Gene disruption of the pcbAB gene encoding ACV synthetase in Cephalosporium acremonium

1990 ◽  
Vol 18 (6) ◽  
pp. 523-530 ◽  
Author(s):  
Jo Ann Hoskins ◽  
Niamh O'Callaghan ◽  
Stephen W. Queener ◽  
Cathleen A. Cantwell ◽  
John S. Wood ◽  
...  
Keyword(s):  
Gene Disruption ◽  
Cephalosporium Acremonium ◽  
Gene Encoding

scholarly journals The Schizosaccharomyces pombe cho1+ Gene Encodes a Phospholipid Methyltransferase

Genetics ◽  
1998 ◽  
Vol 150 (2) ◽  
pp. 553-562
Author(s):  
Margaret I Kanipes ◽  
John E Hill ◽  
Susan A Henry
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sequence Analysis ◽  
Schizosaccharomyces Pombe ◽  
Gene Disruption ◽  
Structure And Function ◽  
Biosynthetic Enzyme ◽  
Gene Encoding ◽  
Complementation Groups ◽  
Eukaryotic Organisms ◽  
And Function

Abstract The isolation of mutants of Schizosaccharomyces pombe defective in the synthesis of phosphatidylcholine via the methylation of phosphatidylethanolamine is reported. These mutants are choline auxotrophs and fall into two unlinked complementation groups, cho1 and cho2. We also report the analysis of the cho1+ gene, the first structural gene encoding a phospholipid biosynthetic enzyme from S. pombe to be cloned and characterized. The cho1+ gene disruption mutant (cho1Δ) is viable if choline is supplied and resembles the cho1 mutants isolated after mutagenesis. Sequence analysis of the cho1+ gene indicates that it encodes a protein closely related to phospholipid methyltransferases from Saccharomyces cerevisiae and rat. Phospholipid methyltransferases encoded by a rat liver cDNA and the S. cerevisiae OPI3 gene are both able to complement the choline auxotrophy of the S. pombe cho1 mutants. These results suggest that both the structure and function of the phospholipid N-methyltransferases are broadly conserved among eukaryotic organisms.

scholarly journals Antigen 84, an Effector of Pleiomorphism in Mycobacterium smegmatis

2007 ◽  
Vol 189 (21) ◽  
pp. 7896-7910 ◽  
Author(s):  
Liem Nguyen ◽  
Nicole Scherr ◽  
John Gatfield ◽  
Anne Walburger ◽  
Jean Pieters ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Smegmatis ◽  
Cell Shape ◽  
Gene Disruption ◽  
Gene Encoding ◽  
Peptidoglycan Biosynthesis ◽  
Asymmetrically Distributed ◽  
Cell Wall Expansion

ABSTRACT While in most rod-shaped bacteria, morphology is based on MreB-like proteins that form an actin-like cytoskeletal scaffold for cell wall biosynthesis, the factors that determine the more flexible rod-like shape in actinobacteria such as Mycobacterium species are unknown. Here we show that a Mycobacterium smegmatis protein homologous to eubacterial DivIVA-like proteins, including M. tuberculosis antigen 84 (Ag84), localized symmetrically to centers of peptidoglycan biosynthesis at the poles and septa. Controlled gene disruption experiments indicated that the gene encoding Ag84, wag31, was essential; when overexpressed, cells became longer and wider, with Ag84 asymmetrically distributed at one pole. Many became grossly enlarged, bowling-pin-shaped cells having up to 80-fold-increased volume. In these cells, Ag84 accumulated predominantly at a bulbous pole that was apparently generated by uncontrolled cell wall expansion. In some cells, Ag84 was associated with exceptional sites of cell wall expansion (buds) that evolved into branches. M. bovis BCG Ag84 was able to form oligomers in vitro, perhaps reflecting its superstructure in vivo. These data suggested a role for Ag84 in cell division and modulating cell shape in pleiomorphic actinobacteria.

scholarly journals Isolation of the gene encoding the Saccharomyces cerevisiae centromere-binding protein CP1.

