scholarly journals Phenotypic and genotypic consequences of CRISPR/Cas9 editing of the replication origins in the rDNA of Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Joseph C. Sanchez ◽  
Anja Ollodart ◽  
Christopher R. L. Large ◽  
Courtnee Clough ◽  
Gina M. Alvino ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Life Span ◽  
Genetic Manipulation ◽  
Consensus Sequence ◽  
Complex Structure ◽  
Rdna Locus ◽  
Origin Of Replication ◽  
Cellular Functions ◽  
Rdna Variation

AbstractThe complex structure and repetitive nature of eukaryotic ribosomal DNA (rDNA) is a challenge for genome assembly, and thus, the consequences of sequence variation in rDNA remain unexplored. However, renewed interest in the role that rDNA variation may play in diverse cellular functions, aside from ribosome production, highlights the need for a method that would permit genetic manipulation of the rDNA. Here, we describe a CRISPR/Cas9 based strategy to edit the rDNA locus in the budding yeast Saccharomyces cerevisiae. Using this approach, we modified the endogenous rDNA origin of replication in each repeat by deleting or replacing its consensus sequence. We characterized the transformants that have successfully modified their rDNA locus and propose a mechanism for how CRISPR/Cas9 mediated editing of the rDNA occurs. In addition, we carried out extended growth and life span experiments to investigate the long-term consequences that altering the rDNA origin of replication has on cellular health. We find that long-term growth of the edited clones results in faster growing suppressors that have acquired segmental aneusomy of the rDNA containing region of chr XII or aneuploidy of chromosomes XII, II, or IV. Furthermore, we find that all edited isolates suffer a reduced life span, irrespective of their levels of extrachromosomal rDNA circles. Our work demonstrates that it is possible to quickly, efficiently and homogeneously edit the rDNA locus via CRISPR/Cas9. It serves as a model for modifying other parts of the rDNA and, more generally, for editing other tandemly repeated sequences in higher eukaryotes.

scholarly journals A Novel Yeast Silencer: The 2μ Origin of Saccharomyces cerevisiae Has HST3-, MIG1- and SIR-Dependent Silencing Activity

Genetics ◽  
2002 ◽  
Vol 162 (1) ◽  
pp. 59-71
Author(s):  
Arnold Grünweller ◽  
Ann E Ehrenhofer-Murray
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Origin Recognition Complex ◽  
Rdna Locus ◽  
Yeast Genome ◽  
Origin Of Replication ◽  
Sir Proteins ◽  
Sequence Elements ◽  
Silencer Element ◽  
Subtelomeric Regions

Abstract Silencing in Saccharomyces cerevisiae is found at the mating-type loci HMR and HML, in subtelomeric regions, and at the rDNA locus. Repressed chromatin is built up by the recruitment of the Sir proteins via their interaction with DNA-binding proteins that bind to silencers. Here, we have performed a genetic screen for novel sequence elements within the yeast genome that display silencing activity. We isolated as a novel silencer element the origin of replication from the endogenous 2μ plasmid (2μARS). 2μARS-mediated silencing was dependent upon the Sir proteins, the origin recognition complex (ORC), and Hst3, a Sir2 histone deacetylase homolog, suggesting that it constituted a novel class of silencing in yeast. Moreover, 2μARS carried a binding site for Mig1, a transcriptional repressor of glucose-regulated genes. Both the Mig1-binding site and the MIG1 gene were necessary for full silencing activity of 2μARS. Furthermore, Hst3 was physically present at 2μARS in a silencing context as well as at the endogenous 2μ plasmid. Also, Hst3 regulated the repression of the flipase gene, although this was likely an indirect effect of HST3 on FLP1 expression.

Saccharomyces cerevisiae SSD1-VConfers Longevity by a Sir2p-Independent Mechanism

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1661-1672 ◽  
Author(s):  
Matt Kaeberlein ◽  
Alex A Andalis ◽  
Gregory B Liszt ◽  
Gerald R Fink ◽  
Leonard Guarente
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Life Span ◽  
Cell Cycle Progression ◽  
Polymorphic Locus ◽  
Cell Integrity ◽  
Cycle Progression ◽  
Cellular Processes ◽  
Maximal Life Span ◽  
Independent Mechanism ◽  
Yeast Aging

