CLONING & SEQUENCING OF THE U MAYDIS KILLER TOXIN GENE

一种由麦氏黑穗病菌分泌的致命毒素，由

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.