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Liang Y, Luo H, Lin Y, Gao F. Recent advances in the characterization of essential genes and development of a database of essential genes. IMETA 2024; 3:e157. [PMID: 38868518 PMCID: PMC10989110 DOI: 10.1002/imt2.157] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 09/29/2023] [Accepted: 10/09/2023] [Indexed: 06/14/2024]
Abstract
Over the past few decades, there has been a significant interest in the study of essential genes, which are crucial for the survival of an organism under specific environmental conditions and thus have practical applications in the fields of synthetic biology and medicine. An increasing amount of experimental data on essential genes has been obtained with the continuous development of technological methods. Meanwhile, various computational prediction methods, related databases and web servers have emerged accordingly. To facilitate the study of essential genes, we have established a database of essential genes (DEG), which has become popular with continuous updates to facilitate essential gene feature analysis and prediction, drug and vaccine development, as well as artificial genome design and construction. In this article, we summarized the studies of essential genes, overviewed the relevant databases, and discussed their practical applications. Furthermore, we provided an overview of the main applications of DEG and conducted comprehensive analyses based on its latest version. However, it should be noted that the essential gene is a dynamic concept instead of a binary one, which presents both opportunities and challenges for their future development.
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Affiliation(s)
| | - Hao Luo
- Department of PhysicsTianjin UniversityTianjinChina
| | - Yan Lin
- Department of PhysicsTianjin UniversityTianjinChina
| | - Feng Gao
- Department of PhysicsTianjin UniversityTianjinChina
- Frontiers Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education)Tianjin UniversityTianjinChina
- SynBio Research PlatformCollaborative Innovation Center of Chemical Science and Engineering (Tianjin)TianjinChina
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2
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Dede M, Hart T. Recovering false negatives in CRISPR fitness screens with JLOE. Nucleic Acids Res 2023; 51:1637-1651. [PMID: 36727483 PMCID: PMC9976895 DOI: 10.1093/nar/gkad046] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2022] [Revised: 01/09/2023] [Accepted: 01/16/2023] [Indexed: 02/03/2023] Open
Abstract
It is widely accepted that pooled library CRISPR knockout screens offer greater sensitivity and specificity than prior technologies in detecting genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the assumption that CRISPR screens are saturating has been largely untested. Through integrated analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we show that a typical CRISPR screen has a ∼20% false negative rate, in addition to library-specific false negatives. Replicability falls sharply as gene expression decreases, while cancer subtype-specific genes within a tissue show distinct profiles compared to false negatives. Cumulative analyses across tissues improves our understanding of core essential genes and suggest only a small number of lineage-specific essential genes, enriched for transcription factors that define pathways of tissue differentiation. To recover false negatives, we introduce a method, Joint Log Odds of Essentiality (JLOE), which builds on our prior work with BAGEL to selectively rescue the false negatives without an increased false discovery rate.
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Affiliation(s)
- Merve Dede
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Traver Hart
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.,Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
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Campos TL, Korhonen PK, Hofmann A, Gasser RB, Young ND. Harnessing model organism genomics to underpin the machine learning-based prediction of essential genes in eukaryotes - Biotechnological implications. Biotechnol Adv 2021; 54:107822. [PMID: 34461202 DOI: 10.1016/j.biotechadv.2021.107822] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2021] [Revised: 08/17/2021] [Accepted: 08/24/2021] [Indexed: 12/17/2022]
Abstract
The availability of high-quality genomes and advances in functional genomics have enabled large-scale studies of essential genes in model eukaryotes, including the 'elegant worm' (Caenorhabditis elegans; Nematoda) and the 'vinegar fly' (Drosophila melanogaster; Arthropoda). However, this is not the case for other, much less-studied organisms, such as socioeconomically important parasites, for which functional genomic platforms usually do not exist. Thus, there is a need to develop innovative techniques or approaches for the prediction, identification and investigation of essential genes. A key approach that could enable the prediction of such genes is machine learning (ML). Here, we undertake an historical review of experimental and computational approaches employed for the characterisation of essential genes in eukaryotes, with a particular focus on model ecdysozoans (C. elegans and D. melanogaster), and discuss the possible applicability of ML-approaches to organisms such as socioeconomically important parasites. We highlight some recent results showing that high-performance ML, combined with feature engineering, allows a reliable prediction of essential genes from extensive, publicly available 'omic data sets, with major potential to prioritise such genes (with statistical confidence) for subsequent functional genomic validation. These findings could 'open the door' to fundamental and applied research areas. Evidence of some commonality in the essential gene-complement between these two organisms indicates that an ML-engineering approach could find broader applicability to ecdysozoans such as parasitic nematodes or arthropods, provided that suitably large and informative data sets become/are available for proper feature engineering, and for the robust training and validation of algorithms. This area warrants detailed exploration to, for example, facilitate the identification and characterisation of essential molecules as novel targets for drugs and vaccines against parasitic diseases. This focus is particularly important, given the substantial impact that such diseases have worldwide, and the current challenges associated with their prevention and control and with drug resistance in parasite populations.
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Affiliation(s)
- Tulio L Campos
- Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia; Bioinformatics Core Facility, Instituto Aggeu Magalhães, Fundação Oswaldo Cruz (IAM-Fiocruz), Recife, Pernambuco, Brazil
| | - Pasi K Korhonen
- Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia
| | - Andreas Hofmann
- Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia
| | - Robin B Gasser
- Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia.
| | - Neil D Young
- Department of Veterinary Biosciences, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria 3010, Australia.
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de Souza ID, Reis CF, Morais DAA, Fernandes VGS, Cavalcante JVF, Dalmolin RJS. Ancestry analysis indicates two different sets of essential genes in eukaryotic model species. Funct Integr Genomics 2021; 21:523-531. [PMID: 34279742 DOI: 10.1007/s10142-021-00794-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2020] [Revised: 06/02/2021] [Accepted: 06/10/2021] [Indexed: 11/28/2022]
Abstract
Essential genes are so-called because they are crucial for organism perpetuation. Those genes are usually related to essential functions to cellular metabolism or multicellular homeostasis. Deleterious alterations on essential genes produce a spectrum of phenotypes in multicellular organisms. The effects range from the impairment of the fertilization process, disruption of fetal development, to loss of reproductive capacity. Essential genes are described as more evolutionarily conserved than non-essential genes. However, there is no consensus about the relationship between gene essentiality and gene age. Here, we identified essential genes in five model eukaryotic species (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster, Caenorhabditis elegans, and Mus musculus) and estimate their evolutionary ancestry and their network properties. We observed that essential genes, on average, are older than other genes in all species investigated. The relationship of network properties and gene essentiality convey with previous findings, showing essential genes as important nodes in biological networks. As expected, we also observed that essential orthologs shared by the five species evaluated here are old. However, all the species evaluated here have a specific set of young essential genes not shared among them. Additionally, these two groups of essential genes are involved with distinct biological functions, suggesting two sets of essential genes: (i) a set of old essential genes common to all the evaluated species, regulating basic cellular functions, and (ii) a set of young essential genes exclusive to each species, which perform specific essential functions in each species.
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Affiliation(s)
- Iara D de Souza
- Bioinformatics Multidisciplinary Environment - IMD, Federal University of Rio Grande Do Norte, Av. Odilon Gomes de Lima, 1722, Capim Macio, Natal, RN, 59078-400, Brazil
| | - Clovis F Reis
- Bioinformatics Multidisciplinary Environment - IMD, Federal University of Rio Grande Do Norte, Av. Odilon Gomes de Lima, 1722, Capim Macio, Natal, RN, 59078-400, Brazil
| | - Diego A A Morais
- Bioinformatics Multidisciplinary Environment - IMD, Federal University of Rio Grande Do Norte, Av. Odilon Gomes de Lima, 1722, Capim Macio, Natal, RN, 59078-400, Brazil
| | - Vítor G S Fernandes
- Bioinformatics Multidisciplinary Environment - IMD, Federal University of Rio Grande Do Norte, Av. Odilon Gomes de Lima, 1722, Capim Macio, Natal, RN, 59078-400, Brazil
| | - João Vitor F Cavalcante
- Bioinformatics Multidisciplinary Environment - IMD, Federal University of Rio Grande Do Norte, Av. Odilon Gomes de Lima, 1722, Capim Macio, Natal, RN, 59078-400, Brazil
| | - Rodrigo J S Dalmolin
- Bioinformatics Multidisciplinary Environment - IMD, Federal University of Rio Grande Do Norte, Av. Odilon Gomes de Lima, 1722, Capim Macio, Natal, RN, 59078-400, Brazil. .,Department of Biochemistry - CB, Federal University of Rio Grande Do Norte, Campus Universitário UFRN, Lagoa Nova, Natal, RN, 59078-970, Brazil.