AbstractThe SSD1 gene of Saccharomyces cerevisiae is a polymorphic locus that affects diverse cellular processes including cell integrity, cell cycle progression, and growth at high temperature. We show here that the SSD1-V allele is necessary for cells to achieve extremely long life span. Furthermore, addition of SSD1-V to cells can increase longevity independently of SIR2, although SIR2 is necessary for SSD1-V cells to attain maximal life span. Past studies of yeast aging have been performed in short-lived ssd1-d strain backgrounds. We propose that SSD1-V defines a previously undescribed pathway affecting cellular longevity and suggest that future studies on longevity-promoting genes should be carried out in long-lived SSD1-V strains.

scholarly journals Distribution of a Limited Sir2 Protein Pool Regulates the Strength of Yeast rDNA Silencing and Is Modulated by Sir4p

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1205-1219 ◽  
Author(s):  
Jeffrey S Smith ◽  
Carrie Baker Brachmann ◽  
Lorraine Pillus ◽  
Jef D Boeke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Simple Model ◽  
Regulatory Mechanism ◽  
Transcriptional Silencing ◽  
Rdna Locus ◽  
Genetic Manipulations ◽  
Protein Levels ◽  
Endogenous Level ◽  
Protein Pool ◽  
Negative Effect

Abstract Transcriptional silencing in Saccharomyces cerevisiae occurs at the silent mating-type loci HML and HMR, at telomeres, and at the ribosomal DNA (rDNA) locus RDN1. Silencing in the rDNA occurs by a novel mechanism that depends on a single Silent Information Regulator (SIR) gene, SIR2. SIR4, essential for other silenced loci, paradoxically inhibits rDNA silencing. In this study, we elucidate a regulatory mechanism for rDNA silencing based on the finding that rDNA silencing strength directly correlates with cellular Sir2 protein levels. The endogenous level of Sir2p was shown to be limiting for rDNA silencing. Furthermore, small changes in Sir2p levels altered rDNA silencing strength. In rDNA silencing phenotypes, sir2 mutations were shown to be epistatic to sir4 mutations, indicating that SIR4 inhibition of rDNA silencing is mediated through SIR2. Furthermore, rDNA silencing is insensitive to SIR3 overexpression, but is severely reduced by overexpression of full-length Sir4p or a fragment of Sir4p that interacts with Sir2p. This negative effect of SIR4 overexpression was overridden by co-overexpression of SIR2, suggesting that SIR4 directly inhibits the rDNA silencing function of SIR2. Finally, genetic manipulations of SIR4 previously shown to promote extended life span also resulted in enhanced rDNA silencing. We propose a simple model in which telomeres act as regulators of rDNA silencing by competing for limiting amounts of Sir2 protein.

scholarly journals Regulatory Mechanisms Controlling Expression of the DAN/TIR Mannoprotein Genes During Anaerobic Remodeling of the Cell Wall in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1169-1177
Author(s):  
Natalia E Abramova ◽  
Brian D Cohen ◽  
Odeniel Sertil ◽  
Rachna Kapoor ◽  
Kelvin J A Davies ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Consensus Sequence ◽  
Ectopic Expression ◽  
Constitutive Expression ◽  
Anaerobic Growth ◽  
Zinc Cluster ◽  
Sterol Transport ◽  
Genes Encoding ◽  
Altered Regulation ◽  
Repression Domain

Abstract The DAN/TIR genes of Saccharomyces cerevisiae encode homologous mannoproteins, some of which are essential for anaerobic growth. Expression of these genes is induced during anaerobiosis and in some cases during cold shock. We show that several heme-responsive mechanisms combine to regulate DAN/TIR gene expression. The first mechanism employs two repression factors, Mox1 and Mox2, and an activation factor, Mox4 (for mannoprotein regulation by oxygen). The genes encoding these proteins were identified by selecting for recessive mutants with altered regulation of a dan1::ura3 fusion. MOX4 is identical to UPC2, encoding a binucleate zinc cluster protein controlling expression of an anaerobic sterol transport system. Mox4/Upc2 is required for expression of all the DAN/TIR genes. It appears to act through a consensus sequence termed the AR1 site, as does Mox2. The noninducible mox4Δ allele was epistatic to the constitutive mox1 and mox2 mutations, suggesting that Mox1 and Mox2 modulate activation by Mox4 in a heme-dependent fashion. Mutations in a putative repression domain in Mox4 caused constitutive expression of the DAN/TIR genes, indicating a role for this domain in heme repression. MOX4 expression is induced both in anaerobic and cold-shocked cells, so heme may also regulate DAN/TIR expression through inhibition of expression of MOX4. Indeed, ectopic expression of MOX4 in aerobic cells resulted in partially constitutive expression of DAN1. Heme also regulates expression of some of the DAN/TIR genes through the Rox7 repressor, which also controls expression of the hypoxic gene ANB1. In addition Rox1, another heme-responsive repressor, and the global repressors Tup1 and Ssn6 are also required for full aerobic repression of these genes.

