High Throughput Methods For Functional Genomics In Saccharomyces Cerevisiae

The Saccharomyces cerevisiae deletion collection has revolutionized yeast genetics by allowing genome-wide deletion screens to be performed in pooled cultures. The inclusion of unique, 20-bp DNA sequences known as "tags" or "barcodes" in each strain allows all ~6000 yeast deletion mutants to be analyzed in a single culture by using a microarray to detect changes in tag abundance. Here we discuss improvements and extensions made to the pooled-yeast-deletion assay. This includes a redesign of the barcode array that is used for detecting the tags included in the deletion collection. Our analysis of this barcode array examines whether it is better to use available microarray surface area on a single feature for each probe, or to instead include multiple smaller features. It also covers aspects of data analysis such as the correction of hybridization defects and array saturation, tag-sequence repairs, and estimation of background hybridization. In addition to these microarray changes, we also examine the impact of experimental design on the quality of pooled growth data. Each step in both the experimental protocol and the data-analysis pipeline is examined for possible improvement. Finally, we discuss preliminary experiments testing the application of pooled fitness profiling to the study of epistasis. Data are shown that demonstrate the feasibility of a pooled-double-mutant fitness assay that is compatible with existing barcode array technology. This assay has the potential to become a valuable functional-genomics tool that may provide an improved understanding of pathway relationships in yeast.

This fully updated edition of the bestselling three-part Methods in Enzymology series, Guide to Yeast Genetics and Molecular Cell Biology is specifically designed to meet the needs of graduate students, postdoctoral students, and researchers by providing all the up-to-date methods necessary to study genes in yeast. Procedures are included that enable newcomers to set up a yeast laboratory and to master basic manipulations. This volume serves as an essential reference for any beginning or experienced researcher in the field. Provides up-to-date methods necessary to study genes in yeast Includes proceedures that enable newcomers to set up a yeast laboratory and to master basic manipulations Serves as an essential reference for any beginning or experienced researcher in the field

This collection of robust, readily reproducible methods for microarray-based studies includes expert guidance in the optimal data analysis and informatics. On the methods side are proven techniques for monitoring subcellular RNA localization en masse, for mapping chromosomes at the resolution of a single gene, and for surveying the steady-state genome-wide distribution of DNA binding proteins in vivo. For those workers dealing with massive data sets, the book discusses the methodological aspects of data analysis and informatics in the design of microarray experiments, the choice of test statistic, and the assessment of observational significance, data reduction, and clustering.

One of the holy grails in biology is the ability to predict functional characteristics from an organism's genetic sequence. Despite decades of research since the first sequencing of an organism in 1995, scientists still do not understand exactly how the information in genes is converted into an organism's phenotype, its physical characteristics. Functional genomics attempts to make use of the vast wealth of data from "-omics" screens and projects to describe gene and protein functions and interactions. A February 2020 workshop was held to determine research needs to advance the field of functional genomics over the next 10-20 years. Speakers and participants discussed goals, strategies, and technical needs to allow functional genomics to contribute to the advancement of basic knowledge and its applications that would benefit society. This publication summarizes the presentations and discussions from the workshop.

This volume provides a collection of protocols for the study of DNA-DNA contact maps, replication profiles, transcription rates, RNA secondary structures, protein-RNA interactions, ribosome profiling and quantitative proteomes and metabolomes. Written for the Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Yeast Functional Genomics: Methods and Protocols aims to ensure successful results in the further study of this vital field.

