Transformation in fungi.

Transformation with exogenous deoxyribonucleic acid (DNA) now appears to be possible with all fungal species, or at least all that can be grown in culture. This field of research is at present dominated by Saccharomyces cerevisiae and two filamentous members of the class Ascomycetes, Aspergillus nidulans and Neurospora crassa, with substantial contributions also from fission yeast (Schizosaccharomyces pombe) and another filamentous member of the class Ascomycetes, Podospora anserina. However, transformation has been demonstrated, and will no doubt be extensively used, in representatives of most of the main fungal classes, including Phycomycetes, Basidiomycetes (the order Agaricales and Ustilago species), and a number of the Fungi Imperfecti. The list includes a number of plant pathogens, and transformation is likely to become important in the analysis of the molecular basis of pathogenicity. Transformation may be maintained either by using an autonomously replicating plasmid as a vehicle for the transforming DNA or through integration of the DNA into the chromosomes. In S. cerevisiae and other yeasts, a variety of autonomously replicating plasmids have been used successfully, some of them designed for use as shuttle vectors for Escherichia coli as well as for yeast transformation. Suitable plasmids are not yet available for use in filamentous fungi, in which stable transformation is dependent on chromosomal integration. In Saccharomyces cerevisiae, integration of transforming DNA is virtually always by homology; in filamentous fungi, in contrast, it occurs just as frequently at nonhomologous (ectopic) chromosomal sites. The main importance of transformation in fungi at present is in connection with gene cloning and the analysis of gene function. The most advanced work is being done with S. cerevisiae, in which the virtual restriction of stable DNA integration to homologous chromosome loci enables gene disruption and gene replacement to be carried out with greater precision and efficiency than is possible in other species that show a high proportion of DNA integration events at nonhomologous (ectopic) sites. With a little more trouble, however, the methodology pioneered for S. cerevisiae can be applied to other fungi too. Transformation of fungi with DNA constructs designed for high gene expression and efficient secretion of gene products appears to have great commercial potential.     

Replicative transformation of the filamentous fungus Ashbya gossypii with plasmids containing Saccharomyces cerevisiae ARS elements.

We have developed a transformation system for the filamentous ascomycete fungus Ashbya gossypii. Mycelial protoplasts were transformed to geneticin-resistance with plasmids containing the Escherichia coli kanamycin-resistance gene as a selectable marker and autonomously replicating sequences (ARS) from Saccharomyces cerevisiae (ARS1, 2 mu ARS). Transformation frequencies of up to 63 transformants per microgram of plasmid DNA were obtained. The transformants were unstable under nonselective conditions. Southern analysis of DNA separated by conventional and pulsed-field-gel electrophoresis showed that the transforming DNA was present as autonomously replicating plasmid. Plasmid integration into chromosomal DNA was not detected. We concluded that the S. cerevisiae ARS elements are functional in A. gossypii, since vectors lacking such elements did not yield transformants.     

Pathways of Transformation in Ustilago Maydis Determined by DNA Conformation

Ustilago maydis was transformed by plasmids bearing a cloned, selectable gene but lacking an autonomously replicating sequence. Transformation was primarily through integration at nonhomologous loci when the plasmid DNA was circular. When the DNA was made linear by cleavage within the cloned gene, the spectrum of integration events shifted from random to targeted recombination at the resident chromosomal allele. In a large fraction of the transformants obtained using linear DNA, the plasmid DNA was not integrated but was maintained in an extrachromosomal state composed of a concatameric array of plasmid units joined end-to-end. The results suggest the operation of several pathways for transformation in U. maydis, and that DNA conformation at the time of transformation governs choice of pathways.     

The plasmids of the morels: characterization and prerequisites for vector development

Summary In several Morchella species linear dsDNA plasmids have been discovered and analyzed. Two linear plasmids of the strain Morchella conica 3 analyzed in detail are associated with the mitochondria but not as an integrative part of the high molecular weight mt DNA. In addition, homologies between different plasmids from different species could be detected. These homologies extend to all parts of the plasmids. Differences in size are due to different lengths of nonhomologous inner parts rather than to differences at the termini. Fragments comprising the entire plasmids were cloned into bacterial vectors and a selectable marker gene for eukaryotes was added to the resulting hybrid plasmids. Transformation of yeast led to the identification of autonomously replicating sequences, this being a prerequisite for the development of autonomously replicating vectors for Morchella.Dedicated to Prof. Dr. H.-J. Rehm on the occasion of his 60th birthday     

Replication of a linear mini-chromosome with terminal inverted repeats from the Kluyveromyces lactis linear DNA plasmid k2 in the cytoplasm of Saccharomyces cerevisiae

