Molecular cloning of the imidazoleglycerolphosphate dehydratase gene of Trichoderma harzianum by genetic complementation in Saccharomyces cerevisiae using a direct expression vector

Summary The Trichoderma harzianum imidazoleglycerolphosphate dehydratase gene (igh) has been isolated by complementation of a Saccharomyces cerevisiae his3 mutant using a direct expression vector. This Escherichia coli-yeast shuttle vector was developed to allow efficient cloning and expression of cDNA libraries. The cDNA is 627 nucleotides long and codes for a protein of 209 amino acids with an apparent molecular mass of 22 466 daltons. The predicted protein sequence showed 63.6%, 58.7%, and 38.4% identity respectively to the corresponding enzymes from S. cerevisiae, Pichia pastoris and E. coli. Northern analysis showed that the expression of the igh gene in T. harzianum is not inhibited by external histidine and the level of igh mRNA was about threefold higher in cells starved of histidine.     

The protein disulphide isomerase gene of the fungus Trichoderma reesei is induced by endoplasmic reticulum stress and regulated by the carbon source

The gene pdi1 encoding protein disulphide isomerase was isolated from the filamentous fungus Trichoderma reesei by degenerate PCR based on a consensus PDI active-site sequence. It was shown that the Trichoderma pdi1 cDNA is able to complement a yeast mutant with a disrupted PDI1 gene. The putative T. reesei PD1I protein has a predicted 20-amino acid N-terminal signal sequence and the C-terminal fungal consensus ER retention signal HDEL. The mature protein shows strong conservation relative to other fungal protein disulphide isomerases. The T. reesei pdi1 promoter has two possible unfolded protein response (UPR) elements and it was shown by treatments with dithiothreitol and tunicamycin that the gene is under the control of the UPR pathway. Expression of a heterologous protein, an IgG antibody Fab fragment, in Trichoderma increases pdi1 expression, probably by inducing the UPR. The level of T. reesei pdi1 mRNA is also regulated by the carbon source, being lowest in glucose-containing media and highest on carbon sources that induce the genes encoding extracellular enzymes. The mechanism of this regulation was studied by examining pdi1 mRNA levels under conditions where the extracellular enzymes are induced by sophorose, as well as in the strain RutC-30, which is mutant for the glucose repressor gene cre1. The results suggest that neither sophorose induction nor glucose repression by the CREI protein affect the pdi1 promoter directly. Received: 4 May 1998 / Accepted: 23 April 1999     

Characterization of the pyruvate kinase-encoding gene (pki1) of Trichoderma reesei

The pyruvate kinase-encoding gene (pki1) from Trichoderma reesei was isolated by hybridization to the corresponding Aspergillus nidulans pkiA gene. The 1614-bp nucleotide (nt) sequence of the cloned gene codes for a 538-amino-acid protein. The coding sequence contains a single intron of 246 nt at a position identical to that of intron E in the A. nidulans gene. The PKI protein shows extensive homology to the PKIs of A. nidulans and A. niger (67%) and Saccharomyces cerevisiae (59%). The 5' non-coding sequence contains a number of motifs typical for yeast glycolytic genes, but so far only rarely found in filamentous fungi.     

Identification, cloning and sequence of the Streptococcus faecium infB (translational initiation factor IF2) gene

Summary The structural gene for translational initiation factor IF2 (infB) from Streptococcus faecium was identified by cross-hybridization with DNA probes derived from the corresponding gene of Bacillus stearothermophilus. The entire infB gene (ca. 2.8 kb) was cloned and sequenced. The amino acid sequence deduced from the nucleotide sequence shows that S. faecium initiation factor IF2 (785 amino acids, M_r 86,415) displays extensive homology (ca. 69% and 53%) with the region comprising three-quarters of the molecule from the carboxy-terminus of B. stearothermophilus and Escherichia coli IF2, respectively. The region comprising one-quarter of the molecule from the amino-terminus, on the other hand, does not display any significant homology.     

The isolation of Ant1, a transposable element from Aspergillus niger

A transposable element has been isolated from the industrially important fungus Aspergillus niger (strain N402). The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. It had inserted at a TA site and appeared to have duplicated the target site upon insertion. The isolated element was found to be 4798 by in length and contained 37-bp inverted, imperfect, terminal repeats (ITRs). The sequence of the central region of the element revealed an open reading frame (designated ORF1) which showed similarity, at the amino acid level, to the transposase of the Tc1/mariner class of DNA transposons. Another sequence within the central region of the element showed similarity to the 3 coding and downstream untranslated region of the amyA gene of A. niger. Sequence homology and structural features indicate that this element, which has been named Ant1 (A. niger transposon 1), is related to the Tc1/mariner group of DNA transposons. Ant1 is apparently present as a single copy in strain N402 of A. niger.     