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Pyatnitskiy MA, Karpov DS, Moshkovskii SA. [Searching for essential genes in cancer genomes]. BIOMEDIT︠S︡INSKAI︠A︡ KHIMII︠A︡ 2018; 64:303-314. [PMID: 30135277 DOI: 10.18097/pbmc20186404303] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
The concept of essential genes, whose loss of functionality leads to cell death, is one of the fundamental concepts of genetics and is important for fundamental and applied research. This field is particularly promising in relation to oncology, since the search for genetic vulnerabilities of cancer cells allows us to identify new potential targets for antitumor therapy. The modern biotechnology capacities allow carrying out large-scale projects for sequencing somatic mutations in tumors, as well as directly interfering the genetic apparatus of cancer cells. They provided accumulation of a considerable body of knowledge about genetic variants and corresponding phenotypic manifestations in tumors. In the near future this knowledge will find application in clinical practice. This review describes the main experimental and computational approaches to the search for essential genes, concentrating on the application of these methods in the field of molecular oncology.
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Affiliation(s)
- M A Pyatnitskiy
- Institute of Biomedical Chemistry, Moscow, Russia; Higher School of Economics, Moscow, Russia
| | - D S Karpov
- Institute of Biomedical Chemistry, Moscow, Russia; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| | - S A Moshkovskii
- Institute of Biomedical Chemistry, Moscow, Russia; Pirogov Russian National Research Medical University, Moscow, Russia
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Lawrence SJ, Wimalasena TT, Nicholls SM, Box WG, Boulton C, Smart KA. Incidence and Characterization of Petites Isolated from Lager Brewing YeastSaccharomyces CerevisiaePopulations. JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 2018. [DOI: 10.1094/asbcj-2012-0917-01] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Affiliation(s)
- Stephen J. Lawrence
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK
| | - Tithira T. Wimalasena
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK
| | - Sarah M. Nicholls
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK
| | - Wendy G. Box
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK
| | - Chris Boulton
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK
| | - Katherine A. Smart
- School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK
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Jenkins CL, Lawrence SJ, Kennedy AI, Thurston P, Hodgson JA, Smart KA. Incidence and Formation of Petite Mutants in Lager Brewing YeastSaccharomyces Cerevisiae(Syn.S. Pastorianus) Populations. JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 2018. [DOI: 10.1094/asbcj-2009-0212-01] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Affiliation(s)
- Cheryl L. Jenkins
- School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford, UK
| | - Stephen J. Lawrence
- Division of Food Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, UK
| | | | - Pat Thurston
- Scottish & Newcastle UK Ltd., Royal Brewery, Manchester, UK
| | - Jeff A. Hodgson
- Scottish & Newcastle UK Ltd., John Smith's Brewery, Tadcaster, UK
| | - Katherine A. Smart
- Division of Food Sciences, School of Biosciences, University of Nottingham, Loughborough, UK
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Abstract
Gene essentiality is a founding concept of genetics with important implications in both fundamental and applied research. Multiple screens have been performed over the years in bacteria, yeasts, animals and more recently in human cells to identify essential genes. A mounting body of evidence suggests that gene essentiality, rather than being a static and binary property, is both context dependent and evolvable in all kingdoms of life. This concept of a non-absolute nature of gene essentiality changes our fundamental understanding of essential biological processes and could directly affect future treatment strategies for cancer and infectious diseases.
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Korona R. GENETIC LOAD OF THE YEAST SACCHAROMYCES CEREVISIAE UNDER DIVERSE ENVIRONMENTAL CONDITIONS. Evolution 2017; 53:1966-1971. [PMID: 28565447 DOI: 10.1111/j.1558-5646.1999.tb04577.x] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/1998] [Accepted: 06/10/1999] [Indexed: 11/30/2022]
Abstract
Fitness effect of spontaneous mutations accumulated in mismatch-repair deficient strains of yeast was estimated by measuring their maximum growth rate. Several environments with different energetic substrates, nutritional conditions, and temperature were tested. Genetic load of haploid strains was about 20-30% under most of these conditions. Because such a pronounced effect was caused by relatively small lesions (point mutations) affecting probably less than 1% of genes, resistance of the yeast genome to DNA damage appears to be rather limited. Fitness transitions among environments were orderly, in the sense that some strains tended to be more or less fit than others in all circumstances. One of the environments (an extremely high temperature, 38°C) was stressful to the strains that accumulated mutations, as some of them stopped to grow, whereas the mutation-free strains were only moderately affected. These results imply that the impact of random point mutations is substantial and generally not dependent on a particular environment. Under stressful conditions, however, natural selection may be especially effective in purging mutations that, if commonly met, could slow down the rate of mutation accumulation.
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Affiliation(s)
- Ryszard Korona
- Institute of Environmental Sciences, Jagiellonian University, Ingardena 6, 30-060, Krakow, Poland
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Lu X, Wu Q, Zhang Y, Xu Y. Genomic and transcriptomic analyses of the Chinese Maotai-flavored liquor yeast MT1 revealed its unique multi-carbon co-utilization. BMC Genomics 2015; 16:1064. [PMID: 26666414 PMCID: PMC4678718 DOI: 10.1186/s12864-015-2263-0] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2015] [Accepted: 11/30/2015] [Indexed: 12/02/2022] Open
Abstract
BACKGROUND Revealing genetic mechanisms behind specific physiological characteristics of Saccharomyces cerevisiae from specific environments is important for industrial applications and requires precise understanding. RESULTS Maotai strain MT1 was isolated from the complicated Chinese Maotai-flavored liquor-making environment with extremely high temperatures, and acidic and ethanol stresses. Compared with the type strain S288c, MT1 can tolerate high acidity (pH 2.0), high ethanol levels (16 %) and high temperatures (44 °C). In addition, MT1 can simultaneously utilize various sugars, including glucose, sucrose, galactose, maltose, melibiose, trehalose, raffinose and turanose. Genomic comparisons identified a distinct MT1 genome, 0.5 Mb smaller than that of S288c. There are 145 MT1-specific genes that are not in S288c, including MEL1, MAL63, KHR1, BIO1 and BIO6. A transcriptional comparison indicated that HXT5 and HXT13, which are theoretically repressed by glucose, were no longer inhibited in MT1 and were highly expressed even in a medium containing 70 g/L glucose. Thus, other sugars may be co-utilized with glucose by MT1 without diauxic growth. CONCLUSIONS Based on a functional genomics analysis, we revealed the genetic basis and evolutionary mechanisms underlying the traits of the Chinese Maotai-flavored yeast MT1. This work provides new insights for the genetic breeding of yeast and also enriches the genetic resources of yeast.
