CLONING & SEQUENCING OF THE U MAYDIS KILLER TOXIN GENE

克隆

• 批准号: 3438861
• 负责人: WILLIAM FLURKEY
• 金额: $ 9.48万
• 依托单位: INDIANA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3438861
• 关键词: DNA directed DNA polymerase; Escherichia coli; RNA directed DNA polymerase; complementary DNA; gene expression; molecular cloning; mycotoxins; nucleic acid sequence; plant virus; protein sequence; virus RNA; western blottings

A killer toxin secreted by the fungus Ustilago maydis and coded by a virus, the U. maydis virus (UmV), is highly specific to cells of the same and closely related fungi. Each of three known killer strains is infected with the same virus, but the toxins secreted are somewhat different in that each will kill the other two killer strains. The virus has a segmented dsRNA genome and the M2 segment has been reported to code for the killer toxin. The M2 segments of the three killer strains will be isolated. cDNAs complimentary to these dsRNA segments will be prepared using reverse transcriptase and the Klenow fragments of DNA polymerase I. The cDNA will be ligated into pBR322 and transformed into E. coli HB101 cells. Positive clones will be identified using antibiotic sensitivity/resistance tests and colony hybridization tests with labeled M2 dsRNA. Full length inserts will be sequenced using M13 and the Sanger dideoxy DNA sequencing method. Computer analysis of the derived DNA sequences will be used to compare the DNA sequence and protein sequences of the three killer toxins. The results will provide direct evidence that the M2 segment codes for the toxin, detect any differences in the primary structure of the toxin-coding genes isolated from different virus strains, establish the molecular weight of the primary translation product, indicate open-reading frames, and permit predictions regarding the properties of the toxic proteins. U. maydis infects a primary food product. Its airborne spores have been reported to be the most important allergen in corn producing states during the pollination season. Toxins from fungi have been suggested to play a role in allergic responses and may have unsuspected significance with regard to allergy related health problems.

玉米黑粉菌分泌的一种致命毒素， 一种病毒，美国。maydis病毒（UmV），是高度特异性的细胞， 相同的和密切相关的真菌。 三个已知的杀手 菌株感染相同的病毒，但毒素分泌 有些不同的是，每一个将杀死其他两个杀手 菌株 该病毒具有分段的dsRNA基因组和M2片段 被报告为致命毒素的编码 的M2区段 三种致命菌株的其中一种将被分离出来 cDNA互补 这些dsRNA片段将使用反向 转录酶和DNA聚合酶I的Klenow片段。 的 将cDNA连接到pBR 322中，转化E.杆菌hb 101 细胞 将使用抗生素鉴定阳性克隆 敏感性/耐药性试验和菌落杂交试验， 标记的M2 dsRNA。 将使用M13对全长插入物进行测序 和桑格双脱氧DNA测序法。 计算机分析 的衍生DNA序列将用于比较DNA 三种致命毒素的基因序列和蛋白质序列 的 结果将提供直接证据表明，M2段代码为 毒素，检测在一级结构的任何差异， 从不同病毒株分离的毒素编码基因， 主要翻译产物的分子量，表明 开放阅读框架，并允许预测 有毒蛋白质的特性。 联合玉米淀粉会感染初级食品。 它的空气孢子 据报道，在玉米生产中， 国家在授粉季节。 真菌的毒素 建议在过敏反应中发挥作用， 对过敏相关健康的意外意义 问题