Functional Yeast Genomics

What is the main focus of the review by Anuj Kumar and Michael Snyder?

[ ]The review focuses on summarizing the most promising technologies in yeast genomics, particularly those that have emerged from the study of the budding yeast Saccharomyces cerevisiae.

What is functional genomics?

[ ]Functional genomics is the development and systematic application of experimental methodologies to analyze gene function on a genome-wide scale.

When did the genomic era begin, and what catalyzed it?

[ ]The genomic era began in earnest during the late 1980s, catalyzed by advances in DNA-sequencing technology.

What were the first two organisms to have their complete genomes sequenced?

[ ]The first two organisms to have their complete genomes sequenced were the bacterium Haemophilus influenzae in 1995 and the yeast Saccharomyces cerevisiae in 1996.

Why is Saccharomyces cerevisiae considered an ideal model organism for genomic studies?

[ ]Saccharomyces cerevisiae is considered an ideal model organism due to its compact genome, ease of experimental manipulation, stability in both haploid and diploid states, and the fact that nearly 50% of human genes implicated in heritable diseases have yeast homologues.

What percentage of yeast genes had been functionally characterized when the complete genome sequence first became available?

[ ]Only one-third of all predicted yeast genes had been functionally characterized when the complete genome sequence first became available.

What is transposon tagging, and how is it used in yeast genomics?

[ ]Transposon tagging involves using transposable elements as mutagenic agents to study gene function. In yeast genomics, transposons are engineered to carry reporter genes, epitope tags, and regulatory elements, allowing for large-scale analysis of gene expression, gene disruption, and protein localization.

What is shuttle mutagenesis?

[ ]Shuttle mutagenesis is a method in which cloned yeast genes are mutated by bacterial transposition in Escherichia coli, and the mutant alleles are subsequently introduced into yeast where they integrate at their corresponding genomic loci by homologous recombination.

What is the purpose of using Cre-lox recombination in transposon tagging?

[ ]Cre-lox recombination is used to reduce the size of the transposon to a smaller element that encodes an epitope tag, which can then be used for immunolocalization or immunoprecipitation studies.

What is the PCR-based gene-deletion strategy in yeast?

[ ]The PCR-based gene-deletion strategy involves generating deletion cassettes by PCR, where each cassette is flanked by short regions of yeast DNA sequence that correspond to the intended deletion target. These cassettes are then introduced into yeast, where they integrate at their corresponding genomic loci, resulting in precise gene replacements.

What are UPTAG and DOWNTAG sequences in the context of yeast gene deletions?

[ ]UPTAG and DOWNTAG sequences are unique 20-bp sequences incorporated into each deletion cassette that serve as molecular barcodes to identify individual deletion strains in a mixed population.

How are barcoded deletion strains analyzed for fitness?

[ ]Barcoded deletion strains are analyzed for fitness by comparative hybridization of their genomic DNA to a microarray of UPTAG and DOWNTAG sequences. Strains deleted for a gene required for growth under a given condition will be underrepresented, yielding lower intensity signals upon hybridization.

What are some limitations of the PCR-based gene-deletion strategy?

[ ]Limitations include the potential for aneuploidy in some deletion strains, the dependence on correctly annotated gene sequences, the high cost and labor involved in synthesizing primers, and the fact that deletions are provided in a single genetic background.

What is the primary use of DNA microarrays in functional genomics?

[ ]DNA microarrays are primarily used to analyze the relative levels of mRNA transcripts on a genome-wide scale, allowing for the identification of genes that are differentially expressed under specific conditions.

What is hierarchical clustering in the context of microarray data analysis?

[ ]Hierarchical clustering is a method used to group genes with similar expression patterns into a single tree, where the branch lengths reflect the degree of similarity between individual expression profiles.

How can microarray data be used to infer gene function?

[ ]Microarray data can be used to infer gene function by comparing expression profiles of deletion mutants with those of known genes. If a strain deleted for an uncharacterized gene shows an expression profile similar to that of a gene with a known function, the uncharacterized gene may have a similar function.

What is chromatin immunoprecipitation (ChIP) used for in yeast genomics?

[ ]Chromatin immunoprecipitation is used to map the binding sites of chromosomal proteins, such as transcription factors, on a genome-wide scale by crosslinking proteins to DNA, immunoprecipitating the DNA-protein complexes, and analyzing the DNA using microarrays.

What is the yeast two-hybrid assay, and how is it used on a genome-wide scale?

[ ]The yeast two-hybrid assay is a method to identify physical interactions between binary protein pairs. On a genome-wide scale, it involves systematically mating haploid yeast strains carrying bait and prey plasmids to identify protein-protein interactions across the entire yeast proteome.

What is biochemical genomics?

[ ]Biochemical genomics is an approach that involves generating a ready supply of purified proteins corresponding to the entire protein complement of an organism, allowing for large-scale biochemical analysis of protein function.

What are protein microarrays, and what are their potential applications?

[ ]Protein microarrays are high-density arrays of purified proteins that can be used for various assays, including identifying protein-protein interactions, measuring enzyme kinetics, and screening for potential drug targets.

What is the significance of mass spectrometry (MS) in yeast genomics?

[ ]Mass spectrometry is used to identify and quantify proteins, analyze protein complexes, and detect post-translational modifications. It is a powerful tool for characterizing the yeast proteome and understanding protein function.

What are some online resources for yeast genomics mentioned in the review?

[ ]Online resources include the Saccharomyces Genome Database (SGD), Yeast Proteome Database (YPD), Munich Information Centre for Protein Sequences (MIPS), and various functional genomic projects such as the yeast genome deletion project and microarray data sets.

What is the ultimate goal of the genomic methods discussed in the review?

[ ]The ultimate goal is to achieve a comprehensive understanding of eukaryotic cellular function, with Saccharomyces cerevisiae serving as a key model organism for genomic studi