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.

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.

The Aa1-Pri4 gene was cloned from the edible mushroom Agrocybe aegerita. The gene, specifically expressed during fruiting initiation, encodes a glycine-rich protein of 116 amino acids, with no homology to already known proteins. Homologous genes were amplified from two other strains belonging to the Agr. aegerita complex and originating from South-East Asia; and a comparison of the three genes revealed a high conservation of the coding sequences (72.8–97.8%). The PRI4 putative protein sequences were highly similar (87.5–100.0%); and all of them contained two protein kinase C sites, suggesting a potential supplementary regulation by phosphorylation at the protein level. The 5′ uncoding regions all presented a leader intron, very variable in sequence (45.7% identity), but with a high C+T content (74.5–79.0%). The presence of such CT-rich sequences previously described in the promoter of highly expressed fungal genes suggests that the leader intron of the Aa1-Pri4 gene could be involved in the high-level, stage-specific expression.

In Saccharomyces cerevisiae, the HAP transcriptional complex is involved in the fermentation–respiration shift. This complex is composed of four subunits. Three subunits are necessary for DNA-binding, whereas the Hap4p subunit, glucose-repressed, contains the transcriptional activation domain. Hap4p is the key regulator of the complex activity in response to carbon sources in S. cerevisiae. To date, no HAP4 homologue has been identified, except in Kluyveromyces lactis. Examination of these two HAP4 sequences led to the identification of two very short conserved peptides also identified in other yeasts. In the yeast Hansenula polymorpha, two possible HAP4 homologues have been found. Their deduced amino acid sequences are similar to the ScHap4p and KlHap4p proteins only in the N-terminal 16-amino-acid basic motif. Since molecular genetic tools exist and complete genome sequence is known for this yeast, we expressed one of these putative HpHap4 proteins in S. cerevisiae and showed that this protein is able to restore the growth defect of the S. cerevisiae hap4-deleted strain. A set of experiments was performed to confirm the functional homology of this new gene with ScHAP4. The discovery of a Hap4-regulatory protein in H. polymorpha with only the N-terminal conserved domain of the S. cerevisiae protein indicates that this domain may play a crucial role during evolution.

Small GTP-binding proteins of the Rab family are involved in the vesicular traffic inside eukaryotic cells. A gene library from the yeast Yarrowia lipolytica was screened with an oligonucleotide deduced from a highly conserved sequence in the Rab family. Four different genes were isolated. One of them, RYL1, was shown to be essential for cell viability. RYL1p displayed a high similarity with and tight phylogenetic relationships to SEC4p. When placed under the control of the GAL10 promoter, RYL1 was able to specifically relieve the thermosensitivity of a sec4–8 mutant of Saccharomyces cerevisiae. Therefore, it is proposed that RYL1 is a functional homologue of the S. cerevisiae SEC4 gene and is involved in the fusion of secretory vesicles with the plasma membrane in the general protein secretion pathway.

The sequence of the Neocallimastix frontalis enolase gene promoter was determined up to 1800 nucleotides 5′ to the major transcriptional start point. The base composition of the enolase upstream sequence revealed a very A+T-rich profile (13.5% G+C) leading to many putative hairpin structures. The functional organization of the N. frontalis enolase promoter was investigated by heterologous transient-expression assays. DNA fragments obtained by the sequential removal of sequences upstream of the translation start codon were fused to the Escherichia coli lacZ gene and the resulting plasmids were used to transform the ascomycetes Aspergillus nidulans and Penicillium roqueforti and the oomycete Saprolegnia monoica. Transient expression of the lacZ reporter gene was observed in regenerating protoplasts of S. monoica when using the 0.3 kb or 1 kb upstream of the enolase coding region. In contrast no β-galactosidase activity was detected in ascomycete protoplasts. DNA hybridization analysis revealed the integration of vector DNA in the genomic DNA of S. monoica and the presence of free copies of the transformation plasmid which could be rescued in E. coli. Our results indicate that the transcriptional machinery of the anaerobic chytrid N. frontalis may differ significantly from that of ascomycetes but that enough conservation exists within the lower fungi to allow a transient-driven expression of a reporter gene in an oomycete fungus.