1990 ◽  
Vol 10 (6) ◽  
pp. 2458-2467 ◽  
Author(s):  
R E Baker ◽  
D C Masison
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Disruption ◽  
Mitotic Chromosome ◽  
Binding Protein ◽  
Chromosome Loss ◽  
Chromosome X ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Unexpected Consequence ◽  
Cell Doubling Time

CP1 is a sequence-specific DNA-binding protein of the yeast Saccharomyces cerevisiae which recognizes the highly conserved DNA element I (CDEI) of yeast centromeres. We cloned and sequenced the gene encoding CP1. The gene codes for a protein of molecular weight 39,400. When expressed in Escherichia coli, the CP1 gene directed the synthesis of a CDEI-binding protein having the same gel mobility as purified yeast CP1. We have given the CP1 gene the genetic designation CEP1 (centromere protein 1). CEP1 was mapped and found to reside on chromosome X, 2.0 centimorgans from SUP4. Strains were constructed in which most of CEP1 was deleted. Such strains lacked detectable CP1 activity and were viable; however, CEP1 gene disruption resulted in a 35% increase in cell doubling time and a ninefold increase in the rate of mitotic chromosome loss. An unexpected consequence of CP1 gene disruption was methionine auxotrophy genetically linked to cep1. This result and the recent finding that CDEI sites in the MET25 promoter are required to activate transcription (D. Thomas, H. Cherest, and Y. Surdin-Kerjan, J. Mol. Biol. 9:3292-3298, 1989) suggest that CP1 is both a kinetochore protein and a transcription factor.

scholarly journals Cytosine Deaminase as a Negative Selection Marker for Gene Disruption and Replacement in the Genus Streptomyces and Other Actinobacteria

2008 ◽  
Vol 75 (4) ◽  
pp. 1211-1214 ◽  
Author(s):  
Marie-Pierre Dubeau ◽  
Mariana Gabriela Ghinet ◽  
Pierre-�tienne Jacques ◽  
Nancy Clermont ◽  
Carole Beaulieu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Disruption ◽  
Negative Selection ◽  
Cytosine Deaminase ◽  
Synthetic Gene ◽  
Selection Marker ◽  
Selection System ◽  
Gene Encoding ◽  
Genus Streptomyces ◽  
Negative Selection Marker

ABSTRACT We developed a novel negative selection system for actinobacteria based on cytosine deaminase (CodA). We constructed vectors that include a synthetic gene encoding the CodA protein from Escherichia coli optimized for expression in Streptomyces species. Gene disruption and the introduction of an unmarked in-frame deletion were successfully achieved with these vectors.

scholarly journals Molecular characterization of the Aspergillus nidulans yA locus.

Genetics ◽  
1989 ◽  
Vol 121 (2) ◽  
pp. 249-254 ◽  
Author(s):  
E B O'Hara ◽  
W E Timberlake
Keyword(s):  
Aspergillus Nidulans ◽  
Gene Disruption ◽  
Molecular Organization ◽  
Dna Fragments ◽  
Developmentally Regulated ◽  
Gene Encoding ◽  
Mutant Strains ◽  
Low Levels ◽  
Chromosome I

Abstract We investigated the molecular organization of the region of Aspergillus nidulans chromosome I containing yA, a gene encoding the developmentally regulated enzyme conidial laccase. DNA fragments were identified that complemented the yA2 mutation and were shown to correspond to yA by genetic mapping and gene disruption experiments. The molecular map of the region was oriented to the genetic map by testing DNA fragments for their ability to complement a mutation in the tightly linked adE gene. The yA gene codes for a 2200 nucleotide mRNA that is present at low levels in vegetative cells and mature conidia, but accumulates to high levels in sporulating cultures. yA mRNA appears shortly after differentiation of sporogenous phialide cells. It accumulates in two developmentally abnormal mutant strains that produce phialides but is absent from two mutant strains that do not produce phialides. Thus, yA transcription is probably restricted to phialides. This result is discussed in relationship to the physiological roles played by phialides in spore differentiation.

