During meiotic chromosome pairing, a loop of unpaired DNA induces the silencing of all paired and unpaired homologous DNA via meiotic silencing, an RNA-mediated post-transcriptional gene-silencing mechanism. To test the effect of unpaired DNA on adjacent genes, we constructed strains containing the DNA of a transformation marker integrated immediately downstream of the Ascospore maturation-1 (Asm-1) gene and tested whether this unpaired DNA silences asm-1⁺. We conclude that unpaired downstream DNA has no effect on Asm-1 expression during meiosis or ascospore development, which suggests that the silencing signal produced by unpaired DNA does not propagate onto adjacent paired regions.

Plasmids containing the AMA1 replicon are capable of autonomous maintenance in Aspergillus nidulans. It has been reported previously that these plasmids can form concatenates by recombination in a transformed mycelium, and up to 10% of molecules are involved in such events. The present study demonstrates that plasmid recombination, although frequent during transformation, rarely occurs during vegetative growth. As a result, the structure and phenotypic stability of AMA1 plasmids generally remains unaltered for many asexual (conidial) generations. It is also evident that plasmid replication does not require specific recombination events in the AMA1 palindrome. However, during sexual reproduction, autonomous plasmids exhibit increased recombination, which results in both plasmid concatenation and integration into the chromosome.

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.

Sexual reproduction of fungi is governed by genes located on the mating-type (MAT) locus. To analyze the MAT locus of the genus Diaporthe (anamorph: Phomopsis), a large genera within the ascomycetous class Sordariomycetes, we cloned and sequenced loci MAT1-1 and MAT1-2 from two heterothallic Diaporthe species, designated as Diaporthe W- and G-types (four isolates in total). The mating-type loci structures of Diaporthe W- and G-types were similar; MAT1-1 isolates had a MAT locus containing three genes, MAT1-1-1, MAT1-1-2 and MAT1-1-3, as was the case with other Sordariomycetes, and in contrast to other Sordariomycetes, MAT1-2 isolates had genes homologous to MAT1-1-2 and MAT1-1-3, in addition to MAT1-2-1. Expression analysis by RT-PCR revealed that all the mating-type genes of Diaporthe W-type were transcriptionally active during vegetative growth. The structure of MAT loci of Diaporthe W- and G-types is distinct from that in other heterothallic filamentous ascomycetes, which have dissimilar gene structure in opposite mating-type loci. This unique structure is informative to discussing the evolutionary history and function of mating-type genes of Sordariomycete fungi.

We disrupted the Aspergillus fumigatus argB gene, encoding ornithine transcarbamylase, using a novel in vitro transposon-based mutagenesis approach. This approach utilizes a modified transposon containing the Neurospora crassa pyr4 gene, which is randomly inserted in vitro into a target sequence of interest. Clones in which the gene of interest has been disrupted are identified by PCR and used to transform a pyrG-deficient strain of A. fumigatus. Using this approach, we obtained arginine auxotrophs of A. fumigatus. Full characterization of the argB insertion was performed by Southern blot analysis. These strains can be supplemented by addition of arginine into the culture medium and can be fully rescued to arginine prototrophy by transformation with the intact A. fumigatus argB gene.

Destruxins are among the most exhaustively researched secondary metabolites of entomopathogenic fungi, yet definitive evidence for their roles in pathogenicity and virulence has yet to be shown. To establish the genetic bases for the biosynthesis of this family of depsipeptides, we identified a 23,792-bp gene in Metarhizium robertsii ARSEF 2575 containing six complete nonribosomal peptide synthetase modules, with an N-methyltransferase domain in each of the last two modules. This domain arrangement is consistent with the positioning of the adjacent amino acids N-methyl-l-valine and N-methyl-l-alanine within the depsipeptide structure of destruxin. DXS expression levels in vitro and in vivo exhibited comparable patterns, beginning at low levels during the early growth phases and increasing with time. Targeted gene knockout using Agrobacterium-mediated transformation produced mutants that failed to synthesize destruxins, in comparison with wild type and ectopic control strains, indicating the involvement of this gene in destruxin biosynthesis. The destruxin synthetase (DXS) disruption mutant was as virulent as the control strain when conidial inoculum was topically applied to larvae of Spodoptera exigua, Galleria mellonella, and Tenebrio molitor indicating that destruxins are dispensable for virulence in these insect hosts. The DXS mutants exhibited no other detectable changes in morphology and development.

The organisation of the URA1 gene of Schizosaccharomyces pombe was determined from the entire cDNA cloned by the transformation of an ATCase-deficient strain of Saccharomyces cerevisiae. The URA1 gene encodes the bifunctional protein GLNase/CPSase-ATCase which catalyses the first two steps of the pyrimidine biosynthesis pathway. The complete nucleotide sequence of the URA1 cDNA was elucidated and the deduced amino-acid sequence was used to define four domains in the protein; three functional domains, corresponding to GLNase (glutamine amidotransferase), CPSase (carbamoylphosphate synthetase) and ATCase (aspartate transcarbamoylase) activities, and one cryptic DHOase (dihydroorotase) domain. Genetic investigations confirmed that both GLNase/CPSase and ATCase activities are carried out by the same polypeptide. They are also both feedback-inhibited by UTP (uridine triphosphate). Its organization and regulation indicate that the S. pombe URA1 gene product appears very similar to the S. cerevisiae URA2 gene product.

Pyrenochaeta lycopersici, as other soil-transmitted fungal pathogens, generally received little attention compared to the pathogens affecting the aerial parts of the plants, although causing stunt and important fruit yield reduction of agronomic relevant crops. The scope of this study was to develop a system allowing to investigate the functional role of P. lycopersici genes putatively involved in the corky root rot of tomato. A genetic transformation system based on a split-marker approach was developed and tested to knock out a P. lycopersici gene encoding for a lytic polysaccharide monooxygenase (Plegl1) induced during the disease development. The regions flanking Plegl1 gene were fused with the overlapping parts of hygromycin marker gene, to favour homologous recombination. We were able to obtain four mutants not expressing the Plegl1 gene though, when tested on a susceptible tomato cultivar, Plegl1 mutants showed unaltered virulence, compared with the wild-type strain. The strategy illustrated in the present work demonstrated for the first time that homologous recombination occurs in P. lycopersici. Moreover, a transformation system mediated by Agrobacterium tumefaciens was established and stable genetic transformants have been obtained. The transformation systems developed represent important tools for investigating both the role of genes putatively involved in P. lycopersici interaction with host plant and the function of other physiological traits which emerged to be genetically expanded from the recent genome sequencing of this fungus.

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.

Carboxypeptidase Z is a serine carboxypeptidase secreted by Absidia zychae NRIC 1199. The cDNA and genomic DNA carrying the scpZ gene encoding carboxypeptidase Z were cloned and sequenced. The nucleotide sequences of the cDNA (1.4 kb) and the genomic DNA (3.3 kb) were analyzed and the intervening sequences were located by a comparison of the two. It was found that the scpZ gene was interrupted by 11 short introns, 50–75 nucleotides in length. Genomic Southern analysis showed that there was only one scpZ gene in the genome of A. zychae. The gene encoded a putative pre-proenzyme composed of 409 amino-acid residues of the mature carboxypeptidase Z (Mr 45 421) and an additional N-terminal sequence of 51 amino-acid residues. The amino-acid sequence around the active serine residue of carboxypeptidase Z (-G-E-S-Y-G-G-) differed from the consensus (-G-E-S-Y-A-G-) which is conserved in most of the serine carboxypeptidases so far analyzed.