Heterologous gene expression in the basidiomycete fungus Coprinus cinereus

Summary Direct selection for TRP⁺ transformants of a trp-2 mutant of Coprinus cinereus showed that the gene mutation could be complemented by the heterologous gene from two other basidiomycete species, Schizophyllum commune (trp1) and Phanerochaete chrysosporium (trpC) but not by an ascomycete gene from Aspergillus nidulans (trpC). Cotransformation was used to confirm that the Aspergillus nidulans gene could be integrated but not expressed. Cotransformations were also used to show that the Aspergillus nidulans isocitrate lyase gene (acuD) and a construct containing the bacterial HygB gene fused to a hemibasidiomycete gene promotor from Ustilago maydis were also unexpressed in Coprinus cinereus.     

Biased inheritance of optional insertions following mitochondrial genome recombination in the basidiomycete fungus Coprinus cinereuss

Summary Two mitochondrial genomes of Coprinus cinereus, H and J, were found to have alternative 1.23 kb insertions. Using the Neurospora crassa cytochrome oxidase-1 (co-1) gene as a probe, the J insertion site was shown to be located within the Coprinus co-1 gene, whereas the H insertion was some 2 kb distant. The insertions showed biased inheritance following mitochondrial genome recombination. Recombination between H and J genomes was detected using the mitochondrial gene mutations acu-10, which causes a cytochrome oxidase defect, and cap-1, which confers chloramphenicol resistance. Fourteen of fifteen independently derived recombinants for these two genes were shown to have both DNA insertions. In a second series of H x J crosses, intragenic recombination between different cap-1 alleles was detected. These mutations are assumed to be in the large ribosomal RNA gene some 6 kb distant from the nearest insertion site. Each of eight independently derived cap-1 ⁺ recombinants had both DNA insertions. Despite their similar size and similar behaviour following recombination the insertions do not share extensive sequence homology.     

Polymorphism of the mitochondrial L-RNA gene of the basidiomycete Coprinus cinereus

Summary Two unexpectedly small mitochondrial (mt) genomes of Coprinus cinereus, P and S, were compared with the H and J genomes we have described previously. H and J are 42 kb in size and differ in having alternative 1.23 kb insertions in or adjacent to the co-1 gene. P and S DNAs lacked both insertions and had an identical 4.4 kb deletion between the co-1 and L-RNA gene. P DNA contained a 700 by insertion and S DNA a 300 by deletion within a sequence coding the L-RNA gene. This was shown by Southern blot analysis using probes containing the 5 or the 3 exon sequences of the L-RNA gene of Neurospora crassa. These hybridisations showed also that the L-RNA gene and co-1 gene in the C. cinereus mt genome are oppositely orientated and must be transcribed from different DNA strands. No DNA homology was detected using probes containing intron sequences from the L-RNA genes of Saccharomyces cerevisiae or N. crassa. There was no evidence of respiratory deficiency in P and S strains and transfer of nuclei by dikaryon formation made it possible to recombine H nuclei with P and S mitochondria, S nuclei with H and P mitochondria and P nuclei with H mitochondria with no apparent detrimental effect on growth. We conclude that P and S mtDNAs represent naturally occurring variants of the C. cinereus mt genome.     

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.     

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.     

Structure and regulation of the isocitrate lyase gene ICL1 from the yeast Saccharomyces cerevisiae

The ICL1 gene encoding the isocitrate lyase from Saccharomyces cerevisiae was cloned and sequenced. A reading frame of 557 amino acids showing significant similarity to isocitrate lyases from seven other species could be identified. Construction of icl1 null mutants led to growth defects on C₂ carbon sources while utilization of sugars or C₃ substrates remained unaffected. Using an ICL1-lacZ fusion integrated at the ICL1 locus, a more than 200-fold induction of -galactosidase activity was observed after growth on ethanol when compared with glucose-repressed conditions. A preliminary analysis of the ICL1 upstream region identified a 364-bp fragment necessary and sufficient for this regulatory phenotype. Sequence motifs also present in the upstream regions of co-regulated genes were found within this region.     

Mitochondrial genetics of Coprinus: Recombination of mitochondrial genomes

Summary The formation of the sexual mycelium or dikaryon in the basidiomycete Coprinus cinereus involves exchange and migration of nuclei without accompanying exchange of mitochondria. The dikaryotic growth which appears around the periphery of mated monokaryons has exclusively the mitochondrial genome of the recipient cells. Recombination of mitochondrial genomes is not, however, precluded during dikaryosis. Using monokaryons with different mitochondrial gene mutations, [acu-10] causing cytochrome aa ₃ deficiency and[cap-1.1] conferring resistance to chloramphenicol, it was shown that recombinant mitochondria arise in the zone of contact of mated monokaryons.     

