Genetic Manipulation Of Yeast Research Articles

An Overview on Selection Marker Genes for Transformation of Saccharomyces cerevisiae.

For genetic manipulation of yeast, numerous selection marker genes have been employed. These include prototrophic markers, markers conferring drug resistance, autoselection markers, and counterselectable markers. This chapter describes the different classes of selection markers and provides a number of examples for different applications.

High-Throughput Rapid Yeast Chronological Lifespan Assay.

The budding yeast is a valuable model system for discovering molecular mechanisms underlying cellular aging. This is due to the ease of performing genetic manipulations in yeast and the vast number of evolutionarily conserved genes that have been found to regulate cellular health and lifespan from yeast to humans. Lifespan assays are an essential tool for examining the effects of these genes on longevity. There are two ways lifespan is measured in yeast: replicative lifespan (RLS) and chronological lifespan (CLS). RLS is a measure of how many divisions an individual mother cell will undergo. CLS measures the length of time nondividing cells survive. Previously described CLS assays involved diluting and plating cells of a culture and counting the colonies that arose. While effective, this method is both time and labor intensive. Here, we describe a method for a high-throughput rapid CLS assay that is both time- and cost-efficient.

Marker-free genetic manipulations in yeast using CRISPR/CAS9 system.

The budding yeast is currently one of the major model organisms for the study of a wide variety of biological processes. Genetic manipulation of yeast involves the extensive usage of selectable markers that can lead to undesired effects. Thus, marker-free genetic manipulation in yeast is highly desirable for gene/promoter replacement and various other applications. Here we combine the power of selectable markers followed by CRISPR/CAS9 genome editing for common genetic manipulations in yeast in a marker-free manner. We demonstrate our approach for whole gene and promoter replacements and for high-efficiency operator array integration. Our approach allows the utilization of many thousands of existing strains including library strains for the generation of significant genetic changes in yeast in a marker-free and cloning-free fashion.

21] Genetic strategies for identification of mitochondrial translation factors in Saccharomyces cerevisiae

This chapter discusses general strategies employed to study yeast mitochondrial translation genetically. Detailed descriptions of procedures for the isolation of mutants, genetic manipulation of yeast, and phenotypic analysis of mitochondrial functions are described. Although the specific strategies discussed in the chapter are directly applicable only to S. cerevisiae at the present time, it is worth considering how they might be extended to other organisms. The mechanisms of mitochondrial (mt) translation initiation are poorly understood. To a large extent, this is because translation systems of mitochondria have resisted detailed in vitro analysis: although isolated intact organelles carries out protein synthesis, no extract of mitochondria with reproducible mRNA-dependent translational activity is described in the chapter. In the absence of in vitro systems, genetic analysis of mitochondrial translation in the yeast Saccharomyces cerevisiae provides information on organellar gene expression. To date, it has allowed the demonstration of in vivo functional roles for some general translation factors homologous to those of other biological systems and the demonstration of the existence of important regulatory proteins for which no homologs in other systems have been found. This chapter summarizes general strategies employed to study yeast mitochondrial transition genetically.

A hit-and-run system for targeted genetic manipulations in yeast.

A hit-and-run system for targeted genetic manipulations in yeast.

High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules.

A set of vector DNAs (Y vectors) useful for the cloning of DNA fragments in Saccharomyces cerevisiae (yeast) and in Escherichia coli are characterized. With these vectors, three modes of yeast transformation are defined. (i) Vectors containing yeast chromosomal DNA sequences (YIp1, YIp5) transform yeast cells at low frequency (1--10 colonies per microgram) and integrate into the genome by homologous recombination; this recombination is reversible. (ii) Hybrids containing endogenous yeast plasmid DNA sequences (YEp2, YEp6) transform yeast cells at much higher frequency (5000--20,000 colonies per microgram). Such molecules replicate autonomously with an average copy number of 5--10 covalently closed circles per yeast cell and also replicate as a chromosomally integrated structure. This DNA may be physically isolated in intact form from either yeast or E. coli and used to transform either organism at high frequency. (iii) Vectors containing a 1.4-kilobase yeast DNA fragment that includes the centromere linked trp1 gene (YRp7) transform yeast with an efficiency of 500--5000 colonies per microgram; such molecules behave as minichromosomes because they replicate autonomously but do not integrate into the genome. The uses of Y vectors for the following genetic manipulations in yeast are discussed: isolation of genes; construction of haploid strains that are merodiploid for a particular DNA sequence; and directed alterations of the yeast genome. General methods for the selection and the analysis of these events are presented.