生物信息学技术

The problems often experienced with DNA isolated from yeast are mostly owing to the impurities remaining in DNA isolated according to certain isolation protocols. Incomplete cutting of chromosomal DNA with restriction endonucleases, or poor transformation of Escherichia coli w ...

Plasma membranes are often prepared from yeast by initially spheroplasting the cells (1–3). Procedure 1 following uses spheroplasting, gives high yields, and is ideally suited to the large-scale isolation of plasma membranes. Modified after Schmidt et al. (2) and Cartwright et al. (4) it coats ...

The study of sterols is of both practical and theoretical interest, being important in the analysis of the action of antifungal compounds, in vitamin D production and investigating the role of sterols in cell division, mating (1), and ethanol tolerance. The extraction of sterols from yeast is both ...

The group of De Duve first described the isolation of peroxisomes from rat liver tissue. These organelles can be separated from other sub-cellular organelles, such as lysosomes and mitochondria, because of their relatively high equilibrium density in sucrose (approx 1.24 g/cm3). The isol ...

Transformation of yeast cells can be achieved using lithium-treated or spheroplasted cells. Spheroplast transformation (1,2) is a high efficiency method yielding up to 104–105 transformants per �g of DNA (but more typically, in practice approximately 103 per �g). The preparation of sph ...

Techniques for constructing genomic yeast DNA libraries and for isolating yeast genes from them, mainly by complementation, have been known for over 15 yr. The first method (1) involved making the library in an Escherichia coli plasmid vector, but as soon as “shuttle” vectors—capable of stable ...

Biolistic transformation is a unique process in which DNA or RNA is introduced into cells on micron-sized particles. These microparticles are accelerated to supersonic speeds utilizing forces generated by a gunpowder discharge or cold gas explosion. This technique was first develo ...

Yeasts are relatively easy to isolate from natural habitats, the technique being to weigh out a sample of the substrate, suspend it in sterile water, and streak or spread a suitable dilution on either a nonselective medium (usually acidified malt extract agar to suppress bacterial growth) to obt ...

Yeast colony hybridization is a method for determining the presence or absence of a sequence of DNA, be it of chromosomal or plasmid location, in an individual (single cell-derived) colony. It can be used, for example, to screen transformants for a nonselectable sequence or to estimate the percent ...

Autonomously replicative plasmids are widely employed as vectors for the expression of cloned genes in yeast (1). A number of different kinds of such plasmid vectors exist, which fall into three classes: ARS (autonomously replicating sequence) based plasmids, 2-�m-based plasmids, and ARS ...

The targeted deletion or inversion of a large chromosomal segment and targeted recombination between two nonhomologous chromosomes can be created in Saccharomyces cerevisiae by using the site-specific recombination system from a heterologous yeast plasmid (1). The method con ...

Wild-type and industrial strains of Saccharomyces cerevisiae appear to be typically polyploid/aneuploid. Such strains are also essentially sterile, but will on rare occasions produce spores. The mating of such spores with reference laboratory strains can be used to give a general est ...

The development of genetically marked Ty elements that transpose at high levels has made Ty mutagenesis a useful tool in yeast genetics (1,2). Ty mutagenesis is useful for several reasons: 1. Mutations made by insertion of a marked Ty e

Reporter gene constructs are used to monitor the expression of genes whose natural product is not readily assayed. The most convenient reporters to use encode enzymes that catalyze a reaction that is easily monitored. Three useful reporter systems for intracellular expression will be de ...

The yeast Saccharomyces cerevisiae is now a standard model organism for the dissection of eukaryotic transcriptional and translational mechanisms (e.g., 1). The study of the mechanism and control of eukaryotic translation requires an in vitro system that will reflect in vivo events and y ...

Polyacrylamide gel electrophoresis (PAGE) of proteins provides a powerful qualitative and quantitative tool for studying yeast protein synthesis. Complex mixtures of proteins can be separated purely on the basis of molecular weight by means of sodium dodecylsulfate-polyacr ...

The expression of many genes is regulated at multiple levels. For most genes, the predominant control is at transcription, and this is usually inferred from changes in mRNA levels that respond to specific stimuli. However, changes in mRNA stability may contribute to observed variation in mRNA ...

RNA analysis is central to a detailed understanding of gene expression. The expression of many nuclear genes from a wide range of organisms has been studied at the protein level using reporter genes (lacZ, for example), and changes in expression levels in response to specific stimuli are frequen ...

During the cyclic process of translation, a small (40S) and large (60S) ribosomal subunit associate with mRNA to form an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation, and then dissociates into the 40S and 60S subunits on termination. During elon ...

Yeasts generally are very easy to maintain, store, cultivate in almost any size of batch, and isolate from most habitats, but if a few elementary methods are understood, their isolation, storage, and culture, for day-to-day use in the laboratory is greatly simplified. Furthermore, the same metho ...