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Purification of Mitochondrial Uracil-DNA Glycosylase Using Ugi-Sepharose Affinity Chromatography
Time of Update: 2021-02-22
Separation is achieved through a highly specific, but reversible, interaction with a complementary binding substrate (ligand) that is immobilized on a solid support (matrix).
This purification approach has been developed to incorporate protein-protein interactions into the design of the affinity matrix ( 3 – 3 ).
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DNA Extraction from Natural Environments
Time of Update: 2021-02-22
Until recently, studies on microbial communities present in natural environments relied on conventional optical microscopic observation and cultivation-based approaches.
The most commonly cited limitation stems from the finding that the majority of microbial cells in natural environments cannot be cultured in the laboratory—the phenomenon of nonculturability.
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Fast Sampling of the Cellular Metabolome
Time of Update: 2021-02-22
For prokaryotic cells this had not yet been accomplished, so the application of a differential method whereby metabolites are measured in the culture supernatant as well as in total broth samples, to calculate the intracellular levels by subtraction, seems to be the most suitable approach.
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Murine Intestinal Dendritic Cell Isolation
Time of Update: 2021-02-22
This chapter describes techniques for the isolation of dendritic cell-enriched single-cell suspensions from the lamina propria (LP) of both small and large intestine and from Peyer’s patches (PP).
This technique can also be used to obtain intestinal macrophage-enriched populations.
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Insect Cell- Culture Techniques in Serum- Containing Medium
Time of Update: 2021-02-22
The establishment of new lines is now routine, and the culture of hundreds of cell lines from a variety of insect species have to date been reported ( 2 ,4 ,5 ,6 ).
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Detection and Differentiation of Ruminant Mycoplasmas
Time of Update: 2021-02-22
While a considerable part of this group is conceived to be of minor epidemiological relevance, it contains some important pathogenic agents that have specific host ranges.
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Immunological Methods for the Detection of Human Cytomegalovirus
Time of Update: 2021-02-22
In addition, HCMV has been implicated as a co-etiological agent in cervical cancer ( 2 ) and has been found associated with a wide range of other tumors ( 1 ).
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Generalized Transduction by Lytic Bacteriophages
Time of Update: 2021-02-22
As interest in lytic phages as antimicrobial therapies or as treatments to reduce environmental contamination with pathogenic bacteria has increased, so has the need to determine if the use of lytic phages may lead to dissemination of virulence factors through generalized transduction, as occurs with temperate phages.
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Transformation of Candida maltosa by Electroporation
Time of Update: 2021-02-22
Candida species are diploid or aneuploid in their genetic constitution and have proven to be difficult organisms to study in terms of their genetics and molecular biology.
Moreover, electroporation permits the introduction of DNA into organisms that are refractory to other transformation techniques ( 2 ).
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A Method for Short-Term Culture of Human Gastric Epithelial Cells to Study the Effects of Helicobacter pylori
Time of Update: 2021-02-22
Here we describe a method for isolation and short-term culture of human primary gastric epithelial cells obtained from gastric biopsy specimens, and the use of these cells to evaluate the effect ofH.
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Cell Lineage Studies Using Retroviruses
Time of Update: 2021-02-22
By means of this label, the subsequent fate of the cell and its progeny can be followed.
This has made retroviruses invaluable in studying cell lineage in vivo, particularly in mammalian embryos, which are too small and inaccessible to be amenable to study with conventional lineage labels.
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Evaluation of Rotavirus Vaccines in Small Animal Models
Time of Update: 2021-02-22
Because RV is a localized enteric infection, and induction of intestinal mucosal immune responses was expected to be required for protection, live orally administered vaccines were pursued first.
The nonreplicating immunogen must be able to induce protective immune responses against the target virus.
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Gene Replacement in Mycobacterium intracellulare
Time of Update: 2021-02-22
Much of the present knowledge of the bacterial cell emanates from studies of spontaneous mutants and mutants obtained as a result of random or directed mutagenesis.
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Reproducible Quantitative PCR of Mitochondrial and Nuclear DNA Copy Number Using the LightCycler
Time of Update: 2021-02-22
Recent developments inPCR fluorimetry have allowed for quick quantification of target molecules. Before the invention of fluorimetric quantitative PCR, researchers who wanted to quantify the amount
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Measuring Oxidative mtDNA Damage and Repair Using Quantitative PCR
Time of Update: 2021-02-22
The human mitochondrial genome was completely sequenced in 1981 by Anderson and co-workers ( 1 ) and consists of a closed circular supercoiledDNA molecule of 16,569 base pairs.
The mitochondrial genome encodes 13 polypeptides, 22 transfer RNAs (tRNAs), and 2 rRNA.
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Biosynthesis, Extraction, Purification, and Analysis of Trisporoid Sexual Communication Compounds from Mated Cultures of Blakeslea trispora
Time of Update: 2021-02-22
The zygomyceteBlakeslea trispora produces high amounts of the general zygomycete β-carotene-derived sexual signal compounds, the trisporoids.
The effect of the extraction and activity of the isolated compounds is best tested physiologically, exploiting the ability of trisporoids to induce the formation of sexually committed hyphae, the zygophores, in other zygomycete species.
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Monitoring Microbial Activities Using Membrane Inlet Mass Spectrometry
Time of Update: 2021-02-22
Currently used methods include manometric techniques, microsensors, chemical assays, gas chromatography and high-performance liquid chromatography, but all have their limitations and usually require substantial disruption to the environment being studied.
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Analysis of Chromatin by Scanning Force Microscopy
Time of Update: 2021-02-22
The most widely used probe microscopy for biological applications, the scanning force microscope (SFM), also known as the atomic force microscope, is capable of imaging samples under very mild conditions (for recent reviews,see refs.
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Preparation of Chromatin Assembly Extracts from Xenopus Oocytes
Time of Update: 2021-02-22
The majority ofDNA in eukaryotic cells is packaged by histones and many poorly characterized nonhistone proteins to form a dynamic structure known as chromatin.
Moreover, histones, via protein-protein interactions or by playing an architectural role, can facilitate or inhibit the transcriptional activation process ( 1 , 2 ).
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Extraction of Yeast Lipids
Time of Update: 2021-02-22
Quantitative extraction of lipids from the tissue or microorganism of choice is key to their subsequent analysis. In this chapter, we describe a simple and rapid protocol that relies on glass bead di