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Affiliation(s)
- Xiaowei Lu
- State Key Laboratory of Food Science and Technology; The Key Laboratory of Industrial Biotechnology, Ministry of Education; Synergetic Innovation Center of Food Safety and Nutrition; School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
| | - Qun Wu
- State Key Laboratory of Food Science and Technology; The Key Laboratory of Industrial Biotechnology, Ministry of Education; Synergetic Innovation Center of Food Safety and Nutrition; School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
| | - Yan Zhang
- Ministry of Education Key Laboratory of Systems Biomedicine, Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, China.
| | - Yan Xu
- State Key Laboratory of Food Science and Technology; The Key Laboratory of Industrial Biotechnology, Ministry of Education; Synergetic Innovation Center of Food Safety and Nutrition; School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
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11
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Simultaneous gene inactivation and promoter reporting in cyanobacteria. Appl Microbiol Biotechnol 2014; 99:1779-93. [DOI: 10.1007/s00253-014-6209-2] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2014] [Revised: 10/28/2014] [Accepted: 10/31/2014] [Indexed: 10/24/2022]
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12
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The Ess1 prolyl isomerase: traffic cop of the RNA polymerase II transcription cycle. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2014; 1839:316-33. [PMID: 24530645 DOI: 10.1016/j.bbagrm.2014.02.001] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/04/2014] [Revised: 02/01/2014] [Accepted: 02/03/2014] [Indexed: 11/23/2022]
Abstract
Ess1 is a prolyl isomerase that regulates the structure and function of eukaryotic RNA polymerase II. Ess1 works by catalyzing the cis/trans conversion of pSer5-Pro6 bonds, and to a lesser extent pSer2-Pro3 bonds, within the carboxy-terminal domain (CTD) of Rpb1, the largest subunit of RNA pol II. Ess1 is conserved in organisms ranging from yeast to humans. In budding yeast, Ess1 is essential for growth and is required for efficient transcription initiation and termination, RNA processing, and suppression of cryptic transcription. In mammals, Ess1 (called Pin1) functions in a variety of pathways, including transcription, but it is not essential. Recent work has shown that Ess1 coordinates the binding and release of CTD-binding proteins that function as co-factors in the RNA pol II complex. In this way, Ess1 plays an integral role in writing (and reading) the so-called CTD code to promote production of mature RNA pol II transcripts including non-coding RNAs and mRNAs.
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13
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Teng X, Dayhoff-Brannigan M, Cheng WC, Gilbert CE, Sing CN, Diny NL, Wheelan SJ, Dunham MJ, Boeke JD, Pineda FJ, Hardwick JM. Genome-wide consequences of deleting any single gene. Mol Cell 2013; 52:485-94. [PMID: 24211263 DOI: 10.1016/j.molcel.2013.09.026] [Citation(s) in RCA: 144] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2013] [Revised: 08/18/2013] [Accepted: 09/19/2013] [Indexed: 11/19/2022]
Abstract
Loss or duplication of chromosome segments can lead to further genomic changes associated with cancer. However, it is not known whether only a select subset of genes is responsible for driving further changes. To determine whether perturbation of any given gene in a genome suffices to drive subsequent genetic changes, we analyzed the yeast knockout collection for secondary mutations of functional consequence. Unlike wild-type, most gene knockout strains were found to have one additional mutant gene affecting nutrient responses and/or heat-stress-induced cell death. Moreover, independent knockouts of the same gene often evolved mutations in the same secondary gene. Genome sequencing identified acquired mutations in several human tumor suppressor homologs. Thus, mutation of any single gene may cause a genomic imbalance, with consequences sufficient to drive adaptive genetic changes. This complicates genetic analyses but is a logical consequence of losing a functional unit originally acquired under pressure during evolution.
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Affiliation(s)
- Xinchen Teng
- Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205, USA
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Abstract
One of the top things on a geneticist's wish list has to be a set of mutants for every gene in their particular organism. Such a set was produced for the yeast, Saccharomyces cerevisiae near the end of the 20th century by a consortium of yeast geneticists. However, the functional genomic analysis of one chromosome, its smallest, had already begun more than 25 years earlier as a project that was designed to define most or all of that chromosome's essential genes by temperature-sensitive lethal mutations. When far fewer than expected genes were uncovered, the relatively new field of molecular cloning enabled us and indeed, the entire community of yeast researchers to approach this problem more definitively. These studies ultimately led to cloning, genomic sequencing, and the production and phenotypic analysis of the entire set of knockout mutations for this model organism as well as a better concept of what defines an essential function, a wish fulfilled that enables this model eukaryote to continue at the forefront of research in modern biology.
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Abstract
Genetic interactions are functional crosstalk among different genetic loci that lead to phenotypic changes, such as health or viability alterations. A disease or lethal phenotype that results from the combined effects of gene mutations at different loci is termed a synthetic sickness or synthetic lethality, respectively. Studies of genetic interaction have provided insight on the relationships among biochemical processes or pathways. Cancer results from genetic interactions and is a major focus of current studies in genetic interactions. Various basic and translational cancer studies have explored the concept of genetic interactions, including studies of the mechanistic characterization of genes, drug discovery, biomarker identification and the rational design of combination therapies. This review discusses the implications of genetic interactions in the development of personalized cancer therapies, the identification of treatment-responsive genes, the delineation of mechanisms of chemoresistance and the rational design of combined therapeutic strategies to overcome drug resistance.
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16
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Motter AE. Improved network performance via antagonism: From synthetic rescues to multi-drug combinations. Bioessays 2010; 32:236-245. [PMID: 20127700 PMCID: PMC2841822 DOI: 10.1002/bies.200900128] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Recent research shows that a faulty or sub-optimally operating metabolic network can often be rescued by the targeted removal of enzyme-coding genes – the exact opposite of what traditional gene therapy would suggest. Predictions go as far as to assert that certain gene knockouts can restore the growth of otherwise nonviable gene-deficient cells. Many questions follow from this discovery: What are the underlying mechanisms? How generalizable is this effect? What are the potential applications? Here, I approach these questions from the perspective of compensatory perturbations on networks. Relations are drawn between such synthetic rescues and naturally occurring cascades of reaction inactivation, as well as their analogs in physical and other biological networks. I specially discuss how rescue interactions can lead to the rational design of antagonistic drug combinations that select against resistance and how they can illuminate medical research on cancer, antibiotics, and metabolic diseases.
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Affiliation(s)
- Adilson E Motter
- Department of Physics and Astronomy and Northwestern Institute on Complex Systems, Northwestern University, Evanston, IL, USA
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17
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Chronic oxidative DNA damage due to DNA repair defects causes chromosomal instability in Saccharomyces cerevisiae. Mol Cell Biol 2008; 28:5432-45. [PMID: 18591251 DOI: 10.1128/mcb.00307-08] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Oxidative DNA damage is likely to be involved in the etiology of cancer and is thought to accelerate tumorigenesis via increased mutation rates. However, the majority of malignant cells acquire a specific type of genomic instability characterized by large-scale genomic rearrangements, referred to as chromosomal instability (CIN). The molecular mechanisms underlying CIN are not entirely understood. We utilized Saccharomyces cerevisiae as a model system to delineate the relationship between genotoxic stress and CIN. It was found that elevated levels of chronic, unrepaired oxidative DNA damage caused chromosomal aberrations at remarkably high frequencies under both selective and nonselective growth conditions. In this system, exceeding the cellular capacity to appropriately manage oxidative DNA damage resulted in a "gain-of-CIN" phenotype and led to profound karyotypic instability. These results illustrate a novel mechanism for genome destabilization that is likely to be relevant to human carcinogenesis.
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18
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Kim PJ, Lee DY, Kim TY, Lee KH, Jeong H, Lee SY, Park S. Metabolite essentiality elucidates robustness of Escherichia coli metabolism. Proc Natl Acad Sci U S A 2007; 104:13638-42. [PMID: 17698812 PMCID: PMC1947999 DOI: 10.1073/pnas.0703262104] [Citation(s) in RCA: 101] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Complex biological systems are very robust to genetic and environmental changes at all levels of organization. Many biological functions of Escherichia coli metabolism can be sustained against single-gene or even multiple-gene mutations by using redundant or alternative pathways. Thus, only a limited number of genes have been identified to be lethal to the cell. In this regard, the reaction-centric gene deletion study has a limitation in understanding the metabolic robustness. Here, we report the use of flux-sum, which is the summation of all incoming or outgoing fluxes around a particular metabolite under pseudo-steady state conditions, as a good conserved property for elucidating such robustness of E. coli from the metabolite point of view. The functional behavior, as well as the structural and evolutionary properties of metabolites essential to the cell survival, was investigated by means of a constraints-based flux analysis under perturbed conditions. The essential metabolites are capable of maintaining a steady flux-sum even against severe perturbation by actively redistributing the relevant fluxes. Disrupting the flux-sum maintenance was found to suppress cell growth. This approach of analyzing metabolite essentiality provides insight into cellular robustness and concomitant fragility, which can be used for several applications, including the development of new drugs for treating pathogens.
