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1.
Yo Suzuki Jason Stam Mark Novotny Nozomu Yachie Roger S. Lasken Frederick P. Roth 《Journal of visualized experiments : JoVE》2012,(70)
Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.The first step is to prepare haploid single-mutants termed ''ProMonsters,'' each of which carries a GFP reporter in a deleted locus and one of the ''toolkit'' loci—either Green Monster GMToolkit-a or GMToolkit-α at the can1Δ locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or α-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation. 相似文献
2.
Mating-Type Functions for Meiosis and Sporulation in Yeast Act through Cytoplasm 总被引:6,自引:1,他引:5
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Klar AJ 《Genetics》1980,94(3):597-605
Given a nutritional regime marked by a low nitrogen level and the absence of fermentable carbon sources, conventional a/α diploid cells of Saccharomyces cerevisiae exhibit a complex developmental sequence that includes a round of premeiotic DNA replication, commitment to meiosis and the elaboration of mature tetrads containing viable ascospores. Ordinarily, haploid cells and diploid cells of genotype a/a and α/α fail to display these reactions under comparable conditions. Here, we describe a simple technique for sporulation of α/α and a/a cells. Cells of genotype α/α are mated to haploid a cells carrying the kar1 (karyogamy defective) mutation to yield heterokaryons containing the corresponding diploid and haploid nuclei. The kar1 strains mate normally, but nuclei in the resultant zygotes do not fuse. When heterokaryotic cells are inoculated into sporulation media, they produce asci with six spores. Four spores carry genotypes derived from the diploid nucleus and the other two possess the markers originating from the haploid nucleus, i.e., the diploid nucleus divides meiotically while the haploid nucleus apparently divides mitotically. Similarly, the a/a genome is "helped" to sporulate as a consequence of mating with α kar1 strains. The results allow us to conclude that the mating-type functions essential for meiosis and sporulation are communicated and act through the cytoplasm and that sporulation can be dissociated from typical meiosis. This procedure will facilitate the genetic analysis of strains that are otherwise unable to sporulate. 相似文献
3.
Cells of the fission yeast Schizosaccharomyces pombe normally reproduce by mitotic division in the haploid state. When subjected to nutrient starvation, two haploid cells fuse and undergo karyogamy, forming a diploid cell that initiates meiosis to form four haploid spores. Here, we show that deletion of the mal3 gene, which encodes a homolog of microtubule regulator EB1, produces aberrant asci carrying more than four spores. The mal3 deletion mutant cells have a disordered cytoplasmic microtubule structure during karyogamy and initiate meiosis before completion of karyogamy, resulting in twin haploid meiosis in the zygote. Treatment with anti-microtubule drugs mimics this phenotype. Mutants defective in karyogamy or mutants prone to initiate haploid meiosis exaggerate the phenotype of the mal3 deletion mutant. Our results indicate that proper microtubule structure is required for ordered progression through the meiotic cycle. Furthermore, the results of our study suggest that fission yeast do not monitor ploidy during meiosis. 相似文献
4.
Jeanne P. Margolskee 《Molecular & general genetics : MGG》1988,211(3):430-434
Summary We have used the special properties of the spo13-1 mutation in order to study the regulation of yeast meiosis by the mating type loci. We have found that both the rme1-1 mutation and the sca mutation allow haploid meiosis in spo13-1 strains. Therefore, haploid meiosis is regulated in the same manner as diploid meiosis. Unlike rme1-1, the sca mutation allows meiosis through derepression of the silent mating type cassettes; sca strains can sporulate only because they express both MAT
a and MAT information. We have found further that sca is an allele of SIR2, one of the genes involved in repression of the silent cassettes. Therefore, the RME1 gene is the only known candidate for a master negative regulator through which the MAT locus controls meiosis. 相似文献
5.
Two successive rounds of chromosome segregation following a single round of DNA replication enable the production of haploid gametes during meiosis. In the fission yeast Schizosaccharomyces pombe, karyogamy is the process where the nuclei from 2 haploid cells fuse to create a diploid nucleus, which then undergoes meiosis to produce 4 haploid spores. By screening a collection of S. pombe deletion strains, we found that the deletion of 2 genes, mal3 and mto1, leads to the production of asci containing up to 8 spores. Here, we show that Mal3, the fission yeast member of the EB1 family of conserved microtubule plus-end tracking proteins, is required for karyogamy, oscillatory nuclear movement, and proper segregation of chromosomes during meiosis. In the absence of Mal3, meiosis frequently initiates before the completion of karyogamy, thus producing up to 8 nuclei in a single ascus. Our results provide new evidence that fission yeast can initiate meiosis prior to completing karyogamy. 相似文献
6.
