Use of a bacterial hygromycin B resistance gene as a dominant selectable marker in Neurospora crassa transformation

Stäben, Chuck; Jensen, Benny; Singer, Michael J.; Pollock, J. A.; Schechtman, Michael G.; Kinsey, John A.; Selker, Eric U.

doi:10.4148/1941-4765.1519

Cited by 242 publications

(220 citation statements)

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“…Plasmids pBluescript SK ϩ (Stratagene) and pGEM (Promega) were generally used for construction of new vectors. Plasmids pBARGEM7-1 (23) and pCSN43 (24) and cosmids G7H3 and G8B12 were obtained from the Fungal Genetics Stock Center, University of Kansas Medical School (Kansas City).…”

Section: Methodsmentioning

confidence: 99%

Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining

Ninomiya

Suzuki

Ishii

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

536

453

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Gene disruption and overexpression play central roles in the analysis of gene function. Homologous recombination is, in principle, the most efficient method of disrupting, modifying, or replacing a target gene. Although homologous integration of exogenous DNA into the genome occurs readily in Saccharomyces cerevisiae , it is rare in many other organisms. We identified and disrupted Neurospora crassa genes homologous to human KU70 and KU80 , which encode proteins that function in nonhomologous end-joining of double-stranded DNA breaks. The resulting mutants, named mus - 51 and mus - 52 , showed higher sensitivity to methyl methanesulfonate, ethyl methanesulfonate, and bleomycin than wild type, but not to UV, 4-nitroquinoline 1-oxide, camptothecin, or hydroxyurea. Vegetative growth, conidiation, and ascospore production in homozygous crosses were normal. The frequency of integration of exogenous DNA into homologous sequences of the genome in the KU disruption strains of N. crassa was compared with that in wild type, mei-3 , and mus - 11 . In mei-3 and mus - 11 , which are defective in homologous recombination, none or few homologous integration events were observed under any conditions. When mtr target DNA with ≈2-kb 5′ and 3′ flanking regions was used for transformation of the KU disruption strains, 100% of transformants exhibited integration at the homologous site, compared to 10 to 30% for a wild-type recipient. Similar results were obtained when the ad-3A gene was targeted for disruption. These results indicate that KU disruption strains are efficient recipients for gene targeting.

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Section: Methodsmentioning

confidence: 99%

Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining

Ninomiya

Suzuki

Ishii

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

536

453

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“…The pelD gene was disrupted by protoplast transformation with a construct made from a 2.7-kb pelD gene fragment with the replacement of a 250-bp internal coding segment of pelD with the hyg gene (24). The hygromycinresistant transformants were screened by PCR with spores directly as the source of the template.…”

Section: Resultsmentioning

confidence: 99%

“…The EcoRI site in the pelD gene clone (20), pX55, was eliminated, and a fragment of pX55 with a deletion of 250 bp from the coding region of pelD was amplified by an inverse PCR with a sense primer, 5Ј-GCG CGC GAA TTC GTC TTC ATC CTC GAG GAG-3Ј, and an antisense primer, 5Ј-GGC CGG ACT AGT CGA GGA CAG TGA CGA TGC-3Ј, with Expand High Fidelity Taq DNA polymerase. The 5-kb PCR product was treated with T4 DNA polymerase and dNTP to remove any overhang A-nucleotide, and a SalI fragment carrying the hyg gene with Aspergillus nidulans trpC promoter from pCSN43 (24) was ligated to generate pPELD::hph (Fig. 1B).…”

Section: Methodsmentioning

confidence: 99%

Requirement for either a host- or pectin-induced pectate lyase for infection of Pisum sativum by Nectria hematococca

Rogers

Kim

Guo

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

127

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Fungal pathogens usually have multiple genes that encode extracellular hydrolytic enzymes that may degrade the physical barriers in their hosts during the invasion process. Nectria hematococca, a plant pathogen, has two inducible pectate lyase (PL) genes (pel) encoding PL that can help degrade the carbohydrate barrier in the host. pelA is induced by pectin, whereas pelD is induced only in planta. We show that the disruption of either the pelA or pelD genes alone causes no detectable decrease in virulence. Disruption of both pelA and pelD drastically reduces virulence. Complementation of the double disruptant with pelD gene, or supplementation of the infection droplets of the double disruptant with either purified enzyme, PLA, or PLD, caused a recovery in virulence. These results show that PL is a virulence factor. Thus, we demonstrate that disruption of all functionally redundant genes is required to demonstrate the role of host barrier-degrading enzymes in pathogenesis and that dismissal of the role of such enzymes based on the effects of single-gene disruption may be premature.

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“…4 kb) was subcloned in plasmid pGEM4 [19] and characterized by restriction mapping (see Figure 1). A smaller EcoRV DNA fragment (1.3 kb), containing the entire coding region of nuo-21, was then ligated into pCSN44 [20] for the transformation of N. crassa. The protocols for the general manipulation of N. crassa wild-type strains 74-OR23-1A and 74-OR8-1a [21], fungal transformation and the selection and analysis of transformants [22][23][24] were used as published.…”

Section: Methodsmentioning

confidence: 99%

Effects of disrupting the 21 kDa subunit of complex I from Neurospora crassa

Ferreirinha

Duarte

Melo

et al. 1999

Biochemical Journal

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We have cloned and inactivated in vivo, by repeat-induced point mutations, the nuclear gene encoding a 21 kDa subunit of complex I from Neurospora crassa. Mitochondria from the nuo21 mutant lack this specific protein but retain other subunits of complex I in approximately normal amounts. In addition, this mutant is able to assemble an almost intact enzyme. The electron transfer activities from NADH to artificial acceptors of mitochondrial membranes from nuo21 differ from those of the wild-type strain, suggesting that the absence of the 21 kDa polypeptide results in conformational changes in complex I. Nevertheless, complex I of nuo21 is able to perform NADH:ubiquinone reductase activity, as judged by the observation that the respiration of mutant mitochondria is sensitive to inhibition by rotenone. We discuss these findings in relation to the involvement of complex I in mitochondrial diseases.

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Use of a bacterial hygromycin B resistance gene as a dominant selectable marker in Neurospora crassa transformation

Abstract: (1989) "Use of a bacterial hygromycin B resistance gene as a dominant selectable marker in Neurospora crassa transformation,"

Cited by 242 publications

References 0 publications

Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining

Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining

Requirement for either a host- or pectin-induced pectate lyase for infection of Pisum sativum by Nectria hematococca

Effects of disrupting the 21 kDa subunit of complex I from Neurospora crassa

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