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Affiliation(s)
- Pan-Jun Kim
- *Center for Systems and Synthetic Biotechnology, Institute for the BioCentury
- Department of Physics
| | - Dong-Yup Lee
- Bioinformatics Research Center
- Metabolic and Biomolecular Engineering National Research Laboratory, BioProcess Engineering Research Center
- Department of Chemical and Biomolecular Engineering (BK21 Program), and
| | - Tae Yong Kim
- *Center for Systems and Synthetic Biotechnology, Institute for the BioCentury
- Metabolic and Biomolecular Engineering National Research Laboratory, BioProcess Engineering Research Center
- Department of Chemical and Biomolecular Engineering (BK21 Program), and
| | - Kwang Ho Lee
- *Center for Systems and Synthetic Biotechnology, Institute for the BioCentury
- Metabolic and Biomolecular Engineering National Research Laboratory, BioProcess Engineering Research Center
- Department of Chemical and Biomolecular Engineering (BK21 Program), and
| | - Hawoong Jeong
- *Center for Systems and Synthetic Biotechnology, Institute for the BioCentury
- Department of Physics
- Bioinformatics Research Center
- To whom correspondence may be addressed. E-mail: or
| | - Sang Yup Lee
- *Center for Systems and Synthetic Biotechnology, Institute for the BioCentury
- Bioinformatics Research Center
- Metabolic and Biomolecular Engineering National Research Laboratory, BioProcess Engineering Research Center
- Department of Chemical and Biomolecular Engineering (BK21 Program), and
- Department of BioSystems, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea
- To whom correspondence may be addressed. E-mail: or
| | - Sunwon Park
- Bioinformatics Research Center
- Department of Chemical and Biomolecular Engineering (BK21 Program), and
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19
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Neyfakh AA, Baranova NN, Mizrokhi LJ. A system for studying evolution of life-like virtual organisms. Biol Direct 2006; 1:23. [PMID: 16916465 PMCID: PMC1569368 DOI: 10.1186/1745-6150-1-23] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2006] [Accepted: 08/17/2006] [Indexed: 11/25/2022] Open
Abstract
Background Fitness landscapes, the dependences of fitness on the genotype, are of critical importance for the evolution of living beings. Unfortunately, fitness landscapes that are relevant to the evolution of complex biological functions are very poorly known. As a result, the existing theory of evolution is mostly based on postulated fitness landscapes, which diminishes its usefulness. Attempts to deduce fitness landscapes from models of actual biological processes led, so far, to only limited success. Results We present a model system for studying the evolution of biological function, which makes it possible to attribute fitness to genotypes in a natural way. The system mimics a very simple cell and takes into account the basic properties of gene regulation and enzyme kinetics. A virtual cell contains only two small molecules, an organic nutrient A and an energy carrier X, and proteins of five types – two transcription factors, two enzymes, and a membrane transporter. The metabolism of the cell consists of importing A from the environment and utilizing it in order to produce X and an unspecified end product. The genome may carry an arbitrary number of genes, each one encoding a protein of one of the five types. Both major mutations that affect whole genes and minor mutations that affect individual characteristics of genes are possible. Fitness is determined by the ability of the cell to maintain homeostasis when its environment changes. The system has been implemented as a computer program, and several numerical experiments have been performed on it. Evolution of the virtual cells usually involves a rapid initial increase of fitness, which eventually slows down, until a fitness plateau is reached. The origin of a wide variety of genetic networks is routinely observed in independent experiments performed under the same conditions. These networks can have different, including very high, levels of complexity and often include large numbers of non-essential genes. Conclusion The described system displays a rich repertoire of biologically sensible behaviors and, thus, can be useful for investigating a number of unresolved issues in evolutionary biology, including evolution of complexity, modularity and redundancy, as well as for studying the general properties of genotype-to-fitness maps. Reviewers This article was reviewed by Drs. Eugene Koonin, Shamil Sunyaev and Arcady Mushegian.
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Affiliation(s)
- Alex A Neyfakh
- Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, IL 60607, USA
| | - Natalya N Baranova
- Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, IL 60607, USA
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20
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Rodríguez-Trelles F, Tarrío R, Ayala FJ. Is ectopic expression caused by deregulatory mutations or due to gene-regulation leaks with evolutionary potential? Bioessays 2005; 27:592-601. [PMID: 15892118 DOI: 10.1002/bies.20241] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
It has long been thought that gene expression is tightly regulated in multicellular eukaryotes, so that expression profiles match functional profiles. This conception emerged from the assumption that gene activity is synonymous with gene function. This paradigm was first challenged by comparative protein electrophoresis studies showing extensive differences in expression patterns among related species. The paradigm is now being challenged by evolutionary transcriptomics using microarray technologies. Most gene expression profiles display features that lack any obvious functional significance. The so-called "ectopic" expression refers to the expression of genes at times and locations where the target gene is not known to have a function. However, ectopic expression might be associated with genuine function even if this function is not essential or has yet to be ascertained. Alternatively, ectopic expression might come about as a superfluous by-product of regulatory systems, which would call for a revision of prevailing ideas about the specificity of gene regulation. We herein review available evidence for ectopic expression and the hypotheses proposed for its origin and evolution. We propose that ectopic expression must be regarded as part of an integrated phenotypic whole. It seems likely that ectopic expression represents a leak in the evolution of regulatory systems, but one that is endowed with considerable evolutionary possibilities.
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21
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Tcheperegine SE, Gao XD, Bi E. Regulation of cell polarity by interactions of Msb3 and Msb4 with Cdc42 and polarisome components. Mol Cell Biol 2005; 25:8567-80. [PMID: 16166638 PMCID: PMC1265733 DOI: 10.1128/mcb.25.19.8567-8580.2005] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2005] [Revised: 03/04/2005] [Accepted: 07/14/2005] [Indexed: 11/20/2022] Open
Abstract
In Saccharomyces cerevisiae, polarized growth depends on interactions between the actin cytoskeleton and the secretory machinery. Here we show that the Rab GTPase-activating proteins (GAPs) Msb3 and Msb4 interact directly with Spa2, a scaffold protein of the "polarisome" that also interacts with the formin Bni1. Spa2 is required for the polarized localization of Msb3 and Msb4 at the bud tip. We also show that Msb3 and Msb4 bind specifically to Cdc42-GDP and Rho1-GDP in vitro and that Msb3 and Rho GDP dissociation inhibitor act independently but oppositely on Cdc42. Finally, we show that Msb3 and Msb4 are involved in Bni1-nucleated actin assembly in vivo. These results suggest that Msb3 and Msb4 regulate polarized growth by multiple mechanisms, directly regulating exocytosis through their GAP activity toward Sec4 and potentially coordinating the functions of Cdc42, Rho1, and Bni1 in the polarisome through their binding to these GTPases. A functional equivalent of the polarisome probably exists in other fungi and mammals.
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Affiliation(s)
- Serguei E Tcheperegine
- Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, 421 Curie Blvd., Room 1012, BRB II/III, Philadelphia, PA 19104-6058, USA
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22
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Feder ME, Walser JC. The biological limitations of transcriptomics in elucidating stress and stress responses. J Evol Biol 2005; 18:901-10. [PMID: 16033562 DOI: 10.1111/j.1420-9101.2005.00921.x] [Citation(s) in RCA: 186] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Global analysis of mRNA abundance via genomic arrays (i.e. transcriptomics or transcriptional profiling) is one approach to finding the genes that matter to organisms undergoing environmental stress. In evolutionary analyses of stress, mRNA abundance is often invoked as a proxy for the protein activity that may underlie variation in fitness. To provoke discussion of the utility and sensible application of this valuable approach, this manuscript examines the adequacy of mRNA abundance as a proxy for protein activity, fitness and stress. Published work to date suggests that mRNA abundance typically provides little information on protein activity and fitness and cannot substitute for detailed functional and ecological analyses of candidate genes. While the transcriptional profile can be an exquisitely sensitive indicator of stress, simpler indicators will often suffice. In view of this outcome, transcriptomics should undergo careful cost-benefit analysis before investigators deploy it in studies of stress responses and their evolution.
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Affiliation(s)
- M E Feder
- Department of Organismal Biology and Anatomy, The University of Chicago, Chicago, IL 60637, USA.
