A deletion of the PDC1 gene for pyruvate decarboxylase of yeast causes a different phenotype than previously isolated point mutations

Summary We deleted most of the pyruvate decarboxylase structural gene PDC1 from the genome of Saccharomyces cerevisiae. Surprisingly, mutants carrying this deletion allele showed a completely different phenotype than previously described point mutations. They were able to ferment glucose and their specific pyruvate decarboxylase activity was only reduced to 45% of the wild type level. Northern blot analysis revealed that a sequence in the yeast genome homologous to PDC1 and formerly designated as a possible pseudogene is expressed and may code for a different but closely related pyruvate decarboxylase. The products of the two PDC genes seem to form hybrid oligomers, however both homooligomers have enzyme activity. Thus, the product of the PDC1 gene is not absolutely neccessary for glucose fermentation in yeast.     

Endopolygalacturonase genes and enzymes from Saccharomyces cerevisiae and Kluyveromyces marxianus

The gene encoding endopolygalacturonase (EC 3.2.1.15) has been cloned, sequenced and expressed from three strains of Saccharomyces cerevisiae (including non-secretors) and three strains of Kluyveromyces marxianus. Both control and coding regions showed small differences within each species, one including loss of a potential glycosylation site. Two non-secreting S. cerevisiae strains (FY1679 and var. uvarum) had non-transcribed copies of functional genes. Maximum enzyme activity was achieved with the S. cerevisiae FY1679 gene in an expressing vector, with an enzyme activity of 51 μmol of reducing sugar released from polygalacturonic acid μg protein⁻¹ min⁻¹, the highest so far reported for a yeast. Received: 19 May 2000 / Accepted: 6 August 2000     

The isolation and nucleotide sequence of the pyruvate decarboxylase gene from Kluyveromyces marxianus

We have determined the nucleotide sequence of a 2360-basepair (bp) region of the Kluyveromyces marxianus genome containing the structural gene for the enzyme pyruvate decarboxylase (PDC). Comparison of the deduced amino-acid sequence of this gene to that of the Saccharomyces cerevisiae PDC genes reveals extensive homology including a motif common to thiamin diphosphate-dependent enzymes.     

Cloning and characterisation of the ribosomal RNA genes of the dimorphic yeast,Yarrowia lipolytica

Summary The ribosomal RNA genes of Yarrowia lipolytica have been identified, both in restriction digests of total genomic DNA and in a pBR322 gene bank, by hybridisation with cloned Saccharomyces cerevisiae rDNA. The Y. lipolytica rDNA repeat unit is 8.9 kb in size and contains the genes for the 25S and 18S, but not the 5S, rRNA species. The number of copies of these repeat units is approx. 50 per haploid genome. Several clones were found which did not conform to the standard restriction map due to differences outside the coding region. It appears that there is either heterogeneity of the spacer sequence within a strain or that the Y. lipolytica rDNA genes may be present as a number of separate clusters within this yeast's genome.     

Mitochondrial DNA of Schizophyllum commune: restriction map,genetic map,and mode of inheritance

Summary Mitochondrial DNA (mtDNA) found in the basidiomycete Schizophyllum commune (strain 4–40) is a circular molecule 49.75 kbp in lenght. A physical map containing 61 restriction sites revealed no repeat structures. Cloned genes from Neurospora crassa, Aspergillus nidulans, and Saccharomyces cerevisiae were used in Southern hybridizations to locate nine mitochondrial genes, including a possible pseudogene of ATPase 9, on the restriction map. A probe from a functional ATPase 9 gene identified homologous fragments only in the nuclear genome of S. commune. Restriction fragment length polymorphisms (RFLPs) between mtDNA isolated from different strains of S. commune were used to show that mitochondria do not migrate with nuclei during dikaryosis.     

Evolution of mitochondrial DNA in yeast: gene order and structural organization of the mitochondrial genome of Saccharomyces uvarum

We have determined the size, the restriction map and the gene order of the mitochondrial genome of the yeast Saccharomyces uvarum. Sequence analysis of the mitochondrial COXII gene confirmed the position of this yeast in the Saccharomyces cerevisiae-like group, near Saccharomyces cerevisiae and Saccharomyces douglasii. Most mitochondrial genes have been positioned on this approximately 57-kb long genome and three regions containing putative replication origins have been identified. The gene order of S. uvarum suggests that the mitochondrial genome of the S.cerevisiae-like yeasts could have evolved from an ancestral molecule, similar to that of S. uvarum, through specific genome rearrangements. Received: 22 April / 2 September 1997     

Cloning and expression of Hormoconis resinae glucoamylase P cDNA in Saccharomyces cerevisiae

A cDNA coding for glucoamylase P of Hormoconis resinae was cloned using a synthetic oligonucleotide probe coding for a peptide fragment of the purified enzyme and polyclonal anti-glucoamylase antibodies. Nucleotide-sequence analysis revealed an open reading frame of 1848 base pairs coding for a protein of 616 amino-acid residues. Comparison with other fungal glucoamylase amino-acid sequences showed homologies of 37–48%. The glucoamylase cDNA, when introduced into Saccharomyces cerevisiae under the control of the yeast ADC1 promoter, directed the secretion of active glucoamylase P into the growth medium.     

