ABSTRACT
The standard method to evaluate capsid integrity of DNA-based viruses, which depends on access to DNase I, relies on the removal of the capsid by solvent extraction and then the evaluation of the nucleic acid products by electrophoresis. Our method, which is based on the direct detection of capsid-borne DNA directly through the use of epifluorescent microscopy, negates the requirement of DNA extraction.
Subject(s)
Bacteriophages/ultrastructure , Capsid/ultrastructure , DNA, Viral/ultrastructure , Image Enhancement/methods , Microscopy, Fluorescence/methods , Organic Chemicals , Benzothiazoles , Diamines , Quinolines , Staining and Labeling/methodsABSTRACT
An assay modeled on a known polymorphism in the PE_PGRS9 gene of Mycobacterium tuberculosis was designed to assess the mutability of a sequence containing interspersed PGRS repeats. Application of the assay in Mycobacterium smegmatis revealed sequence plasticity: in addition to recapitulating the mutation on which it was based, other mutations likely mediated by replication slippage between PGRS repeats were detected. However, the mutation rates argued against marked hypermutability of such sequences in mycobacteria.
Subject(s)
Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Membrane Proteins/genetics , Mutation , Mycobacterium tuberculosis/genetics , Base Composition , Base Sequence , Gene Rearrangement , Genotype , Molecular Sequence DataABSTRACT
One of the cellular consequences of nitrosative stress is alkylation damage to DNA. To assess whether nitrosative stress is registered on the genome of Mycobacterium tuberculosis, mutants lacking an alkylation damage repair and reversal operon were constructed. Although hypersensitive to the genotoxic effects of N-methyl-N'-nitro-N-nitrosoguanidine in vitro, the mutants displayed no phenotype in vivo, suggesting that permeation of nitrosative stress to the level of cytotoxic DNA damage is restricted.