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23
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Blanc VM, Adams J. Evolution in Saccharomyces cerevisiae: Identification of Mutations Increasing Fitness in Laboratory Populations. Genetics 2003; 165:975-83. [PMID: 14668358 PMCID: PMC1462841 DOI: 10.1093/genetics/165.3.975] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Abstract
Since the publication of the complete sequence of the genome of Saccharomyces cerevisiae, a number of comprehensive investigations have been initiated to gain insight into cellular function. The focus of these studies has been to identify genes essential for survival in specific environments or those that when mutated cause gross phenotypic defects in growth. Here we describe Ty1-based mutational approaches designed to identify genes, which when mutated generate evolutionarily significant phenotypes causing small but positive increments on fitness. As expected, Ty1 mutations with a positive fitness effect were in the minority. However, mutations in two loci, one inactivating FAR3 and one upstream of CYR1, identified in evolving populations, were shown to have small but significantly positive fitness effects.
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Affiliation(s)
- Victoria M Blanc
- Department of Biology, University of Michigan, Ann Arbor, Michigan 48109-1048, USA
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24
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Abstract
Recent large-scale studies of protein complexes in yeast have demonstrated that the wide majority of proteins exist in the cell as parts of multicomponent assemblies, mostly novel and of unknown function. The structural and functional analysis of these complexes should be a priority for structural biologists in coming years. In silico methods such as docking simulations, which may contribute to this analysis, are being tested in the CAPRI community-wide experiment, which assesses blind predictions of the structure of protein-protein complexes.
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Affiliation(s)
- Joël Janin
- Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France.
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25
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Glöckner G, Eichinger L, Szafranski K, Pachebat JA, Bankier AT, Dear PH, Lehmann R, Baumgart C, Parra G, Abril JF, Guigó R, Kumpf K, Tunggal B, Cox E, Quail MA, Platzer M, Rosenthal A, Noegel AA. Sequence and analysis of chromosome 2 of Dictyostelium discoideum. Nature 2002; 418:79-85. [PMID: 12097910 DOI: 10.1038/nature00847] [Citation(s) in RCA: 132] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The genome of the lower eukaryote Dictyostelium discoideum comprises six chromosomes. Here we report the sequence of the largest, chromosome 2, which at 8 megabases (Mb) represents about 25% of the genome. Despite an A + T content of nearly 80%, the chromosome codes for 2,799 predicted protein coding genes and 73 transfer RNA genes. This gene density, about 1 gene per 2.6 kilobases (kb), is surpassed only by Saccharomyces cerevisiae (one per 2 kb) and is similar to that of Schizosaccharomyces pombe (one per 2.5 kb). If we assume that the other chromosomes have a similar gene density, we can expect around 11,000 genes in the D. discoideum genome. A significant number of the genes show higher similarities to genes of vertebrates than to those of other fully sequenced eukaryotes. This analysis strengthens the view that the evolutionary position of D. discoideum is located before the branching of metazoa and fungi but after the divergence of the plant kingdom, placing it close to the base of metazoan evolution.
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Affiliation(s)
- Gernot Glöckner
- IMB Jena, Department of Genome Analysis, Beutenbergstr. 11, 07745 Jena, Germany.
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26
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Rokyta D, Badgett MR, Molineux IJ, Bull JJ. Experimental genomic evolution: extensive compensation for loss of DNA ligase activity in a virus. Mol Biol Evol 2002; 19:230-8. [PMID: 11861882 DOI: 10.1093/oxfordjournals.molbev.a004076] [Citation(s) in RCA: 59] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Deletion of the viral ligase gene drastically reduced the fitness of bacteriophage T7 on a ligase-deficient host. Viral evolution recovered much of this fitness during long-term passage, but the final fitness remained below that of the intact virus. Compensatory changes occurred chiefly in genes involved in DNA metabolism: the viral endonuclease, helicase, and DNA polymerase. Two other compensatory changes of unknown function also occurred. Using a method to distinguish compensatory mutations from other beneficial mutations, five additional substitutions from the recovery were shown to enhance adaptation to culture conditions and were not compensatory for the deletion. In contrast to the few previous studies of viral recovery from deletions, the compensatory changes in T7 did not restore the deletion or duplicate major regions of the genome. The ability of this deleted genome to recover much of the lost fitness via mutations in its remaining genes reveals a considerable evolutionary potential to modify the interactions of its elements in maintaining an essential set of functions.
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Affiliation(s)
- D Rokyta
- Section of Integrative Biology, University of Texas, Austin, TX 78712, USA
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27
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Zeyl C, DeVisser JA. Estimates of the rate and distribution of fitness effects of spontaneous mutation in Saccharomyces cerevisiae. Genetics 2001; 157:53-61. [PMID: 11139491 PMCID: PMC1461475 DOI: 10.1093/genetics/157.1.53] [Citation(s) in RCA: 124] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
The per-genome, per-generation rate of spontaneous mutation affecting fitness (U) and the mean fitness cost per mutation (s) are important parameters in evolutionary genetics, but have been estimated for few species. We estimated U and sh (the heterozygous effect of mutations) for two diploid yeast strains differing only in the DNA mismatch-repair deficiency used to elevate the mutation rate in one (mutator) strain. Mutations were allowed to accumulate in 50 replicate lines of each strain, during 36 transfers of randomly chosen single colonies (approximately 600 generations). Among wild-type lines, fitnesses were bimodal, with one mode showing no change in mean fitness. The other mode showed a mean 29.6% fitness decline and the petite phenotype, usually caused by partial deletion of the mitochondrial genome. Excluding petites, maximum-likelihood estimates adjusted for the effect of selection were U = 9.5 x 10(-5) and sh = 0.217 for the wild type. Among the mutator lines, the best fit was obtained with 0.005 < or = U < or = 0.94 and 0.049 > or = sh > or = 0.0003. Like other recently tested model organisms, wild-type yeast have low mutation rates, with high mean fitness costs per mutation. Inactivation of mismatch repair increases the frequency of slightly deleterious mutations by approximately two orders of magnitude.
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Affiliation(s)
- C Zeyl
- Department of Biology, Wake Forest University, Winston-Salem, North Carolina 27109, USA.
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28
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Abstract
What is the minimum number of genes or functions necessary to support cellular life? The concept of a 'minimal genome' has become popular, but is it a useful concept, and if so, what might a minimal genome encode? We argue that the concept may be useful, even though the goal of defining a general minimal genome may never be attained.
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Affiliation(s)
- S N Peterson
- The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, Maryland 20850, USA.
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29
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Abstract
Population genetics is a highly theoretical field in which many models and theories of broad significance have received little experimental testing. Microbes are well-suited for empirical population genetics since populations of almost any size may be studied genetically, and because many have easily controlled life cycles. Saccharomyces cerevisiae is almost ideal for such studies as the growing body of knowledge and techniques that have made it the best characterized eukaryote genome also allow the experimental manipulation and analysis of its population genetics. In experiments to date, the evolution of laboratory yeast populations has been observed for up to 1000 generations. In several cases, adaptation has occurred by gene duplications. The interaction between mutation, selection and genetic drift at varying population sizes is a major area of theoretical study in which yeast experiments can provide particularly valuable data. Conflicts between gene-level and among-cell selection, and co-evolution between genes within a genome, are additional topics in which a population genetics perspective may be particularly helpful. The growing field of genomics is increasingly complementary with that of population genetics. The characterization of the yeast genome presents unprecedented opportunities for the detailed study of evolutionary and population genetics. Conversely, the redundancy of the yeast genome means that, for many open reading frames, deletion has only a quantitative effect that is most readily observed in competitions with a wild-type strain.
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Affiliation(s)
- C Zeyl
- Department of Biology, Wake Forest University, Winston-Salem, NC 27109, USA.
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30
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Abstract
The bacterial genomic era began with the publication of the chromosomal sequence of Haemophilus influenzae. As few of the observed genes had been examined experimentally, functional assignments were made by comparative analysis and for many genes no annotation could be made. This mini-review briefly describes the genomic-scale experimental approaches being used to identify genes required for the growth of microorganisms. Identifying 'essential genes', the simplest possible annotation for the unknown open reading frames, is important for antibacterial and antifungal research and is a first step to defining the minimum functional requirement for autonomous growth.
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Affiliation(s)
- K A Reich
- Abbott Laboratories, Department 46Y, Abbott Park, IL 60064-6100, USA.
