May G S
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
J Cell Biol. 1989 Nov;109(5):2267-74. doi: 10.1083/jcb.109.5.2267.
An internal 1.4-kb Bst EII fragment was used to disrupt the benA gene and establish heterokaryons. The heterokaryons demonstrated that the molecular disruption of benA results in a recessive benA null mutation. Conidia from a heterokaryon swell and germinate but cannot undergo nuclear division and are thus inviable. A chimeric beta-tubulin gene was constructed with the benA promoter driving the tubC structural gene. This chimeric gene construction was placed on a plasmid containing a selectable marker for Aspergillus transformation and the gene disrupting fragment of benA. Integration of this plasmid at benA by the internal gene disrupting fragment of benA simultaneously disrupts the benA gene and replaces it with the chimeric beta-tubulin gene, rescuing the benA null generated by the integration. Strains generated by this procedure contain only tubC beta-tubulin for all beta-tubulin functions. Strains having only tubC beta-tubulin are viable and exhibit no detectable microtubule dysfunction though they are more sensitive than wild-type strains to the antimicrotubule drug benomyl. It is concluded that the two beta-tubulin genes of Aspergillus nidulans, though highly divergent, are interchangeable.
一个1.4 kb的内部Bst EII片段被用于破坏benA基因并建立异核体。这些异核体表明benA的分子破坏会导致隐性benA无效突变。来自异核体的分生孢子会膨胀并萌发,但无法进行核分裂,因此无法存活。构建了一个嵌合β-微管蛋白基因,其中benA启动子驱动tubC结构基因。这个嵌合基因构建体被置于一个含有用于曲霉转化的选择标记和benA基因破坏片段的质粒上。通过benA的内部基因破坏片段将该质粒整合到benA处,同时破坏benA基因并用嵌合β-微管蛋白基因取代它,挽救了由整合产生的benA无效突变。通过该程序产生的菌株所有β-微管蛋白功能仅含有tubCβ-微管蛋白。仅含有tubCβ-微管蛋白的菌株是可存活的,并且尽管它们比野生型菌株对抗微管药物苯菌灵更敏感,但未表现出可检测到的微管功能障碍。得出的结论是,构巢曲霉的两个β-微管蛋白基因虽然高度不同,但却是可互换的。