MOLECULAR GENETIC ANALYSIS OF PNEUMOCYSTIS

我们的长期目标是了解并最终控制致病性 人类肺孢子虫感染。我们在此提出的方法是 使用克隆的pc DNA片段来表征基因结构， 组织，并分离DNA探针，以探索 大鼠肺孢子虫与人的关系。 这种分析将是使用脉冲场进行染色体作图 电泳我们根据两个标准选择克隆目标， 可及性，以及与长期目标的相关性。首先，我们将 追求基因，由于它们在真核生物中的保守性， 最容易接近。编码核糖体RNA的基因和 钙调素基因将是我们最初克隆的主要目标。这些 初步研究将确定pc的基本分子遗传方案， 提供启动分子遗传表征所需的工具 在实验室老鼠和病人身上的病原体。一致 在这些实验中，我们将分离出一组染色体特异性探针。 这样的实验也应该产生分散的重复DNA元件，如果 都存在于PC基因组中。rRNA基因克隆，染色体 标记和重复元件将用作杂交探针， 检测大鼠和人类PC分离株之间的基因组变异 hosts.最后，我们将寻找我们预期会更多的基因， 很难克隆，但与化疗问题最相关 肺孢子虫感染这些将包括编码阳离子的基因 ATP酶、鸟氨酸脱羧酶和二氢叶酸易位 后两种基因将用作与DNA还原酶的杂交探针。 这是为了探索遗传变异作为一种反应的可能作用， 药物暴露

Our long term goal is to understand ultimately control the pathogenicity of pneumocystis infections in humans. The approach we propose here is to use cloned pc DNA fragments to characterize gene structure and organization, and to isolate DNA probes with which to explore the relationship between pneumocystis of rats and man. A principal tool in such analysis will be chromosomal mapping using pulsed field electrophoresis. We have selected cloning targets based on two criteria, accessibility, and relevance to the long term goal. Initially, we will pursue genes which by virtue of their conservation among eucaryotes, are most readily accessible. The genes encoding ribosomal RNAs and the calmodulin gene will be our primary initial cloning targets. These initial studies will define the basic molecular genetic scheme in pc, and provide the tools needed to initiate molecular genetic characterization of the pathogen both in laboratory rats and in patients. In concert with these experiments, we will isolate a panel of chromosome-specific probes. Such experiments should also yield dispersed repetitive DNA elements, if any are present in the pc genome. Clones of rRNA genes, chromosome markers and repetitive elements will be used as hybridization probes to examine genomic variation among isolates of pc form both rat and human hosts. Finally, we will pursue genes that we anticipate will be more difficult to clone, but are most pertinent to the problem of chemotherapy of pneumocystis infections. These will include genes encoding cation translocation ATPAses, ornithine decarboxylase, and dihydrofolate reductase, The latter two genes will be used as hybridzation probes with which to explore the possible role of genetic variation as a response to drug exposure.