scholarly journals In-Frame and Frameshift Mutations in Zebrafish presenilin 2 Affect Different Cellular Functions in Young Adult Brains

2021 ◽  
pp. 1-10
Author(s):  
Karissa Barthelson ◽  
Stephen Martin Pederson ◽  
Morgan Newman ◽  
Haowei Jiang ◽  
Michael Lardelli
Keyword(s):  
Cell Cycle ◽  
Young Adult ◽  
Oxidative Phosphorylation ◽  
Frameshift Mutation ◽  
Long Term Potentiation ◽  
Cellular Functions ◽  
Reading Frame ◽  
Term Potentiation ◽  
Presenilin 2

Background: Mutations in PRESENILIN 2 (PSEN2) cause early onset familial Alzheimer’s disease (EOfAD) but their mode of action remains elusive. One consistent observation for all PRESENILIN gene mutations causing EOfAD is that a transcript is produced with a reading frame terminated by the normal stop codon—the “reading frame preservation rule”. Mutations that do not obey this rule do not cause the disease. The reasons for this are debated. Objective: To predict cellular functions affected by heterozygosity for a frameshift, or a reading frame-preserving mutation in zebrafish psen2 using bioinformatic techniques. Methods: A frameshift mutation (psen2N140fs) and a reading frame-preserving (in-frame) mutation (psen2T141 _ L142delinsMISLISV) were previously isolated during genome editing directed at the N140 codon of zebrafish psen2 (equivalent to N141 of human PSEN2). We mated a pair of fish heterozygous for each mutation to generate a family of siblings including wild type and heterozygous mutant genotypes. Transcriptomes from young adult (6 months) brains of these genotypes were analyzed. Results: The in-frame mutation uniquely caused subtle, but statistically significant, changes to expression of genes involved in oxidative phosphorylation, long-term potentiation and the cell cycle. The frameshift mutation uniquely affected genes involved in Notch and MAPK signaling, extracellular matrix receptor interactions and focal adhesion. Both mutations affected ribosomal protein gene expression but in opposite directions. Conclusion: A frameshift and an in-frame mutation at the same position in zebrafish psen2 cause discrete effects. Changes in oxidative phosphorylation, long-term potentiation and the cell cycle may promote EOfAD pathogenesis in humans.

scholarly journals Cell surface anchorage and ligand-binding domains of the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin, a member of the immunoglobulin superfamily.

1993 ◽  
Vol 13 (4) ◽  
pp. 2554-2563 ◽  
Author(s):  
D Wojciechowicz ◽  
C F Lu ◽  
J Kurjan ◽  
P N Lipke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Adhesion ◽  
Amino Acid ◽  
Cell Surface ◽  
Consensus Sequence ◽  
Binding Domain ◽  
Immunoglobulin Superfamily ◽  
Cell Contact ◽  
Amino Acid Residues ◽  
Adhesion Proteins

alpha-Agglutinin is a cell adhesion glycoprotein expressed on the cell wall of Saccharomyces cerevisiae alpha cells. Binding of alpha-agglutinin to its ligand a-agglutinin, expressed by a cells, mediates cell-cell contact during mating. Analysis of truncations of the 650-amino-acid alpha-agglutinin structural gene AG alpha 1 delineated functional domains of alpha-agglutinin. Removal of the C-terminal hydrophobic sequence allowed efficient secretion of the protein and loss of cell surface attachment. This cell surface anchorage domain was necessary for linkage to a glycosyl phosphatidylinositol anchor. A construct expressing the N-terminal 350 amino acid residues retained full a-agglutinin-binding activity, localizing the binding domain to the N-terminal portion of alpha-agglutinin. A 278-residue N-terminal peptide was inactive; therefore, the binding domain includes residues between 278 and 350. The segment of alpha-agglutinin between amino acid residues 217 and 308 showed significant structural and sequence similarity to a consensus sequence for immunoglobulin superfamily variable-type domains. The similarity of the alpha-agglutinin-binding domain to mammalian cell adhesion proteins suggests that this structure is a highly conserved feature of adhesion proteins in diverse eukaryotes.

scholarly journals A set of novel CRISPR-based integrative vectors for Saccharomyces cerevisiae

2018 ◽  
Vol 3 ◽  
pp. 72
Author(s):  
Peter W Daniels ◽  
Anuradha Mukherjee ◽  
Alastair SH Goldman ◽  
Bin Hu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Manipulation ◽  
Strand Break ◽  
Integration Strategy ◽  
System A ◽  
Target Dna ◽  
Biosynthesis Gene ◽  
Dna Fragment ◽  
Integrative Vectors ◽  
Yeast Genomes

