The isolation of specific genes from the basidiomycete Schizophyllum commune

Summary We have developed a routine way to isolate genes directly from the basidiomycete fungus, Schizophyllum commune. Plasmid DNA from a genomic gene library was used to isolate five specific genes by complementation of Schizophyllum mutations via transformation. The mutant strains were deficient in the ability to synthesize either adenine (ade2 and ade5), uracil (ural, encoding orotidine-5-phosphate decarboxylase; OMPdecase), tryptophan (rpl, encoding indole-3-glycerol phosphate synthetase; IGPS) or para aminobenzoic acid (pab1). In each case, Southern analysis revealed that transformation to prototrophy was concomitant with the integration of vector sequence into the genome of the S. commune mutant. Total DNA from transformants was restricted, religated, and used to transform E. coli. Ampicillin resistant plasmids were recovered from E. coli and tested for their ability to transform the corresponding mutant of S. commune. Plasmids complementing the ade2, adeS, pabl, trpl, and ural mutations were recovered.     

The ade4 gene of Schizosaccharomyces pombe: cloning,sequence and regulation

We report the isolation and sequence of the Schizosaccharomyces pombe ade4 gene which encodes the glutamine phosphoribosylpyrophosphate amidotransferase, the first enzyme of the purine nucleotide de-novo biosynthetic pathway. The enzyme contains 533 amino acids and its sequence exhibits homologies to the corresponding enzymes of Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis, chicken, rat, and human. In contrast to the situation in S. cerevisiae, adenine does not repress ade4 expression at the mRNA level and also other nutritional signals seem not to affect its expression.     

The red/white colony color assay in the yeast Saccharomyces cerevisiae: epistatic growth advantage of white ade8-18, ade2 cells over red ade2 cells

In the yeast Saccharomyces cerevisiae the ade2, and/or the ade1, mutation in the adenine biosynthetic pathway leads to the accumulation of a cell-limited red pigment, while epistatic mutations in the same pathway, i.e. ade8, preclude this phenomenon, resulting in normal white colonies. The shift in color from red to white (or vice versa) with a combination of appropriate wild-type and mutant alleles of the adenine-pathway genes has been widely utilized as a non-selective phenotype to visualise and quantify the occurrence of various genetic events such as recombination, conversion and aneuploidy. It has provided an invaluable tool for the study of gene dosage and plasmid stability. In competition experiments between disrupted ade2, ade8-18 transformants carrying either a functional or non-functional episomal ADE8 gene, we verified that white ade8 ade2 cells show a remarkable selective advantage over red ade2 cells, with important implications on the use of this assay for the monitoring of genetic events. The accumulation of the red pigment in ade2 cells is likely to be the cause for impaired growth in these cells. Received: 15 June / 15 August 1996     

Homologous transformation of the lignin-degrading basidiomycete Phanerochaete chrysosporium

Summary A clone containing the Phanerochaete chrysosporium ade1 gene was isolated from a EMBL3 genomic library using the ade5 gene encoding aminoimidazole ribonucleotide synthetase, from Schizophyllum commune, as a probe. A 6.0 kb fragment incorporating the ade1 gene was subcloned into pUC18 (pADE1) and used to transform the P. chrysosporium ade1 auxotrophic strain. Transformation frequencies were similar to those obtained previously with the S. commune ade5 gene; however, homologous transformants arose earlier than heterologous transformants. The transformants were mitotically and meiotically stable and Southern blot analysis indicated that the plasmid, pADE1, integrated ectopically in single or multiple copies. The pADE1 insert was mapped for restriction sites and the approximate location of the ade1 gene within the insert was determined.     

Multiple-copy integration in the yeast Yarrowia lipolytica

Using an EcoRI-BglII fragment of the G unit of the rDNA of Y. lipolytica and a set of 11 deletions in the URA3 promoter, we have constructed several plasmids to test gene amplification in the rDNA. These plasmids contain the rDNA fragment for integration, defective versions of the URA3 gene, the XPR2 gene encoding alkaline extracellular protease (AEP) as a reporter gene, and part of the pBR322 plasmid for selection and replication in E. coli. Among these plasmids, one corresponds to a deletion which allows multiple integration into the rDNA (plasmid pINA773). Two other plasmids (pINA767 and pINA772) give multiple integration only with a mutated URA3 gene. Transformants carrying these three plasmids were tested for copy number, stability, chromosomal localization and AEP secretion. Transformants containing plasmids pINA767, 772 and 773 displayed an average copy number of 5, 12 and 25–60 copies respectively of the plasmid, as estimated by PCR and DNA hybridization. Integrations occurred in only one chromosome except for transformants containing 60 copies where copies were observed at least in two different chromosomes. Multiple integrations were found both as tandem repeats and as dispersed copies. Plasmid copy number was stable in both minimum and rich media, for strains containing less than ten copies per cells. However, for higher copy number, multiple integrations were stable only when AEP synthesis was not induced, while in inducing medium stability of the multiple integrations was dramatically affected.     