CAN1-SUC2 gene fusion studies in Saccharomyces cerevisiae

Summary Various gene fusions between the arginine permease and invertase have been constructed in order to obtain information about whether part of the CAN1 gene product can induce secretion of biologically active invertase missing its own signal sequence. A construction containing 30 N-terminal amino acid residues of the CAN1 gene product fused to invertase was not secreted. When the CAN1 portion was elongated to 477 or 560 amino acid residues, secretion of the fusion proteins was observed. A fusion lacking 59 amino acids at the amino-terminal end of the arginine permease was also secreted. These results indicate that the amino-terminal end of the arginine permease is neither sufficient nor essential for membrane insertion; instead this enzyme should contain an internal targeting sequence facilitating secretion. Some general implications on the biosynthesis and topology of membrane proteins are also discussed as well as the homology with histidine permease.     

Cloning and expression of the UGA4 gene coding for the inducible GABA-specific transport protein of Saccharomyces cerevisiae

Japanese pear (Pyrus serotina Rehd.) exhibits gametophytic self-incompatibility. Following our previous findings that basic ribonucleases in the styles of Japanese pear are associated with self-incompatibility genes (S-RNases), stylar proteins with high pI values were analyzed by two-dimensional gel electrophoresis further to characterize S-RNases. A group of basic proteins of about 30 kDa associated with self-incompatibility genes were identified. These proteins contained sugar chains which reacted with concanavalin A and wheat germ agglutinin, and thus were designated as S-glycoproteins of Japanese pear. The fact that the S-glycoprotein was expressed at a much lower level in a self-compatible mutant than in the original variety suggested a role of S-glycoproteins in mediating self-incompatibility of Japanese pear. Immunoblot analysis indicated that S-glycoproteins are identical to previously identified S-RNases. The S-glycoproteins were predominantly expressed in the style, in the ovary in trace amounts, and not in leaf, pollen or germinated pollen. The N-terminal amino acid sequences of the S-glycoproteins showed homology not only with each other but also with those of the S-allele-associated proteins from plants of the family Solanaceae at levels of about 30–50%.     

Isolation and analysis of functional homologues of the secretion-related SAR1 gene of Saccharomyces cerevisiae from Aspergillus niger and Trichoderma reesei

The Aspergillusniger and Trichodermareesei genes encoding the functional homologues of the small GTP-binding protein SAR1p, which is involved in the secretion pathway in Saccharomyces cerevisiae, have been cloned and characterised. The A. niger gene (sarA) contains five introns, whereas the T. reesei gene (sar1) has only four. In both cases the first intron is at the same position as the single S. cerevisiae SAR1 intron. The encoded proteins show 70–80% identity to the SAR1 protein. Complementation of S. cerevisiaesar1 and sec12 mutants by expression vectors carrying the A. nigersarA and T. reesei sar1 cDNA clones confirmed that the cloned genes are functional homologues of the S. cerevisiae SAR1 gene. Three mutant alleles of the A. nigersarA gene (D29G, E109K, D29G/E109K), generated by site-directed mutagenesis, revealed a thermosensitive dominant-negative phenotype in the presence of the wild-type sarA allele. This result contrasts with the situation in S. cerevisiae, where similar mutations have a thermosensitive phenotype. Taken together, our results indicate that the sarA gene is involved in an essential function in A. niger.     

Cloning and characterization of a gene cluster from Bacillus stearothermophilus comprising infC, rpmI and rplT

Summary Using two synthetic deoxyribonucleotide probes encoding segments of the primary structure of initiation factor IF3 from Bacillus stearothermophilus, we identified and cloned a segment of DNA which carries the infC gene. As in Escherichia coli, the infC gene begins with the unusual initiation triplet AUU, and is followed by the structural genes for ribosomal proteins L35 and L20 (rpmI and rplT, respectively).     

Functional analysis of an Aspergillus ficuum phytase gene in Saccharomyces cerevisiae and its root-specific, secretory expression in transgenic soybean plants

Phytases release inorganic phosphates from phytate in soil. A gene encoding phytase (AfPhyA) was isolated from Aspergillus ficuum and its ability to degrade phytase and release phosphate was demonstrated in Saccharomyces cerevisiae. A promoter from the Arabidopsis Pky10 gene and the carrot extensin signal peptide were used to drive the root-specific and secretory expression of the AfPhyA gene in soybean plants. The phytase activity and inorganic phosphate levels in transgenic soybean root secretions were 4.7 U/mg protein and 439 μM, respectively, compared to 0.8 U/mg protein and 120 μM, respectively, in control soybeans. Our results demonstrated the potential usefulness of the root-specific promoter for the exudation of recombinant phytases and offered a new perspective on the mobilization of phytate in soil to inorganic phosphates for plant uptake. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Guilan Li and Shaohui Yang authors contribute equally to the paper.