PDC6, a weakly expressed pyruvate decarboxylase gene from yeast,is activated when fused spontaneously under the control of the PDC1 promoter

Summary Three structural genes encode the pyruvate decarboxylase isoenzymes in the yeast Saccharomyces cerevisiae. PDC1 and PDC5 are active during glucose fermentation where PDC1 is expressed about six times more strongly than PDC5. Expression of PDC6 is weak and seems to be induced in ethanol medium. Consequently, pdc1 pdc5 double mutants do not ferment glucose and do not grow on glucose medium. Spontaneous mutants, derived from such a pdc1 pdc5 strain, were isolated which could again ferment glucose. They showed pyruvate decarboxylase activity due to a duplication of PDC6. The second copy of PDC6 was expressed under the control of the PDC1 promoter, which was still present in the pdc1 strain. However, the resulting PDC1-PDC6 fusion gene could only partially substitute for PDC1: to achieve normal growth and high pyruvate decarboxylase activity strains carrying PDC1-PDC6 required a functional PDC5 gene which is dispensable in a PDC1 wild-type background. Thus, expression of PDC5 depends on the state of the PDC1 locus: low in the PDC1 wild-type background and high in PDC1-PDC6 fusion strains and, as shown previously, in pdc1 mutants. The activation of PDC5 expression in PDC1-PDC6 strains may be due to particular properties of the PDC1-PDC6 fusion protein or simply to the weaker expression of PDC1-PDC6 in comparison to the wild-type PDC1 gene.     

Identification of an upstream activation site in the pyruvate decarboxylase structural gene (PDC1) of Saccharomyces cerevisiae

Summary The upstream region of the Saccharomyces cerevisiae pyruvate decarboxylase structural gene, PDC1, has been isolated and fused to the indicator gene Escherichia coli lacZ. 1.2 kb of the upstream region has been sequenced. The PDC1-lacZ fusion has been integrated at the ura3-52 locus in the yeast genome, and has a basal level of expression on ethanol. On glucose media this level is increased 30–50 fold. An upstream activation site, UAS_pdc, between 793 and 535 by upstream from the ATG of PDC1, which mediates the response to glucose has been identified by deletion analysis. The UAS_pdc contains a consensus RPG box, originally identified in ribosomal protein genes (Leer et al. 1985). The function of UAS_pdc is independent of distance from the ATG. There is also an upstream repressing sequence located between 535 and 385 by upstream from the translational start of PDC1.     

Characterization of the <Emphasis Type="Italic">Arxula adeninivorans AHOG1</Emphasis> gene and the encoded mitogen-activated protein kinase

Arxula adeninivorans is an osmo-resistant yeast species that can tolerate high levels of osmolytes like NaCl, PEG400 and ethylene glycol. As in other yeast species, this tolerance is elicited by components of the high osmolarity glycerol (HOG) response pathway. In the present study, we isolated and characterized as a key component of this pathway the A. adeninivorans AHOG1 gene encoding the mitogen-activated protein (MAP) kinase Ahog1p, an enzyme of 45.9 kDa. The gene includes a coding sequence of 1,203 bp disrupted by a 57-bp intron. The identity of the gene was confirmed by complementation of a hog1 mutation in a Saccharomyces cerevisiae mutant strain and the high degree of homology of the derived amino acid sequence with that of MAP kinases from other yeasts and fungi. Under stress-free conditions, the inactive Ahog1p is present in low levels. When exposed to osmotic stress, Ahog1p is rendered active by phosphorylation. In addition, AHOG1 expression is increased. Assessment of the AHOG1 promoter activity with a lacZ reporter gene confirmed its inducibility by osmolytes, a characteristic not observed in homologous HOG1 genes of other yeast species. This specific property could account for the fast adaptation and high osmo-resistance encountered in this species.     

Structural heterozygosis at genes IL V2 and IL V5 in Saccharomyces carlsbergensiss

Summary Chromosomes XII and XIII of a Saccharomyces carlsbergensis brewing strain were analysed after their transfer into Saccharomyces cerevisiae by kar1-mediated single chromosome transfer. The lager yeast was found to be heterozygous for the isoleucine-valine biosynthesis genes IL V2 (encoding acetohydroxy acid synthase) and IL V5 (encoding acetohydroxy acid reductoisomerase). In both cases, Southern analysis showed restriction site polymorphisms, and that one allele hybridizes more strongly to that of S. cerevisiae than the other. The alleles with limited nucleotide sequence homology are located on chromosomes which recombine poorly with the corresponding S. cerevisiae chromosomes (XIII and XII) during meiosis. A cluster of ribosomal RNA genes is located on the chromosome XII with the S. cerevisiae-like IL V5, but not on the homoeologous chromosome. The present analysis supports the view that S. carlsbergensis is an amphiploid hybrid.     