J-B Leducq V Llaurens V Castric P Saumitou-Laprade O J Hardy X Vekemans 《Heredity》2011,106(2):319-329
The effect of selection on patterns of genetic structure within and between populations may be studied by contrasting observed patterns at the genes targeted by selection with those of unlinked neutral marker loci. Local directional selection on target genes will produce stronger population genetic structure than at neutral loci, whereas the reverse is expected for balancing selection. However, theoretical predictions on the intensity of this signal under precise models of balancing selection are still lacking. Using negative frequency-dependent selection acting on self-incompatibility systems in plants as a model of balancing selection, we investigated the effect of such selection on patterns of spatial genetic structure within a continuous population. Using numerical simulations, we tested the effect of the type of self-incompatibility system, the number of alleles at the self-incompatibility locus and the dominance interactions among them, the extent of gene dispersal, and the immigration rate on spatial genetic structure at the selected locus and at unlinked neutral loci. We confirm that frequency-dependent selection is expected to reduce the extent of spatial genetic structure as compared to neutral loci, particularly in situations with low number of alleles at the self-incompatibility locus, high frequency of codominant interactions among alleles, restricted gene dispersal and restricted immigration from outside populations. Hence the signature of selection on spatial genetic structure is expected to vary across species and populations, and we show that empirical data from the literature as well as data reported here on three natural populations of the herb Arabidopsis halleri confirm these theoretical results. 相似文献
7.
Gabrielle L. Harrow John A. Lees William P. Hanage Marc Lipsitch Jukka Corander Caroline Colijn Nicholas J. Croucher 《The ISME journal》2021,15(5):1523
Streptococcus pneumoniae can be divided into many strains, each a distinct set of isolates sharing similar core and accessory genomes, which co-circulate within the same hosts. Previous analyses suggested the short-term vaccine-associated dynamics of S. pneumoniae strains may be mediated through multi-locus negative frequency-dependent selection (NFDS), which maintains accessory loci at equilibrium frequencies. Long-term simulations demonstrated NFDS stabilised clonally-evolving multi-strain populations through preventing the loss of variation through drift, based on polymorphism frequencies, pairwise genetic distances and phylogenies. However, allowing symmetrical recombination between isolates evolving under multi-locus NFDS generated unstructured populations of diverse genotypes. Replication of the observed data improved when multi-locus NFDS was combined with recombination that was instead asymmetrical, favouring deletion of accessory loci over insertion. This combination separated populations into strains through outbreeding depression, resulting from recombinants with reduced accessory genomes having lower fitness than their parental genotypes. Although simplistic modelling of recombination likely limited these simulations’ ability to maintain some properties of genomic data as accurately as those lacking recombination, the combination of asymmetrical recombination and multi-locus NFDS could restore multi-strain population structures from randomised initial populations. As many bacteria inhibit insertions into their chromosomes, this combination may commonly underlie the co-existence of strains within a niche.Subject terms: Population genetics, Microbial ecology, Microbial genetics, Bacterial genetics, Phylogenetics 相似文献
8.
Carl JW Liu JQ Joshi PS El-Omrani HY Yin L Zheng X Whitacre CC Liu Y Bai XF 《Journal of immunology (Baltimore, Md. : 1950)》2008,181(1):320-328
Despite negative selection in the thymus, significant numbers of autoreactive T cells still escape to the periphery and cause autoimmune diseases when immune regulation goes awry. It is largely unknown how these T cells escape clonal deletion. In this study, we report that CD24 deficiency caused deletion of autoreactive T cells that normally escape negative selection. Restoration of CD24 expression on T cells alone did not prevent autoreactive T cells from deletion; bone marrow chimera experiments suggest that CD24 on radio-resistant stromal cells is necessary for preventing deletion of autoreactive T cells. CD24 deficiency abrogated the development of experimental autoimmune encephalomyelitis in transgenic mice with a TCR specific for a pathogenic autoantigen. The role of CD24 in negative selection provides a novel explanation for its control of genetic susceptibility to autoimmune diseases in mice and humans. 相似文献
9.
