A novel endochitinase agar-plate assay has been developed and used to identify 11 full-length cDNAs encoding endochitinase I (ENC I) from aTrichoderma harzianum cDNA library by expression in yeast. The 1473-bpchil cDNA encodes a 424-residue precursor protein including both a signal sequence and a propeptide. The deduced ENC I amino-acid sequence is homologous to other fungal and bacterial chitinases, and the enzyme cross-reacts with a polyclonal antiserum raised against chitinase A1 fromBacillus circulans. TheT. harzianum endochitinase I was secreted into the culture medium by the yeastSaccharomyces cerevisiae in a functionally active form. The purified recombinant enzyme had a molecular mass of 44 kDa, an isoelectric point of 6.3, a pH optimum of 7.0 and a temperature optimum of 20 °C.

We have isolated the 3-phosphoglycerate kinase (PGK) gene of the yeastYarrowia lipolytica by probing a genomic library with a PCR fragment amplified with primers deduced from two highly conserved regions of various PGKs. It is a unique sequence encoding a polypeptide of 417 residues with extensive homology to other PGKs, especially to that ofAspergillus nidulans (76% identity). The expression of theY. lipolytica PGK1 gene proved to be higher on gluconeogenic substrates than on glycolytic ones. Haploid strains harboring a disrupted allele were able to grow on mixtures of a gluconeogenic carbon source and of a glycolytic one, but required proline supplementation in the presence of glucose, and were inhibited by glycerol.

A genomic library from the yeastPichia anomala has been constructed and employed to clone the gene encoding the sucrose-hydrolysing enzyme invertase by complementation of a sucrose non-fermenting mutant ofSaccharomyces cerevisiae. The cloned gene,INV1, was sequenced and found to encode a polypeptide of 550 amino acids which contained a 22 amino-acid signal sequence and ten potential glycosylation sites. The amino-acid sequence shows significant identity with other yeast invertases and also withKluyveromyces marxianus inulinase, a yeast β-fructofuranosidase which has a different substrate specificity. The nucleotide sequences of the 5′ and 3′ non-coding regions were found to contain several consensus motifs probably involved in the initiation and termination of gene transcription.

Transgenic filamentous fungi of the speciesAspergillus niger, A. nidulans andA. awamori expressing and secretingErwinia carotovora subsp.atroseptica pectate lyase 3 (PL3) were generated. Correct processing of the pre-enzyme was achieved using theA. niger pectin lyase A (PEL A) signal peptide. With the prepro-peptide ofA. niger polygalacturonase II, secreted enzymes still possessed the 6- aa pro-sequence, indicating the importance of the conformation of the precursor protein for correct cleavage of the signal sequence. PL3 expression was markedly increased in media optimized for limited protease activity, and reached 0.4, 0.8 and 2.0 mg/l for expression inA. niger, A. awamori andA. nidulans, respectively. Glycans attached to the PL3 enzymes exhibited species-specific differences, and an increase of molecular mass coincided with reduced specific activities of the enzymes.

A gene encoding a putative GABA aminotransferase (ugatA) was isolated from the basidiomyceteUstilago maydis via heterologous hybridization to the GABA aminotransferase gene (gatA) ofAspergillus nidulans. The derived amino-acid sequence ofugatA shows strong identity throughout the protein to the GABA aminotransferase enzymes fromA. nidulans andSaccharomyces cerevisiae. Northern analysis inU. maydis indicated that theugatA transcript is inducible by the ω-amino acids GABA and β-alanine, and is not subject to nitrogen catabolite repression. With the use ofugatA promoter-lacZ fusion constructs, it was demonstrated that the removal of sequences located approximately 250 by 5′ to the translational start site ofugatA (including multiple copies of a 7-bp direct repeat) resulted in the loss of induction by ω-amino acids. While theugatA gene under the control of theA. nidulans gatA promoter was able to fully complement agatA⁻ phenotype inA. nidulans, the full-lengthugatA gene was not, suggesting a lack of expression from theU. maydis promoter inA. nidulans. AU. maydis strain with a gene disruption at theugatA locus showed decreased growth on β-alanine as a sole nitrogen source, but was able to grow on GABA as a sole nitrogen source, indicating an alternative pathway for the utilization of GABA inU. maydis.