Comparison of the lignin-degrading white rot fungi Phanerochaete chrysosporium and Sporotrichum pulverulentum at the DNA level

Summary DNA-hybridisation studies showed a close relationship between Phanerochaete chrysosporium ME446, most used in lignin degradation studies, and Sporotrichum pulverulentum Novobranova, the other standard lignin degrading strain. Two other strains of P. chrysosporium were both less related. We show that P. chrysosporium ME446 and S. pulverulentum Novobranova both have a GC-content of 59% for chromosomal DNA with the rRNA genes present as an AT-rich satellite; the mitochondrial DNA has a GC-content of 33%. The genome size estimated for P. chrysosporium ME446 is about 4–5 × 10⁷ bp.     

Genomic organization of a cellulase gene family in Phanerochaete chrysosporium

Summary Southern blot and nucleotide sequence analysis of Phanerochaete chrysosporium BKM-F-1767 genomic clones indicate that this wood-degrading fungus contains at least six genes with significant homology to the Trichoderma reesei cellobiohydrolase I gene (cbh1). Using pulsed-field gel electrophoresis to separate P. chrysosporium chromosomes, the six cellulase genes were found to hybridize to at least three different chromosomes, one of which is dimorphic. The organization of these genes was similar in another P. chrysosporium strain, ME 446. It is clear that, unlike T. reesei, the most well-studied cellulolytic fungus, P. chrysosporium contains a complex, cbh1-like gene family.     

Transformations of Penicillium islandicum and Penicillium frequentans that produce anthraquinone-related compounds

Wild-type strains of Penicillium islandicum and Penicillium frequentans, which produce anthraquinone and related compounds, were transformed to benomyl and hygromycin B resistance. Plasmids pSV50 and pBT6, with benomyl-resistant -tublin genes, and plasmids pAN7-1 and pDH25, with a bacterial hygromycin phosphotransferase gene under the control of Aspergillus nidulans sequences, were used respectively. Transformation frequencies with these plasmids were 10–20 transformants per g of DNA per 4-8×10⁷ viable protoplasts. Intergration of plasmid DNAs into chromosomal DNAs was confirmed by Southern-blot analysis. Copy numbers and sites of integration varied among transformants. The integrated plasmid DNAs conferring a drug-resistant phenotype were mitotically stable with or without selection. The demonstration of such transformation systems is the essential first step in the application of recombinant DNA technology to study the biosynthetic genes of anthraquinone and related compounds in P. islandicum and P. frequentans.     

Concurrent transfer and recombination between plasmids encoding for heat-stable enterotoxin and drug resistance in porcine enterotoxigenicEscherichia coli

The frequency of and genetic mechanisms for simultaneous transfer of genes encoding for tetracycline and sulpha-streptomycin resistance, heat-labile (LT) and heat-stable (ST-mouse) enterotoxin production in porcine enterotoxigenicEscherichia coli toEscherichia coli K12 were investigated. SevenE.coli strains of 0-group 149 were studied by conjugation and transformation experiments. All strains transferred tetracycline-resistance plasmids at a high frequency. No interaction was observed between these plasmids and those encoding for LT production. However, most tetracycline-resistant recipient cells were ST-mouse⁺ following recombination events between plasmids encoding for colicin B and ST-mouse production and plasmids encoding for tetracycline resistance. Alternatively, when selecting for sulpha or streptomycin resistance a majority of the transconjugants were also ST-mouse⁺, as plasmids coding for sulpha and streptomycin were mobilized by the colicin B and ST-mouse encoding plasmid. Since the simultaneous transfer of genes encoding for drug resistance, colicin B and ST-mouse production are common events in vitro, they might also occur frequently in vivo during antibiotic selective pressure.     

