CLONING & SEQUENCING OF THE U MAYDIS KILLER TOXIN GENE

一种由玉米黑粉菌真菌分泌的致命毒素，编码为 一种病毒，即玉米 U. 玉米病毒 (UmV)，对下列细胞具有高度特异性： 相同且密切相关的真菌。 三个已知杀手中的每一个 菌株感染同一种病毒，但分泌的毒素 有所不同，每个人都会杀死另外两个杀手 菌株。 该病毒具有分段的 dsRNA 基因组和 M2 片段 据报道已编码为致命毒素。 M2 段 三种杀手菌株中的一种将被分离出来。 互补cDNA 这些 dsRNA 片段将使用反向制备 转录酶和 DNA 聚合酶 I 的 Klenow 片段。 将cDNA连接至pBR322并转化至大肠杆菌HB101 细胞。 将使用抗生素鉴定阳性克隆 敏感性/抗性测试和菌落杂交测试 标记的M2 dsRNA。 全长插入片段将使用 M13 进行测序 以及桑格双脱氧DNA测序方法。 计算机分析 衍生的 DNA 序列将用于比较 DNA 三种杀手毒素的序列和蛋白质序列。 的 结果将提供直接证据表明 M2 段代码 毒素，检测一级结构的任何差异 从不同病毒株中分离出毒素编码基因，建立 初级翻译产物的分子量，表明 开放阅读框架，并允许预测 有毒蛋白质的特性。 玉米 U. maydis 感染初级食品。 其空气中的孢子具有 据报道是玉米生产中最重要的过敏原 授粉季节期间的州。 来自真菌的毒素已被 建议在过敏反应中发挥作用，并可能有 对于过敏相关的健康有着意想不到的重要性 问题。

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.