ESTABLISHMENT OF HIGH-THROUGHPUT SCREENING FOR IMMUNOSUPPRESSANTS USING YEAST MODEL SYSTEM

需要钙调磷酸酶活性生长的裂殖酵母突变体的分离及其在免疫抑制剂高通量筛选中的应用。当钙调磷酸酶基因被破坏或FK506（他氯莫司）加入到培养基中，野生型裂殖酵母细胞是活的。我们诱变野生型裂殖酵母细胞，发现几个免疫抑制剂敏感的突变体。这表明，这些突变体是合成致命的钙调神经磷酸酶中断和突变的基因是共享一个基本功能的钙调神经磷酸酶。一些免疫抑制剂敏感突变体也表现出对高温的敏感性，表明这些突变基因是必需的，并命名为免疫抑制剂和温度敏感突变体（其突变体）。我们分离了8个突变体并鉴定了突变基因。所有这些突变体都需要钙调磷酸酶活性来维持其生存能力。筛选程序使用野生型及其几种突变株。如果候选药物抑制其突变体的生长而不是野生型细胞的生长，则表明该药物特异性抑制钙调磷酸酶活性，并且应该是免疫抑制剂的良好候选物。我们还开发了一种新的筛选系统，用于MAP激酶途径的抑制剂，使用钙调磷酸酶破坏的裂殖酵母菌株。这些筛选系统可随时用于学术和工业用途。

Isolation of fission yeast mutants that requires calcineurin phosphatse activity for their growth and their application for a high-throughput screening of immunosuppressants. When calcineurin gene was disrupted or FK506 (taclolimus) was added to the media, wild-type fission yeast cells were viable. We mutagenized wild-type fission yeast cells and found several immunosuppressant-sensitive mutants. It is indicated that these mutants are synthetically lethal with calcineurin disruption and the mutated genes are sharing an essential function with calcineurin. Several immunosuppressant-sensitive mutants also showed sensitivity to high temperature, suggesting those mutated genes are essential, and named immunosuppressant- and temperature-sensitive mutants (its mutants). We isolated 8 its mutants and identified mutated genes. All of these mutants required calcineurin activity for their viability. The screening procedure uses wild-type and several its mutant strains. If candidate drug inhibits the growth of its mutants but not of wild-type cells, it is indicated that the drug specifically inhibits calcineurin activity and should be a good candidate for a immunosuppressant. We also developed a novel screening system for an inhibitor of MAP kinase pathway using calcineurin disrupted fission yeast strain. These screening systems are readily available for both academic and industrial use.