Auswahl der wissenschaftlichen Literatur zum Thema „In-yeast genome cloning“

Abstract We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Escherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura+ transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences. Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement the mutant phenotype. This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E. coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3) they provide phenotypes that are laborious to score. We demonstrate the utility of this technique by isolating three genes, GAL83, SSN2 and MAK7, each of which presents one of these problems for cloning.

Fission yeast, a single-cell eukaryotic organism, shares many fundamental cellular processes with higher eukaryotes, including gene transcription and regulation, cell cycle regulation, vesicular transport and membrane trafficking, and cell death resulting from the cellular stress response. As a result, fission yeast has proven to be a versatile model organism for studying human physiology and diseases such as cell cycle dysregulation and cancer, as well as autophagy and neurodegenerative diseases like Alzheimer’s, Parkinson’s, and Huntington’s diseases. Given that viruses are obligate intracellular parasites that rely on host cellular machinery to replicate and produce, fission yeast could serve as a surrogate to identify viral proteins that affect host cellular processes. This approach could facilitate the study of virus–host interactions and help identify potential viral targets for antiviral therapy. Using fission yeast for functional characterization of viral genomes offers several advantages, including a well-characterized and haploid genome, robustness, cost-effectiveness, ease of maintenance, and rapid doubling time. Therefore, fission yeast emerges as a valuable surrogate system for rapid and comprehensive functional characterization of viral proteins, aiding in the identification of therapeutic antiviral targets or viral proteins that impact highly conserved host cellular functions with significant virologic implications. Importantly, this approach has a proven track record of success in studying various human and plant viruses. In this protocol, we present a streamlined and scalable molecular cloning strategy tailored for genome-wide and comprehensive functional characterization of viral proteins in fission yeast.

ABSTRACT Plasmid pJM81 contains a Herpes simplex virus thymidine kinase (TK) gene that is expressed in yeast. Cells containing the plasmid utilize thymidine (TdR) and the analogue 5-bromodeoxyuridine (BUdR) for specific incorporation into DNA. TdR auxotrophs, harboring plasmid pJM81 and a mutation in the yeast gene TMP1 require high concentrations of TdR (300 μg/ml) to support normal growth rates and the wild-type mitochondrial genome (ρ+) cannot be maintained. We have identified a yeast gene, TUT1, in which recessive mutations allow efficient utilization of lower concentrations of TdR. Strains containing the mutations tmp1 and tut1, as well as plasmid pJM81, form colonies at 2 μg/ml TdR, grow at nearly normal rates and maintain the ρ+ genome at 50 μg/ml TdR. These strains can be used to radiolabel DNA specifically and to synchronize DNA replication by TdR starvation. In addition, the substitution of BUdR for TdR allows the selective killing of DNA-synthesizing cells by 310-nm irradiation and allows the separation of replicated and unreplicated forms of DNA by CsCl equilibrium density banding. We also describe a unique, generally applicable system for cloning mutant alleles that exploits the fact that Tk+ yeast cells are sensitive to 5-fluorodeoxyuridine (FUdR) and that gene conversions can occur between a yeast chromosome and a TK-containing plasmid.

Mitochondrial FAS (fatty acid synthesis) of type II is a widely conserved process in eukaryotic organisms, with particular importance for respiratory competence and mitochondrial morphology maintenance in Saccharomyces cerevisiae. The recent characterization of three missing enzymes completes the pathway. Etr1p (enoyl thioester reductase) was identified via purification of the protein followed by molecular cloning. To study the link between FAS and cell respiration further, we also created a yeast strain that has FabI enoyl-ACP (acyl-carrier protein) reductase gene from Escherichia coli engineered to carry a mitochondrial targeting sequence in the genome, replacing the endogenous ETR1 gene. This strain is respiratory competent, but unlike the ETR1 wild-type strain, it is sensitive to triclosan on media containing only non-fermentable carbon source. A colony-colour-sectoring screen was applied for cloning of YHR067w/RMD12, the gene encoding mitochondrial 3-hydroxyacyl-ACP dehydratase (Htd2/Yhr067p), the last missing component of the mitochondrial FAS. Finally, Hfa1p was shown to be the mitochondrial acetyl-CoA carboxylase.

