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1.
PLoS Genet ; 19(10): e1010985, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37844074

RESUMO

UPF-1-UPF-2-UPF-3 complex-orchestrated nonsense-mediated mRNA decay (NMD) is a well-characterized eukaryotic cellular surveillance mechanism that not only degrades aberrant transcripts to protect the integrity of the transcriptome but also eliminates normal transcripts to facilitate appropriate cellular responses to physiological and environmental changes. Here, we describe the multifaceted regulatory roles of the Neurospora crassa UPF complex in catalase-3 (cat-3) gene expression, which is essential for scavenging H2O2-induced oxidative stress. First, losing UPF proteins markedly slowed down the decay rate of cat-3 mRNA. Second, UPF proteins indirectly attenuated the transcriptional activity of cat-3 gene by boosting the decay of cpc-1 and ngf-1 mRNAs, which encode a well-studied transcription factor and a histone acetyltransferase, respectively. Further study showed that under oxidative stress condition, UPF proteins were degraded, followed by increased CPC-1 and NGF-1 activity, finally activating cat-3 expression to resist oxidative stress. Together, our data illustrate a sophisticated regulatory network of the cat-3 gene mediated by the UPF complex under physiological and H2O2-induced oxidative stress conditions.


Assuntos
Peróxido de Hidrogênio , Neurospora , Peróxido de Hidrogênio/farmacologia , Catalase/genética , Degradação do RNAm Mediada por Códon sem Sentido , Estresse Oxidativo/genética
2.
Curr Genet ; 66(4): 835-847, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32152733

RESUMO

Neurospora crassa is an excellent model fungus for studies on molecular genetics, biochemistry, physiology, and molecular cell biology. Along with the rapid progress of Neurospora research, new tools facilitating more efficient and accurate genetic analysis are in high demand. Here, we tested whether the dominant selective makers widely used in yeasts are applicable in N. crassa. Among them, we found that the strains of N. crassa are sensitive to the aminoglycoside antibiotics, G418 and nourseothricin. 1000 µg/mL of G418 or 50 µg/mL of nourseothricin is sufficient to inhibit Neurospora growth completely. When the neomycin phosphotransferase gene (neo) used in mammalian cells is expressed, N. crassa shows potent resistance to G418. This establishes G418-resistant marker as a dominant selectable marker to use in N. crassa. Similarly, when the nourseothricin acetyltransferase gene (nat) from Streptomyces noursei is induced by qa-2 promoter in the presence of quinic acid (QA), N. crassa shows potent resistance to nourseothricin. When nat is constitutively expressed by full-length or truncated versions of the promoter from the N. crassa cfp gene (NCU02193), or by the trpC promoter of Aspergillus nidulans, the growth of N. crassa in the presence of nourseothricin is proportional to the expression levels of Nat. Finally, these two markers are used to knock-out wc-2 or al-1 gene from the N. crassa genome. The successful development of these two markers in this study expands the toolbox for N. crassa and very likely for other filamentous fungi as well.


Assuntos
Farmacorresistência Fúngica/genética , Marcadores Genéticos , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/genética , Acetiltransferases/genética , Antibacterianos/farmacologia , Elementos de DNA Transponíveis , Farmacorresistência Fúngica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Genes Dominantes , Gentamicinas/farmacologia , Canamicina Quinase/genética , Microrganismos Geneticamente Modificados , Regiões Promotoras Genéticas , Ácido Quínico/farmacologia , Estreptotricinas/farmacologia
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