实验基础

Transcriptional profiling methods have been utilized in the analysis of various biological processes in Dictyostelium. Recent advances in high-throughput sequencing have increased the resolution and the dynamic range of transcriptional profiling. Here we describe the uti ...

Micrococcal nuclease (MNase) is an endonuclease that cleaves native DNA at high frequency, but is blocked in chromatin by sites of intimate DNA–protein interaction, including nucleosomal regions. Protection from MNase cleavage has often been used to map transcription factor bindi ...

The increasing emergence of drug-resistant Mycobacterium tuberculosis poses significant threat to the treatment of tuberculosis (TB). Conventional drug susceptibility testing is time-consuming and takes several weeks because of the slow growth rate of M. tuberculosis and the ...

DNA-based typing has contributed to the understanding of M. tuberculosis epidemiology and evolution. IS6110 RFLP was the first method described and has been used in many epidemiologic investigations. Technological difficulties have hampered the widespread establishment of t ...

DNA fingerprinting techniques are based on genome variation and form the basis of molecular epidemiology studies of tuberculosis. A number of markers are in use for the molecular differentiation of Mycobacterium tuberculosis isolates by DNA fingerprinting. One of these markers is the ...

The identification of essential genes is of major importance to mycobacterial research, and a number of essential genes have been identified in mycobacteria, however confirming essentiality is not straightforward, as deletion of essential genes results in a lethal phenotype. In this ...

Conditional expression–specialized transduction essentiality test (CESTET) is a genetic tool used to determine essentiality of individual genes in Mycobacterium smegmatis. CESTET combines specialized transduction, a highly efficient gene knockout method, with the ut ...

Mycobacteria produce an effective permeability layer that consists of a mycolic acid–containing cell wall. This protection confers a natural resistance to many chemical agents and results in a low permeability toward both hydrophilic and lipophilic agents. The permeability of ce ...

Two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry (MS) is the classic proteomics approach used to monitor the dynamics of protein abundance and posttranslational modifications in biological systems. In this chapter, we provide detailed protoc ...

DNA microarray technology represents an extremely powerful tool to understand the biology of Myobacterium tuberculosis and its interaction with the host. It opens up the possibility of monitoring the expression level of thousands of genes in parallel, thus the ability to test the effect on ...

The development of microarray technology has allowed the genomes of mycobacteria to be directly compared to identify DNA regions that differ between strains due to deletion, insertion, or sequence divergence. The use of microarrays in comparative genomics has proved to be a valuable tool ...

A procedure for metabolic labeling of all cellular lipids starting with a culture of mycobacteria is described in this chapter using either a pulse-chase or a simple labeling experimental design. Three fractions are produced for subsequent lipid analysis: (1) the culture filtrate; (2) a rea ...

There is massive gene replication predicted for the activation of fatty acids and their entry into the β-oxidation cycle for fatty acid oxidation. These two steps in fatty acid metabolism are catalyzed by FadD and FadE enzymes with 36 genes predicted for each of these respective activities in Myc ...

In this chapter, we describe in detail the steps involved in isolation and characterization of lipoglycans from Mycobacterium tuberculosis and Mycobacterium smegmatis. In addition, procedures involved in structural analysis such as immunoblotting with mAb CS-35 or CS-40, gas chr ...

This chapter describes two protocols for isolating total RNA from mycobacteria: one for extraction from in vitro cultures and one for extraction from in vivo. In these protocols, RNA is liberated from mycobacteria by disruption with small glass beads in the presence of Trizol to stabilize the R ...

Environmental amoebae have been shown to be a host to pathogenic mycobacteria. Mycobacterium avium, Mycobacterium marinum, and Mycobacterium peregrinum can all grow inside Acanthamoeba and other environmental amoebae. Once ingested by Acanthamoeba, M. avium upregulates a num ...

MycoDB (http://myco.bham.ac.uk) is an online resource designed to facilitate genomic analyses of Mycobacterium spp. and related genera. Regions of interest can be found by searching the annotation, BLAST searching against the sequence data, or specifying genomic coordinates. Tools ...

Phage transduction is an attractive method of genetic manipulation in mycobacteria. ΦMycoMarT7 is well suited for transposon mutagenesis as it is temperature sensitive for replication and contains T7 promoters that promote transcription, a highly active transposase gene, and an ...

The ability to select genes to knock out of mycobacterial genomes has greatly improved our understanding of mycobacteria. This chapter describes a method for doing this. The gene (including a 1-kb flanking region) is cloned into a pNIL series vector and disrupted by deletion or insertion of a cass ...

A myriad of methods has been reported for the isolation of genomic DNA from Mycobacterium spp.; some methods use mechanical disruption of the bacterial cells, whereas others use some form of chemical or enzymatic lysis. Regardless of the approach, the end points remain efficient breaking of the ...