Highly-efficient transformation of the homobasidiomycete Schizophyllum commune to phleomycin resistance

Regulatory sequences of the glyceraldehyde-3-phosphate-dehydrogenase (GPD) gene from the homobasidiomycete Schizophyllum commune were fused to the coding sequence of the ble gene from Streptoalloteichus hindustanus, which codes for a phleomycin-binding protein. The resulting construct transformed S. commune to phleomycin resistance at a high frequency (up to 10⁴ transformants/g DNA per 10⁷ protoplasts) when regeneration was done in 0.5 M MgSO₄. A similar construct with regulatory sequences from Aspergillus nidulans failed to give transformants, showing the importance of homologous regulatory sequences for the expression of genes in S. commune. The homologous GPD promoter could be deleted up to position -130 without any effect on the number of phleomycin-resistant transformants. This is the first effective stable transformation system in a homobasidiomycete employing antibiotic resistance.     

Pulsed-field gel electrophoretic analysis of Schizophyllum commune chromosomal DNA

Summary Six chromosomal DNA bands of Schizophyllum commune have been resolved using transverse alternating field electrophoresis. The estimated sizes of the chromosomal DNAs ranged from 5.1 to 1.2 megabase pairs (Mb), the total genome size being approximately 35–36 Mb. Chromosomal length polymorphisms were found between the two S. commune isolates examined. The DNA bands corresponding to the two chromosomes containing the A and B mating-type loci were identified in hybridization experiments using probes specific to their respective linkage groups. The utility of eluted chromosomal DNAs as hybridization probes to select clones from genomic libraries, and the use of these clones in transformation experiments to identify genes of interest, are discussed.     

Nucleotide base sequence of the mitochondrial COIII gene of Schizophyllum commune

Summary The cytochrome oxidase subunit III gene (COIII) of the Badidiomycete S. commune has been identified, cloned, and sequenced. The gene contains no introns, is AT-rich (69%) and exhibits a high degree of similarity to sequences from Ascomycetes. While most mitochondrial genes use both TGA and TGG to specify tryptophan, the COIII gene of Schizophyllum uses TGG exclusively. Translation requires no deviation from the universal code.     

Transformation of Phanerochaete chrysosporium and Neurospora crassa with adenine biosynthetic genes from Schizophyllum commune

Summary Protoplasted basidiospores of two different adenine auxotrophs of the lignin-degrading basidiomycete Phanerochaete chrysosporium were transformed to prototrophy using plasmids containing genes encoding adenine biosynthetic enzymes from Schizophyllum commune. Fragments containing these genes were subcloned into pUC18 and P. chrysosporium transformants obtained with these subclones were analyzed. The subclones were mapped for restriction sites and the approximate locations of the complementing genes were determined. One of these plasmids was used to transform the Neurospora crassa auxotrophic strain ade2, thereby identifying the S. commune ade5 biosynthetic gene as encoding phosphoribosylaminoimidazole synthetase.     

Isolation of the DNA sequence coding indole-3-glycerol phosphate synthetase and Phosphoribosylanthranilate isomerase of Schizophyllum commune

Summary A Schizophyllum gene library was made in plasmid pRK9, Plasmids from this library were tested for their ability to complement several auxotrophic mutations of Escherichia coli. The goal was to isolate a Schizophyllum auxotrophic gene that could be used to transform a corresponding Schizophyllum auxotrophic mutant to prototrophy. Complementation was observed only for E. coli trpC indole 3-glycerol phosphate synthetase (IGPS) and phosphoribosyl-anthranilate isomerase (PRAI) mutations. Plasmids with a Schizophyllum sequence coding for both IGPS and PRAI activities were recovered from E. coli transformants. Expression of the Schizophyllum gene (TRP1) in E. coli is probably dependent on the Serratia marcescens promoter of plasmid pRK9. The DNA sequence containing the Schizophyllum TRP1 gene was not obviously rearranged in cloning.     

