Isolation and analysis of a corprinus cinereus DNA fragment showing homology to a fimbrial cDNA from ustilago violacea

摘要

Surface proteinaceous fibrils, termed fimbriae, were firstidentified on gram negative bacteria in the 1940s. Fungal fimbriae,discovered some 25 years later, are found on members of all fungalclasses. In the present study, polyclonal antiserum raised against thefimbrial proteins of U. vio/acea were used in order to identifyantigenically related proteins from Coprinus cinereus andSchizophy//um commune. Two polypeptides with molecular masses of37 and 39 kDa from C. cinereus were observed and confirm earlierresults. A single previously unidentified 50 kDa polypeptide in S.commune crossreacted with the antiserum. The 50 kDa protein wasfound to consist of 3 isoforms with isoelectric points ranging from 5.6to 5.8.A fimbrial cDNA derived from U. vio/acea was used to identifyDNA restriction fragments from C. cinereus and S. commune showinghomology to the fimbrial transcript of U. vio/acea. Heterologoushybridization with this cDNA was used in order to screen a C. cinereusgenomic DNA library. A single clone, A2-3A, with a 14 kbp insertshowed strong homology to the pfim3-1 cDNA. The region of homology,a 700 bp Xba I fragment, was subcloned into pUG19. This plasmid wasrefered to as pXX8. DNA sequence determinations of pXX8 and adjacentfragments from A2-3A suggested that the cloned DNA was a portion ofthe rONA repeat encoding the small subunit rRNA.DNA sequence analysis of pfim3-1 yielded an incomplete openreading frame. The predicted amino acid sequence codes for a 206amino acid, 22 kDa polypeptide which contains a domain similar to atransmembrane domain from rat leukocyte antigen, GDS3. As well, anuntranslated 576 nucleotide domain showed 81 % homology to pXX8 and830/0 homology to the 188 rRNA sequence of Ustilago maydis. Thissequence was found adjacent to a region of adenine-thymine base pairspresumed to represent the polyadenylation sequence of the fimbrialtranscript. The size and extent of homology is sufficient to account forthe hybridization of pfim3-1 to rDNA. It is suggested that this domainrepresents a completely novel regulatory domain within eukaryotesthat may enable the observed rapid regeneration of fimbriae in U.violacea.