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Study of Pneumocystis carinii : from Molecular Microbiology to Drug Discovery

卡氏肺孢子虫研究：从分子微生物学到药物发现

基本信息

批准号：
07557027
负责人：
NAKAMURA Yoshikazu
金额：
$ 13.44万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

The major cell surface glycoprotein (MSG) of Pneumocystis carinii plays a crucial role in the host-parasite interaction. We have discovered that genes encoding MSGs are repeated, highly polymorphic, distributed among all of the 14-15 chromosomes, and are expressed from a unique expression site termed UCS,leading to antigenic variation to evade the host immune system. We have shown further that a UCS site is telomeric and that antigenic variation may be generated by site-specific or homologous recombination between the UCS site and multiple MSG repertoires. Telomeric fragments other than the UCS were cloned by screening with the telomere sequence. Non-UCS telomeric clones that were sequenced also contained MSG gene (s) regardless of having been screened with non-MSG probes. Frequent occurrences of these telomeric MSG clones indicate that they represent silent MSG repertoires localized in the telomere regions. The UCS contained the two transcription start sites. The promoter activity of UCS was examined using a UCS-lacZ (beta-galactosidase gene) fusion in the budding yeast Saccharomyces cerevisiae and the fission yeast Shizosaccharomyces pombe because P.carinii is phylogenically close to yeast. UCS allowed beta-galactosidase synthesis in S.pombe but not in S.cerevisiae. The transcript start sites determined by 5'RACE analysis are located slightly upstream of the two authentic start sites in P.carinii. The promoter activity of UCS itself is about one fifth of that of a widely used promoter of S.pombe, nmtl. Three other putative promoter sequences of P.carinii also expressed a reporter gene in S.pombe. These findings indicate that Shizosaccharomyces pombe is useful as a heterologous expression host for native Pneumocystis carinii genes.

卡氏肺囊虫的主要细胞表面糖蛋白（MSG）在宿主与寄生虫的相互作用中发挥着至关重要的作用。我们发现编码MSG的基因是重复的、高度多态性的、分布在所有14-15条染色体上，并且从称为UCS的独特表达位点表达，导致抗原变异以逃避宿主免疫系统。我们进一步表明，UCS 位点是端粒，并且抗原变异可以通过 UCS 位点和多个 MSG 库之间的位点特异性或同源重组产生。通过用端粒序列筛选来克隆除UCS之外的端粒片段。测序的非 UCS 端粒克隆也含有 MSG 基因，无论是否用非 MSG 探针进行筛选。这些端粒 MSG 克隆的频繁出现表明它们代表了位于端粒区域的沉默 MSG 库。 UCS 包含两个转录起始位点。使用 UCS-lacZ（β-半乳糖苷酶基因）融合体在芽殖酵母酿酒酵母和裂殖酵母裂殖酵母中检查 UCS 的启动子活性，因为 P.carinii 在系统发育上与酵母接近。 UCS 允许在粟酒裂殖酵母中合成 β-半乳糖苷酶，但不能在酿酒酵母中合成。通过 5'RACE 分析确定的转录起始位点位于 P.carinii 中两个真实起始位点的稍上游。 UCS本身的启动子活性约为广泛使用的粟酒裂殖酵母启动子nmtl的五分之一。 P.carinii 的其他三个推定启动子序列也在粟酒裂殖酵母中表达报告基因。这些发现表明裂殖酵母可用作天然卡氏肺囊虫基因的异源表达宿主。