Brettin Thomas, Altherr Michael R, Du Ying, Mason Roxie M, Friedrich Alexandra, Potter Laura, Langford Chris, Keller Thomas J, Jens Jason, Howie Heather, Weyand Nathan J, Clary Susan, Prichard Kimberly, Wachocki Susi, Sodergren Erica, Dillard Joseph P, Weinstock George, So Magdalene, Arvidson Cindy Grove
Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
BMC Microbiol. 2005 Sep 1;5:50. doi: 10.1186/1471-2180-5-50.
The sexually transmitted disease, gonorrhea, is a serious health problem in developed as well as in developing countries, for which treatment continues to be a challenge. The recent completion of the genome sequence of the causative agent, Neisseria gonorrhoeae, opens up an entirely new set of approaches for studying this organism and the diseases it causes. Here, we describe the initial phases of the construction of an expression-capable clone set representing the protein-coding ORFs of the gonococcal genome using a recombination-based cloning system.
The clone set thus far includes 1672 of the 2250 predicted ORFs of the N. gonorrhoeae genome, of which 1393 (83%) are sequence-validated. Included in this set are 48 of the 61 ORFs of the gonococcal genetic island of strain MS11, not present in the sequenced genome of strain FA1090. L-arabinose-inducible glutathione-S-transferase (GST)-fusions were constructed from random clones and each was shown to express a fusion protein of the predicted size following induction, demonstrating the use of the recombination cloning system. PCR amplicons of each ORF used in the cloning reactions were spotted onto glass slides to produce DNA microarrays representing 2035 genes of the gonococcal genome. Pilot experiments indicate that these arrays are suitable for the analysis of global gene expression in gonococci.
This archived set of Gateway entry clones will facilitate high-throughput genomic and proteomic studies of gonococcal genes using a variety of expression and analysis systems. In addition, the DNA arrays produced will allow us to generate gene expression profiles of gonococci grown in a wide variety of conditions. Together, the resources produced in this work will facilitate experiments to dissect the molecular mechanisms of gonococcal pathogenesis on a global scale, and ultimately lead to the determination of the functions of unknown genes in the genome.
性传播疾病淋病，在发达国家和发展中国家都是一个严重的健康问题，其治疗仍然是一项挑战。致病因子淋病奈瑟菌基因组序列的近期完成，为研究该生物体及其引起的疾病开辟了一整套全新的方法。在此，我们描述了使用基于重组的克隆系统构建一个能够表达的克隆集的初始阶段，该克隆集代表淋病奈瑟菌基因组的蛋白质编码开放阅读框（ORF）。
到目前为止，该克隆集包括淋病奈瑟菌基因组2250个预测ORF中的1672个，其中1393个（83%）经序列验证。该集合中包括菌株MS11淋病奈瑟菌遗传岛61个ORF中的48个，这些在菌株FA1090的测序基因组中不存在。从随机克隆构建了L -阿拉伯糖诱导型谷胱甘肽 - S -转移酶（GST）融合体，并且每个在诱导后都显示表达预测大小的融合蛋白，证明了重组克隆系统的用途。将克隆反应中使用的每个ORF的PCR扩增产物点样到载玻片上，以产生代表淋病奈瑟菌基因组2035个基因的DNA微阵列。初步实验表明这些阵列适用于分析淋病奈瑟菌中的全局基因表达。
这个存档的Gateway入门克隆集将有助于使用各种表达和分析系统对淋病奈瑟菌基因进行高通量基因组和蛋白质组学研究。此外，所产生的DNA阵列将使我们能够生成在各种条件下生长的淋病奈瑟菌的基因表达谱。总之，这项工作中产生的资源将有助于在全球范围内剖析淋病奈瑟菌致病机制的分子机制的实验，并最终导致确定基因组中未知基因的功能。