Physical mapping of genes for chloroplast DNA encoded subunit polypeptides of the ATPsynthase complex from Petunia hybrida

Summary Escherichia coli minicells harbouring the cloned restriction fragment Sall S9 from P. hybrida chloroplast DNA synthesize the beta and epsilon polypeptide subunits of the CF₁ component of the chloroplast ATPsynthase complex. The polypeptides were identified by molecular weight determination and immunoprecipitation. The position of the atpB and the atpE gene, encoding respectively the beta and epsilon subunit, on the Sall S9 fragment was determined in more detail by studying polypeptide synthesis directed by subclones of the S9 fragment in E. coli minicells. The atpB and atpE genes are located close to the rbcL gene, the distance between the rbcL gene and atpB gene being approximately 770 bp. Analysis of the expression of subclones of the S9 fragment in E. coli minicells also revealed that the atpE gene can be transcribed and translated independently of the expression of the atpB gene.The location of the genes coding for the alpha subunit (atpA gene) and the proteolipid subunit III of CF₀ (atpH) of the ATPsynthase complex on the physical map of P. hybrida cpDNA was determined by hybridization of restriction enzyme digests of petunia cpDNA with cloned cpDNA fragments from Spirodela and wheat, containing internal parts of respectively the atpA and the atpH gene. The two genes map close together within a region of 5.2 kbp on the physical map of P. hybrida cpDNA. The distance between the atpA gene and the atpB and atpE genes is approximately 42 kbp.Abbreviations cpDNA chloroplast DNA - rbcL gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase - CF₁ coupling factor, extrinsic part of the chloroplast ATPsynthase complex - kbp kilo base pairs     

Centromeric DNA of Kluyveromyces lactis

Summary A direct selection method was used to isolate centromeres from a genomic library of the yeast Kluyveromyces lactis. The method is based on the lethality at high copy number of the ochre-suppressing tRNA gene SUP11. Five different chromosomal fragments were found that confer mitotic stability to plasmids containing a replication origin of K. lactis (KARS). In addition, KARS plasmids containing these fragments have a copy number or approximately one, and each of the five fragments hybridizes to a different chromosome of K. lactis. From these results we conclude that five of the six centromeres of K. lactis have been isolated. These centromeres do not function in S. cerevisiae.     

Cordycepin in<Emphasis Type="Italic"> Schizosaccharomyces pombe</Emphasis>: effects on the wild type and phenotypes of mutants resistant to the drug

The adenosine analogue cordycepin (3-deoxyadenosine) inhibits growth and causes aberrant cell morphology in the fission yeast, Schizosaccharomyces pombe. Exogenously added thiamine, the pyrimidine moiety of the thiamine molecule, and adenine alleviate its growth-disturbing effect. At concentrations that do not inhibit growth, the drug reduces mating and sporulation and causes a decrease in the mRNA level of gene ste11 and the ste11-dependent gene, mei2. The mating- and sporulation-inhibiting effect of cordycepin is overcome by adenine. A mutant disrupted for the ado1 gene encoding adenosine kinase exhibits a cordycepin-resistant and methionine-sensitive phenotype, excretes adenosine into the medium and mates and sporulates poorly in the presence of adenine. A S. pombe mutant containing a frameshift mutation at the beginning of the carboxy-terminal half of gene ufd1 (the Saccharomyces cerevisiae UFD1 homologue) is cordycepin-resistant and sterile. Strains disrupted for the ufd1 gene only form microcolonies.Communicated by M. Yamamoto     

Mapping of rRNA genes by integration of hybrid plasmids in Schizosaccharomyces pombe

Summary The major rRNA genes of the fission yeast Schizosaccharomyces pombe were mapped on chromosome III by plasmid integration. The integration vector YIp33 containing S. cerevisiae LEU2 gene was combined with the S. pombe rDNA. Since LEU2 complements S. pombe leu1 deficiency, it could be used as the genetic marker for integration. The 10.4 kb rDNA repeat contained ARS sequence, and therefore 2.4 kb and 0.7 kb subfragments not containing ARS were subcloned into YIp33 and transformed leu1 S. pombe cells to Leu⁺. Genetic analyses of the transformants indicated that the integrated rDNA resides in the long arm of the shortest chromosome III, tightly linked to ade5 (1.4 cM). This result is consistent with our previous finding that the DAPI-stained smallest chromosomes were associated with the nucleolus (Umesono et al. 1983).Abbreviations ARS autonomously replicating sequence - DAPI 4,6-diamidino-2-phenylindole - kb kilo base pairs - rDNA DNA segment containing ribosomal RNA genes - rRNA ribosomal RNA     

Whole cell transformation of the alfalfa fungal pathogen Colletotrichum trifolii

Summary A transformation system for Colletotrichum trifolii, a fungal pathogen of alfalfa, has been developed using whole cells as recipients. Hygromycin B and benomyl resistant colonies were isolated following treatment of fungal tissue with lithium acetate and separate plasmids containing the respective genes which confer resistance to these antibiotics. The DNA was stably integrated into the fungal chromosome. This approach to transformation has general utility for phytopathogenic fungi and represents an initial step in the molecular analysis of virulence determinants in this race specific fungus.     

