scholarly journals Application of the Saccharomyces cerevisiae FLP/FRT Recombination System in Filamentous Fungi for Marker Recycling and Construction of Knockout Strains Devoid of Heterologous Genes

2010 ◽  
Vol 76 (14) ◽  
pp. 4664-4674 ◽  
Author(s):  
Katarina Kopke ◽  
Birgit Hoff ◽  
Ulrich Kück
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Filamentous Fungi ◽  
Genetic Manipulation ◽  
Direct Repeat ◽  
Flp Recombinase ◽  
Marker Recycling ◽  
Recombination System ◽  
One Step ◽  
Experimental Approaches ◽  
Recombinase Gene

ABSTRACT To overcome the limited availability of antibiotic resistance markers in filamentous fungi, we adapted the FLP/FRT recombination system from the yeast Saccharomyces cerevisiae for marker recycling. We tested this system in the penicillin producer Penicillium chrysogenum using different experimental approaches. In a two-step application, we first integrated ectopically a nourseothricin resistance cassette flanked by the FRT sequences in direct repeat orientation (FRT-nat1 cassette) into a P. chrysogenum recipient. In the second step, the gene for the native yeast FLP recombinase, and in parallel, a codon-optimized P. chrysogenum flp (Pcflp) recombinase gene, were transferred into the P. chrysogenum strain carrying the FRT-nat1 cassette. The corresponding transformants were analyzed by PCR, growth tests, and sequencing to verify successful recombination events. Our analysis of several single- and multicopy transformants showed that only when the codon-optimized recombinase was present could a fully functional recombination system be generated in P. chrysogenum. As a proof of application of this system, we constructed a ΔPcku70 knockout strain devoid of any heterologous genes. To further improve the FLP/FRT system, we produced a flipper cassette carrying the FRT sites as well as the Pcflp gene together with a resistance marker. This cassette allows the controlled expression of the recombinase gene for one-step marker excision. Moreover, the applicability of the optimized FLP/FRT recombination system in other fungi was further demonstrated by marker recycling in the ascomycete Sordaria macrospora. Here, we discuss the application of the optimized FLP/FRT recombination system as a molecular tool for the genetic manipulation of filamentous fungi.

scholarly journals Flippase (FLP) recombinase-mediated marker recycling in the dermatophyte Arthroderma vanbreuseghemii

Microbiology ◽  
2014 ◽  
Vol 160 (10) ◽  
pp. 2122-2135 ◽  
Author(s):  
Yohko Yamada ◽  
Mari Maeda ◽  
Mohamed Mahdi Alshahni ◽  
Michel Monod ◽  
Peter Staib ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Trichophyton Mentagrophytes ◽  
Pathogenic Yeast ◽  
Selectable Markers ◽  
Gene Deletions ◽  
Flp Recombinase ◽  
Marker Recycling ◽  
Recombination System ◽  
One Step ◽  
Recessive Mutant

Biological processes can be elucidated by investigating complex networks of relevant factors and genes. However, this is not possible in species for which dominant selectable markers for genetic studies are unavailable. To overcome the limitation in selectable markers for the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes), we adapted the flippase (FLP) recombinase-recombination target (FRT) site-specific recombination system from the yeast Saccharomyces cerevisiae as a selectable marker recycling system for this fungus. Taking into account practical applicability, we designed FLP/FRT modules carrying two FRT sequences as well as the flp gene adapted to the pathogenic yeast Candida albicans (caflp) or a synthetic codon-optimized flp (avflp) gene with neomycin resistance (nptII) cassette for one-step marker excision. Both flp genes were under control of the Trichophyton rubrum copper-repressible promoter (PCTR4 ). Molecular analyses of resultant transformants showed that only the avflp-harbouring module was functional in A. vanbreuseghemii. Applying this system, we successfully produced the Ku80 recessive mutant strain devoid of any selectable markers. This strain was subsequently used as the recipient for sequential multiple disruptions of secreted metalloprotease (fungalysin) (MEP) or serine protease (SUB) genes, producing mutant strains with double MEP or triple SUB gene deletions. These results confirmed the feasibility of this system for broad-scale genetic manipulation of dermatophytes, advancing our understanding of functions and networks of individual genes in these fungi.

scholarly journals Novel Template Plasmids pCyaA’-Kan and pCyaA’-Cam for Generation of Unmarked Chromosomal cyaA’ Translational Fusion to T3SS Effectors in Salmonella

2021 ◽  
Vol 9 (3) ◽  
pp. 475
Author(s):  
Paulina A. Fernández ◽  
Marcela Zabner ◽  
Jaime Ortega ◽  
Constanza Morgado ◽  
Fernando Amaya ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Western Blot ◽  
Eukaryotic Cells ◽  
Intracellular Camp ◽  
Gene Fusions ◽  
Secretion Systems ◽  
Translational Fusion ◽  
Flp Recombinase ◽  
Recombination System ◽  
Camp Levels