Strain specific differences in ribosomal DNA from the fungus Schizophyllum commune

Summary CsCl-bisbenzimide gradients were used to purify ribosomal DNA (rDNA) from Schizophyllum commune total DNA. Southern hybridizations demonstrate that this DNA codes for rRNA. Restriction mapping of the rDNA from four strains revealed strain variation with repeat lengths of 9.2–9.6 kbp. Specific differences in the length of the rDNA repeat in different strains are due to insertions of 0.2 or 0.4 kbp of DNA at a single site. Different strains also show restriction site polymorphisms. Our analysis demonstrates the caution that must be exercised when interpreting restriction data from genomes containing restriction polymorphisms. Restriction digests with MspI and HpaII indicate that the rDNA contains 5-methylcytosine and that the unit repeats are not methylated identically, but rather differentially. This is the first report of methylated rDNA in fungi.     

The Aα mating-type locus ofSchizophyflum commune: Structure and function of geneX

GeneX maps immediately right of geneY at the A mating-type locus inSchizophyllum commune. AllelesX1, X3 andX4 were isolated, subcloned, and sequenced. The structure of the alleles and their relationship to the A locus is described. The deducedX isoforms possess no recognized motifs common to other polypeptides and no significant similarity to any sequences in the protein databases.X alleles do not activate A-regulated development when transformed into recipient strains possessing different A mating types or when these transformants are mated with tester strains. AnX1-disrupted construction yielded a high frequency (33%) of homologous gene replacements.X-disrupted mutants have wild-type phenotypes and mate normally. Both the functional analyses and sequence data forX1,X3, andX4 suggest that the right boundary of the A mating-type locus falls betweenAY andX. We propose that theZ andY genes constitute the Aoc locus in its entirety.     

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.     

An expression system for the functional analysis of pheromone genes in the tetrapolar basidiomycete Schizophyllum commune

The investigation of putative pheromone genes of basidiomycetes has been difficult since the small open reading frames are essentially annotated on the basis of a C-terminal farnesylation signal. In order to identify the functional reading frame, expression of small DNA fragments in the fungus is necessary. The expression system developed in the presented paper allows fusion to the promoter of the tef1 gene encoding the constitutively and highly expressed translation elongation factor EF1alpha. This system has been shown to be functional using an easily selectable gene, ura1. The application to identification of functional pheromone genes has been shown with the newly detected bap2(4) gene. The Bap2(4) pheromone is the first Balpha pheromone gene activating only a single receptor specificity.     

Transformation as a method of increasing gene copy number and gene expression in the basidiomycete fungus Coprinus cinereuss

Summary Seven transformants having varying numbers of non-homologously integrated copies of the isocitrate lyase gene, acu-7, were analysed for enzyme activity. Maximum levels of activity, 3.8 times that of the wild type, were observed in a transformant with only two gene copies whereas eight gene copies in another transformant led to only 25% wild type activity. Acu⁺ transformants were not selected directly for expression of acu-7 but as cotransformants. Analysis of 14 transformants not expressing acu-7 showed that four contained transforming DNA sequences and significantly, two had evidence of non-homologously integrated tandem duplications of the entire acu-7 plasmid DNA. The site of integration of the gene was thus important in determining whether or not it was. expressed and to what level it was expressed. A comparison of induced and uninduced levels of enzyme activity confirmed that the enzyme was still tightly regulated.     

Single oligonucleotide nested PCR: a rapid method for the isolation of genes and their flanking regions from expressed sequence tags

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 predecessors 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.     

Developmental regulation of the methylation of the ribosomal DNA in the basidiomycete fungusSchizophyllum commune

Summary We started partial overlapping hybridization on chromosome V from the centromere-linked gene SU8 towards centromere 5. Transformation of Podospora anserina with the cloned sequences and subsequent genetic analysis reveal that cosmids issuing from a 60 kb region never integrate near centromere 5, whereas cosmids from either side return mostly to this locus. Molecular analysis shows that heterologous integrations of cosmids spanning the 60 kb region occurs in a specific sequence of 11 kb common to all the inserts. We propose that this sequence contains a gene or a structure which is lethal in a duplicated form. Straightforward interpretation of genetic data suggests that this structure does not correspond to centromere 5. Its function is still unknown.     