A putative mitochondrial fission gene from the ectomycorrhizal ascomycete<Emphasis Type="Italic"> Tuber borchii</Emphasis> Vittad.: cloning,characterisation and phylogeny

Mitochondrial binary division is a complex process occurring in multiple steps, mediated by several proteins. In Saccharomyces cerevisiae, a mitochondrial membrane protein, Fis1p, is required for the proper assembly of the mitochondrial division apparatus. In this study, we report the cloning, characterisation and phylogenetic analysis of Tbfis1, a gene from the ectomycorrhizal ascomycetous truffle Tuber borchii, encoding for an orthologue of S. cerevisiae Fis1p. The Tbfis1 coding region consists of a 468-nucleotide open reading frame interrupted by four introns, which encodes for a polypeptide of 155 amino acids, having a predicted transmembrane domain structure typical of the Fis1p Family. Southern blot analysis revealed that Tbfis1 is a single-copy gene in the T. borchii genome. Tbfis1 is highly expressed during the first stages of T. borchii fruit body ripening, while its expression decreases during T. borchii mycelium ageing. Also, Virtual Northern blot analysis revealed Tbfis1 expression in the symbiotic phase of the fungus life cycle. Phylogenetic analysis allowed the identification of Tbfis1 orthologues in filamentous fungi, yeasts, plants, worms, flies and mammals, indicating that the function of the protein coded by this gene has been conserved during evolution.     

Mitochondrial DNA in the aquatic fungus Allomyces

Summary The mitochondrial DNA from seven species of the aquatic phycomycete Allomyces has been isolated and characterized by restriction enzyme analysis. Comparison of the mitochondrial DNA restriction enzyme fragmentation patterns showed pronounced differences not only among species but also among four isolates of A. arbuscula. The mitochondrial DNAs range in size from 39 kbp in A. neo-moniliformis to 56 kbp in A. macrogynus.A physical map of the mitochondrial DNA of Allomyces arbuscula strain Costa Rica 21 has been constructed. The genome is circular and has a size of 49.2 kbp. The genes coding for the small and large ribosomal RNAs, cytochrome oxidase subunits 1, 2, and 3, apocytochrome b, and ATPase subunits 6 and 9 were localized in the mitochondria) DNA by heterologous hybridization with specific mitochondria) gene probes from Saccaromyces cerevisiae and Neurospora crassa. Comparison of the gene map of the closely related species Blastocladiella emersonii with that of A. arbuscula indicates a similar gene order in the two organisms.     

Localization of yeast glucoamylase genes by PFGE and OFAGE

Summary Chromosomes of two closely related yeast strains, the amylolytic Saccharomyces diastaticus and the non-amylolytic Saccharomyces cerevisiae, were resolved by pulsed field gel electrophoresis (PFGE) and orthological field alteration gel electrophoresis (OFAGE). Electrophoretic karyotypes of these two strains are identical. Sixteen cloned Saccharomyces genes of known chromosomal location were used to identify individual chromosomes by Southern hybridization analyses. The Southern blots were reprobed with a cloned fragment of the STA2 glucoamylase gene of S. diastaticus. STA2 exhibits homology to STA1 and STA3 as well as the sporulation-specific glucoamylase (SGA) gene from both Saccharomyces strains. The three unlinked, homologous genes, STA1 (DEX2, MAL5), STA2 (DEX1) and STA3 (DEX3) encoding the extracellular glucoamylase isozymes GAI, GAII and GAIII in S. diastaticus were then assigned to chromosomes IV, II and XIV, respectively. The SGA gene, encoding an intracellular glucoamylase in both S. diastaticus and S. cerevisiae, was assigned to chromosome IX. Electrophoretic mapping of the STA and SGA genes is at present the only way to localize these genes, since glucoamylase repressor gene(s) (STA10, INH1 and/or IST2) are present in most laboratory strains of S. cerevisiae and the SGA phenotype is only detectable during sporulation.     