An intronless form of thebgl1 gene encoding an extracellularβ-glucosidase fromTrichoderma reesei was expressed in the yeast Saccharomyces cerevisiae under the control of the yeast GAL 1 promoter. Transformation of a yeast strain with this vector resulted in transformants that produce and secrete activeβ-glucosidase into the growth medium. Additionally, active recombinantβ-glucosidase protein was shown to be localized predominantly in the periplasmic space by using ap-nitrophenylβ-D-glycoside hydrolysis assay against fractionated yeast cells. The apparent size of the recombinant enzyme was 10–15 kDa larger than that of the native form. Treatment of the recombinantβ-glucosidase with endoglycosidase-H indicated the apparent increase in size was due to N-linked glycosylation.

Aspergillus niger var.awamori contains multiple copies of a transposable element, Vader. This element was detected as a 437-bp insertion in four independently isolated spontaneous mutants of theniaD (nitrate reductase) gene. The Vader element is present in approximately 15 copies in bothA. niger var.awamori andA. niger. A single copy of Vader was detected from only one of the two laboratory strains ofA. nidulans which were also examined. Insertion of the Vader element into theniaD gene ofA. niger var.awamori caused a 2-bp duplication (TA) of the target sequence. The Vader element is flanked by a 44-bp inverted repeat. The genetic stabilities of the inserted Vader elements atniaD were examined by studying reversion frequencies resulting in colonies able to grow on nitrate as a sole nitrogen source. MutantsniaD392 andniaD436 reverted at a frequency of 9x10^-3 and 4x10^-2, respectively. Two of the mutants,niaD587 andniaD410, reverted at a lower frequency of 6x10^-4.

We have isolated mutants ofSaccharomyces cerevisiae with an increased sensitivity to oxidative stress. Allpos9 mutants (pos for peroxide sensitivity) were hypersensitive to methylviologene, hyperbaric oxygen or hydrogen peroxide, but grew similarly to the wild-type under all other conditions tested. Isolation and sequencing of the respectivePOS9 gene revealed that it was identical toSKN7. The predicted Skn7/Pos9 protein possesses a domain with high homology to prokaryotic response regulators. These regulatory proteins are part of a simple signalling cascade termed a “two-component system”, where a phosphorylation signal of a histidine kinase is transferred to a conserved aspartate residue of the response regulator. To test the functional role of the respective aspartate residue of Skn7/Pos9 protein in oxidative stress, we mutagenized this residue in vitro to alanine, arginine and glutamate. Only the glutamate allele (D⁴²⁷ to E) was able to rescue the hydrogen peroxide-sensitivity ofpos9 mutants. By fusion experiments with the Gal4 DNA-binding domain we identified the isolated response regulator-like domain as a novel eukaryotic domain sufficient for gene activation. Whereas this hybrid protein activated transcription of alacZ reporter gene under aerobic conditions, no activation was observed under anaerobic conditions, indicating that the response regulator domain is involved in a signalling reaction. Two-hybrid investigations also suggest an oligomerization of the Pos9 protein. Our results indicate that a two-component system is involved in the oxidative-stress response of yeast.

Alcohol dehydrogenase II (ADH II, structural genealcB) was purified from a strain H1035,biA1; alcE1; alc500 alcD1, which produces 100-times more ADH II activity than thealcAalcR deletion strain (alc500). Antibodies were raised against this ADH, and were used to screen a cDNA library in γgt11. We have isolated the gene for an ADH which is over-expressed in H1035, and which we believe to be thealcB gene; cDNA and genomic clones were sequenced. The sequence contains three introns and encodes a protein of 367 amino acids. This protein shows a clear level of identity to a range of alcohol dehydrogenases, but is no more closely related to the ADH I and ADH III previously described inA. nidulans than to the ADHs ofS. pombe andS. cerevisiae. The significance of consensus sequences found in the 5′ region of the gene is discussed in relation to the regulation of the gene.

Orthogonal field agarose-gel electrophoresis (OFAGE) of chromosomes from translocation-bearing and normalNeurospora crassa strains was utilized, first, to recover cosmids from a translocated region, and second, to map translocation breakpoints. Surprisingly, the right breakpoints in two independently derived, interstitial translocations,T(II → III) AR18 andT(II → VI)P2869, are within about 5.6 kbp of each other suggesting that this region of linkage group (LG) II may be fragile or otherwise subject to chromosome breakage. Mapping translocation breakpoints through OFAGE, or other similar methods, should allow for DNA sequencing across breakpoints that are not associated with mutant phenotypes or that are not within walking distance of cloned markers.