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Abstract. We have characterized Neurospora crassa transformants obtained with plasmid pJR2, which consists of the Neurospora glutamate dehydrogenase (am) gene cloned in pUC8 and an am132 host strain which contains a …
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formed with pJR2–41U-Png1-His 6 and pREP1–3FLAG-Mst1 and cultured in the EMM medium without uracil and leucine overnight. 100 A 600 cells were harvested and washed twicewithcoldphosphate-bufferedsaline,treatedwithZymol-yase (Sigma) for 30 min, and then lysed in TPER lysis buffer (Pierce) (23). After analyzing the expression of Png1-His 6 and
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pJR2-3XL-asp1C879S were generated according to the same protocol. In brief, PCR fragments were generated by directed mutagenesis using a QuikChange II site-directed mutagen-esis kit (Agilent Technologies) and cloned into pJR2-3XL [26] via homologous recombination in S. cerevisiae25 []. The quadruple mutant pJR2-3XL-asp1365−920, …
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