Cloning and expression of a chitinase gene from the hyperparasitic fungus Aphanocladium album

Summary Recombinant clones from a cDNA library of an Aphanocladium album chitinase-overproducing mutant strain were isolated by screening with antiserum against a 39 kDa chitinase purified from this hyperparasitic fungus. Analysis of the isolated positive clones indicated that most of them carried the same cDNA. A cDNA from this group was used as a hybridization probe to isolate an 8 kb DNA fragment from a genomic library of the wild-type strain. The chitinase 1 gene was mapped to this fragment by two independent approaches. Its partial DNA sequence was in perfect agreement with an amino-terminal peptide sequence obtained by sequencing 23 amino acids of the 39 kDa chitinase. Its transfer in Fusarium oxysporum resulted in a transformant producting both a protein of about 39 kDa that cross-reacted with the chitinase antiserum and a chitinase activity that was inhibited by the same antiserum. Northern blot analysis indicates that the cloned chitinase gene was subject to catabolite repression and appeared inducible by chitin.     

Unusual organization and lack of recombination in the ribosomal RNA genes of Coprinus cinereus

Summary We find three interesting characteristics of the genes encoding the ribosomal RNA (rRNA) in the basidiomycete Coprinus cinereus. First, there are only 60 to 90 copies of the genes, fewer than in other fungi. Second, the genes are organized in an unusual arrangement. The 5S rRNA genes are located in the repeat unit which encodes the other rRNAs and all four rRNAs are transcribed in the same direction. Third, meiotic recombination is inhibited within the ribosomal DNA.     

Transformation of Rhizopus niveus using a bacterial blasticidin S resistance gene as a dominant selectable marker

Summary Rhizopus niveus has been transformed to blasticidin S resistance by vectors containing the bacterial blasticidin S resistance gene under the control of a Rhizopus promoter. Southern analysis of the total DNA from transformants indicated that the introduced DNA was rearranged, and that one of the transformants harbored extrachromosomal plasmids with rearranged DNA. Using this transformation system, the introduction of pUBSR101, a plasmid carrying the Escherichia coli lacZ gene fused to the promoter and the N-terminal region of the R. niveus aspartic proteinase-II (RNAP-II) gene, resulted in an increase of -galactosidase activity in the cell extract, indicating expression of the lacZ fusion gene in R. niveus. This is the first report of a transformation system for filamentous fungi using the blasticidin S resistance gene as a dominant selectable marker.     

A method to sharply delimit a yeast nuclear gene in a cloned DNA fragment

Summary We have developped a procedure to delimit the boundaries of a cloned gene carried on a DNA fragment as large as 4 to 5 kilobases. The method consists in the following. Two series of limit digest products generated with a tetranucleotide recognition sequence endonuclease and originating from either of the two ends of this DNA segment are tested for their complementing capacity by yeast transformation. The gene is then delimited by the overlap of the two shortest complementing fragments.     

G418-resistance as a dominant marker and reporter for gene expression in Saccharomyces cerevisiae

Summary Coding sequence cartridges for aminoglycoside phosphotransferase (APT) were isolated from bacterial transposon Tn903. When incorporated into a heterologous gene construction utilising the PGK1 promoter and terminator, the heterologous APT gene provided a G418-resistance determinant that functioned efficiently as a dominant marker for yeast in both multiple- and single-copy. Transformant colonies on selective medium appeared rapidly, within 36–48 h, and growth rate of the transformed cells was normal. A simple and highly sensitive radiolabelling assay for APT enzyme activity was developed for use with crude cell protein extracts. Enzyme activity units were equated to the amount of APT protein present in the cells, and the APT protein was shown to be stable in yeast. Heterologous APT expression was 130-fold reduced compared with homologous PGK1. This resulted from an estimated two-fold decrease in mRNA level and a 65-fold decrease in translation efficiency. The latter was unaffected by AUG sequence context change, but corresponded with a high frequency of minor codons in the APT-coding sequence. APT can be used as a semi-quantitative reporter of gene expression, whose useful features are in vivo detection via the G418-resistance phenotype and powerful cell-free assay.     

Primary structure and expression pattern of the 33-kDa chitinase gene from the mycoparasitic fungus Trichoderma harzianum

A gene (chit33) from the mycoparasitic fungus Trichoderma harzianum, coding for a chitinase of 33 kDa, has been isolated and characterized. Partial amino-acid sequences from the purified 33-kDa chitinase were obtained. The amino-terminal peptide sequence was employed to design an oligonucleotide probe and was used as a primer to isolate a 1.2-kb cDNA. The cDNA codes for a protein of 321 amino acids, which includes a putative signal peptide of 19 amino acids. All microsequenced peptides found in this sequence, indicate that this cDNA codes for the 33-kDa chitinase. A high homology (approximately 43% identity) was found with fungal and plant chitinases, including yeast chitinases. However enzyme characteristics suggest a nutritional (saprophytic or mycoparasitic), rather than a morphogenetic, role for this chitinase. The chit33 gene appears as a single copy in the T. harzianum genome, is strongly suppressed by glucose, and de-repressed under starvation conditions as well as in the presence of autoclaved mycelia and/or fungal cell walls. The 33-kDa chitinase seems to be very stable except under starvation conditions. The independent regulation of each of the chitinases in T. harzianum indicates different specific roles.     