Integrating a desired DNA sequence into yeast genomes is a widely-used genetic manipulation in the budding yeast Saccharomyces cerevisiae. The conventional integration method is to use an integrative plasmid such as pRS or YIplac series as the target DNA carrier. The nature of this method risks multiple integrations of the target DNA and the potential loss of integrated DNA during cell proliferation. In this study, we developed a novel yeast integration strategy based on the widely used CRISPR-Cas9 system and created a set of plasmids for this purpose. In this system, a plasmid bearing Cas9 and gRNA expression cassettes will induce a double-strand break (DSB) inside a biosynthesis gene such as Met15 or Lys2. Repair of the DSB will be mediated by another plasmid bearing upstream and downstream sequences of the DSB and an integration sequence in between. As a result of this repair the sequence is integrated into genome by replacing the biosynthesis gene, the disruption of which leads to a new auxotrophic genotype. The newly-generated auxotroph can serve as a traceable marker for the integration. In this study, we demonstrated that a DNA fragment up to 6.3 kb can be efficiently integrated into the Met15 or Lys2 locus using this system. This novel integration strategy can be applied to various yeasts, including natural yeast isolated from wild environments or different yeast species such as Candida albicans.

scholarly journals Loss of Ubp3 increases silencing, decreases unequal recombination in rDNA, and shortens the replicative life span in Saccharomyces cerevisiae

2014 ◽  
Vol 25 (12) ◽  
pp. 1916-1924 ◽  
Author(s):  
David Öling ◽  
Rehan Masoom ◽  
Kristian Kvint
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Life Span ◽  
Rna Polymerase Ii ◽  
Ribosomal Dna ◽  
Genetic Evidence ◽  
Wild Type ◽  
Replicative Life Span ◽  
Unequal Recombination

Ubp3 is a conserved ubiquitin protease that acts as an antisilencing factor in MAT and telomeric regions. Here we show that ubp3∆ mutants also display increased silencing in ribosomal DNA (rDNA). Consistent with this, RNA polymerase II occupancy is lower in cells lacking Ubp3 than in wild-type cells in all heterochromatic regions. Moreover, in a ubp3∆ mutant, unequal recombination in rDNA is highly suppressed. We present genetic evidence that this effect on rDNA recombination, but not silencing, is entirely dependent on the silencing factor Sir2. Further, ubp3∆ sir2∆ mutants age prematurely at the same rate as sir2∆ mutants. Thus our data suggest that recombination negatively influences replicative life span more so than silencing. However, in ubp3∆ mutants, recombination is not a prerequisite for aging, since cells lacking Ubp3 have a shorter life span than isogenic wild-type cells. We discuss the data in view of different models on how silencing and unequal recombination affect replicative life span and the role of Ubp3 in these processes.

Mathematical modeling of fluid movement inside the eyeball during intravitreal injection

2021 ◽  
pp. 106-109
Author(s):  
D.V. Lipatov ◽  
◽  
S.A. Skladchikov ◽  
N.P. Savenkova ◽  
V.V. Novoderezkin ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Mathematical Model ◽  
Residence Time ◽  
Complex Structure ◽  
Posterior Vitreous Detachment ◽  
Vitreous Body ◽  
Complete Removal ◽  
Intravitreal Injections ◽  
The World

Background. The avalanche-like growth of intravitreal injections in the world has significantly increased interest in the hemodynamics of the processes that occur in the eye when a drug is injected into the vitreous cavity. Every year, the number of intravitreally used drugs and promising areas in which they can be used is growing. This also applies to the creation of new combined medicines and the development of drugs with a long-term therapeutic effect. Aims. Create mathematical model of eyeball to evaluate the movement of the drug substance in it; to estimate the time of the drug's presence in the eye cavity before its complete removal, to characterize the ways of its removal from the eye cavity; to assess the significance of posterior vitreous detachment during the time when the drug is present in the eye cavity; to evaluate the effect on the hydrodynamics of the depth of drug administration. Results. When the drug is administered closer to the center of the eyeball, its residence time increases in comparison with the parietal administration. With a complete posterior detachment of the vitreous body, the time of finding the drug in the eye is prolonged compared to its absence. The obtained results of mathematical modeling of the movement of the drug administered intravitreally cannot be mechanically transferred to the human eye, due to the more complex structure of the latter. Key words: intravitreal injections, vitreous body, mathematic computing.

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