Abstract Yeast DNA fragments that confer multiple drug resistance when amplified were isolated. Cells containing a yeast genomic library cloned in the high copy autonomously replicating vector, YEp24, were plated on medium containing cycloheximide. Five out of 100 cycloheximide-resistant colonies were cross-resistant to the unrelated inhibitor, sulfometuron methyl, due to a plasmid-borne resistance determinant. The plasmids isolated from these resistant clones contained two nonoverlapping regions in the yeast genome now designated PDR4 and PDR5 (for pleiotropic drug resistant). PDR4 was mapped to chromosome XIII, 31.5 cM from LYS7 and 9 cM from the centromere. PDR4 was mapped to chromosome XV between ADE2 and H1S3. Genetic analysis demonstrated that at least three tightly linked genes (PDR5, PDR2 and SMR3) that mediate resistance to inhibitors are located in this region. Insertion mutations in the either PDR4 or PDR5 genes are not lethal, but the insertion in PDR5 results in a drug-hypersensitive phenotype.

Auxotrophic markers are useful tools in cloning and genome editing, enable a large spectrum of genetic techniques, as well as facilitate the study of metabolite exchange interactions in microbial communities. If unused background auxotrophies are left uncomplemented however, yeast cells need to be grown in nutrient supplemented or rich growth media compositions, which precludes the analysis of biosynthetic metabolism, and which leads to a profound impact on physiology and gene expression. Here we present a series of 23 centromeric plasmids designed to restore prototrophy in typicalSaccharomyces cerevisiaelaboratory strains. The 23 single-copy plasmids complement for deficiencies inHIS3, LEU2, URA3, MET17 or LYS2genes and in their combinations, to match the auxotrophic background of the popular functional-genomic yeast libraries that are based on the S288c strain. The plasmids are further suitable for designing self-establishing metabolically cooperating (SeMeCo) communities, and possess a uniform multiple cloning site to exploit multiple parallel selection markers in protein expression experiments.

Abstract In the yeast Saccharomyces cerevisiae, certain mutant alleles of YME4, YME6, and MDM10 cause an increased rate of mitochondrial DNA migration to the nucleus, carbon-source-dependent alterations in mitochondrial morphology, and increased rates of mitochondrial DNA loss. While single mutants grow on media requiring mitochondrial respiration, any pairwise combination of these mutations causes a respiratory-deficient phenotype. This double-mutant phenotype allowed cloning of YME6, which is identical to MMM1 and encodes an outer mitochondrial membrane protein essential for maintaining normal mitochondrial morphology. Yeast strains bearing null mutations of MMM1 have altered mitochondrial morphology and a slow growth rate on all carbon sources and quantitatively lack mitochondrial DNA. Extragenic suppressors of MMM1 deletion mutants partially restore mitochondrial morphology to the wild-type state and have a corresponding increase in growth rate and mitochondrial DNA stability. A dominant suppressor also suppresses the phenotypes caused by a point mutation in MMM1, as well as by specific mutations in YME4 and MDM10.

The various thermophilic fungi like Chaetomium thermophile has potential to secrete xylanase and cellulase enzymes. Inthe present study eukaryotic expression system of Pichia pastoris (yeast) was used to express xylanase gene. Thexylanase (Xyn 11-A) gene was isolated from C. thermophile strain NIBGE-1. Primers were designed to amplify the gene,ligated into P. pastoris pPIC3.5K vector, the resultant recombinant clone pSSZ810 was transformed into the genome ofP. pastoris GS115 strain through electroporation. Transformants were selected on yeast peptone dextrose medium(YPD) plates containing antibiotic geneticin (100 mg/mL) upto final concentration of 0.75 mg/mL. The maximum activityof xylanase 2.04 U/mL after incubation of 2 hrs at 50ºC was observed in the presence of 100% methanol inducer uptofinal concentration of 30μL (0.5%) as compared to control. HPLC analysis represented high peak of xylose as comparedto control. SDS-PAGE indicated approx. 28 kDa protein of expressed xylanase gene.