Control of the expression of the ADE2 gene of the yeast Saccharomyces cerevisiae

The ADE2 gene encodes AIR-carboxylase which catalyzes the sixth step of the purine biosynthetic pathway in Saccharomyces cerevisiae. We have analyzed the effect of deletions in the promoter region of this gene on the expression of the enzyme using a fusion of the ADE2 gene promoter to the bacterial lacZ gene. Adenine added to the growth medium repressed the expression of the fusion at the level of mRNA. The ADE2-lacZ fusion expression can be slightly activated in response to amino-acid starvation, but only in Gcn4 ⁺ strains and in an adenine-supplemented medium. In the absence of adenine in the medium ADE2 gene expression is derepressed, and neither starvation for histidine nor a gcd1 general control regulatory mutation leads to additional derepression. Our experiments indicate that the ADE2 gene of the purine biosynthetic pathway is under both specific adenine control and the general amino-acid control system. The cis-acting promoter elements mediating both modes of regulation overlap each other and are located around the proximal TGACTC sequence.     

Physical mapping of the Schizosaccharomyces pombe histone genes

The histone-encoding genes in Schizosaccharomyces pombe were physically mapped by hybridisation to filters containing cosmid and P1 genomic libraries. The H2A.2 gene and the H2A.1-H2B.1 gene pair mapped between the ade6 and rikl genes on chromosome III. The three H4–H3 gene pairs were mapped to three different regions by a H4.1 probe. Southern analysis of clones from each region revealed the positions of the three H4–H3 gene paris. H4.1–H3.1 was localised to chromosome I between the mei2 and rad1 genes; H4.2–H3.2 mapped between rad3 and cdc2 on chromosome II; H4.3–H3.3 was localised to a region between the nuc1 and puc1 genes on chromosome II.     

Two amino-acid biosynthetic genes are encoded on the plastid genome of the red alga Porphyra umbilicalis

Summary To isolate the gene encoding the amino-acid biosynthetic enzyme acetolactate synthase (ALS) from the red alga Porphyra umbilicalis, PCR experiments were carried out using P. umbilicalis DNA as the template and degenerate oligonucleotides representing conserved regions of ALS amino-acid sequences. Interestingly, the PCR product (0.9 kb) hybridized exclusively to the plastid DNA of this red alga. DNA sequencing of two contiguous EcoRI plastid DNA clones revealed a 590 aminoacid open reading frame with 55 to 61% identity to cyanobacterial ALS sequences. A second gene (argB) encoding another amino-acid biosynthetic enzyme, Nacetylglutamate kinase, was identified upstream of, and on the opposite strand to the gene encoding ALS (ilvB). This is the first molecular characterization of a gene for an arginine biosynthetic enzyme from any plant. In addition, two tRNA genes, trnT(GGU) and trnY(GUA), were detected downstream from ilvB while four tRNA genes, trnfM(CAU), trnA(GGC), trnA(GGC), trnS(GCU) and trnD(GUC), were found downstream from argB. trnA(GGC) is not found in the chloroplast genomes of land plants.     

An abnormal cell division cycle in an AIR carboxylase-deficient mutant of the fission yeast Schizosaccharomyces pombe

Summary Adenine-requiring mutant strains of S. pombe enter the stationary phase after depleting a culture medium of adenine or its analogues. Stationary phase cells of six mutants defective at different stages of the purine nucleotide synthetic pathway were examined for cell volume and DNA content, and then compared in these respects with those of a prototrophic wild-type strain. The cell cycle of the wild-type strain was arrested in the G₂ phase (2C state) in the nitrogen rich medium, as is evident from DNA content per cell (0.0425 pg) and cell volume (47.7 m³). An AIR carboxylase-deficient (ade6) mutant strain was found to have an unusual cell volume (307.4 m³) and DNA content (0.1187 pg). By DAPI fluorescence microscopy, each mutant cell was seen to contain only one enlarged nucleus, which indicates the absence of cell populations containing cells in the 4C state of the S phase following nuclear division. It then follows that in ade6 mutant cells, DNA synthesis occurs in the absence of a completed nuclear division. Thus in S. pombe cells, the completion of nuclear division is not necessarily required for the next cycle initiation of DNA synthesis under certain physiological conditions.Abbreviation AIR aminoimidazole ribonucleotide - DAPI 4,6-diamidino-2-phenylindole - PCA perchloric acid - DABA 3,5-diaminobenzoic acid     

An addition to the chromosome map of Schizosaccharomyces pombe: the localization of gua genes

Summary The gua1 and gua2 genes, encoding enzymes for the biosynthesis of guanylic acid (GMP) in Schizosaccharomyces pombe, were found to be located on chromosomes II and I respectively, and the sites of these genes were precisely determined on the chromosome maps. With this additional finding, the order of all the genes involved in the biosynthesis of purine nucleotides in S. pombe has now been established.