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/farmacologiaRESUMO
UBR5 is a nuclear phosphoprotein of obscure functions. Clinical analyses reveal that UBR5 amplifications and overexpression occur in over 20% cases of human breast cancers. Breast cancer patients carrying UBR5 genetic lesions with overexpression have significantly reduced survival. Experimental work in vitro and in vivo demonstrates that UBR5, functioning as an oncoprotein, plays a profound role in breast cancer growth and metastasis. UBR5 drives tumor growth largely through paracrine interactions with the immune system, particularly through inhibiting the cytotoxic response mediated by CD8+ T lymphocytes, whereas it facilitates metastasis in a tumor cell-autonomous manner via its transcriptional control of key regulators of the epithelial-mesenchymal transition, ID1 and ID3. Furthermore, simultaneous targeting of UBR5 and PD-L1 yields strong therapeutic benefit to tumor-bearing hosts. This work significantly expands our scarce understanding of the pathophysiology and immunobiology of a fundamentally important molecule and has strong implications for the development of novel immunotherapy to treat highly aggressive breast cancers that resist conventional treatment.