L’ingénierie des génomes des micro-organismes est devenue un standard dans les biotechnologies microbiennes. En 2010, des technologies prometteuses de biologie de synthèse utilisant la levure comme plateforme pour l’assemblage et l’ingénierie de génomes synthétiques bactériens suivi de leur transplantation dans une cellule receveuse ont vu le jour. Ces technologies ont conduit à la création des premières cellules synthétiques et ouvert de nouvelles voies vers la construction de cellules aux propriétés biologiques entièrement contrôlées. Le transfert de ces outils à des micro-organismes d’intérêt industriel comme la bactérie Gram+ Bacillus subtilis (Bsu), modèle dans le secteur des biotechnologies, constituerait une avancée majeure. C’est précisément l’objectif du projet ANR « Bacillus 2.0 », qui réunit deux équipes INRAE et qui se propose d’adapter l’ensemble de ces outils de biologie de synthèse à Bsu afin d’être en mesure de partir d’une conception, assistée par ordinateur, de génomes semi-synthétiques de Bsu jusqu’à l’obtention de nouvelles souches industrielles. Cependant, les premiers travaux réalisés sur ce projet ont montré que le génome entier de Bsu ne pouvait pas être cloné et maintenu en l’état dans la levure. Ces résultats risquaient de remettre en question la faisabilité du projet dans son ensemble et en particulier la pertinence d’utiliser la levure comme plateforme d’assemblage du génome semi-synthétique de Bsu.L’objectif de ma thèse a consisté à démontrer que la levure restait un hôte pertinent pour le projet « Bacillus 2.0 ». Elle s’est déclinée en 3 parties. Dans la première partie, une méthode de clonage de génome récemment développée au laboratoire et dénommée CReasPy-Fusion, a progressivement été adaptée à Bsu. Les résultats obtenus ont montré (i) le transfert possible d'ADN plasmidique entre protoplastes bactériens et sphéroplastes de levure, (ii) l'efficacité d'un système CRISPR-Cas9 porté par les cellules de levure pour capturer/modifier cet ADN plasmidique pendant la fusion Bsu/levure, puis (iii) l'efficacité de ce même système pour capturer des fragments de génome d’une centaine de kb à partir de trois souches différentes. Des observations en microscopie à fluorescence ont également été réalisées et ont mis en évidence deux types d’interactions qui permettraient de passer d’un contact protoplastes/sphéroplastes à un ADN bactérien cloné dans la levure. Dans la seconde partie de ma thèse, la méthode CReasPy-Fusion a été mise à profit pour tenter de cloner de grands fragments du génome de Bsu dans la levure. Des fragments génomiques jusqu’à ~1 Mb ont pu être clonés dans la levure, mais leur capture a nécessité l’ajout préalable d’un grand nombre d’ARS sur le génome de Bsu pour stabiliser les constructions génétiques. La dernière partie a été l’adaptation de la méthode RAGE à Bsu. Cette méthode permet le transfert, non pas d’un génome entier mais de portions de génomes bactériens depuis la levure vers la bactérie à éditer. Une preuve de concept a été réalisée avec l’échange d’un premier fragment de génome de 155 kb par une version réduite de 44 kb.En conclusion, les travaux réalisés au cours de cette thèse ont montré la pertinence d’utiliser la levure comme plateforme d’ingénierie dans les modifications à grande échelle du génome de Bsu. D’une part, nous avons montré que des fragments d’une centaine de kb peuvent être clonés dans la levure, modifiés et transférés dans une cellule receveuse de façon à générer des Bsu mutants. Cette stratégie offre une véritable alternative à la transplantation de génome. D’autre part, nous avons montré que de grands fragments du génome de Bsu (jusqu’à 1Mb) peuvent également être clonés dans la levure à condition de contenir de nombreux ARS dans leurs séquences. Grâce à ces résultats, le clonage d’un génome réduit de Bsu chez la levure est redevenu un objectif réalisable
