Tsukagoshi N
Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-0814, Japan.
Nihon Ishinkin Gakkai Zasshi. 1998;39(2):85-90. doi: 10.3314/jjmm.39.85.
The Aspergillus oryzae Taka-amylase A (TAA) gene has been used as a model gene to characterize the regulatory mechanisms of gene expression in Aspergilli. TAA gene contained a typical eukaryotic promoter with a TATA box and putative regulatory elements such as a CCAAT sequence in its 5'-noncoding region. A nuclear protein designated AnCP bound to the CCAAT sequence. Replacement of the CCAAT sequence with CGTAA was found to abolish the binding of AnCP and to have an inhibitory effect on taa promoter activity. Although AnCP was not purified to homogeneity, AnCP appeared to have an apparent molecular mass of approximately 120 kDa. In Saccharomyces cerevisiae, the CCAAT-binding HAP complex is a heteromeric protein comprising at least four subunits (yHAP2, yHAP3, yHAP4 and yHAP5) and is involved in the regulation of genes responsible for oxidative phosphorylation. A gene designated hapC with significant homology to yHAP3 has been isolated from A. nidulans by Hynes et al. We expressed hapC gene as a fusion protein with MalE in E. coli, purified HapC protein and prepared anti-HapC antiserum. The MalE-HapC fusion protein was able to substitute for the authentic HapC in AnCP. Furthermore, addition of the anti-HapC antiserum to the DNA binding reaction mixture retarded the mobility of the shifted band. These indicate clearly that HapC protein acts as a subunit of AnCP and that AnCP is a counterpart of the yeast Hap complex.
米曲霉Taka淀粉酶A(TAA)基因已被用作模型基因来表征曲霉属中基因表达的调控机制。TAA基因在其5'非编码区包含一个带有TATA盒的典型真核启动子以及推定的调控元件,如CCAAT序列。一种名为AnCP的核蛋白与CCAAT序列结合。发现用CGTAA取代CCAAT序列会消除AnCP的结合,并对taa启动子活性产生抑制作用。尽管AnCP未被纯化至同质,但AnCP的表观分子量似乎约为120 kDa。在酿酒酵母中,CCAAT结合HAP复合物是一种异源蛋白,至少由四个亚基(yHAP2、yHAP3、yHAP4和yHAP5)组成,并参与负责氧化磷酸化的基因的调控。Hynes等人从构巢曲霉中分离出了一个与yHAP3具有显著同源性的基因hapC。我们在大肠杆菌中将hapC基因表达为与MalE的融合蛋白,纯化了HapC蛋白并制备了抗HapC抗血清。MalE-HapC融合蛋白能够替代AnCP中的真实HapC。此外,向DNA结合反应混合物中加入抗HapC抗血清会使迁移带的迁移率减慢。这些清楚地表明HapC蛋白作为AnCP的一个亚基起作用,并且AnCP是酵母Hap复合物的对应物。