Versatile fungal transformation vectors carrying the selectable bar gene of Streptomyces hygroscopicus

Straubinger, Bernhard; Straubinger, E.; Wirsel, Stefan G. R.; Turgeon, G.; Yoder, O. C.

doi:10.4148/1941-4765.1441

Cited by 33 publications

(30 citation statements)

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“…We have also used a vector (pMT1612) conferring glufosinate resistance, in which the bar (glufosinate resistance) gene of Streptomyces hygroscopicus from plasmid pBP1T (Straubinger et al 1992 NkuAD dramatically improves the frequency of correct gene targeting: We initially tested the effects of nkuAD on gene targeting using a linear DNA fragment generated by fusion PCR to create a C-terminal histone H1-monomeric red fluorescent protein (mRFP) (Campbell et al 2002;Toews et al 2004) fusion. We chose this test system because histone H1-mRFP fusions are easily scored by fluorescence microscopy.…”

Section: Resultsmentioning

confidence: 99%

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A Versatile and Efficient Gene-Targeting System for Aspergillus nidulans

et al. 2006

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Aspergillus nidulans is an important experimental organism, and it is a model organism for the genus Aspergillus that includes serious pathogens as well as commercially important organisms. Gene targeting by homologous recombination during transformation is possible in A. nidulans, but the frequency of correct gene targeting is variable and often low. We have identified the A. nidulans homolog (nkuA) of the human KU70 gene that is essential for nonhomologous end joining of DNA in double-strand break repair. Deletion of nkuA (nkuAD) greatly reduces the frequency of nonhomologous integration of transforming DNA fragments, leading to dramatically improved gene targeting. We have also developed heterologous markers that are selectable in A. nidulans but do not direct integration at any site in the A. nidulans genome. In combination, nkuAD and the heterologous selectable markers make up a very efficient genetargeting system. In experiments involving scores of genes, 90% or more of the transformants carried a single insertion of the transforming DNA at the correct site. The system works with linear and circular transforming molecules and it works for tagging genes with fluorescent moieties, replacing genes, and replacing promoters. This system is efficient enough to make genomewide gene-targeting projects feasible.

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Section: Resultsmentioning

confidence: 99%

“…The bar gene encoding glufosinate resistance was taken from the plasmid pBP1T (Straubinger et al 1992). The bar gene was then placed between the amdS promoter [containing the I9 and the I66 mutations that give increased expression (see Hynes and Davis 2004)] and the Aspergillus niger glucoamylase terminator.…”

Section: Strainsmentioning

confidence: 99%

A Versatile and Efficient Gene-Targeting System for Aspergillus nidulans

et al. 2006

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“…A 1.0-kb ClaI and AgeI fragment of the CTB3 gene was excised from plasmid pCTB3G and replaced with the end-filled EcoRI/HindIII phosphinothricin acetyltransferase gene (BAR) gene under the Cochliobolus heterostrophus promoter 1 (P1) from pBP1T (Straubinger et al, 1992) to result in pCTB3/Bar6 ( Fig. 2A).…”

Section: Gene Disruption and Genetic Complementationmentioning

confidence: 99%

The Cercospora nicotianae gene encoding dual O-methyltransferase and FAD-dependent monooxygenase domains mediates cercosporin toxin biosynthesis

Dekkers

You

Gowda

et al. 2007

Fungal Genetics and Biology

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“…After addition of an A residue to both ends, the amplification product was cloned into pGEM-T-Easy for sequence analysis. It was then recovered from pGEM-T-Easy for cloning into the fungal vector pBARKS1 (28) for transformation into C. nicotianae mutant strains.…”

Section: Methodsmentioning

confidence: 99%

Isolation of PDX2 , a Second Novel Gene in the Pyridoxine Biosynthesis Pathway of Eukaryotes, Archaebacteria, and a Subset of Eubacteria

Ehrenshaft

Daub

2001

J Bacteriol

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In this paper we describe the isolation of a second gene in the newly identified pyridoxine biosynthesis pathway of archaebacteria, some eubacteria, fungi, and plants. Although pyridoxine biosynthesis has been thoroughly examined in Escherichia coli, recent characterization of the Cercospora nicotianae biosynthesis gene PDX1 led to the discovery that most organisms contain a pyridoxine synthesis gene not found in E. coli. PDX2 was isolated by a degenerate primer strategy based on conserved sequences of a gene specific to PDX1-containing organisms. The role of PDX2 in pyridoxine biosynthesis was confirmed by complementation of two C. nicotianae pyridoxine auxotrophs not mutant in PDX1. Also, targeted gene replacement of PDX2 in C. nicotianae results in pyridoxine auxotrophy. Comparable to PDX1, PDX2 homologues are not found in any of the organisms with homologues to the E. coli pyridoxine genes, but are found in the same archaebacteria, eubacteria, fungi, and plants that contain PDX1 homologues. PDX2 proteins are less well conserved than their PDX1 counterparts but contain several protein motifs that are conserved throughout all PDX2 proteins.Recent work in our laboratory with the filamentous, phytopathogenic fungus Cercospora nicotianae revealed that a highly conserved group of gene homologues found in eubacteria, archaebacteria, fungi, and plants play a role in a divergent pyridoxine (vitamin B 6 ) biosynthesis pathway (8). PDX1 was originally identified as a gene required for resistance of this fungus to a singlet-oxygen-generating toxin, cercosporin, which it produces to parasitize plants (9, 10). During characterization of this gene, however, we discovered that it rescued both C. nicotianae and Aspergillus flavus pyridoxine auxotrophs to prototrophy (8). This observation was subsequently confirmed in Aspergillus nidulans, in which a PDX1 homologue, PYROA, also rescued pyridoxine auxotrophy (24). Interestingly, despite this direct evidence for the involvement of PDX1 homologues in pyridoxine synthesis, PDX1 shows no homology to any of the known Escherichia coli pyridoxine biosynthesis genes or to any gene in the completely sequenced E. coli genome. Database analysis determined that organisms with homologues to the E. coli genes (some eubacteria) lacked PDX1 homologues and that organisms with PDX1 homologues (other eubacteria, archaebacteria, fungi, and plants) lacked homologues to the E. coli genes. These data suggested that a divergence in the pyridoxine synthesis pathway occurred sometime during the evolution of the eubacteria (8).The advent of genomic and other large-scale sequencing projects allows homology comparisons on an organismal level. Saccharomyces cerevisiae contains three unlinked PDX1 homologues, one of which (SNZ1, for snooze) was extensively studied because its expression increases dramatically during stationary phase (5). Analyses by Galperin and Koonin (12) uncovered that PDX1-containing organisms also contained a copy of a second homologous gene and that three of these organisms (S. cerevi...

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Versatile fungal transformation vectors carrying the selectable bar gene of Streptomyces hygroscopicus

Cited by 33 publications

References 0 publications

A Versatile and Efficient Gene-Targeting System for Aspergillus nidulans

A Versatile and Efficient Gene-Targeting System for Aspergillus nidulans

The Cercospora nicotianae gene encoding dual O-methyltransferase and FAD-dependent monooxygenase domains mediates cercosporin toxin biosynthesis

Isolation of PDX2 , a Second Novel Gene in the Pyridoxine Biosynthesis Pathway of Eukaryotes, Archaebacteria, and a Subset of Eubacteria

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