Genome engineering of microorganisms has become a standard in microbial biotechnology. In 2010, promising synthetic biology technologies using yeast as a platform for the assembly and engineering of synthetic bacterial genomes followed by their transplantation into a recipient cell have emerged. These technologies have led to the creation of the first synthetic cells and opened new avenues towards the construction of cells with fully controlled biological properties. Transferring these tools to microorganisms of industrial interest such as the Gram+ bacterium Bacillus subtilis (Bsu), a model in the biotechnology sector, would be a major step forward. This is precisely the aim of the ANR "Bacillus 2.0" project, which brings together two INRAE teams and aims to adapt all these synthetic biology tools to Bsu so as to be able to go from computer-aided design of semi-synthetic Bsu genomes to the production of new industrial strains. However, initial work on this project showed that the entire Bsu genome could not be cloned and maintained in yeast in its current state. These results threatened to call into question the feasibility of the entire project and, in particular, the relevance of using yeast as a platform for assembling the semi-synthetic Bsu genome.The goal of my thesis was to demonstrate that yeast remained a relevant host for the Bacillus 2.0 project. It was divided into 3 parts. In the first part, a genome cloning method recently developed in the laboratory, called CReasPy-Fusion, was progressively adapted to Bsu. The results obtained showed (i) the possible transfer of plasmid DNA between bacterial protoplasts and yeast spheroplasts, (ii) the efficiency of a CRISPR-Cas9 system carried by yeast cells to capture/modify this plasmid DNA during Bsu/yeast fusion, and then (iii) the efficiency of the same system to capture genomic fragments of about a hundred kb from three different strains. Fluorescence microscopy observations were also carried out revealing two types of interaction that would enable the transition from protoplast/spheroplast contact to cloned bacterial DNA in yeast. In the second part of my thesis, the CReasPy-Fusion method was used in an attempt to clone large Bsu genome fragments in yeast. Genomic fragments of up to ~1 Mb could be cloned in yeast, but their capture required the prior addition of a large number of ARS to the Bsu genome to stabilize the genetic constructs. The final part was the adaptation of the RAGE method to Bsu. This method allow the transfer, not of a whole genome, but of portions of bacterial genomes from yeast to the bacteria to be edited. Proof of concept was achieved by exchanging a 155 kb genome fragment with a reduced 44 kb version.In conclusion, the work carried out during this thesis has shown the relevance of using yeast as an engineering platform for large-scale modifications of the Bsu genome. On the one hand, we have shown that fragments of around 100 kb can be cloned in yeast, modified and transferred into a recipient cell to generate Bsu mutants. This strategy offers a real alternative to genome transplantation. On the other hand, we have shown that large fragments of the Bsu genome (up to 1 Mb) can also be cloned in yeast, provided they contain numerous ARS in their sequences. Thanks to these results, cloning a reduced Bsu genome in yeast has once again become an achievable goal

