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1. 发明授权

US5527687A Enzyme induction screen for ergosterol biosynthesis inhibitors 失效
标题翻译：麦角甾醇生物合成抑制剂的酶诱导筛选
公开(公告)号：US5527687A
公开(公告)日：1996-06-18
申请号：US68986
申请日：1993-05-25
申请人： Donald R. Kirsch , Margaret H. K. Lai
发明人： Donald R. Kirsch , Margaret H. K. Lai
IPC分类号： C12Q1/18 , C12Q1/26 , C12Q1/34 , C12Q1/54 , C12Q1/48
CPC分类号： C12Q1/34 , C12Q1/18 , C12Q1/26 , C12Q2334/52 , G01N2333/395 , G01N2333/924 , Y10S435/942
摘要： A method for screening for sterol biosynthesis inhibitors of potential use as fungicides or antihypercholesterolemic agents identifies agents by the induction of lanosterol 14-.alpha.-demethylase, an enzyme in the biosynthetic pathway of ergosterol and cholesterol, in cultures containing the agents. In one screening test, test samples are incubated in a culture of a Saccharomyces cerevisiae strain sensitive to ergosterol biosynthesis and containing a gene fusion of a lanosterol 14-.alpha.-demethylase clone with a gene for bacterial .beta.-galactosidase. After incubation of the culture, an increase in lancsterol 14-.alpha.-demethylase activity is determined indirectly by measuring .beta.-galactosidase activity. The culture media contains a chromogenic substrate of .beta.-galactosidase such as orthonitrophenyl-.beta.-D-galactoside or 5-bromo-4-chloro-3-indoyl-.beta.-D-galactoside, so that active samples are identified by the production of colored product. For comparison purposes, screening tests may employ a lanosterol 14-.alpha.-demethylase inhibitor such as dinaconazole as a positive control.
摘要翻译：用于筛选潜在用作杀真菌剂或抗高胆固醇血症药的甾醇生物合成抑制剂的方法通过诱导羊毛甾醇14-α-甲基化酶（在麦角甾醇和胆固醇的生物合成途径中的酶）在含有该试剂的培养物中鉴定药剂。在一次筛选试验中，将测试样品在对麦角甾醇生物合成敏感的酿酒酵母菌株的培养物中培养，并含有羊毛甾醇14-α-甲基化酶克隆与细菌β-半乳糖苷酶基因的基因融合物。培养物培养后，通过测量β-半乳糖苷酶活性间接测定兰曲星14-α-甲基化酶活性的增加。培养基含有β-半乳糖苷酶如邻硝基苯基-β-D-半乳糖苷或5-溴-4-氯-3-吲哚基-β-D-半乳糖苷的显色底物，使活性样品通过生产有色产品。为了比较目的，筛选试验可以使用羊毛甾醇14-α-甲基化酶抑制剂如依替康唑作为阳性对照。

2. 发明授权

US5512472A DNA sequence encoding sterol .DELTA.14 reductase 失效
标题翻译：编码固醇DELTA14还原酶的DNA序列
公开(公告)号：US5512472A
公开(公告)日：1996-04-30
申请号：US439131
申请日：1995-05-11
申请人： Margaret H. K. Lai , Donald R. Kirsch , Martin Bard
发明人： Margaret H. K. Lai , Donald R. Kirsch , Martin Bard
IPC分类号： C12N9/02 , C12N15/53 , C12N15/09 , C12N15/81
CPC分类号： C12N9/001
摘要： A gene encoding Saccharomyces cerevisiae sterol .DELTA.14 reductase of the ergosterol biosynthetic pathway is isolated and cloned by selecting strains carrying sequences on a 2.mu. based vector for resistance to a morpholine fungicide such as fenpropimorph. Four distinct plasmid inserts which produce morpholine resistance are obtained, and one of these is characterized and sequenced. The purified and isolated DNA sequence encoding sterol .DELTA.14 reductase encodes a polypeptide exhibiting homology to the S. cerevisiae sterol C-24(28) reductase enzyme in the ergosterol biosynthetic pathway.
摘要翻译：通过选择在2基于载体上携带序列的菌株来分离和克隆麦角甾醇生物合成途径的酿酒酵母甾醇DELTA 14还原酶的基因，以抵抗吗啉杀真菌剂例如茴香霉素。获得产生吗啉抗性的四种不同的质粒插入片段，其中之一被表征和测序。编码固醇DELTA14还原酶的纯化和分离的DNA序列编码与麦角甾醇生物合成途径中的酿酒酵母甾醇C-24（28）还原酶具有同源性的多肽。

