Isolation and characterization of the EF-1? gene of the filamentous fungus Puccinia graminis f. sp. tritici

Abstract

A gene of Puccinia graminis f. sp. tritici, coding for the translation elongation factor 1 alpha (EF-1 alpha), was isolated from a P. graminis genomic library using the EF-1 alpha gene sequence of Absidia glauca. The coding region of 1389 nucleotides encodes a polypeptide of 463 amino acids and is interrupted by eight introns. An additional intron is located in the 5' untranslated region. A single transcription start point (tsp) was mapped by primer extension. A cDNA fragment corresponding to P. graminis EF-1 alpha mRNA hybridized with a 1.9-kb-long poly(A+)RNA, sufficient to encode the EF-1 alpha protein. Southern hybridization of digested genomic DNA revealed that two copies of the EF-1 alpha gene exist in the genome of P. graminis.

An EST library from Puccinia graminis f. sp. tritici reveals genes potentially involved in fungal differentiation

“…The same is possible, e.g. for the three ESTs PGT_654 , PGT_671 , and PGT_721 , exhibiting similarity to a major alcohol dehydrogenase from Uromyces fabae (Voegele et al , 2005) and ESTs PGT_778 and PGT_727 with similarity to translation elongation factor 1‐α from Puccinia graminis (Schillberg et al , 1995).…”

“…Backlund & Szabo (1993) estimated the genome size of Puccinia graminis to be around 67 Mb and determined a G/C content of 45.3%. Sequences of only a few genes of Puccinia graminis are known, among these sequences are elongation factor 1‐α (Schillberg et al , 1995), actin (X77857), usp1 (L08126) and usp2 (L08127) (Liu et al , 1993), hss1 (U26597), and some ribosomal RNA genes. Small EST libraries have been described for two other rust fungi, namely Puccinia triticina (Thara et al , 2003) and Uromyces fabae (Hahn & Mendgen, 1997).…”

Transient transformation of the rust fungus Puccinia graminis f. sp. tritici

“…For heterologous gene expression in basidiomycetes, homologous promoter sequences or promoters from other basidiomycetes perform well, demonstrating the importance of promoter choice for the expression of heterologous genes (Schuren and Wessels 1994;Ogawa et al 1998). Our rationale for heterologous gene expression in Pgt was to fuse the Pgt EF-1a promoter (Schillberg et al 1995) to the heterologous hpt or GUS gene from E. coli. Biolistic transformation of Pgt uredospores with a vector containing the hpt gene under the control of the Pgt EF-1a promoter led to hygromycin B-resistant germlings.…”

“…To generate pKLHyg14, the 1400-bp EcoRI-BamHI fragment containing the promoter of the Pgt EF-1a gene (Schillberg et al 1995), including 162 bp of the coding region interrupted by a 80-bp intron, was ligated into the EcoRI/BamHI sites of the pBluescript KS(+) vector (Stratagene, Heidelberg, Germany), giving the intermediate construct pG2-43. The BamHI-XbaI fragment of pKBcosHT (kindly provided by Dr. Weltring, University of MuÈ nster, Germany), carrying the E. coli hpt gene followed by the trpC termination region from Aspergillus nidulans, was cloned downstream of the EF-1a gene fragment in pG2-43.…”

“…Recently, we isolated the gene for the Pgt translation elongation factor 1a (EF-1a), together with its promoter (Schillberg et al 1995). EF-1a genes are known to possess highly active regulatory sequences (Merrick 1992) which have been used for heterologous gene expression in plants (Curie et al 1993) and fungi (Steiner et al 1990;Burmester 1992).…”