. Samples from Polish travellers returning from areas where simian malaria is endemic were examined with the use of polymerase chain reaction (PCR). The small subunit of ribosomal RNA (SSU rRNA) genes was subjected to analysis using nested PCR reaction. No positive results of P. knowlesi were obtained. Due to morphological similarities to P. malariae, potentially severe clinical course of infection and P. knowlesi endemic regions being a common tourist destination, diagnostic and clinical vigilance is necessary, including molecular methods use for precise parasite identification
Real-Time Observation of Backtracking by Bacterial RNA Polymerase
RNA
polymerase (RNAP) backtracking is a backward sliding of the
enzyme along DNA and RNA. It plays important roles in many essential
processes in bacteria and in eukaryotes. We describe here a fluorescence-based
approach that allows a real-time observation of bacterial RNAP backtracking.
A Cy3 fluorescence probe, when incorporated into a specific site in
the nontemplate strand near the site of backtracking, allows RNAP
movements to be monitored near the probe because of a robust enhancement
of fluorescence caused by protein proximity. Using this approach,
we showed that binding of NTP to the active site prior to phosphodiester
bond formation inhibited backtracking, consistent with the coupling
of NTP binding to translocation. The extent and the kinetics of backtracking
did not show a simple correlation with the instability of the DNA–RNA
hybrid, indicating a more complex dependence of backtracking on DNA
template sequence. Experiments with transcription through an abasic
site in DNA template or neutravidin bound to biotinylated template
strand base illustrated an important role of backtracking in defining
how RNAP reacts to such obstacles in the DNA template. The described
approach will be a useful tool in deciphering the mechanism of backtracking
and in studying factors that
affect the backtracking
Detection Methodology Based on Target Molecule-Induced Sequence-Specific Binding to a Single-Stranded Oligonucleotide
We have recently developed a mix-and-read format homogeneous
antigen peptide based assay for detection of the antibodies (Tian,
L.; Heyduk, T. <i>Anal. Chem.</i> <b>2009</b>, <i>81</i>, 5218–5225) that employed for target detection
a simple biophysical mechanism of target antibody induced annealing
between two complementary oligonucleotides attached to the antigen
peptide. In this work, we propose and experimentally validate an alternative
variant of this assay format in which target antibody binding to antigen
peptide–oligonucleotide conjugate produces a complex with high
sequence-specific binding affinity to a single-stranded capture oligonucleotide.
This new assay format can be used for preparing various solid-surface
based assays by immobilizing the capture oligonucleotide. This assay
design is not limited to antibody detection. We demonstrate that it
can also be employed for detecting proteins or pathogenic bacteria
using oligonucleotide-labeled antibodies as target recognition elements.
Preparation of these solid-surface based assays is simplified because
all interactions with the solid surfaces are mediated by well-understood
oligonucleotide–oligonucleotide interactions and because of
the relative ease of immobilizing oligonucleotides on various solid
surfaces. These unique aspects of the assay design also allow microarray-style
multiplexing that could be most useful for multiplexed antibody profiling
for diagnosis and analysis of cancer, autoimmune, and infectious diseases
RNA polymerase motions during promoter melting.
All cellular RNA polymerases (RNAPs), from those of bacteria to those of man, possess a clamp that can open and close, and it has been assumed that the open RNAP separates promoter DNA strands and then closes to establish a tight grip on the DNA template. Here, we resolve successive motions of the initiating bacterial RNAP by studying real-time signatures of fluorescent reporters placed on RNAP and DNA in the presence of ligands locking the clamp in distinct conformations. We report evidence for an unexpected and obligatory step early in the initiation involving a transient clamp closure as a prerequisite for DNA melting. We also present a 2.6-angstrom crystal structure of a late-initiation intermediate harboring a rotationally unconstrained downstream DNA duplex within the open RNAP active site cleft. Our findings explain how RNAP thermal motions control the promoter search and drive DNA melting in the absence of external energy sources