3. 发明授权

US5591576A Sterol .DELTA.14 reductase screen 失效
标题翻译： Sterol DELTA 14还原酶屏
公开(公告)号：US5591576A
公开(公告)日：1997-01-07
申请号：US107348
申请日：1993-08-16
申请人： Margaret H. K. Lai , Donald R. Kirsch , Martin Bard
发明人： Margaret H. K. Lai , Donald R. Kirsch , Martin Bard
IPC分类号： C12N9/02 , C12N15/53 , C12Q1/18 , C12Q1/26 , C12N15/63 , C12Q1/02 , C12Q1/68
CPC分类号： C12N9/001 , C12Q1/18 , C12Q1/26 , C12Y103/0107
摘要： A binary assay identifies agents that inhibit sterol .DELTA.14 reductase involved in ergosterol biosynthesis. In the primary screen, sterol .DELTA.14 reductase inhibition by a test sample is assayed by adding the test sample to a culture of Neurospora crassa having an erg-3 mutation and also to a culture of a strain having an erg-1 mutation, comparing the extent of growth inhibition after incubation in the two cultures, and identifying as positives those samples that show growth inhibition in the erg-3 culture exceeding that in the erg-1 culture. In the secondary screen, samples that test positive in the primary screen are reassayed by adding the test sample to a culture of a Saccharomyces cerevisiae strain into which has been introduced multiple copies of a gene encoding sterol .DELTA.14 reductase and also to a strain of S. cerevisiae that does not have the introduced gene; positive samples are identified after incubation by observation that growth inhibition in the culture having no introduced reductase gene exceeds growth inhibition in the culture having the introduced reductase gene. In preferred embodiments, a known inhibitor of sterol .DELTA.14 reductase is employed in solidified media in both the primary and the secondary screens, resulting in an assay that is highly sensitive and specific for the detection of sterol .DELTA.14 reductase inhibitors.
摘要翻译：二元测定法鉴定了抑制参与麦角固醇生物合成的甾醇DELTA14还原酶的药剂。在主筛网中，通过将测试样品加入到具有erg-3突变的粗糙脉孢菌培养物中，并将其与具有erg-1突变的菌株的培养物进行比较，来测定测试样品的固醇DELTA 14还原酶抑制在两种培养物中孵育后的生长抑制程度，并且鉴定出在erg-3培养物中显示生长抑制的样品超过erg-1培养物中的那些样品的阳性。在次级屏幕中，通过将测试样品加入到已经引入了多拷贝的编码固醇DELTA14还原酶的基因的Saccharomyces cerevisiae菌株的培养物中，并且还添加到S的菌株没有引入基因的酿酒酵母; 通过观察，通过观察到没有引入的还原酶基因的培养物中的生长抑制超过在具有引入的还原酶基因的培养物中的生长抑制的情况下，鉴定阳性样品。在优选的实施方案中，在初级和次级筛中的固化培养基中使用已知的甾醇DELTA 14还原酶抑制剂，导致对检测固醇DELTA14还原酶抑制剂具有高度敏感性和特异性的测定。

4. 发明授权

US5418144A Spindle pole body screen for fungicides 失效
标题翻译：主轴杆体屏幕为杀菌剂
公开(公告)号：US5418144A
公开(公告)日：1995-05-23
申请号：US66764
申请日：1993-05-25
申请人： Donald R. Kirsch , Margaret H. K. Lai
发明人： Donald R. Kirsch , Margaret H. K. Lai
IPC分类号： C12Q1/18 , C12N1/00
CPC分类号： C12Q1/18 , Y10S435/942
摘要： A method for the identification of agents which inhibit spindle pole body formation or function, thus exhibiting selective fungicidal activity, involves the incubation of test samples in cultures of a Saccharomyces cerevisiae strain that produces excess numbers of spindle pole bodies. Cultures containing samples that inhibit spindle pole bodies exhibit enhanced growth because the growing yeast is rescued from the adverse effects of excess spindle bodies. In the preferred practice of the invention, the test sample is added to a S. cerevisiae culture or culture area containing a strain that has a conditional mutation producing excess spindle pole bodies, such as diploid esp1-1 strains. The culture or culture area is preincubated under permissive conditions wherein the strain can grow to some extent, and then conditions are shifted to restrictive conditions so that the mutant strain either cannot grow or grows poorly. The extent of growth in the culture or culture area containing test sample is then compared with the extent of growth in the absence of test sample, and the presence of spindle pole body inhibition is determined by observation of whether culture growth in the presence of test sample exceeds growth in its absence. Preferred embodiments employ test samples on disks or in wells in solidified cultures, facilitating easy visual inspection of growth. A presumptive spindle pole body inhibitor, identified in random screening, is used as a positive control which can be compared to the test sample.
摘要翻译：用于鉴定抑制纺锤体体形成或功能，从而显示出选择性杀真菌活性的试剂的方法涉及在产生过量纺锤体的酿酒酵母菌株的培养物中孵育测试样品。含有抑制锭子极体的样品的培养物表现出增强的生长，因为生长的酵母被从多余的纺锤体的不利影响中拯救出来。在本发明的优选实践中，将测试样品加入到含有产生过量纺锤体的条件突变的菌株的酿酒酵母培养物或培养物区域中，例如二倍体esp1-1菌株。培养或培养区域在允许条件下进行预培养，其中菌株可以在一定程度上生长，然后将条件转移到限制条件，使得突变菌株不能生长或生长差。然后将含有测试样品的培养或培养区域的生长程度与不存在测试样品的生长程度进行比较，并通过观察测试样品存在培养生长来确定主轴极体抑制的存在超过其缺席时的增长。优选实施例在盘或在固化培养物中的孔中采用测试样品，便于生长的容易的视觉检查。使用随机筛选鉴定的推定的主轴极体抑制剂作为阳性对照，可与测试样品进行比较。

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