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.     

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.     

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.     

Integrative transformation of the yeast Yarrowia lipolytica

Summary An EcoR1 shotgun of Yarrowia lipolytica DNA was inserted into the plasmid YIp333 which carries the LYS2 gene of S. cerevisiae. The resulting plasmid pool was transformed in both S. cerevisiae and Y. lipolytica. Whereas numerous replicating plasmids could be isolated from the S. cerevisiae Lys⁺ transformants, all transformants of Y. lipolytica so far analyzed were found to result from integrative transformation. This occurred at a frequency of 1 to 10 transformants per g of input DNA. Co-transformation occurred at high frequency and resulted in tandem integration of 2 to 10 copies of the incoming DNA. Structural and segregational stability of the transforming DNA were both high.     

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.     

The ftr cistron of Coprinus cinereus is the structural gene for a multifunctional transport carrier molecule

Summary Mutants of the basidiomycete Coprinus cinereus which were selected for their resistance to growth inhibitions caused by hexose analogues are all alleles of the ftr cistron. They are shown to have approximately normal levels of activity of enzymes involved in intracellular sugar metabolism and to accumulate normal levels of sugar phosphates. However, the mutants show greatly depressed rates of sugar uptake. Uptake rates from 0.01 mM solutions of 2-deoxy-D-glucose were only 1 to 4% of the wild type rate, and from 15 mM solutions the mutant rates were between 16 and 40% of normal. Kinetic analysis showed that the mutant V_max values were reduced to a few per-cent of normal while K_m values were relatively little changed and in some cases the mutants had an increased affinity for the substrate. Reverse mutations restored the V_max value and the K_m to about the wild type level. Previous data had shown that position of mutants within the allele map depended on selection conditions in a way that implied some interaction between the ftr gene product and the substrate. Since the mutants are defective in transport from both high and low sugar concentrations, and since they exhibit coordinated alterations in K_m and V_max, it is concluded that the ftr cistron is the structural gene for a product involved in sugar translocation (both as carrier and energisation link) in both high and low affinity glucose transport systems.     

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.     

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.     

Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi

Summary The yeast Saccharomyces cerevisiae has been engineered to synthesize and secrete desulfato-hirudin (hirudin), a thrombin inhibitor from the leech Hirudo medicinalis. The synthetic gene coding for hirudin was expressed constitutively under the control of four size-variants of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) and cloned into a 2 based multicopy yeast vector. The constitutive action of the four promoter variants was confirmed by demonstrating that the expression and secretion of hirudin is growth-related. The different efficiencies of the promoter variants not only affected hirudin expression but also led to changes in several cellular parameters, such as cell growth, average plasmid copy number and plasmid stability. The observed changes show that yeast cells establish a specific equilibrium for each promoter variant. We conclude, that the adjustment of cellular parameters in response to the expression levels of a heterologous protein is regulated by two counteracting selective forces: (1) the need for complementation of the auxotrophic host marker by the plasmid-encoded selection gene which, in the case of dLEU2, requires several plasmid copies; and (2) a selective advantage of cells with a lower copy number enabling them to escape the burden of heterologous protein production.     

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 nature of extra-chromosomal maintenance of transforming plasmids in the filamentous basidiomycete Phanerochaete chrysosporium

Summary The nature of extra-chromosomal maintenance of the transforming plasmid p12-6 in Phanerochaete chrysosporium was studied. Our results indicate that the plasmid is maintained in the fungal transformants extra-chromosomally as part of a larger endogenous plasmid (designated pME) of P. chrysosporium. Using the total DNA of p12-6 fungal transformants, p12-6, as well as a larger plasmid, p511, were recovered in recA ^– E. coli strains while only p12-6 was recovered in recA ⁺ E. coli strains. The results also showed that the cytosine methylation system has no apparent effect on the strain-dependent recovery of p12-6 and p511 in E. coli from the total DNA of fungal transformants.     

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.     