The k1 and k2 linear DNA plasmids of Kluveromyces lactis replicate in the cytoplasm under the control of plasmid-encoded genes. These plasmids can also replicate autonomously in the cytoplasm of mitochondrial DNA-deficient strains of Saccharomyces cerevisiae. Essential for replication are plasmid-specific terminal inverted repeats (TIRs) to which a terminal protein (TP) is attached at the 5' ends. A plasmid was constructed with k2 TIRs in opposite orientations and with a selectable marker (URA3) under the control of k1UCS2 (upstream conserved sequence 2, the promoter of k1 open reading frame 2) in between the TIRs. Transformation of k1- and k2-containing S. cerevisiae with a fragment generated by releasing the TIR-flanked fragment from the plasmid by restriction digestion was very efficient, despite the absence of a TP. Transformation was also achieved with a fragment generated by PCR. Southern blotting demonstrated that transformants contained multiple copies of DNA fragments with the same size as the transforming DNA, supporting the hypothesis that these were replicating linear mini-chromosomes. The high frequency of transformation strongly suggests that these mini-chromosomes readily replicate supported by k2. Derivatives with a heterologous gene, firefly luciferase (LUC), expressed luciferase at high levels provided the gene was adjacent to a cytoplasmic plasmid promoter (k2UCS5).     

The development of a transformation system for the dimorphic plant pathogen Holleya sinecauda based on Ashbya gossypii DNA elements

We have developed a transformation system for the dimorphic plant pathogenic fungus Holleya sinecauda based on an electroporation protocol used for the closely related filamentous fungus Ashbya gossypii. DNA-mediated transformation of the dominant selection marker kanMX generated H. sinecauda transformants that were resistant to the antibiotic drug G418/geneticin. Freely replicating plasmids could be established in H. sinecauda using an A. gossypii autonomously replicating sequence (ARS) element, whereas Saccharomyces cerevisiae ARS elements, which are functional in A. gossypii, were not functional in H. sinecauda. In addition, centromeric DNA of A. gossypii stabilized the maintenance of plasmids in H. sinecauda under non-selective conditions. We isolated a fragment of the HsLEU2 gene and used this locus for targeted integration of kanMX3, consisting of the kanMX gene flanked by direct repeats. This allowed the construction of a Hsleu2 strain which became G418 sensitive after direct repeat-induced marker excision. The Hsleu2 strain can be complemented by the ScLEU2 gene. Finally, we constructed high- and low-copy shuttle vectors for H. sinecauda.     

High-efficiency transformation of Pichia stipitis based on its URA3 gene and a homologous autonomous replication sequence, ARS2.

This paper describes the first high-efficiency transformation system for the xylose-fermenting yeast Pichia stipitis. The system includes integrating and autonomously replicating plasmids based on the gene for orotidine-5'-phosphate decarboxylase (URA3) and an autonomous replicating sequence (ARS) element (ARS2) isolated from P. stipitis CBS 6054. Ura- auxotrophs were obtained by selecting for resistance to 5-fluoroorotic acid and were identified as ura3 mutants by transformation with P. stipitis URA3. P. stipitis URA3 was cloned by its homology to Saccharomyces cerevisiae URA3, with which it is 69% identical in the coding region. P. stipitis ARS elements were cloned functionally through plasmid rescue. These sequences confer autonomous replication when cloned into vectors bearing the P. stipitis URA3 gene. P. stipitis ARS2 has features similar to those of the consensus ARS of S. cerevisiae and other ARS elements. Circular plasmids bearing the P. stipitis URA3 gene with various amounts of flanking sequences produced 600 to 8,600 Ura+ transformants per micrograms of DNA by electroporation. Most transformants obtained with circular vectors arose without integration of vector sequences. One vector yielded 5,200 to 12,500 Ura+ transformants per micrograms of DNA after it was linearized at various restriction enzyme sites within the P. stipitis URA3 insert. Transformants arising from linearized vectors produced stable integrants, and integration events were site specific for the genomic ura3 in 20% of the transformants examined. Plasmids bearing the P. stipitis URA3 gene and ARS2 element produced more than 30,000 transformants per micrograms of plasmid DNA. Autonomously replicating plasmids were stable for at least 50 generations in selection medium and were present at an average of 10 copies per nucleus.     

