Novel insights into the unfolded protein response using Pichia pastoris specific DNA microarrays.
Novel insights into the unfolded protein response using Pichia pastoris specific DNA microarrays.
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DOI：
10.1186/1471-2164-9-390
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发表时间：
2008-08-19
期刊：
影响因子：
4.4
通讯作者：
Mattanovich D
中科院分区：
文献类型：
--
作者：
Graf A;Gasser B;Dragosits M;Sauer M;Leparc GG;Tüchler T;Kreil DP;Mattanovich D
DNA微阵列被认为是微生物学基础研究和应用研究的重要工具。然而，对于许多工业上重要的微生物，缺乏商业上可用的微阵列仍然阻碍了生理学研究。例如，我们对毕氏酵母的蛋白质折叠和分泌的理解目前广泛依赖于与酿酒酵母的类比得出的结论。为了弥补这一差距，我们开发了酵母的全基因组DNA微阵列，并分析了该酵母物种的未折叠蛋白响应（UPR），与酿酒酵母相比。通过将部分注释的巴斯德酵母基因列表与de novo基因发现相结合，生成了假定开放阅读框列表，并使用探针设计工具TherMODO（基于热力学模型的寡核苷酸设计优化器）设计了寡核苷酸探针集。为了评估新型阵列设计的性能，将携带寡核苷酸集的微阵列与双硫苏糖醇（DTT）处理或过表达UPR转录因子HAC1的菌株进行杂交，并与正常培养基中的野生型菌株进行比较。比较了DTT处理对酿酒酵母的文献数据，发现了两种酵母的相似之处，但也有重要的差异。作为UPR基因最直接的控制基因，HAC1的过表达使人们对P. pastoris和酵母中这一重要调控途径有了重要的新认识。观察到的巴氏酵母和酿酒酵母之间的差异强调了DNA微阵列对工业生产菌株的重要性。P. pastoris对DTT的反应主要是通过调控化学刺激、电子传递和呼吸相关基因，而HAC1的过表达诱导了许多涉及翻译、核糖体生物发生和细胞器生物合成的基因，说明DTT处理触发的调控事件与HAC1过表达的反应仅部分重叠。用两个不同的寡集获得的结果的高再现性很好地表明了它们的鲁棒性，并强调了不太严格选择调节特征的重要性，以避免大量假阴性结果。
DNA Microarrays are regarded as a valuable tool for basic and applied research in microbiology. However, for many industrially important microorganisms the lack of commercially available microarrays still hampers physiological research. Exemplarily, our understanding of protein folding and secretion in the yeast Pichia pastoris is presently widely dependent on conclusions drawn from analogies to Saccharomyces cerevisiae. To close this gap for a yeast species employed for its high capacity to produce heterologous proteins, we developed full genome DNA microarrays for P. pastoris and analyzed the unfolded protein response (UPR) in this yeast species, as compared to S. cerevisiae. By combining the partially annotated gene list of P. pastoris with de novo gene finding a list of putative open reading frames was generated for which an oligonucleotide probe set was designed using the probe design tool TherMODO (a thermodynamic model-based oligoset design optimizer). To evaluate the performance of the novel array design, microarrays carrying the oligo set were hybridized with samples from treatments with dithiothreitol (DTT) or a strain overexpressing the UPR transcription factor HAC1, both compared with a wild type strain in normal medium as untreated control. DTT treatment was compared with literature data for S. cerevisiae, and revealed similarities, but also important differences between the two yeast species. Overexpression of HAC1, the most direct control for UPR genes, resulted in significant new understanding of this important regulatory pathway in P. pastoris, and generally in yeasts. The differences observed between P. pastoris and S. cerevisiae underline the importance of DNA microarrays for industrial production strains. P. pastoris reacts to DTT treatment mainly by the regulation of genes related to chemical stimulus, electron transport and respiration, while the overexpression of HAC1 induced many genes involved in translation, ribosome biogenesis, and organelle biosynthesis, indicating that the regulatory events triggered by DTT treatment only partially overlap with the reactions to overexpression of HAC1. The high reproducibility of the results achieved with two different oligo sets is a good indication for their robustness, and underlines the importance of less stringent selection of regulated features, in order to avoid a large number of false negative results.
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影响因子：
4.4
作者：
Gasser B;Maurer M;Rautio J;Sauer M;Bhattacharyya A;Saloheimo M;Penttilä M;Mattanovich D
通讯作者：
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影响因子：
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作者：
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影响因子：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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