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31
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Graner E, Mercadante AF, Zanata SM, Forlenza OV, Cabral AL, Veiga SS, Juliano MA, Roesler R, Walz R, Minetti A, Izquierdo I, Martins VR, Brentani RR. Cellular prion protein binds laminin and mediates neuritogenesis. BRAIN RESEARCH. MOLECULAR BRAIN RESEARCH 2000; 76:85-92. [PMID: 10719218 DOI: 10.1016/s0169-328x(99)00334-4] [Citation(s) in RCA: 234] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Laminin (LN) plays a major role in neuronal differentiation, migration and survival. Here, we show that the cellular prion protein (PrPc) is a saturable, specific, high-affinity receptor for LN. The PrPc-LN interaction is involved in the neuritogenesis induced by NGF plus LN in the PC-12 cell line and the binding site resides in a carboxy-terminal decapeptide from the gamma-1 LN chain. Neuritogenesis induced by LN or its gamma-1-derived peptide in primary cultures from rat or either wild type or PrP null mice hippocampal neurons, indicated that PrPc is the main cellular receptor for that particular LN domain. These results point out to the importance of the PrPc-LN interaction for the neuronal plasticity mechanism.
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Affiliation(s)
- E Graner
- Ludwig Institute for Cancer Research, São Paulo Branch, Rua Prof. Antônio Prudente 109/4A, 01509-010, São Paulo, Brazil
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32
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Hutchison CA, Peterson SN, Gill SR, Cline RT, White O, Fraser CM, Smith HO, Venter JC. Global transposon mutagenesis and a minimal Mycoplasma genome. Science 1999; 286:2165-9. [PMID: 10591650 DOI: 10.1126/science.286.5447.2165] [Citation(s) in RCA: 607] [Impact Index Per Article: 23.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Abstract
Mycoplasma genitalium with 517 genes has the smallest gene complement of any independently replicating cell so far identified. Global transposon mutagenesis was used to identify nonessential genes in an effort to learn whether the naturally occurring gene complement is a true minimal genome under laboratory growth conditions. The positions of 2209 transposon insertions in the completely sequenced genomes of M. genitalium and its close relative M. pneumoniae were determined by sequencing across the junction of the transposon and the genomic DNA. These junctions defined 1354 distinct sites of insertion that were not lethal. The analysis suggests that 265 to 350 of the 480 protein-coding genes of M. genitalium are essential under laboratory growth conditions, including about 100 genes of unknown function.
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Affiliation(s)
- C A Hutchison
- The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, USA
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33
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Abstract
To begin genome-wide functional analysis, we analysed the consequences of deleting each of the 265 genes of chromosome VIII of Saccharomyces cerevisiae. For 33% of the deletion strains a growth phenotype could be detected: 18% of the genes are essential for growth on complete glucose medium, and 15% grow significantly more slowly than the wild-type strain or exhibit a conditional phenotype when incubated under one of 20 different growth conditions. Two-thirds of the mutants that exhibit conditional phenotypes are pleiotropic; about one-third of the mutants exhibit only one phenotype. We also measured the level of expression directed by the promoter of each gene. About half of the promoters direct detectable transcription in rich glucose medium, and most of these exhibited only low or medium activity. Only 1% of the genes are expressed at about the same level as ACT1. The number of active promoters increased to 76% upon growth on a non-fermentable carbon source, and to 93% in minimal glucose medium. The majority of promoters fluctuated in strength, depending on the medium.
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Affiliation(s)
- R Niedenthal
- Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, Geb. 26.12.01.64, 40225 Düsseldorf, Germany
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Casalone E, Barberio C, Cavalieri D, Ceccarelli I, Riparbelli M, Ugolini S, Polsinelli M. Disruption and phenotypic analysis of six novel genes from chromosome IV of Saccharomyces cerevisiae reveal YDL060w as an essential gene for vegetative growth. Yeast 1999; 15:1691-701. [PMID: 10572265 DOI: 10.1002/(sici)1097-0061(199911)15:15<1691::aid-yea489>3.0.co;2-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022] Open
Abstract
The disruption of six novel genes (YDL059c, YDL060w, YDL063c, YDL065c, YDL070w and YDL110c), localized on the left arm of chromosome IV in Saccharomyces cerevisiae, is reported. A PCR-based strategy was used to construct disruption cassettes in which the kanMX4 dominant marker was introduced between two long flanking homology regions, homologous to the promoter and terminator sequences of the target gene (Wach et al., 1994). The disruption cassettes were used to generate homologous recombinants in two diploid strains with different genetic backgrounds (FY1679 and CEN. PK2), selecting for geneticin (G418) resistance conferred by the presence of the dominant marker kanMX4. The correctness of the cassette integration was tested by PCR. After sporulation and tetrad analysis of the heterozygous deletant diploids, geneticin-resistant haploids carrying the disrupted allele were isolated. YDL060w was shown to be an essential gene for vegetative growth. A more detailed phenotypic analysis of the non-lethal haploid deletant strains was performed, looking at cell and colony morphology, growth capability on different media at different temperatures, and ability to conjugate. Homozygous deletant diploids were also constructed and tested for sporulation. Only minor differences between parental and mutant strains were found for some deletant haploids.
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Affiliation(s)
- E Casalone
- Department of Biomedical Science, University 'G. D'Annunzio' of Chieti, via dei Vestini 31, I-66100 Chieti, Italy.
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35
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Abstract
The commonly used genetic approaches in yeast are designed to identify defects in cell/colony growth. In order to identify genes which control molecular mechanisms during quiescence ('stationary phase'), different tactics are required. We describe the development of a new genetic approach based on the previous observations that gene expression in quiescent Saccharomyces cerevisiae cells is largely repressed. For studying the mechanism controlling the repression of gene expression in stationary phase, we use UBI4-lacZ as a reporter gene. The product of this fusion gene was shown previously to encode an unstable protein in dividing cells. We show here that it is also unstable in stationary cells. We demonstrate that the relatively short half-life of this reporter protein can be utilized to monitor the dynamics of the repression of gene expression during stationary phase in liquid culture, using ACT1 or SSA3 promoters as the model promoters. By adapting a colony color test, we show that the reporter gene can also be used to monitor gene expression in quiescent colonies, thus serving as a tool to screen for defects in the regulation of this process during growth arrest. The utility of the approach was demonstrated by confirming the defects of top1Delta and bcy1Delta cells to appropriately express the ACT1p-UBI4-lacZ in stationary phase. The mutant colonies were easily discernible from wild-type colonies by our color test. Finally, using SSA3p-UBI4-lacZ as the reporter gene, we found that the 5'-untranslated region of SSA3 mRNA is sufficient to repress translation of the reporter mRNA after entry of the cells into stationary phase. The possibility that the short length of the SSA3 5'-untranslated region is a major determinant of the inefficient translation of SSA3p-UBI4-lacZ in stationary phase is discussed.
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Affiliation(s)
- I Paz
- Department of Molecular Microbiology and Biotechnology, Ramat Aviv 69978, Israel
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36
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Abstract
Twelve different ORFs have been deleted from the right arm of Saccharomyces cerevisiae chromosome II; namely YBR193c, YBR194w, YBR197c, YBR198c, YBR201w, YBR203w, YBR207w, YBR209w, YBR210w, YBR211c, YBR217w and YBR228w. Tetrad analysis of heterozygous deletant strains revealed that YBR193c, YBR198c and YBR211c are essential genes for vegetative growth. No effects were detected in any of the haploid deletion mutants for the rest of the ORFs with respect to growth, gross morphology or mating.
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Affiliation(s)
- S Rodríguez-Navarro
- Departament de Bioquímica i Biología Molecular, Facultat de Ciències Biològiques, Universitat de València, Spain.