Structure and expression of a phosphate deficiency-inducible ribonuclease gene in<Emphasis Type="Italic"> Pholiota nameko</Emphasis>

Thirty-one cDNAs corresponding to pdi genes [inorganic phosphate (Pi) deficiency-inducible genes] were previously isolated through the differential screening of a cDNA library constructed from the mycelium of Pholiota nameko. Among the cDNAs, pdi370 was analyzed here. The deduced amino acid sequence showed high similarity to fungal ribonucleases (RNases) and contained two signature sequences conserved in T2 family RNases: CAS1 and CAS2. Genomic DNA harboring the pdi370 gene was isolated from a genomic library of P. nameko. Sequence analysis showed that the pdi370 gene is interrupted by 16 introns and that the promoter region contains two cis-acting sequences found in Pi deficiency-induced genes from Saccharomyces cerevisiae, together with several known functional elements, such as a TATA box. RNase activity in the mycelium and culture filtrate increased 5.6-fold and 5.2-fold, respectively, under Pi-deficient conditions. Staining for RNase activity showed that at least four RNases are induced and secreted under the conditions. The N-terminal sequence of one of them agreed with that of the pdi370 gene product.Communicated by J. Heitman     

Sulfate assimilation in Aspergillus terreus: analysis of genes encoding ATP-sulfurylase and PAPS-reductase

Two genes for the sulfate assimilation pathway in Aspergillus terreus were cloned. The genes sAT (coding for PAPS-reductase) and sCT (coding for ATP-sulfurylase) form a small gene cluster. Both genes are similar to their homologs in A. nidulans (sA and sC), Penicillium chrysogenum (aps) and Saccharomyces cerevisiae (MET3 and MET16). In the coding sequence of the sCT gene, a typical non-functional APS-kinase-like domain is present. The sCT gene is expressed in A. nidulans, but its expression there is less sensitive to methionine level than in the original species. Two regions 5′ upstream of sAT were found to be similar to those of sA. Received: 23 February 2000 / Accepted: 8 June 2000     

Isolation and characterization of KlUBP2, a ubiquitin hydrolase gene of Kluyveromyces lactis that can suppress a ts-mutation in CBF2, a gene encoding a centromeric protein of Saccharomyces cerevisiae

 The Kluyveromyces lactis UBP2 gene was isolated as a suppressor of a temperature-sensitive mutation in CBF2, a gene coding for a centromere-binding protein of Saccharomyces cerevisiae. The UBP genes are hydrolases than can cleave a ubiquitin moiety from a protein substrate. KlUBP2 is not essential for growth since a disruption of the KlUBP2 gene had little effect, except for a slight decrease in the growth rate. The stability of centromere-containing plasmids was not influenced either. In addition to KlUBP2, five S. cerevisiae genes involved in the ubiquitination pathway could suppress the ts-mutation in the CBF2 gene, namely UBA1, UBA2, UBP1, UBP2 and YUH1, although YUH1 was the only one that could do this like KlUBP2 from a single-copy plasmid. Surprisingly, these genes encode proteins with antagonistic activity as two, UBA1 and UBA2, are ubiquitin-activating enzymes whereas the other three are de-ubiquitinating hydrolases. Received: 6 July 1999 / 29 March 2000     

Isolation and characterization of the 3-phosphoglycerate kinase gene (pgk) from the filamentous fungus Trichoderma reesei

Summary The 3-phosphoglycerate kinase gene (pgk) from Trichoderma reesei was isolated by hybridization with the corresponding Saccharomyces cerevisiae PGK gene. The 1,545 nt long nucleotide sequence of the cloned gene codes for a 416 amino acid protein. The coding sequence contains two introns of 219 and 75 nt, respectively, at positions identical to those corresponding genes from the other filamentous fungi Aspergillus nidulans and Penicillum chrysogenum. This gene codes for two mRNAs of about 1.65 kb and 1.85 kb. The PGK protein of Trichoderma shows extensive homology to the PGKs of other fungi A. nidulans (77%), P. chrysogenum (73%) and Saccharomyces cerevisiae (69%). However, the PGKs of the two other filamentous fungi, A. nidulans and P. chrysogenum, seem to be more closely related to each other than to the T. reesei enzyme.Abbreviations bp Base pair - nt nucleotide - kb kilobase     

The gene for cadmium metallothionein from a cadmium-resistant yeast appears to be identical to CUP 1 in a copper-resistant strain

Summary A cadmium-resistant strain of Saccharomyces cerevisiae produces a cadmium metallothionein with the same characteristics as the copper metallothionein that is encoded by CUP 1 in a copper-resistant strain. The structural gene for metallothionein from the cadmium-resistant strain resembles CUP 1 in terms of the fragmentation patterns generated by restriction enzymes. Furthermore, the gene may be amplified as 2.0 kb repeating units in both the cadmium-resistant and the copperresistant strains. However, transformants with a plasmid that carried the metallothionein gene from the cadmiumresistant strain were resistant to copper but not to cadmium. It appears that the same metallothionein gene, CUP 1, is amplified in both cadmium- and copper-resistant yeasts. However, the mechanism for the cadmiumspecific inducibility of the gene may be restricted to the cadmium-resistant strain.