Charles P. Novotny

A method for extracting high-molecular-weight deoxyribonucleic acid from fungi.

Abstract A new method for isolating high-molecular-weight DNA from mycelium of the basidiomycete Schizophyllum commune is reported. Lyophilized mycelium is broken with mortar and pestle, and DNA extracted during gentle shaking in buffer containing 2% sodium dodecyl sulfate and toluene. DNA with double-strand length in excess of 80 kilobase pairs and singlestrand length of 36 kilobases can be prepared routinely in milligram quantities. This DNA is of high purity and suitable for reassociations and recombinant DNA studies. The procedure has also been used to prepare DNA from another filamentous basidiomycete, Heterobasidion annosum , and from yeast. With slight modification the procedure is attractive for isolating recombinant plasmids from yeast.

Transformation of the basidiomycete, Schizophyllum commune

Protoplasts of a Schizophyllum commune tryptophan auxotroph (trp1), deficient in indole-3-glycerol phosphate synthetase (IGPS), were transformed to trp+ with plasmid DNA containing the Schizophyllum TRP1 sequence. Efficiencies up to 30 transformants per microgram of plasmid DNA were obtained. Southern blots reveal that the transforming DNA is integrated in chromosomal DNA. The trp+ phenotype of transformants is stable in meiosis and mitosis. Transformants possess IGPS activity comparable to wild-type cells.

Cloning and comparison of Aα mating-type alleles of the basidiomycete Schizophyllum commune

SummaryAn Aα mating-type allele (Aα4) was isolated by walking the chromosome from the closely linked PAB1 gene. A cosmid clone containing the Aα1 allele isolated from the walk was used as a probe to recover the Aα1 allele from another cosmid library. Cosmids encoding mating-type activity were identified by transforming Schizophyllum cells and screening for activation of A-regulated development. Putative mating-type transformants were confirmed in mating tests and genetic analyses of progeny. The identity of the specific alleles isolated was demonstrated by showing that their effectiveness in transforming for mating type is limited to recipient strains possessing an Aα allele different from the one encoded by the cloned sequences. Transforming DNA is active in trans, suggesting that Aα encodes a diffusible product. Restriction mapping shows that Aα1 and Aα4 are coded in the same physical region of the genome, but within a subregion that contains extensive sequence divergence. In addition, Southern analyses show that there is only one copy of Aα1 or Aα4 per haploid genome, and that they do not cross-hybridize to one another or to any of the other Aα alleles. Aα1 and Aα4 were subcloned as 2.8 and 1.2 kb fragments, respectively, retaining in transformation all the mating-type activity demonstrated of the original cosmids.

Strain specific differences in ribosomal DNA from the fungus Schizophyllum commune

SummaryCsCl-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.

Transformation ofSchizophyllum commune: An analysis of parameters for improving transformation frequencies

Abstract The procedure used for transforming protoplasts prepared from germinated basidiospores of Schizophyllum commune has been improved to yield 10 3 transformants per microgram of DNA when using 10 7 protoplasts. Important to the protocol are keeping the protoplasts on ice and creating an osmotic shock when mixing DNA and protoplasts. Protoplasts may be stored at −70°C; this eliminates the need to prepare fresh protoplasts for each experiment. Titrations of CaCl 2 , polyethylene glycol, DNA, and protoplasts were tested individually to maximize the effect of each. Transformation does not occur without CaCl 2 or polyethylene glycol. The optimal concentration for CaCl 2 is 50 m M and that for polyethylene glycol ( M r = 3350) is 25% (w/v). At optimal polyethylene glycol and CaCl 2 concentrations, the number of transformants obtained and the calculated transformation frequencies (transformants per microgram of DNA) are dependent on protoplast and DNA concentrations.

Isolation and characterization of mitochondrial DNA from the basidiomyceteSchizophyllum commune

Abstract Mitochondrial DNA was extracted from DNase I-treated mitochondrial preparations from a homokaryotic strain of the woodrotting basidiomycete Schizophyllum commune . An AT-rich satellite DNA was also isolated from both homokaryotic and dikaryotic strains using CsCl-bisbenzimide gradients. The satellite DNA and the DNA from DNase I-treated preparations of mitochondria have identical buoyant densities and hybridize to the same restriction fragments in Southern hybridizations. It is concluded that this satellite DNA is mitochondrial DNA. Restriction analyses of the mitochondrial DNA of strain 4–40 with five endonucleases show the mitochondrial genome to be about 49.85 kbp. Mitochondrial DNA from the 4–39 × 4–40 dikaryon has identical restriction fragments. The satellite, which we have shown to be mitochondrial DNA, represents 3–4% of the total cellular DNA and averages 22–29 copies per haploid cell under our growth conditions. Mitochondrial DNA prepared from three other strains of Schizophyllum revealed restriction enzyme polymorphisms and ranged in size from 50.3 to 52.2 kbp.

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

SummaryMitochondrial 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.

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

SummaryA 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.

The isolation of specific genes from the basidiomycete Schizophyllum commune

SummaryWe 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.

Cloning and characterization of aSchizophyllum gene withAβ6 mating-type activity

The A-pathway of development in the basidiomycete fungusSchizophyllum commune may be activated by either of two mating-type loci,Aξ andAβAα consists of two multiallelic genes,Y andZ. Y contains a putative homeodomain; Z contains a homeodomain-related region. Non- self combinations of Y and Z form heteromultimers which are thought to be transcription factors of developmental genes. To more completely understand A-regulated development it is necessary to address the issue of functional redundancy, i.e., how do two different mating loci,Aα andAβ, both manage to regulate the same pathway. Here we report the structure of a gene withAβ6 activity. This gene, denotedAβV6, encodes a deduced polypeptide of 640 amino-acids with a homeodomain motif. V6 also contains a 20-amino acid sequence that is conserved in Aα Y1, Y3 and Y4. Except for the homeodomain and the conserved sequence, the deduced V6 polypeptide shows no significant identity to AαY, AαZ, or other known proteins. The presence of a homeodomain suggests that V, like Y and Z, may be a regulatory protein for genes in the A-pathway. Thus whileAα andAβ encode different proteins, the general mechanism by which Aα and Aβ components signal A-regulated development may be similar.