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37
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Dueñas E, Vazquez de Aldana CR, de Cos T, Castro C, Henar Valdivieso M. Generation of null alleles for the functional analysis of six genes from the right arm of Saccharomyces cerevisiae chromosome II. Yeast 1999; 15:615-23. [PMID: 10341424 DOI: 10.1002/(sici)1097-0061(199905)15:7<615::aid-yea385>3.0.co;2-x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Using PCR-ligated long flanking homology cassettes, null alleles of six open reading frames (ORFs) from chromosome II have been created in Saccharomyces cerevisiae. Deletants were constructed in three genetic backgrounds: FY1679, W303 and CEN.PK2. Tetrad analysis of heterozygous deletants revealed that none of the ORFs is essential for vegetative growth. Basic phenotypic analysis of haploid deletants showed that deletion of the YBR283c ORF causes a slight growth defect at 30 degrees C and 37 degrees C on glycerol-complete, glucose-complete, and glucose-minimal media only in the FY1679 and W303 backgrounds. Transformation of these deletants with the corresponding cognate gene in a centromeric plasmid complements the defects. Deletion of the YBR287w ORF leads to poor growth on glucose-minimal medium at 15 degrees C in the FY 1679 background. None of the six ORFs seems to be involved in mating or sporulation.
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Affiliation(s)
- E Dueñas
- Departamento de Microbiología y Genética, Universidad de Salamanca, Spain
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38
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Sanjuan R, León M, Zueco J, Sentandreu R. Basic phenotypic analysis of six novel yeast genes reveals two essential genes and one which affects the growth rate. Yeast 1999; 15:351-60. [PMID: 10206193 DOI: 10.1002/(sici)1097-0061(19990315)15:4<351::aid-yea364>3.0.co;2-l] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Phenotypic analysis was performed on six mutants of Saccharomyces cerevisiae deleted in one of the following open reading frames (ORFs), located on chromosome II: YBR254c, YBR255w, YBR257w, YBR258c, YBR259w and YBR266c. Disruption of the ORFs was carried out in the diploid strain FY1679 using the kanMX4 marker flanked by short sequences homologous to the target locus. Tetrad analysis following sporulation of the heterozygous disruptants showed that YBR254c and YBR257w are essential genes. YBR257w was later characterized and renamed POP4, its gene product being involved in 5.8S rRNA and tRNA processing (Chu et al., 1997). The tetrad analysis performed for the heterozygous disruptant for YBR266c showed that two of the four viable spores gave colonies of smaller size, reflecting a slower growth rate. Growth analysis of the disruptant haploids confirmed this defect at 30 degrees C and also at 15 degrees C. However, complementation tests failed to confirm that the deletion of YBR266c is responsible for this growth defect. Growth analysis of the disruptant haploid ybr255w(delta) showed a slower growth rate on YPD and minimal medium at 15 degrees C. Finally, no phenotypic effect could be detected associated to the disruption of ORFs YBR258c and YBR259w.
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Affiliation(s)
- R Sanjuan
- Sección Departamental de Microbiología, Facultad de Farmacia, Universidad de Valencia, Burjasot, Spain
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Dueñas E, Revuelta JL, del Rey F, Vázquez de Aldana CR. Disruption and basic phenotypic analysis of six novel genes from the left arm of chromosome XIV of Saccharomyces cerevisiae. Yeast 1999; 15:63-72. [PMID: 10028186 DOI: 10.1002/(sici)1097-0061(19990115)15:1<63::aid-yea338>3.0.co;2-7] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
We describe here the construction of six deletion mutants and their basic phenotypic analysis in three different backgrounds. The six genes were disrupted in three diploid strains (FY1679, W303 and CEN.PK2) by the long flanking homology (LFH) method (Wach, 1996). Transformants were selected as geneticin (G418)-resistant colonies and correct integration of the kanMX4 cassette was checked by colony PCR. Following sporulation of the heterozygous diploids, tetrads were dissected and scored for segregation of G418-resistance and auxotrophic markers. One of the six ORFs (YNL158w) corresponds to an essential gene which has no homology with other genes present in the databases and has two predicted transmembrane domains. Growth tests performed on different media at 15 degrees C, 30 degrees C or 37 degrees C with haploid deletants of the five non-essential genes revealed no apparent phenotype in any of them.
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Affiliation(s)
- E Dueñas
- Departamento de Microbiología y Genética, Universidad de Salamanca/CSIC, Spain
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40
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Affiliation(s)
- M V Olson
- Departments of Medicine (Division of Medical Genetics) and Genetics, University of Washington, Seattle, WA 98195, USA.
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41
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Fairhead C, Llorente B, Denis F, Soler M, Dujon B. New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using ‘split-marker’ recombination. Yeast 1998. [DOI: 10.1002/(sici)1097-0061(199611)12:14<1439::aid-yea37>3.0.co;2-o] [Citation(s) in RCA: 140] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
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42
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Fairhead C, Thierry A, Denis F, Eck M, Dujon B. 'Mass-murder' of ORFs from three regions of chromosome XI from Saccharomyces cerevisiae. Gene 1998; 223:33-46. [PMID: 9858675 DOI: 10.1016/s0378-1119(98)00171-1] [Citation(s) in RCA: 25] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
The complete sequence of the yeast Saccharomyces cerevisiae reveals the presence of many new genes, many of which are without homologs in databases. Characterisation of these genes by novel methods includes systematic deletion followed by phenotypic analysis of mutant strains. We have developed a hierarchical strategy for such a functional analysis of genes, in which the primary phenotypic screening is performed on groups of contiguous genes which are then reinvestigated down to the single gene level. This strategy is applied to the whole chromosome XI as part of EUROFAN (the EUROpean Functional ANalysis) program, and we present here our results on a group of 22 genes from this chromosome. This sample is representative of the results that are emerging for the whole chromosome. Out of the 22 genes deleted, three were shown to be essential, and another three genes confer a mutant growth phenotype to cells when deleted. All phenotypes have been complemented. These figures are in accordance with the previously published fraction of lethal and growth-defective deletions of single genes. We have found no synthetic phenotypes resulting from a combination of deleted genes and have always been able to attribute a mutant phenotype to a single gene.
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Affiliation(s)
- C Fairhead
- Unité de Génétique Moléculaire des Levures (UFR927 Univ. P. et M. Curie and URA 1300, CNRS), Institut Pasteur, 25 rue du Dr Roux, F-75724, Paris Cedex 15,
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43
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Gawantka V, Pollet N, Delius H, Vingron M, Pfister R, Nitsch R, Blumenstock C, Niehrs C. Gene expression screening in Xenopus identifies molecular pathways, predicts gene function and provides a global view of embryonic patterning. Mech Dev 1998; 77:95-141. [PMID: 9831640 DOI: 10.1016/s0925-4773(98)00115-4] [Citation(s) in RCA: 165] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
In a large-scale gene expression screen 1765 randomly picked cDNAs were analyzed by whole-mount in situ hybridization in Xenopus embryos. Two hundred and seventy three unique, differentially expressed genes were identified, 204 of which are novel in Xenopus. Partial DNA sequences and expression patterns were documented and assembled into a database, 'AXelDB'. Approximately 30% of cDNAs analyzed represent differentially expressed genes and about 5% show highly regionalized expression. Novel marker genes and potential developmental regulators were found. Differential expression of mitochondrial genes was observed. Marker genes were used to study regionalization of the entire gastrula as well as the tail forming region and the epidermis of the tailbud embryo. Four 'synexpression' groups representing genes with shared, complex expression pattern that predict molecular pathways involved in patterning and differentiation were identified. According to their probable functional significance these groups are designated as Delta1, Bmp4, ER-import and Chromatin group. Within synexpression groups, a likely function of genes without sequence similarity can be predicted. The results indicate that synexpression groups have strong prognostic value. A cluster analysis was made by comparing gene expression patterns to derive a novel parameter, 'tissue relatedness'. In conclusion, this study describes a semi-functional approach to investigate genes expressed during early development and provides global insight into embryonic patterning.
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Affiliation(s)
- V Gawantka
- Division of Molecular Embryology, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120, Heidelberg, Germany
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44
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Maftahi M, Gaillardin C, Nicaud JM. Generation of Saccharomyces cerevisiae deletants and basic phenotypic analysis of eight novel genes from the left arm of chromosome XIV. Yeast 1998; 14:271-80. [PMID: 9544246 DOI: 10.1002/(sici)1097-0061(199802)14:3<271::aid-yea218>3.0.co;2-y] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
The disruption of eight novel genes was realized in two genetic backgrounds. Among these open reading frames, NO333, NO348 and NO364 presented homologies with other proteins of yeast or other organisms, whereas NO320, NO325, NO339, NO384 and NO388 showed no similarity with any protein. Tetrad analysis of heterozygous deletant strains revealed that NO348, NO364 and NO388 are essential genes for vegetative growth, whereas NO320, NO325, NO333, NO339 and NO384 are non-essential. Basic phenotypic analyses of the non-lethal deletant strains as suggested in the six-pack B0 programme did not reveal any significant differences between parental and mutant strains.