New yeast vectors containing the autonomously replicating sequences from Candida maltosa genome

Two different DNA sequences from the yeast Candida maltosa confer the ability to replicate autonomously to the yeast integrative vector pLD700 on which they are cloned. The recombinant plasmids pLD701 and pLD702 with autonomously replicating sequences (ARS) from Candida maltosa and LEU2 gene from Saccharomyces cerevisiae transform the auxotrophic strain S. cerevisiae DC5 with the efficiency 3-5 x 10(3) per microgram of DNA. Like other yeast vectors harbouring ARS, these plasmids are not stable in yeast cells. Restriction and hybridization analyses have revealed the pLD701 plasmid to contain ARS from chromosomal DNA of C. maltosa. Plasmid pLD701 appears to be a useful vector for yeast transformation.     

An Aspergillus nidulans uvsC null mutant is deficient in homologous DNA integration

The Aspergillus nidulans uvsC gene was identified as a homolog of RAD51 and recA of Saccharomyces cerevisiae and Escherichia coli, respectively, whose role in genetic recombination and recombinational repair has been extensively studied. Like many other filamentous fungi, A. nidulans shows no bias towards either homologous or ectopic integration of exogenous DNA. Therefore it is a unique and useful organism for the study of the mechanisms of DNA integration. Homologous integration of a 1.7-kb argB gene was not detected in 50 transformants obtained from a uvsC null mutant. In contrast, the frequency of homologous integration in uvsC+ control strains varied from 41 to 86%. Another feature observed with the uvsC null mutant was that an increased number of transformants had undergone ectopic integrations at multiple sites in the genome. These results are consistent with the established function of Rad51/RecA, and further indicate the involvement of redundant pathways in integration of exogenous DNA. This study provides direct evidence for the involvement of uvsC in exogenous DNA integration and should contribute to the improvement of genetic manipulations in general, but particularly in fungi.     

Illegitimate integration of single-stranded DNA in Saccharomyces cerevisiae

We studied illegitimate recombination by transforming yeast with a single-stranded (ss) non-replicative plasmid. Plasmid pCW12, containing the ARG4gene, was used for transformation of yeast strains deleted for the ARG4, either in native (circular) form or after linearization within the vector sequence by the restriction enzyme ScaI. Both circular and linearized ss plasmids were shown to be much more efficient in illegitimate integration than their double-stranded (ds) counterparts and more than two-thirds of the transformants analysed contained multiple tandem integrations of the plasmid. Pulsed-field gel electrophoresis of genomic DNA revealed significant changes in the karyotype of some transformants. Plasmid DNA was frequently detected on more than one chromosome and on mitotically unstable, autonomously replicating elements. Our results show that the introduction of nonhomologous ss DNA into yeast cells can lead to different types of alterations in the yeast genome.     

Cloned mouse DNA fragments can replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro.

Mouse liver DNA was cut out with BamHI and cloned into YIp5, which contained the URA3 gene of Saccharomyces cerevisiae in pBR322. Of the several plasmids isolated, two plasmids, pMU65 and pMU111, could transform S. cerevisiae from the URA- to the URA+ phenotype and could replicate autonomously within the transformant, indicating that mouse DNA fragments present in pMU65 or pMU111 contain autonomously replicating sequences (ARS) for replication in S. cerevisiae. Furthermore, to determine the correlation between ARS function in yeast cells and that in much higher organisms, we tried to challenge these plasmids with the simian virus 40 (SV40) DNA replication system. Of the two plasmids tested, the EcoRI-BglII region of pMU65 could be hybridized with a chemically synthesized 13-nucleotide fragment corresponding to the origin region of SV40 DNA. Both pMU65 (the EcoRI-BglII region cloned in pBR322) and its subclone pMU65EB could replicate semiconservatively, and initiation of DNA replication started from the EcoRI-BglII region when the replicating activity of these plasmids was tested in the in vitro SV40 DNA replication system we have established before. Furthermore, pMU65 and pMU65EB could replicate autonomously within monkey Cos cells which produce SV40 T antigen constitutively. These results show that a 2.5-kilobase fragment of the EcoRI-BglII region in pMU65 contains the ARS needed for replication in the SV40 DNA replication system.     

