GENOMIC ANALYSIS OF CHLAMYDIAL INFECTION

衣原体属由四个种组成，它们都是真核细胞和组织的专性细胞内寄生虫。人类特有的病原体是沙眼衣原体和肺炎衣原体的成员。沙眼衣原体的三个代表(即D、L2和MoPn)和肺炎衣原体(CWL029株、AR39株和J138株)的基因组测序项目已经完成，从而可以在物种内部和跨物种进行基因组比较。比较基因组学可能有助于确定特定的基因产物，这些基因产物在由这些物种和血清变异引起的不同疾病中发挥作用。这些研究中最引人注目的发现是在该属内发现了高度的共性。物种和血清型之间的差异主要是在染色体的遗传区内发现的，这些遗传区定义了复制区域的终止，并基于发现的高水平的基因重排和重组而被称为可塑性区域。我们发起了更密切地分析这些病原体的全球基因表达模式的研究，以了解导致衣原体生长相关发育周期的受控基因表达的潜在主题，以及可能导致引起各种疾病后遗症的感染的性质和严重程度的基因表达差异。已经制作了代表沙眼衣原体D血清型(901个ORFs，包括8个编码在保守的小质粒上的ORFs)整个染色体的微阵列幻灯片。使用从完整基因组序列设计的寡核苷酸，通过聚合酶链式反应扩增ORF，以扩增整个基因。ORF以三份一份的形式出现在玻片上，并用于DNA：DNA和DNA：mRNA杂交实验，以确定(不同沙眼衣原体变种之间的)基因携带和(沙眼衣原体D血清型在感染过程中的)基因表达。为了确定D血清型沙眼衣原体在发育周期中与宿主细胞(即HeLa细胞)相互作用的性质，我们选择从感染细胞中提取总RNA，并将样本分成细菌和宿主细胞特异性的样本。前述细菌微阵列将用于分析衣原体基因的表达，而纯化的宿主细胞mRNA(扩增和生物素标记的)将被送往NCI的同事，在那里宿主细胞表达模式将使用Affymetrix制造的商用人类微阵列芯片(即HG-U95A，代表大约12,000个已经根据功能或疾病相关性进行表征的序列)来确定。这些实验将建立与沙眼衣原体在转化的人类细胞系中正常生长相关的基因表达模式。我们计划使用这些分析为研究相关人类疾病模型中的衣原体生长提供一个比较基础。

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.