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Affiliation(s)
- M Maftahi
- Institut National Agronomique Paris-Grignon, INRA-CNRS, Centre de Biotechnologies Agro-Industrielles, Thiverval-Grignon, France
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45
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Thatcher JW, Shaw JM, Dickinson WJ. Marginal fitness contributions of nonessential genes in yeast. Proc Natl Acad Sci U S A 1998; 95:253-7. [PMID: 9419362 PMCID: PMC18192 DOI: 10.1073/pnas.95.1.253] [Citation(s) in RCA: 135] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/1997] [Accepted: 10/29/1997] [Indexed: 02/05/2023] Open
Abstract
Analysis of the complete genome sequence of Saccharomyces cerevisiae confirms and extends earlier evidence that a majority of yeast genes are not essential, at least under laboratory conditions. Many fail to yield a discernible mutant phenotype even when disrupted. Genes not subject to natural selection would accumulate inactivating mutations, so these "cryptic" genes must have functions that are overlooked by the standard methods of yeast genetics. Two explanations seem possible: (i) They have important functions only in environments not yet duplicated in the laboratory and would have conditional phenotypes if tested appropriately. (ii) They make small, but significant, contributions to fitness even under routine growth conditions, but the effects are not large enough to be detected by conventional methods. We have tested the second "marginal benefit" hypothesis by measuring the fitnesses of a random collection of disruption mutants in direct competition with their wild-type progenitor. A substantial majority of mutant strains that lack obvious defects nevertheless are at a significant selective disadvantage just growing on rich medium under normal conditions. This result has important implications for efforts to understand the functions of novel genes revealed by sequencing projects.
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Affiliation(s)
- J W Thatcher
- Department of Biology, University of Utah, Salt Lake City, UT 84112, USA
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46
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Olivas WM, Muhlrad D, Parker R. Analysis of the yeast genome: identification of new non-coding and small ORF-containing RNAs. Nucleic Acids Res 1997; 25:4619-25. [PMID: 9358174 PMCID: PMC147069 DOI: 10.1093/nar/25.22.4619] [Citation(s) in RCA: 70] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
The genome sequences from increasing numbers of organisms allow for rapid and organized examination of gene expression. Yet current computational-based paradigms for gene recognition are limited and likely to miss genes expressing non-coding RNAs or mRNAs with small open reading frames (ORFs). We have utilized two strategies to determine if there are additional transcripts in the yeast Saccharomyces cerevisiae that were not identified in previous analyses of the genome. In one approach, we identified strong consensus polymerase III promoters based on sequence, and determined experimentally if these promoters drive the expression of an RNA polymerase III transcript. This approach led to the identification of a new, non-essential 170 nt non-coding RNA. An alternative strategy analyzed RNA expression from large sequence gaps>2 kb between predicted ORFs. Fifteen unique RNA transcripts ranging in size from 161 to 1200 nt were identified from a total of 59 sequence gaps. Several of these RNAs contain unusually small potential ORFs, while one is clearly non-coding and appears to be a small nucleolar RNA. These results suggest that there are likely to be additional previously unidentified non-coding RNAs in yeast, and that new paradigms for gene recognition will be required to identify all expressed genes from an organism.
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Affiliation(s)
- W M Olivas
- Department of Molecular and Cellular Biology and Howard Hughes Medical Institute, University of Arizona, Tucson, AZ 85721, USA
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47
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Molecular Cloning of Chromosome I DNA fromSaccharomyces cerevisiae: Characterization of the 54 kb Right TerminalCDC15-FLO1-PHO11 Region. Yeast 1997. [DOI: 10.1002/(sici)1097-0061(199710)13:13<1251::aid-yea174>3.0.co;2-f] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022] Open
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48
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Smith V, Chou KN, Lashkari D, Botstein D, Brown PO. Functional analysis of the genes of yeast chromosome V by genetic footprinting. Science 1996; 274:2069-74. [PMID: 8953036 DOI: 10.1126/science.274.5295.2069] [Citation(s) in RCA: 222] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
Genetic footprinting was used to assess the phenotypic effects of Ty1 transposon insertions in 268 predicted genes of chromosome V of Saccharomyces cerevisiae. When seven selection protocols were used, Ty1 insertions in more than half the genes tested (157 of 268) were found to result in a detectable reduction in fitness. Results could not be obtained for fewer than 3 percent of the genes tested (7 of 268). Previously known mutant phenotypes were confirmed, and, for about 30 percent of the genes, new mutant phenotypes were identified.
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Affiliation(s)
- V Smith
- Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA. Medicine, Stanford, CA 94305, USA.
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49
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Braun EL, Fuge EK, Padilla PA, Werner-Washburne M. A stationary-phase gene in Saccharomyces cerevisiae is a member of a novel, highly conserved gene family. J Bacteriol 1996; 178:6865-72. [PMID: 8955308 PMCID: PMC178587 DOI: 10.1128/jb.178.23.6865-6872.1996] [Citation(s) in RCA: 58] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
The regulation of cellular growth and proliferation in response to environmental cues is critical for development and the maintenance of viability in all organisms. In unicellular organisms, such as the budding yeast Saccharomyces cerevisiae, growth and proliferation are regulated by nutrient availability. We have described changes in the pattern of protein synthesis during the growth of S. cerevisiae cells to stationary phase (E. K. Fuge, E. L. Braun, and M. Werner-Washburne, J. Bacteriol. 176:5802-5813, 1994) and noted a protein, which we designated Snz1p (p35), that shows increased synthesis after entry into stationary phase. We report here the identification of the SNZ1 gene, which encodes this protein. We detected increased SNZ1 mRNA accumulation almost 2 days after glucose exhaustion, significantly later than that of mRNAs encoded by other postexponential genes. SNZ1-related sequences were detected in phylogenetically diverse organisms by sequence comparisons and low-stringency hybridization. Multiple SNZ1-related sequences were detected in some organisms, including S. cerevisiae. Snz1p was found to be among the most evolutionarily conserved proteins currently identified, indicating that we have identified a novel, highly conserved protein involved in growth arrest in S. cerevisiae. The broad phylogenetic distribution, the regulation of the SNZ1 mRNA and protein in S. cerevisiae, and identification of a Snz protein modified during sporulation in the gram-positive bacterium Bacillus subtilis support the hypothesis that Snz proteins are part of an ancient response that occurs during nutrient limitation and growth arrest.
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Affiliation(s)
- E L Braun
- Department of Biology, University of New Mexico, Albuquerque 87131, USA
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50
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Fairhead C, Llorente B, Denis F, Soler M, Dujon B. New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using 'split-marker' recombination. Yeast 1996; 12:1439-57. [PMID: 8948099 DOI: 10.1002/(sici)1097-0061(199611)12:14%3c1439::aid-yea37%3e3.0.co;2-o] [Citation(s) in RCA: 46] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
New tools are needed for speedy and systematic study of the numerous genes revealed by the sequence of the yeast genome. We have developed a novel transformation strategy, based on 'split-marker' recombination, which allows generation of chromosomal deletions and direct gene cloning. For this purpose, pairs of yeast vectors have been constructed which offer a number of advantages for large-scale applications such as one-step cloning of target sequence homologs and combinatorial use. Gene deletions or gap-repair clonings are obtained by cotransformation of yeast by a pair of recombinant plasmids. Gap-repair vectors are based on the URA3 marker. Deletion vectors include the URA3, LYS2 and kanMX selection markers flanked by I-Scel sites, which allow their subsequent elimination from the transformant without the need for counter-selection. The application of the "split-marker' vectors to the analysis of a few open reading frames of chromosome XI is described.
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Affiliation(s)
- C Fairhead
- Unité de Génétique Moléculaire des Levures (UFR 927 Université P. & M. Curie, URA 1149 CNRS), Institut Pasteur, Paris, France
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