BIOCHEMICAL AND GENETIC MECHANISMS OF OBLIGATE INTRACELLULAR PARASITISM

专性细胞内寄生的生化和遗传机制

• 批准号: 3818129
• 负责人: J C WILLIAMS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3818129
• 关键词: Escherichia coli; Q fever; aspartate carbamoyltransferase; bacterial DNA; bacterial genetics; bacterial proteins; cold temperature; communicable diseases; gene expression; genetic library; genetic manipulation; genetic promoter element; genetic strain; heat; histochemistry /cytochemistry; host organism interaction; intracellular parasitism; microorganism culture; molecular cloning; monoclonal antibody; nucleic acid sequence; plasmids; surface antigens; virulence

The objectives of this project were the characterization of two recently cloned genes and the expression of a previously cloned gene under the control of a heat shock promoter (hsp) from Coxiella burnetii. A. Gene cloning. Genomic DNA potentially encoding aspartate transcarbamoylase (ATCase, pyrB) was cloned into the plasmid pEMBL8+. A 7 kb fragment was selected for its ability to complement a pyrB-E. coli auxotroph. The putative C. burnetii pyrB gene was shown to express an active ATCase using cell-free extracts of the recombinant E. coli. DNA sequence analysis of a 2.2 kb subclone revealed a product of approximate size and composition in common with the pyrB gene of E. coli. B. Surface antigen. The product of a 2.5 kb fragment from the same EcoR1 genomic library was selected by monoclonal antibodies raised against a 29.5 kd immunodominant surface protein. DNA sequence analysis revealed an open reading frame encoding a 30 kd lipoprotein. The amino acid composition of the purified 29.5 kd surface protein conflicts with the deduced amino acid composition of the cloned lipoprotein gene product detected by monoclonal antibodies. C. HSP. A recently cloned operon, htpAB, under the control of an hsp from C. burnetii expressed 62 and 14 kd gene products at 42 degrees C in the recombinant E. coli. A similar study in C. burnetii indicated that both of these and at least 5 more genes were maximally expressed at 42 degrees C. In addition a cold-shock gene product was only expressed at 23 degrees C. D. Significance. The C. burnetii pyrB gene resembles structurally the gene of E. coli, but appears to be regulated differently. An immunodominant 29.5 kd surface protein may share a common epitope with other similarly sized lipoproteins. The induction of gene expression during stress responses under the control of hsp may contribute to the survival of C. burnetii in the phagolysosome.

该项目的目标是两个 最近克隆的基因和先前克隆的表达 在Coxiella的热激启动子（HSP）控制下的基因 Burnetii。 A.基因克隆。 基因组DNA可能编码 天冬氨酸转育霉菌（Atcase，pyrb）被克隆到 质粒Pembl8+。 选择了一个7 kb的片段 补充pyrb-e。大肠菌营养。 推定的C. burnetii pyrb 基因显示使用无细胞提取物表达活跃的ATCASE 重组大肠杆菌 2.2 kb的DNA序列分析 亚克隆揭示了大小和组成的产物 常见于大肠杆菌的pyrb基因。 B.表面抗原。 这 来自同一ecor1基因组库的2.5 kb片段的产物 通过针对29.5 kD饲养的单克隆抗体选择 免疫主导表面蛋白。 DNA序列分析显示 编码30 KD脂蛋白的开放阅读框。 氨基酸 纯化的29.5 kd表面蛋白与与 克隆的脂蛋白基因的推导氨基酸组成 单克隆抗体检测到的产品。 C. HSP。 最近 克隆操纵子HTPAB在C. burnetii的HSP控制下 在42度C中表达62和14 KD基因产物。 重组大肠杆菌。 在C. burnetii中进行的类似研究表明 这些和至少5个基因均以最大表达 在42度C下。此外，冷冲基因产物仅是 在23度C. D.显着性下表达。 C. burnetii pyrb 基因在结构上类似于大肠杆菌的基因，但似乎是 受不同的监管。 29.5 kd表面蛋白的免疫降低 可能会与其他大小相似的脂蛋白共享一个共同的表位。 在应力反应中的基因表达诱导 HSP的控制可能有助于burnetii在 吞噬体。