Abstract The major goal of the human genome project includes the isolation of the entire human genome in overlapping clones and the development of physical maps of the cloned DNA. Cloning into yeast artificial chromosomes (YAC) represents the method of choice for genome mapping analysis in the megabase range.

Abstract The analysis of large and complex genomes requires both mapping and cloning of DNA. Until a few years ago the largest fragment of DNA that could be cloned was - 40 kb in length using a cosmid vector (see Chapters I and 2). The development of yeast artificial chromosome (YAC) cloning vectors has greatly extended this cloning range. Following the description of the YAC vector system (1) the size of DNA fragment that could be maintained as a YAC has increased by almost one order of magnitude. This has greatly facilitated the study of complex genomes and has been instrumental in the efforts to construct the first generation physical map of the entire human genome (2). titre plates provides a valuable long-term resource which can be simultaneously accessed by several researchers. Consequently, it is important to plan carefully before embarking on this exercise that requires substantial time and resource. This chapter will mainly concentrate on the construction of a YAC library. For completeness, determination of the average YAC size within the library and a PCR-based library screening method have also been included.

Abstract Most methods for genome mapping rely on some form of cloning to isolate a subfraction of the genome for analysis, whether into yeast or bacterial hosts (as in physical mapping, Chapters 10 and 11), into hybrid cells (as in radiation hybrid mapping, Chapter 4), or as offspring amongst which polymorphic markers segregate (as in linkage mapping, Chapters 1-3).

Abstract A 1000-fold range of DNA sizes may be cloned in the current range of cloning vectors: the ideal vector for genome mapping studies would be one that is easy to use while containing as much DNA as possible. Cosmids now occupy the middle ground: they have a significant capacity, thus reducing the number of steps required to clone an entire gene or region, combined with very simple methods for isolating inserted DNA in pure form. As a consequence, positional cloning strategies frequently involve the use of cosmids as a final cloning vector for reducing a yeast artificial chromosome (YAC) clone to manageably sized DNA fragments (1). Cosmids are units that are very likely to contain an entire gene, are easily mapped with respect to restriction sites, and are amenable to the application of a number of other functional assays, e.g. exon trapping (2, 3), genomic sequencing (4-6), and fingerprinting to generate contig maps (7, 8). As a result, the detailed information that can be obtained from a cosmid clone makes it the ideal medium for genome analysis. Indeed, several genome mapping studies (e.g, Caenorhabditis elegans (9), Escherichia coli (10)), have relied on physical DNA maps built around cosmid clones.

Abstract Genetic analysis of mutants in Saccharomyces cerev1szae offers a unique approach to the study of biological processes. To initiate genetic studies in this organism one can mutagenize a population and screen or select for those mutants which affect the process of interest. Alternatively, one can use techniques selectively to mutagenize a single gene already known to play an important role in this process. The generation of mutant yeast strains starting from a cloned non-essential yeast gene is relatively straightforward. To remove the wild-type gene product, an essential step in analysing recessive alleles, DNA at the wild-type non-essential locus can be deleted entirely from the genome. A collection of mutant alleles can be made in vitro and introduced into the deleted host cell via episomal cloning vectors. The generation of mutant yeast strains from an essential yeast gene is a more complicated task since removal of the wild-type gene in one simple step yields an inviable genotype.

Abstract Many simple but powerful techniques have been developed to investigate gene expression in Saccharomyces cerevisiae, and a number of these have been employed in the analysis of RNA splicing. Test substrates can be transcribed from expression cassettes introduced into yeast by transformation or transplacement, and splicing can be monitored by RNA analysis or assay of a suitable reporter gene product. Gene disruption and inducible expression systems have been invaluable for investigating the roles of components of the splicing machinery in yeast. Many of the genes for yeast splicing factors have recently been isolated by molecular cloning, which is facilitated by the compact nature of the Saccharomyces genome and the fact that these genes are present as single copies. Splicing of yeast mRNA precursors can also be studied in vitro, since for Saccharomyces a simple method has been developed for making cell-free extracts capable of splicing synthetic pre-mRNA substrates. Such studies have shown that the splicing pathway in yeast is very similar to that of higher eukaryotes and that there is considerable conservation of structure and function between splicing factors in yeast and mammalian cells. However, yeast is an extremely valuable system for the detailed analysis of splicing since it allows some approaches which are not possible with mammalian cells, particularly the genetic selection and isolation of splicing mutants.