Isolation of the Bα and Bβ mating-type loci of Schizophyllum commune

The B mating type of the basidiomycete fungus, Schizophyllum commune is determined by two, tightly linked, multi-specificity (also called multi-allelic) loci: B and B. A plasmid library was used in DNA-mediated transformation to obtain transformants that displayed B-directed development. Plasmids that conferred B1 and B1 mating-type specificities were rescued from the transformants. Fragments of DNA from each plasmid hybridized to genomic DNA from the strain used to make the plasmid library; however, they did not hybridize, or hybridized only weakly, to genomic DNA from strains with mating-type specificities different from B1 or B1. The cloned fragments are presumed to correspond to active regions of each B mating-type locus.     

Molecular organisation of an A mating type factor of the basidiomycete fungus Coprinus cinereus

Summary The A3 and A3 genes, which together constitute the A42 mating type factor of Coprinus cinereus, were isolated from a cosmid genomic library by walking 50 kb, a map distance of 0.5 units, from the closely linked metabolic gene pab-1. Cosmid clones having A gene function were identified by transformation into compatible A6 (22) and A5 (11) host cells where either 3 or 3 was expected to elicit the A factor — regulated development of unfused clamp cells. DNAs were digested with various enzymes before transformation in order to identify the smallest fragments containing an active 3 or 3 gene. Two non-overlapping fragments were identified as containing the 3 and 3 genes respectively. Southern hybridisation analyses showed that these two cloned genes had no detectable sequence homology, and that there was little or no hybridisation to the and gene alleles that constitute the A5 and A6 factors. 3 and 3 were shown to be less than 2.0 kb apart and embedded in a DNA sequence extending over 9.0 kb which was unique to our A42 strain and may contain a third A factor gene.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of the antitumor clavaric acid-producing basidiomycete <Emphasis Type="Italic">Hypholoma sublateritium</Emphasis>

The basidiomycete Hypholoma sublateritium produces clavaric acid, an antitumor isoprenoid compound. Arthrospores of this fungus were transformed by Agrobacterium tumefaciens-mediated conjugation. Five plasmids carrying different regulatory sequences to drive expression of the hph (hygromycin phosphotransferase) gene were tested. The promoter used was critically important in order to express heterologous genes in H. sublateritium. Constructions carrying the Agaricus bisporus glyceraldehyde-3-phosphate dehydrogenase promoter (Pgpd) showed a good transformation efficiency, whereas constructions with the gpd promoter from ascomycetes were ineffective. Transformant clones showed a random integration pattern of plasmid DNA. Most transformants showed a single integrated copy of the transforming plasmid, but about 1.5% showed double or multiple integrations. All the analyzed transformants were mitotically stable and maintained the integrated exogenous DNA in the absence of antibiotic. The green fluorescent protein gene was expressed from the A. bisporus gpd promoter, as shown by RT-PCR studies, but no significant fluorescence was observed. Transformation of H. sublateritium opens the way for the genetic manipulation of clavaric acid biosynthesis in this fungus.     

DNA-mediated transformation of the secondarily homothallic basidiomycete Coprinus bilanatus

Summary The Coprinus cinereus trp-2 gene which encodes a trifunctional protein of the tryptophan biosynthetic pathway was used to transform a trp-2 mutant of the secondarily homothallic species Coprinus bilanatus. Southern blot analysis confirmed the presence of transforming DNA in the genome of transformants and indicated the presence of tandemly duplicated copies of the plasmid in some of these. Although these two species of Coprinus are regarded as closely related, no cross-hybridisation between the homologous trp-2 genes was detected.