CLONING & SEQUENCING OF THE U MAYDIS KILLER TOXIN GENE

一种由真菌玉米黑粉菌分泌的致命毒素，由 梅迪斯病毒(UmV)是一种高度特异的病毒。 相同且密切相关的真菌。已知的三个凶手中的每一个 菌株感染了相同的病毒，但毒素分泌 略有不同，因为每个人都会杀死另外两个杀手 菌株。该病毒有一个分段的dsRNA基因组和M2片段 已经被报告为杀手毒素的代号。M2细分市场 三种致命菌株中的一种将被分离出来。免费赠送的DNA 对于这些dsRNA片段，将使用反向 转录酶和DNA聚合酶的Klenow片段I. 将其连接到pBR322中，转化到大肠杆菌HB101中 细胞。阳性克隆将使用抗生素进行鉴定 敏感性/耐药性试验和菌落杂交试验 标记M2 dsRNA。全长插入物将使用M13进行排序 和桑格双脱氧DNA测序法。计算机分析 的DNA序列将被用来比较DNA 三种致命性毒素的序列和蛋白质序列。这个 结果将提供直接证据，证明M2细分市场代码 这种毒素，检测到在初级结构上的任何差异 从不同毒株分离的毒素编码基因，建立 初级翻译产物的相对分子质量表明 开放阅读框架，并允许关于 有毒蛋白质的性质。 梅迪斯杆菌会感染一种主要食品。它在空气中传播的孢子 据报道是玉米生产中最重要的过敏原 在授粉季节。来自真菌的毒素已经被 建议在过敏反应中发挥作用，并可能有 对过敏相关健康的意想不到的意义 有问题。

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.