scholarly journals Induction of a Toxin-Antitoxin Gene Cassette under High Hydrostatic Pressure Enables Markerless Gene Disruption in the Hyperthermophilic Archaeon Pyrococcus yayanosii

2018 ◽  
Vol 85 (4) ◽  
Author(s):  
Qinghao Song ◽  
Zhen Li ◽  
Rouke Chen ◽  
Xiaopan Ma ◽  
Xiang Xiao ◽  
...  
Keyword(s):  
Hydrostatic Pressure ◽  
High Temperatures ◽  
High Hydrostatic Pressure ◽  
Gene Disruption ◽  
Inducible Promoter ◽  
Constitutive Promoter ◽  
Gene Encoding ◽  
Content Type ◽  
Genetic Tools ◽  
Pyrococcus Yayanosii

ABSTRACT The discovery of hyperthermophiles has dramatically changed our understanding of the habitats in which life can thrive. However, the extreme high temperatures in which these organisms live have severely restricted the development of genetic tools. The archaeon Pyrococcus yayanosii A1 is a strictly anaerobic and piezophilic hyperthermophile that is an ideal model for studies of extreme environmental adaptation. In the present study, we identified a high hydrostatic pressure (HHP)-inducible promoter (Phhp) that controls target gene expression under HHP. We developed an HHP-inducible toxin-antitoxin cassette (HHP-TAC) containing (i) a counterselectable marker in which a gene encoding a putative toxin (virulence-associated protein C [PF0776 {VapC}]) controlled by the HHP-inducible promoter was used in conjunction with the gene encoding antitoxin PF0775 (VapB), which was fused to a constitutive promoter (PhmtB), and (ii) a positive marker with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-encoding gene from P. furiosus controlled by the constitutive promoter Pgdh. The HHP-TAC was constructed to realize markerless gene disruption directly in P. yayanosii A1 in rich medium. The pop-out recombination step was performed using an HHP-inducible method. As proof, the PYCH_13690 gene, which encodes a 4-α-glucanotransferase, was successfully deleted from the strain P. yayanosii A1. The results showed that the capacity for starch hydrolysis in the Δ1369 mutant decreased dramatically compared to that in the wild-type strain. The inducible toxin-antitoxin system developed in this study greatly increases the genetic tools available for use in hyperthermophiles. IMPORTANCE Genetic manipulations in hyperthermophiles have been studied for over 20 years. However, the extremely high temperatures under which these organisms grow have limited the development of genetic tools. In this study, an HHP-inducible promoter was used to control the expression of a toxin. Compared to sugar-inducible and cold-shock-inducible promoters, the HHP-inducible promoter rarely has negative effects on the overall physiology and central metabolism of microorganisms, especially piezophilic hyperthermophiles. Previous studies have used auxotrophic strains as hosts, which may interfere with studies of adaptation and metabolism. Using an inducible toxin-antitoxin (TA) system as a counterselectable marker enables the generation of a markerless gene disruption strain without the use of auxotrophic mutants and counterselection with 5-fluoroorotic acid. TA systems are widely distributed in bacteria and archaea and can be used to overcome the limitations of high growth temperatures and dramatically extend the selectivity of genetic tools in hyperthermophiles.