Genetic transformation of the symbiotic basidiomycete fungus Hebeloma cylindrosporum

Summary The pAN7.1 plasmid containing the E. coli hygromycin B phosphotransferase gene was used to transform protoplasts of the ectomycorrhizal fungus Hebeloma cylindrosporum. Hygromycin-resistant transformants were selected at a frequency of one to five per g of transforming DNA. Southern blot analyses revealed multiple copy integration of the transforming plasmid into the genome. The selection system was used to introduce other genes of interest by co-transformation. Two plasmids, one containing tryptophan biosynthesis genes and the other the NADP-glutamate dehydrogenase gene from the saprophytic basidiomycete Coprinus cinereus, were successfully introduced into the H. cylindrosporum genome with up to 70% efficiency of co-transformation. The hygromycin resistance phenotype was stably maintained during growth of transformants on hygromycinfree medium. All tranformants retained their ability to form mycorrhizae with the habitual host plant Pinus pinaster, making them suitable for future physiological studies.     

Identification of a lys2 mutant of Candida maltosa by means of transformation

Summary We have isolated five mutants of Candida maltos, which lack the 2-aminoadipate reductase activity, an enzyme involved in the lysine biosynthesis. By means of complementation analysis using protoplast fusion, the isolated mutants were divided into two complementation groups. Thereof the C. maltosa strain G457 could be transformed by the plasmids pDP12 and pDP13, which contain the L YS2-coding gene of Saccharomyces cerevisiae. On the basis of our presented results obtained by studies on hybridization, stability, and recovery of plasmids from C. maltosa transformants, we suggest that transformation does proceed integratively.Abbreviations AA 2-aminoadipate - PRA phosphoribosylanthranilate     

Cloning of the Rhizopus niveus pyr4 gene and its use for the transformation of Rhizopus delemar

We have cloned a pyr4 gene encoding orotidine-5-monophosphate decarboxylase of the filamentous fungus Rhizopus niveus. The pyr4 gene of R. nivens has an open reading frame composed of 265 amino-acid residues and has two putative introns. We have also isolated a pyr4 mutant of Rhizopus delemar from 5-fluoroorotic acid-resistant mutants and transformed it with the pyr4 gene of R. niveus as a selectable marker. Introduced DNA was integrated into the chromosome in a multiple tandem array. The mitotic stability of the introduced DNA was increased by a repeated sporulation process. The expression of the Escherichia coli -glucuronidase gene in R. delemar was successfully obtained under the control of the pgk2 gene promoter of R. niveus by co-transformation with the pyr4 gene.     

A new,rapid and efficient transformation procedure for Neurospora

Summary A new, rapid, efficient and reliable method for transforming Neurospora crassa is described. In this procedure, germinated conidia are treated with lithium acetate, then incubated with DNA, followed by exposure to polyethylene glycol and then a brief heat shock, prior to plating on selective medium. Optimal conditions to achieve a high transformation rate are reported. Transformation can be obtained with both circular and linear plasmid DNA and also with genomic DNA. Although the rate is substantially decreased, transformation was also obtained with relatively impure DNA preparations, such as that made via rapid miniprep procedures. This transformation technique is simple and reliable and provides a considerable savings in time and materials.     

Expression of the avian gag-myc oncogene in Saccharomyces cerevisiae

Summary We have isolated and characterized three conditional hyporecombination mutants, rec1-1, rec3-1 and rec4-1, that define three REC genes of Saccharomyces cerevisiae required for spontaneous general mitotic interchromosomal recombination. Each MATa/MAT rec/rec diploid is deficient in mitotic single site gene conversion, intragenic recombination, intergenic recombination and sporulation at the restrictive temperature (36°C). The rec1-1 mutation also confers conditional enhanced sensitivity to the killing effects of X-rays. The rec1-1 and rec3-1 mutations have been mapped to chromosome VII. The rec1-1, rec3-1 and rec4-1 mutations exhibit complementation at 36°C for both mitotic recombination and sporulation.