The type III secretion systems (T3SS) encoded in pathogenicity islands SPI-1 and SPI-2 are key virulence factors of Salmonella. These systems translocate proteins known as effectors into eukaryotic cells during infection. To characterize the functionality of T3SS effectors, gene fusions to the CyaA’ reporter of Bordetella pertussis are often used. CyaA’ is a calmodulin-dependent adenylate cyclase that is only active within eukaryotic cells. Thus, the translocation of an effector fused to CyaA’ can be evaluated by measuring cAMP levels in infected cells. Here, we report the construction of plasmids pCyaA’-Kan and pCyaA’-Cam, which contain the ORF encoding CyaA’ adjacent to a cassette that confers resistance to kanamycin or chloramphenicol, respectively, flanked by Flp recombinase target (FRT) sites. A PCR product from pCyaA’-Kan or pCyaA’-Cam containing these genetic elements can be introduced into the bacterial chromosome to generate gene fusions by homologous recombination using the Red recombination system from bacteriophage λ. Subsequently, the resistance cassette can be removed by recombination between the FRT sites using the Flp recombinase. As a proof of concept, the plasmids pCyaA’-Kan and pCyaA’-Cam were used to generate unmarked chromosomal fusions of 10 T3SS effectors to CyaA’ in S. Typhimurium. Each fusion protein was detected by Western blot using an anti-CyaA’ monoclonal antibody when the corresponding mutant strain was grown under conditions that induce the expression of the native gene. In addition, T3SS-1-dependent secretion of fusion protein SipA-CyaA’ during in vitro growth was verified by Western blot analysis of culture supernatants. Finally, efficient translocation of SipA-CyaA’ into HeLa cells was evidenced by increased intracellular cAMP levels at different times of infection. Therefore, the plasmids pCyaA’-Kan and pCyaA’-Cam can be used to generate unmarked chromosomal cyaA’ translational fusion to study regulated expression, secretion and translocation of Salmonella T3SS effectors into eukaryotic cells.

scholarly journals The Spindle Checkpoint of the Yeast Saccharomyces cerevisiae Requires Kinetochore Function and Maps to the CBF3 Domain

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1493-1502
Author(s):  
Richard D Gardner ◽  
Atasi Poddar ◽  
Chris Yellman ◽  
Penny A Tavormina ◽  
M Cristina Monteagudo ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Critical Role ◽  
Spindle Checkpoint ◽  
Essential Genes ◽  
Gene Fusions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Checkpoint Signaling ◽  
One Step ◽  
Diploid Cells ◽  
Kinetochore Function

Abstract We have measured the activity of the spindle checkpoint in null mutants lacking kinetochore activity in the yeast Saccharomyces cerevisiae. We constructed deletion mutants for nonessential genes by one-step gene replacements. We constructed heterozygous deletions of one copy of essential genes in diploid cells and purified spores containing the deletion allele. In addition, we made gene fusions for three essential genes to target the encoded proteins for proteolysis (degron alleles). We determined that Ndc10p, Ctf13p, and Cep3p are required for checkpoint activity. In contrast, cells lacking Cbf1p, Ctf19p, Mcm21p, Slk19p, Cse4p, Mif2p, Mck1p, and Kar3p are checkpoint proficient. We conclude that the kinetochore plays a critical role in checkpoint signaling in S. cerevisiae. Spindle checkpoint activity maps to a discreet domain within the kinetochore and depends on the CBF3 protein complex.

scholarly journals One-step hot formamide extraction of RNA from Saccharomyces cerevisiae

RNA Biology ◽  
2017 ◽  
Vol 14 (12) ◽  
pp. 1722-1726 ◽  
Author(s):  
Daniel Shedlovskiy ◽  
Natalia Shcherbik ◽  
Dimitri G. Pestov
Keyword(s):  
Saccharomyces Cerevisiae ◽  
One Step

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.

Putative GTP-binding protein, Gtr1, associated with the function of the Pho84 inorganic phosphate transporter in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (7) ◽  
pp. 2958-2966
Author(s):  
M Bun-Ya ◽  
S Harashima ◽  
Y Oshima
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid Residues ◽  
Transport Activity ◽  
Reading Frame ◽  
Gtp Binding ◽  
Pi Transport ◽  
Recombination System ◽  
Opposite Strand ◽  
Constitutive Synthesis ◽  
Site Specific Recombination System

We have found an open reading frame which is 1.1 kb upstream of PHO84 (which encodes a Pi transporter) and is transcribed from the opposite strand. In Saccharomyces cerevisiae, this gene is distal to the TUB3 locus on the left arm of chromosome XIII and is named GTR1. GTR1 encodes a protein consisting of 310 amino acid residues containing, in its N-terminal region, the characteristic tripartite consensus elements for binding GTP conserved in GTP-binding proteins, except for histidine in place of a widely conserved aspargine residue in element III. Disruption of the GTR1 gene resulted in slow growth at 30 degrees C and no growth at 15 degrees C; other phenotypes resembled those of pho84 mutants and included constitutive synthesis of repressible acid phosphatase, reduced Pi transport activity, and resistance to arsenate. The latter phenotypes were shown to be due to a defect in Pi uptake, and the Gtr1 protein was found to be functionally associated with the Pho84 Pi transporter. Recombination between chromosome V (at the URA3 locus) and chromosome XIII (in the GTR1-PHO84-TUB3 region) by using a plasmid-encoded site-specific recombination system indicated that the order of these genes was telomere-TUB3-PHO84-GTR1-CENXIII.

Evolution of moonlighting proteins: insight from yeasts

2014 ◽  
Vol 42 (6) ◽  
pp. 1715-1719 ◽  
Author(s):  
Carlos Gancedo ◽  
Carmen-Lisset Flores ◽  
Juana M. Gancedo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Present Article ◽  
Catabolite Repression ◽  
Kluyveromyces Lactis ◽  
Approaches To Studying ◽  
Moonlighting Proteins ◽  
Experimental Approaches ◽  
Gal Genes

The present article addresses the possibilities offered by yeasts to study the problem of the evolution of moonlighting proteins. It focuses on data available on hexokinase from Saccharomyces cerevisiae that moonlights in catabolite repression and on galactokinase from Kluyveromyces lactis that moonlights controlling the induction of the GAL genes. Possible experimental approaches to studying the evolution of moonlighting hexose kinases are suggested.

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