Gene Disruption in Scedosporium aurantiacum: Proof of Concept with the Disruption of SODC Gene Encoding a Cytosolic Cu,Zn-Superoxide Dismutase

2017 ◽  
Vol 183 (1) ◽  
pp. 241-249 ◽  
Author(s):  
Victoire Pateau ◽  
Bienvenue Razafimandimby ◽  
Patrick Vandeputte ◽  
Christopher R. Thornton ◽  
Thomas Guillemette ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Gene Disruption ◽  
Proof Of Concept ◽  
Gene Encoding

Cloning of the PYR4 gene encoding orotidine-5?-phosphate decarboxylase in Cephalosporium acremonium

1990 ◽  
Vol 17 (3) ◽  
pp. 223-227 ◽  
Author(s):  
Alejandro Vian ◽  
Miguel Angel Pe�alva
Keyword(s):  
Cephalosporium Acremonium ◽  
Gene Encoding

scholarly journals Characterization of Saccharomyces cerevisiae deficient in expression of phospholipase D

1996 ◽  
Vol 314 (1) ◽  
pp. 15-19 ◽  
Author(s):  
Krishna M. ELLA ◽  
Joseph W. DOLAN ◽  
Chen QI ◽  
Kathryn E. MEIER
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Phospholipase D ◽  
Gene Disruption ◽  
Carbon Sources ◽  
Gene Encoding ◽  
Sequence Identity ◽  
One Step

A gene encoding phospholipase D (PLD) in Saccharomyces cerevisiae was identified. The 195 kDa product of PLD1 has 24% overall sequence identity with a plant PLD. Expression of yeast PLD activity was eliminated by one-step gene disruption. Yeast haploids lacking PLD activity were deficient in growth on non-fermentable carbon sources. Diploids lacking expression of PLD1 were unable to sporulate.

scholarly journals The 4-Oxalomesaconate Hydratase Gene, Involved in the Protocatechuate 4,5-Cleavage Pathway, Is Essential to Vanillate and Syringate Degradation in Sphingomonas paucimobilisSYK-6

2000 ◽  
Vol 182 (24) ◽  
pp. 6950-6957 ◽  
Author(s):  
Hirofumi Hara ◽  
Eiji Masai ◽  
Yoshihiro Katayama ◽  
Masao Fukuda
Keyword(s):  
Gene Disruption ◽  
Lignin Degradation ◽  
Gel Filtration ◽  
Cysteine Residue ◽  
Open Reading Frame ◽  
Growth Defect ◽  
Sphingomonas Paucimobilis ◽  
Gene Encoding ◽  
Reading Frame ◽  
Semialdehyde Dehydrogenase

ABSTRACT Sphingomonas paucimobilis SYK-6 is able to grow on various dimeric lignin compounds, which are converted to vanillate and syringate by the actions of unique lignin degradation enzymes in this strain. Vanillate and syringate are degraded by theO-demethylase and converted into protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, while the results suggested that 3MGA is degraded through another pathway in which PCA 4,5-dioxygenase is not involved. In a 10.5-kb EcoRI fragment carrying the genes for PCA 4,5-dioxygenase (ligAB), 2-pyrone-4,6-dicarboxylate hydrolase (ligI), and a portion of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (ligC), we found the ligJ gene encoding 4-oxalomesaconate (OMA) hydratase, which catalyzes the conversion of OMA into 4-carboxy-4-hydroxy-2-oxoadipate. The ligJ gene is transcribed in the same direction as ligABC genes and consists of an 1,023-bp open reading frame encoding a polypeptide with a molecular mass of 38,008 Da, which is located 73-bp upstream fromligA. The ligJ gene product (LigJ), expressed in Escherichia coli, was purified to near homogeneity and was estimated to be a homodimer (69.5 kDa) by gel filtration chromatography. The isoelectric point was determined to be 4.9, and the optimal temperature is 30°C. The Km for OMA and the V max were determined to be 138 μM and 440 U/mg, respectively. LigJ activity was inhibited by the addition of thiol reagents, suggesting that some cysteine residue is part of the catalytic site. The ligJ gene disruption in SYK-6 caused the growth defect on and the accumulation of common metabolites from both vanillate and syringate, indicating that the ligJ gene is essential to the degradation of these two compounds. These results indicated that syringate is converted into OMA via 3MGA, and it enters the PCA 4,5-cleavage pathway.

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