We have developed a powerful experimental framework that combines competitive selection and microarray-based genetic footprinting to comprehensively reveal the genetic basis of bacterial behaviors. Application of this method to Escherichia coli motility identifies 95% of the known flagellar and chemotaxis genes, and reveals three dozen novel loci that, to varying degrees and through diverse mechanisms, affect motility. To probe the network context in which these genes function, we developed a method that uncovers genome-wide epistatic interactions through comprehensive analyses of double-mutant phenotypes. This allows us to place the novel genes within the context of signaling and regulatory networks, including the Rcs phosphorelay pathway and the cyclic di-GMP second-messenger system. This unifying framework enables sensitive and comprehensive genetic characterization of complex behaviors across the microbial biosphere. 相似文献
10.
Varnica Khetrapal Kurosh Mehershahi Shazmina Rafee Siyi Chen Chiew Ling Lim Swaine L. Chen 《Nucleic acids research》2015,43(13):e83
Creation of defined genetic mutations is a powerful method for dissecting mechanisms of bacterial disease; however, many genetic tools are only developed for laboratory strains. We have designed a modular and general negative selection strategy based on inducible toxins that provides high selection stringency in clinical Escherichia coli and Salmonella isolates. No strain- or species-specific optimization is needed, yet this system achieves better selection stringency than all previously reported negative selection systems usable in unmodified E. coli strains. The high stringency enables use of negative instead of positive selection in phage-mediated generalized transduction and also allows transfer of alleles between arbitrary strains of E. coli without requiring phage. The modular design should also allow further extension to other bacteria. This negative selection system thus overcomes disadvantages of existing systems, enabling definitive genetic experiments in both lab and clinical isolates of E. coli and other Enterobacteriaceae. 相似文献
11.
The REC104 gene was initially defined by mutations that rescued the inviability of a rad52 spo 13 haploid strain in meiosis. We have observed that rec104 mutant strains undergo essentially no induction of meiotic gene conversion, and we have not been able to detect any meiotic crossing over in such strains. The REC104 gene has no apparent role in mitosis, since mutations have no observable effect on growth, mitotic recombination, or DNA repair. The DNA sequence of REC104 reveals that it is a previously unknown gene with a coding region of 549-bp, and genetic mapping has localized the gene to chromosome VIII near FUR1. Expression of the REC104 gene is induced in meiosis, and it appears that the gene is not transcribed in mitotic cells. Possible roles for the REC104 gene product in meiosis are discussed. 相似文献
12.
Axel Thiel Grégoire Michoud Yann Moalic Didier Flament Mohamed Jebbar 《Applied and environmental microbiology》2014,80(7):2299-2306
In this study, we developed a gene disruption system for Thermococcus barophilus using simvastatin for positive selection and 5-fluoroorotic acid (5-FOA) for negative selection or counterselection to obtain markerless deletion mutants using single- and double-crossover events. Disruption plasmids carrying flanking regions of each targeted gene were constructed and introduced by transformation into wild-type T. barophilus MP cells. Initially, a pyrF deletion mutant was obtained as a starting point for the construction of further markerless mutants. A deletion of the hisB gene was also constructed in the UBOCC-3256 (ΔpyrF) background, generating a strain (UBOCC-3260) that was auxotrophic for histidine. A functional pyrF or hisB allele from T. barophilus was inserted into the chromosome of UBOCC-3256 (ΔpyrF) or UBOCC-3260 (ΔpyrF ΔhisB), allowing homologous complementation of these mutants. The piezophilic genetic tools developed in this study provide a way to construct strains with multiple genetic backgrounds that will allow further genetic studies for hyperthermophilic piezophilic archaea. 相似文献
13.
14.
Mutations in the Myp1 Gene of Ustilago Maydis Attenuate Mycelial Growth and Virulence 总被引:1,自引:0,他引:1
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Mating between haploid, budding cells of the dimorphic fungus Ustilago maydis results in the formation of a dikaryotic, filamentous cell type. Mating compatibility is governed by two mating-type loci called a and b; transformation of genes from these loci (e.g., a1 and b1) into a haploid strain of different mating type (e.g., a2 b2) allows filamentous growth and establishes a pathogenic cell type. Several mutants with a nonmycelial colony morphology were isolated after insertional mutagenesis of a filamentous, pathogenic haploid strain. The mutagenized region in one such mutant was recovered by plasmid rescue and employed to isolate a gene involved in conditioning the mycelial phenotype (myp1). An 1150 amino acid open reading frame is present at the myp1 locus; the predicted polypeptide is rich in serine residues and contains short regions with similarity to SH3 domain ligands. Construction of myp1 disruption and deletion mutants in haploid strains confirmed that this gene plays a role in mycelial growth and virulence. 相似文献
15.