Enhancing the activity of biocontrol agents could be the most important factor in their success in controlling fruit disease and their ultimate acceptance in commercial disease management. Direct manipulation of a biocontrol agent resulting in enhancement of diseases control could be achieved by using recent advances in molecular biology techniques. The objectives of this project were to isolate genes from yeast species that were used as postharvest biocontrol agents against postharvest diseases and to determine their role in biocontrol efficacy. The emphasis was to be placed on the yeast, Candida oleophila, which was jointly discovered and developed in our laboratories, and commercialized as the product, Aspire. The general plan was to develop a transformation system for C . oleophila and either knockout or overexpress particular genes of interest. Additionally, biochemical characterization of the lytic peptides was conducted in the wild-type and transgenic isolates. In addition to developing a better understanding of the mode of action of the yeast biocontrol agents, it was also our intent to demonstrate the feasibility of enhancing biocontrol activity via genetic enhancement of yeast with genes known to code for proteins with antimicrobial activity. Major achievements are: 1) Characterization of extracellular lytic enzymes produced by the yeast biocontrol agent Candida oleophila; 2) Development of a transformation system for Candida oleophila; 3) Cloning and analysis of C.oleophila glucanase gene; 4) Overexpression of and knockout of C. oleophila glucanase gene and evaluating its role in the biocontrol activity of C. oleophila; 5) Characterization of defensin gene and its expression in the yeast Pichiapastoris; 6) Cloning and Analysis of Chitinase and Adhesin Genes; 7) Characterization of the rnase secreted by C . oleophila and its inhibitory activity against P. digitatum. This project has resulted in information that enhanced our understanding of the mode of action of the yeast C . oleophila. This was important step towards enhancing the biocontrol activity of the yeast. Fungal cell wall enzymes produced by the yeast antagonist were characterized. Different substrates were identified to enhance there production in vitro. Exo-b-1, 3 glucanase, chitinase and protease production was stimulated by the presence of cell-wall fragments of Penicillium digitatum in the growing medium, in addition to glucose. A transformation system developed was used to study the role of lytic enzymes in the biocontrol activity of the yeast antagonist and was essential for genetic manipulation of C . oleqphila. After cloning and characterization of the exo-glucanase gene from the yeast, the transformation system was efficiently used to study the role of the enzyme in the biocontrol activity by over-expressing or knocking out the activity of the enzyme. At the last phase of the research (still ongoing) the transformation system is being used to study the role of chitinase gene in the mode of action. Knockout and over expression experiments are underway.

The primary objectives for the US lab included: the characterization of ELF3 transcription and translation; the creation and characterization of various transgenic lines that misexpress ELF3; defining genetic pathways related to ELF3 function regulating floral initiation in Arabidopsis; and the identification of genes that either interact with or are regulated by ELF3. Light quality, photoperiod, and temperature often act as important and, for some species, essential environmental cues for the initiation of flowering. However, there is relatively little information on the molecular mechanisms that directly regulate the developmental pathway from the reception of the inductive light signals to the onset of flowering and the initiation of floral meristems. The ELF3 gene was identified as possibly having a role in light-mediated floral regulation since elj3 mutants not only flower early, but exhibit light-dependent circadian defects. We began investigating ELF3's role in light signalling and flowering by cloning the ELF3 gene. ELF3 is a novel gene only present in plant species; however, there is an ELF3 homolog within Arabidopsis. The Arabidopsis elj3 mutation causes arrhythmic circadian output in continuous light; however, we show conclusively normal circadian function with no alteration of period length in elj3 mutants in dark conditions and that the light-dependent arrhythmia observed in elj3 mutants is pleiotropic on multiple outputs regardless of phase. Plants overexpressing ELF3 have an increased period length in constant light and flower late in long-days; furthermore, etiolated ELF3-overexpressing seedlings exhibit a decreased acute CAB2 response after a red light pulse, whereas the null mutant is hypersensitive to acute induction. This finding suggests that ELF3 negatively regulates light input to both the clock and its outputs. To determine whether ELF3's action is phase dependent, we examined clock resetting by light pulses and constructed phase response curves. Absence of ELF3 activity causes a significant alteration of the phase response curve during the subjective night, and overexpression of ELF3 results in decreased sensitivity to the resetting stimulus, suggesting that ELF3 antagonizes light input to the clock during the night. Indeed, the ELF3 protein interacts with the photoreceptor PHYB in the yeast two-hybrid assay and in vitro. The phase ofELF3 function correlates with its peak expression levels of transcript and protein in the subjective night. ELF3 action, therefore, represents a mechanism by which the oscillator modulates light resetting. Furthermore, flowering time is dependent upon proper expression ofELF3. Scientifically, we've made a big leap in the understanding of the circadian system and how it is coupled so tightly with light reception in terms of period length and clock resetting. Agriculturally, understanding more about the way in which the clock perceives and relays temporal information to pathways such as those involved in the floral transition can lead to increased crop yields by enabling plants to be grown in suboptimal conditions.