Since 1996, when the first Saccharomyces cerevisiae genome sequence was released, a wealth of genomic data has been made available for numerous S. cerevisiae strains, its close relatives, and non-conventional yeast species isolates of diverse origins. Several annotated genomes of interspecific hybrids, both within the Saccharomyces clade and outside, are now also available. This genomic information, together with functional genomics and genome engineering tools, is providing a holistic assessment of the complex cellular responses to environmental challenges, elucidating the processes underlying evolution, speciation, hybridization, domestication, and uncovering crucial aspects of yeasts´ physiological genomics to guide their biotechnological exploitation. S. cerevisiae has been used for millennia in the production of food and beverages and research over the last century and a half has generated a great deal of knowledge of this species. Despite all this, S. cerevisiae is not the best for all uses and many non-conventional yeast species have highly desirable traits that S. cerevisiae does not have. These include tolerance to different stresses (e.g. acetic acid tolerance in Zygosaccharomyces bailii, osmotolerance in Z. rouxii, and thermotolerance in Kluyveromyces marxianus and Ogataea (Hansenula) polymorpha), the capacity of assimilation of diverse carbon sources (e.g. high native capacity to metabolyze xylose and potential for the valorization of agroforest residues by Scheffersomyces (Pichia) stipites), as well as, high protein secretion, fermentation efficiency and production of desirable flavors, capacity to favor respiration over fermentation, high lipid biosynthesis and accumulation, and efficient production of chemicals other than ethanol amongst many. Several non-Saccharomyces species have already been developed as eukaryotic hosts and cell factories. Others are highly relevant as food spoilers or for desirable flavor producers. Therefore, non-conventional yeasts are now attracting increasing attention with their diversity and complexity being tackled by basic research for biotechnological applications. The interest in the exploitation of non-conventional yeasts is very high and a number of tools, such as cloning vectors, promoters, terminators, and efficient genome editing tools, have been developed to facilitate their genetic engineering. Functional and Comparative Genomics of non-conventional yeasts is elucidating the evolution of genome functions and metabolic and ecological diversity, relating their physiology to genomic features and opening the door to the application of metabolic engineering and synthetic biology to yeasts of biotechnological potential. We are entering the era of the non-conventional yeasts, increasing the exploitation of yeast biodiversity and metabolic capabilities in science and industry. In this collection the industrial properties of S. cerevisiae, in particular uses, are explored along with its closely related species and interspecific hybrids. This is followed by comparisons between S. cerevisiae and non-conventional yeasts in specific applications and then the properties of various non-conventional yeasts and their hybrids.

High throughput sequencing (HTS) technologies have conquered the genomics and epigenomics worlds. The applications of HTS methods are wide, and can be used to sequence everything from whole or partial genomes, transcriptomes, non-coding RNAs, ribosome profiling, to single-cell sequencing. Having such diversity of alternatives, there is a demand for information by research scientists without experience in HTS that need to choose the most suitable methodology or combination of platforms and to define their experimental designs to achieve their specific objectives. Field Guidelines for Genetic Experimental Designs in High-Throughput Sequencing aims to collect in a single volume all aspects that should be taken into account when HTS technologies are being incorporated into a research project and the reasons behind them. Moreover, examples of several successful strategies will be analyzed to make the point of the crucial features. This book will be of use to all scientist that are unfamiliar with HTS and want to incorporate such technologies to their research.

In the wake of the development of technology to sequence the complete genome of an organism, it has become expedient to generate methodologies to elucidate and characterize the function of all genes constituting the complete genetic makeup of an organism, whereby the knowledge of the genetic code may be for scientific and intellectual profit. This work consists of an investigation into two possible methods for determining the role of genes involved in the DNA and cellular damage response, though the methods are generally applicable to investigating a wide variety of biological pathways and responses. A library of approximately 4,800 yeast (Saccharomyces cerevisiae) deletion strains produced by the Saccharomyces Genome Deletion Project and consisting essentially of all possible mutants having one non-essential gene deleted (and replaced with unique identification tags called "bar codes") from the genome are employed in this endeavor. The methods focus on gathering phenotype data in a high-throughput manner and in response to the alkylating agent methyl methanesulfonate (MMS). The first method makes use of a new technology called the Living ChipTM, which can hold libraries of compounds or cell cultures in an array of 50-nl channels and which could ideally accommodate all deletion strains on a single array. The second method involves pooling all strains together in a single culture and allowing them to grow competitively to determine their relative fitness based on a specific treatment.

Yeast Metabolic Engineering: Methods and Protocols provides the widely established basic tools used in yeast metabolic engineering, while describing in deeper detail novel and innovative methods that have valuable potential to improve metabolic engineering strategies in industrial biotechnology applications. Beginning with an extensive section on molecular tools and technology for yeast engineering, this detailed volume is not limited to methods for Saccharomyces cerevisiae, but describes tools and protocols for engineering other yeasts of biotechnological interest, such as Pichia pastoris, Hansenula polymorpha and Zygosaccharomyces bailii. Tools and technologies for the investigation and determination of yeast metabolic features are described in detail as well as metabolic models and their application for yeast metabolic engineering, while a chapter describing patenting and regulations with a special glance at yeast biotechnology closes the volume. Written in the highly successful Methods in Molecular Biology series format, most chapters include an introduction to their respective topic, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols and tips on troubleshooting and avoiding known pitfalls. Comprehensive and authoritative, Yeast Metabolic Engineering: Methods and Protocols aims to familiarize researchers with the current state of these vital and increasingly useful technologies.