Multi-User Cyanobacterial Resource (MCR)

聚球藻。PCC6803基因组序列将被用于开发DNA微阵列，从而使得在这个重要的模式系统中能够使用有效的技术来监测差异转录的全球模式。该项目将生产和利用一个代表聚球藻全部3168个基因的聚合酶链式反应生成的基因组。PCC6803基因组是一种单细胞、可转化的蓝藻，是研究光合作用和环境基因调控的重要模式生物。扩增的基因集将被排列在显微镜载玻片上，与从mRNA产生的荧光标记的cDNA进行探测，目的是分析差异转录活动的全球模式。由于聚合酶链式反应扩增寡核苷酸结合了一对与基因特异序列相连的共同适配序列，因此聚合酶链式反应产生的基因集将具有超出差异转录图谱主要应用的用途。因此，聚合酶链式反应产生的基因片段的有序文库的两侧将是适配子序列。这些适配子的存在将具有两个重要功能。首先，它将允许从常见的聚合酶链式反应引物中重新扩增主基因片段集，从而最大限度地提高可打印产品的产量，并最大限度地减少对原始基因特异性聚合酶链式反应引物的消耗。这将为这些阵列提供充足的DNA，并允许进行实验，例如，对DNA杂交微阵列使用不同的偶联化学。其次，这种方法允许框架内定向克隆扩增的基因，从而允许未来将这些基于核酸的程序与高通量蛋白质分析联系起来。由此产生的用于全球转录图谱的DNA微阵列将为研究人员提供一个强大的新工具，以解决许多不同生物学研究领域的基本问题。

The Synechocystis sp. PCC6803 genomic sequence will be used to develop DNA microarrays, thus enabling efficient techniques to monitor global patterns of differential transcription in this important model system. The project will produce and utilize a PCR-generated gene set representing the entire 3168 genes of the Synechocystis sp. PCC6803 genome, a unicellular, transformable cyanobacterium that is an important model organism for the study of photosynthesis and environmental gene regulation. The amplified gene set will be arrayed on microscope slides to be probed with fluorescently-labeled cDNA produced from mRNA for the purpose of analyzing global patterns of differential transcriptional activity. The PCR-generated gene set will have utility beyond the primary application of differential transcriptional profiling, because the PCR amplification oligonucleotides incorporate a pair of common adaptamer sequences connected to the gene-specific sequences. The ordered library of PCR-generated gene fragments will thus be flanked by adaptamer sequences. The presence of these adaptamers will have two important functions. First, it will allow re-amplification of 'master-sets' of the primary gene fragment sets from common PCR primers, thereby maximizing yields of printable product and minimizing the depletion of the original stock of gene-specific PCR primers. This will provide ample DNA for these arrays and allow experimentation, for example, with different coupling chemistries for the DNA hybridization microarrays. Second, this approach allows in-frame directional cloning of the amplified genes, thus permitting the future connection of these nucleic acid-based procedures with high-throughput protein analysis. The resulting availability of DNA microarrays for global transcriptional profiling will provide researchers with a powerful new tool to address fundamental problems in many different areas of biological research.