We report on the development of a new PCR technique for the isolation of genomic fragments that flank known DNA sequences. This technique, single oligonucleotide nested (SON)-PCR, relies on only two amplification reactions with two or three nested sequence-specific primers. It allows the isolation of DNA regions located on either side of a known DNA sequence, with high specificity. DNA products of 2 kb in size can be generated that all contain one copy of the same primer at both ends. Sequence analysis of these products indicates that the binding of the primers to non-specific DNA sites mainly depends on their overall complementarity to the target sequence. Moreover, analysis shows that short extensions of the primers can occur during the first amplification reaction and that a 2-bp overlap between subsequent primers can target their annealing to their predecessor’s sequence. Ninety percent of the DNA products larger than 0.5 kb correspond to fragments of interest and we obtained successful results with various templates and primer sets. SON-PCR therefore seems a very efficient and widely applicable method for the rapid identification of large unknown DNA regions. Based on available expressed sequence tags, this technique was applied to isolate the palH and pacC genes of the phytopathogenic fungus Botrytis cinerea, with their 5′ or 3′ flanking regions.

Thioredoxins (TRXs) are small disulfide oxidoreductases of ca. 12 kDa found in all free living organisms. In plants, two chloroplastic TRXs, named TRX f and TRX m, were originally identified as light dependent regulators of several carbon metabolism enzymes including Calvin cycle enzymes. The availability of genome sequences revealed an unsuspected multiplicity of TRXs in photosynthetic eukaryotes, including new chloroplastic TRX types. Moreover, proteomic approaches and focused studies allowed identification of 90 potential chloroplastic TRX targets. Lately, recent studies suggest the existence of a complex interplay between TRXs and other redox regulators such as glutaredoxins (GRXs) or glutathione. The latter is involved in a post-translational modification, named glutathionylation that could be controlled by GRXs. Glutathionylation appears to specifically affect the activity of TRX f and other chloroplastic enzymes and could thereby constitute a previously undescribed regulatory mechanism of photosynthetic metabolism under oxidative stress. After summarizing the initial studies on TRX f and TRX m, this review will focus on the most recent developments with special emphasis on the contributions of genomics and proteomics to the field of TRXs. Finally, new emerging interactions with other redox signaling pathways and perspectives for future studies will also be discussed.

Candida guilliermondii (teleomorph Meyerozyma guilliermondii) is an ascomycetous species belonging to the Saccharomycotina CTG clade which has been studied over the last 40 years due to its biotechnological interest, biological control potential and clinical importance. Such a wide range of applications in various areas of fundamental and applied scientific research has progressively made C. guilliermondii an attractive model for exploring the potential of yeast metabolic engineering as well as for elucidating new molecular events supporting pathogenicity and antifungal resistance. All these research fields now take advantage of the establishment of a useful molecular toolbox specifically dedicated to C. guilliermondii genetics including the construction of recipient strains, the development of selectable markers and reporter genes and optimization of transformation protocols. This area of study is further supported by the availability of the complete genome sequence of the reference strain ATCC 6260 and the creation of numerous databases dedicated to gene ontology annotation (metabolic pathways, virulence, and morphogenesis). These genetic tools and genomic resources represent essential prerequisites for further successful development of C. guilliermondii research in medical mycology and in biological control by facilitating the identification of the multiple factors that contribute to its pathogenic potential. These genetic and genomic advances should also expedite future practical uses of C. guilliermondii strains of biotechnological interest by opening a window into a better understanding of the biosynthetic pathways of valuable metabolites.

The cDNA coding for the endoglucanase EGII ofP. janthinellum was cloned and sequenced. The open reading frame comprises 1230 nucleotides and the deduced amino-acid sequence shows an overall homology of 63% with theT. reesei egl2. The cellulose-binding domain of EGII represents a typical member of the A family of cellulases. Theegl2 gene is only induced by cellulose or cellobiose and not by sophorose. A promotor fragment including 1 kb was cloned and sequenced. Three major transcription startpoints were identified. Five motifs matching the binding site of the carbon-catabolite repressor CREA ofA. nidulans were detected. Their potential implication in repression was analyzed by bandshift assays.