Transformation of Penicillium chrysogenum using the Aspergillus nidulans amdS gene as a dominant selective marker

Summary The Aspergillus nidulans acetamidase gene (amdS) has been used to transform Penicillium chrysogenum at low frequency. Several transformants were tested and shown to be mitotically stable. Southern blot analysis indicated that transforming DNA had integrated into the chromosomal DNA, possibly at multiple sites.     

Transformants of Neurospora crassa with the nit-4 nitrogen regulatory gene: copy number,growth rate and enzyme activity

Summary nit-4 is a pathway-specific regulatory gene which controls nitrate assimilation in Neurospora crassa, and appears to mediate nitrate induction of nitrate and nitrite reductase. The NIT4 protein consists of 1090 amino-acid residues and possesses a single GAL4-like putative DNA-binding domain plus acidic, glutaminerich, and polyglutamine regions. Several mutants with amino-acid substitutions in the putative DNA-binding domain and a nit-4 deletion mutant, which encodes a truncated NIT4 protein lacking the polyglutamine region, are functional, i.e., they are capable of transforming a nit-4 mutant strain. However, transformants obtained with most of these nit-4 mutant genes possess a markedly reduced level of nitrate reductase and grow only slowly on nitrate, emphasizing the need to examine quantitatively the affects of in vitro-manipulated genes. The possibility that some mutant genes could yield transformants only if multiple copies were integrated was examined. The presence of multiple copies of wild-type or mutant nit-4 genes did not generally lead to increased enzyme activity or growth rate, but instead frequently appeared to be detrimental to nit-4 function. A hybrid nit-4-nirA gene transforms nit-4 mutants but only allows slow growth on nitrate and has a very low level of nitrate reductase.     

The genetic basis for the meiotic disorder of Coprinus congregatus

Summary A pale-mushroom phenotype in the fungus Coprinus congregatus has previously been reported to be caused by a cytoplasmic, infectious agent that inhibits the formation of synaptonemal complexes. After a period of 5 years of successful extraction of infectious material from carrier homokaryons, infectivity has now been lost and even cytoplasmic contact through somatic fusions no longer transmits the pale phenotype. In attempts to obtain new carrier homokaryons, a genetic analysis has been done. This study demonstrates that the pale phenotype in this species is primarily determined by a nuclear gene, symbolized pal ^–. The pale phenotype appears predictably when the dikaryon is homoallelic for the pal ^– allele. Homoallelic wild type (pal ⁺) and heteroallelic dikaryons display the dark phenotype. The former infectious nature of the pale phenotype and the current anomalous behavior of one homokaryon is discussed in the context that the pale phenotype may involve both a nuclear gene and a cytoplasmic element under some conditions.     

Structure of the Aspergillus niger pelA gene and its expression in Aspergillus niger and Aspergillus nidulans

Summary Aspergillus niger pectin lyases are encoded by a multigene family. The complete nucleotide sequence of the pectin lyase PLA-encoding gene pelA has been determined. Comparison of the deduced amino acid sequence with the deduced amino acid sequence of the other characterized pectin lyase, PLD, shows that the proteins share 69% amino acid identity. When grown on media with pectin as the sole carbon source, A. niger transformants containing multiple copies of the pelA gene show raised mRNA levels and overexpression of the gene product PLA compared with the wild-type strain. PLA was purified and characterized. In A. nidulans transformants PLA is also produced in medium containing a high concentration of glucose and no pectin.Deceased April 30, 1988     

Transformation of Trichoderma viride using the Neurospora crassa pyr4 gene and its use in the expression of a Taka-amylase A gene from Aspergillus oryzae

Summary A pyrG mutant of Trichoderma viride, a very efficient cellulase producer, was isolated from among 5-fluoroorotic acid-resistant mutants. The mutation was complemented with the pyr4 gene of Neurospora crassa and used as a selection marker for the transformation of T. viride. A plasmid vector, pDJB1-Taa, carrying both the pyr4 gene and a gene encoding Taka-amylase A from Aspergillus oryzae, was constructed and introduced into protoplasts of T. viride pyrG^-. The transformation frequency was 1–10 transformants (3 on average) per g DNA. One transformant showed highly elevated -amylase production (about 17 times higher than the recipient level) and the integration of more than one copy of the Taka-amylase gene.