GENOMIC ANALYSIS OF CHLAMYDIAL INFECTION

衣原体属由四个物种组成，它们都是真核细胞和组织的专性细胞内寄生虫。 人类特异性病原体是C. trachomatis和C.肺炎。 已完成了三个代表性的C.沙眼衣原体（即血清型D、血清型L2和血清型MoPn）和C.肺炎链球菌（菌株CWL 029、菌株AR 39和菌株J138）的基因组比较，从而允许在物种内和跨物种边界进行基因组比较。 比较基因组学可能有助于确定在这些物种和血清变异体引起的不同疾病中发挥作用的特定基因产物。 这些研究中最引人注目的发现是在属内发现的高度同线性。 种和血清型之间的差异主要存在于染色体的遗传区域内，这些区域定义了“复制终止区”，并基于发现的高水平遗传重排和重组而被称为“可塑性区”。我们发起了研究，以更密切地分析这些病原体的全球基因表达模式，以了解导致基因表达受控制的潜在主题在与衣原体生长相关的发育周期中，以及基因表达的差异可能导致感染的性质和严重程度，从而引起各种疾病的后遗症。 微阵列载玻片已经产生，代表整个染色体的C。沙眼血清型D（901个ORF，包括保守的小质粒上编码的8个ORF）。 使用从完整的基因组序列设计的寡核苷酸通过PCR扩增ORF以扩增整个基因。 将ORF一式三份点在载玻片上，并用于DNA：DNA和DNA：mRNA杂交实验以确定两种基因携带（在不同的C.沙眼衣原体血清型）和基因表达（血清型D在感染过程中）。为了在发育周期过程中将沙眼衣原体血清型D与宿主细胞（即HeLa细胞）分离，我们选择从感染的细胞中纯化总RNA，并将样品破碎成细菌和宿主细胞特异性的。 上述细菌微阵列将用于分析衣原体基因表达，而纯化的宿主细胞mRNA（扩增和生物素标记）将被发送给NCI的同事，其中宿主细胞表达模式将使用市售的人微阵列芯片（即HG-U95 A，代表ca. 12，000个序列，其已经在功能或疾病关联方面进行了表征）。 这些实验将建立与C.沙眼衣原体感染。 我们计划利用这些分析为相关人类疾病模型中衣原体生长的调查提供比较基础。

The genus Chlamydia consists of four species all of which are obligate intracellular parasites of eukaryotic cells and tissues. The human-specific pathogens are members of the species C. trachomatis and C. pneumoniae. Genomic sequencing projects have been completed for three representatives of C. trachomatis (i.e. Serovar D, Serovar L2, and Serovar MoPn) and C. pneumoniae (strain CWL029, strain AR39, and strain J138) allowing for genomic comparisons to be made within and across species boundaries. Comparative genomics may help identify specific gene-products that play a role in the different diseases caused by these species and serovariants. The most striking finding of these studies is the high degree of synteny found within the genus. Differences between the species and serovars are found predominantly within genetic regions of the chromosomes that define the 'termination of replication regions' and have been termed 'plasiticity zones' based on the high level of genetic rearrangement and recombination found.We have originated studies to more closely analyze the global gene expression patterns of these pathogens to understand both the underlying themes of controlled gene expression that result in the developmental cycle associated with chlamydial growth as well the differences in gene expression that may contribute to the nature and severity of infections giving rise to the various disease sequelae. Microarray slides have been generated that represent the entire chromosome of C. trachomatis serovar D (901 ORFs, including 8 ORFs encoded on the conserved small plasmid). The ORFs were amplified by PCR using oligonucleotides designed from the completed genome sequence to amplify entire genes. The ORFs were spotted, in triplicate, on glass slides and are being used in DNA:DNA and DNA:mRNA hybridization experiments to determine both gene carriage (among the different C. trachomatis serovariants) and gene expression (of Serovar D during the course of infection).To determine the nature of the interaction of C. trachomatis serovar D with the host cell (i.e. HeLa cells) during the course of the developmental cycle, we have chosen to purify total RNA from infected cells and fractionate the samples into bacterial and host cell-specific. The aforementioned bacterial microarray will be used to analyze chlamydial gene expression while the purified host cell mRNA (amplified and biotin-labelled) will be sent to colleagues at NCI where host cell expression patterns will be determined using commercially available, human, microarray chips (i.e. HG-U95A, representing ca. 12,000 sequences that have been characterized with respect to function or disease association) manufactured by Affymetrix. These experiments will establish the patterns of gene expression associated with normal growth of C. trachomatis in transformed human cell lines. We plan to use these analyses to provide a comparative basis for the investigation of chlamydial growth in relevant models of human disease.