Study of Pneumocystis carinii : from Molecular Microbiology to Drug Discovery

卡氏肺囊虫的主要细胞表面糖蛋白（MSG）在宿主-寄生虫相互作用中起着至关重要的作用。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条染色体中，并从称为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。我们进一步证明了UCS位点是端粒性的，并且抗原变异可能是由UCS位点和多个味精库之间的位点特异性或同源重组产生的。利用端粒序列筛选，克隆出了UCS以外的端粒片段。无论是否用非味精探针筛选，对非ucs端粒克隆进行测序也含有味精基因。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的MSG谱。UCS包含两个转录起始位点。利用UCS- lacz （β -半乳糖苷酶基因）基因融合在出芽酵母酿酒酵母和裂变酵母Shizosaccharomyces pombe中检测UCS启动子活性，因为P.carinii在系统发育上与酵母接近。UCS允许S.pombe合成β -半乳糖苷酶，而s.c reevisiae不允许。5'RACE分析确定的转录本起始位点位于P.carinii两个真实起始位点的上游。UCS本身的启动子活性约为S.pombe， nmtl广泛使用的启动子活性的五分之一。另外三个被推测为carinii的启动子序列也在S.pombe中表达了一个报告基因。这些结果表明，pombe Shizosaccharomyces pombe可以作为本地卡氏肺囊虫基因的异源表达宿主。

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.