The REC104 gene was initially defined by mutations that rescued the inviability of a rad52 spo13 haploid strain in meiosis. We have observed that rec104 mutant strains undergo essentially no induction of meiotic gene conversion, and we have not been able to detect any meiotic crossing over in such strains. The REC104 gene has no apparent role in mitosis, since mutations have no observable effect on growth, mitotic recombination, or DNA repair. The DNA sequence of REC104 reveals that it is a previously unknown gene with a coding region of 549-bp, and genetic mapping has localized the gene to chromosome VIll near FUR1. Expression of the REC104 gene is induced in meiosis, and it appears that the gene is not transcribed in mitotic cells. Possible roles for the REC104 gene product in meiosis are discussed. © 1993 Wiley-Liss, Inc. 相似文献
16.
Pan X Yuan DS Ooi SL Wang X Sookhai-Mahadeo S Meluh P Boeke JD 《Methods (San Diego, Calif.)》2007,41(2):206-221
Analysis of genetic interactions has been extensively exploited to study gene functions and to dissect pathway structures. One such genetic interaction is synthetic lethality, in which the combination of two non-lethal mutations leads to loss of organism viability. We have developed a dSLAM (heterozygote diploid-based synthetic lethality analysis with microarrays) technology that effectively studies synthetic lethality interactions on a genome-wide scale in the budding yeast Saccharomyces cerevisiae. Typically, a query mutation is introduced en masse into a population of approximately 6000 haploid-convertible heterozygote diploid Yeast Knockout (YKO) mutants via integrative transformation. Haploid pools of single and double mutants are freshly generated from the resultant heterozygote diploid double mutant pool after meiosis and haploid selection and studied for potential growth defects of each double mutant combination by microarray analysis of the "molecular barcodes" representing each YKO. This technology has been effectively adapted to study other types of genome-wide genetic interactions including gene-compound synthetic lethality, secondary mutation suppression, dosage-dependent synthetic lethality and suppression. 相似文献
17.
Mutants that are resistant to α-factor have been isolated from a mating-type haploid strains of yeast by direct selection on agar medium containing partially purified α-factor. All resistant mutants isolated were found to be sterile. They were characterized and compared with mutants previously isolated as nonmating. Among 93 able to mate at low frequency and to sporulate, none showed linkage to the mating-type locus. The results support the hypothesis that the response to α-factor by cells of mating-type a is essential for mating. 相似文献
18.
Systematic quantification of gene interactions by phenotypic array analysis 总被引:2,自引:2,他引:0
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A phenotypic array method, developed for quantifying cell growth, was applied to the haploid and homozygous diploid yeast deletion strain sets. A growth index was developed to screen for non-additive interacting effects between gene deletion and induced perturbations. From a genome screen for hydroxyurea (HU) chemical-genetic interactions, 298 haploid deletion strains were selected for further analysis. The strength of interactions was quantified using a wide range of HU concentrations affecting reference strain growth. The selectivity of interaction was determined by comparison with drugs targeting other cellular processes. Bio-modules were defined as gene clusters with shared strength and selectivity of interaction profiles. The functions and connectivity of modules involved in processes such as DNA repair, protein secretion and metabolic control were inferred from their respective gene composition. The work provides an example of, and a general experimental framework for, quantitative analysis of gene interaction networks that buffer cell growth. 相似文献
19.
20.
Laurence Decourty Christophe Malabat Emmanuel Frachon Alain Jacquier Cosmin Saveanu 《Nucleic acids research》2021,49(15):8535
Gene deletion and gene expression alteration can lead to growth defects that are amplified or reduced when a second mutation is present in the same cells. We performed 154 genetic interaction mapping (GIM) screens with query mutants related with RNA metabolism and estimated the growth rates of about 700 000 double mutant Saccharomyces cerevisiae strains. The tested targets included the gene deletion collection and 900 strains in which essential genes were affected by mRNA destabilization (DAmP). To analyze the results, we developed RECAP, a strategy that validates genetic interaction profiles by comparison with gene co-citation frequency, and identified links between 1471 genes and 117 biological processes. In addition to these large-scale results, we validated both enhancement and suppression of slow growth measured for specific RNA-related pathways. Thus, negative genetic interactions identified a role for the OCA inositol polyphosphate hydrolase complex in mRNA translation initiation. By analysis of suppressors, we found that Puf4, a Pumilio family RNA binding protein, inhibits ribosomal protein Rpl9 function, by acting on a conserved UGUAcauUA motif located downstream the stop codon of the RPL9B mRNA. Altogether, the results and their analysis should represent a useful resource for discovery of gene function in yeast. 相似文献