Efficient synthesis of a 72-kDa mitochondrial polypeptide using the yeast Ty expression system.

Using the Ty system from yeast we report the efficient expression of a heterologous eukaryotic gene encoding a 72 kDa mitochondrial polypeptide. The pFM2IIBgIII expression vector was initially modified for this purpose by inserting the factor X(a) protease cleavage site. The TyA gene, which encodes the structural component of the yeast virus-like particles (VLPs), and the eukaryotic yst1 gene, encoding a 72 kDa mitochondrial tyrosyl-tRNA synthetase from the filamentous fungus Podospora anserina, were subsequently fused to the factor X(a) cleavage site. The resulting chimeric gene, in which the two polypeptide coding sequences are separated by the factor X(a) cleavage site, was expressed in yeast. High yield expression of this foreign protein, which was isolated from yeast transformants as hybrid TyVLPs, was verified after factor X(a) treatment by SDS polyacrylamide gel electrophoresis and antibody detection. The strategy presented here should be useful for expressing a wide variety of eukaryotic genes.

Identification of regulatory elements in the cutinase promoter from Fusarium solani f. sp. pisi (Nectria haematococca).

The cutinase gene from Fusarium solani f. sp. pisi (Nectria haematococca) is induced upon contact with the plant cuticular polymer, cutin, by the unique hydroxy fatty acid monomers released by cutinase carried by virulent strains of the fungus, and this gene is also catabolite-repressed by glucose. Functional elements of the cutinase promoter were studied in vivo by transforming F. solani pisi with fusions of 5'-flanking regions of the cutinase gene and the gene encoding chloramphenicol acetyltransferase (cat). DNA-binding proteins from F. solani pisi were analyzed in vitro by gel shift experiments, methylation interference analysis, and UV-cross-linking experiments. Thus, we identified four promotor elements involved in cutinase gene regulation: a silencer, positive-acting G-rich element, an element that binds a basal transcription factor, and a palindrome necessary for induction by cutin monomer. A silencer between -287 and -249 keeps basal gene expression low but also influences the inducibility of the gene. To restore high levels of induction, a G-rich positive-acting element with sequence similarities to other fungal elements acts as an antagonist to the silencer. Basal transcription is mediated by the first 141 base pairs of the cutinase promoter. The binding site of transcription factor CTF2 was identified between the TATA box and the transcription initiation sites. Gene induction by cutin monomers is regulated by CTF1, most probably a dimeric DNA-binding protein of 49 kDa with a palindromic recognition site at -170.

The mitochondrial tyrosyl-tRNA synthetase of Podospora anserina is a bifunctional enzyme active in protein synthesis and RNA splicing.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mt tyrRS), which is encoded by the nuclear gene cyt-18, functions not only in aminoacylation but also in the splicing of group I introns. Here, we isolated the cognate Podospora anserina mt tyrRS gene, designated yts1, by using the N. crassa cyt-18 gene as a hybridization probe. DNA sequencing of the P. anserina gene revealed an open reading frame (ORF) of 641 amino acids which has significant similarity to other tyrRSs. The yts1 ORF is interrupted by two introns, one near its N terminus at the same position as the single intron in the cyt-18 gene and the other downstream in a region corresponding to the nucleotide-binding fold. The P. anserina yts1+ gene transformed the N. crassa cyt-18-2 mutant at a high frequency and rescued both the splicing and protein synthesis defects. Furthermore, the YTS1 protein synthesized in Escherichia coli was capable of splicing the N. crassa mt large rRNA intron in vitro. Together, these results indicate that YTS1 is a bifunctional protein active in both splicing and protein synthesis. The P. anserina YTS1 and N. crassa CYT-18 proteins share three blocks of amino acids that are not conserved in bacterial or yeast mt tyrRSs which do not function in splicing. One of these blocks corresponds to the idiosyncratic N-terminal domain shown previously to be required for splicing activity of the CYT-18 protein. The other two are located in the putative tRNA-binding domain toward the C terminus of the protein and also appear to be required for splicing. Since the E. coli and yeast mt tyrRSs do not function in splicing, the adaptation of the Neurospora and Podospora spp. mt tyrRSs to function in splicing most likely occurred after the divergence of their common ancestor from yeast.