Expanding the ku70 toolbox for filamentous fungi: establishment of complementation vectors and recipient strains for advanced gene analyses

Mutants with a defective non-homologous-end-joining (NHEJ) pathway have boosted functional genomics in filamentous fungi as they are very efficient recipient strains for gene-targeting approaches, achieving homologous recombination frequencies up to 100%. For example, deletion of the ku70 homologous gene kusA in Aspergillus niger resulted in a recipient strain in which deletions of essential or non-essential genes can efficiently be obtained. To verify that the mutant phenotype observed is the result of a gene deletion, a complementation approach has to be performed. Here, an intact copy of the gene is transformed back to the mutant, where it should integrate ectopically into the genome. However, ectopic complementation is difficult in NHEJ-deficient strains, and the gene will preferably integrate via homologous recombination at its endogenous locus. To circumvent that problem, we have constructed autonomously replicating vectors useful for many filamentous fungi which contain either the pyrG allele or a hygromycin resistance gene as selectable markers. Under selective conditions, the plasmids are maintained, allowing complementation analyses; once the selective pressure is removed, the plasmid becomes lost and the mutant phenotype prevails. Another disadvantage of NHEJ-defective strains is their increased sensitivity towards DNA damaging conditions such as radiation. Thus, mutant analyses in these genetic backgrounds are limited and can even be obscured by pleiotropic effects. The use of sexual crossings for the restoration of the NHEJ pathway is, however, impossible in imperfect filamentous fungi such as A. niger. We have therefore established a transiently disrupted kusA strain as recipient strain for gene-targeting approaches.     

High-frequency transformation of a methylotrophic yeast, Candida boidinii, with autonomously replicating plasmids which are also functional in Saccharomyces cerevisiae.

We have developed a transformation system which uses autonomous replicating plasmids for a methylotrophic yeast, Candida boidinii. Two autonomous replication sequences, CARS1 and CARS2, were newly cloned from the genome of C. boidinii. Plasmids having both a CARS fragment and the C. boidinii URA3 gene transformed C. boidinii ura3 cells to Ura+ phenotype at frequencies of up to 10(4) CFU/micrograms of DNA. From Southern blot analysis, CARS plasmids seemed to exist in polymeric forms as well as in monomeric forms in C. boidinii cells. The C. boidinii URA3 gene was overexpressed in C. boidinii on these CARS vectors. CARS1 and CARS2 were found to function as an autonomous replicating element in Saccharomyces cerevisiae as well. Different portions of the CARS1 sequence were needed for autonomous replicating activity in C. boidinii and S. cerevisiae. C. boidinii could also be transformed with vectors harboring a CARS fragment and the S. cerevisiae URA3 gene.     

Illegitimate integration of single-stranded DNA in Saccharomyces cerevisiae

 We studied illegitimate recombination by transforming yeast with a single-stranded (ss) non-replicative plasmid. Plasmid pCW12, containing the ARG4gene, was used for transformation of yeast strains deleted for the ARG4, either in native (circular) form or after linearization within the vector sequence by the restriction enzyme ScaI. Both circular and linearized ss plasmids were shown to be much more efficient in illegitimate integration than their double-stranded (ds) counterparts and more than two-thirds of the transformants analysed contained multiple tandem integrations of the plasmid. Pulsed-field gel electrophoresis of genomic DNA revealed significant changes in the karyotype of some transformants. Plasmid DNA was frequently detected on more than one chromosome and on mitotically unstable, autonomously replicating elements. Our results show that the introduction of nonhomologous ss DNA into yeast cells can lead to different types of alterations in the yeast genome. Received: 9 February 1996/Accepted: 7 July 1996     

Hairpin plasmid--a novel linear DNA of perfect hairpin structure.

The terminal structures of deletion derivatives of linear DNA killer plasmid from yeast were analyzed. The yeast Kluyveromyces lactis harbors two unique double-stranded linear DNA killer plasmids, pGKL1 of 8.9 kb and pGKL2 of 13.4 kb. The killer toxin and the resistance to the killer are coded by pGKL1, while pGKL2 is required for the maintenance of pGKL1 in the cell. When the pGKL plasmids from K. lactis were transferred into Saccharomyces cerevisiae by transformation, non-killer transformants harboring pGKL2 and new plasmids, F1 of 7.8 kb and F2 of 3.9 kb, were obtained. F2 was shown to be a linear DNA arising from a 5-kb deletion of the right part of pGKL1. F1 was an inverted dimer of F2. Here we show that F2 has two different terminal structures: one end has a protein attached at the 5' terminus whereas the two strands of duplex are linked together at the other end, thus forming a hairpin structure. This is a novel type of autonomously replicating DNA molecule.     

Construction, replication, and chromatin structure of TRP1 RI circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA.

Transformation studies with Saccharomyces cerevisiae (bakers' yeast) have identified DNA sequences which permit extrachromosomal maintenance of recombinant DNA plasmids in transformed cells. It has been hypothesized that such sequences (called ARS for autonomously replicating sequence) serve as initiation sites for DNA replication in recombinant DNA plasmids and that they represent the normal sites for initiation of replication in yeast chromosomal DNA. We have constructed a novel plasmid called TRP1 R1 Circle which consists solely of 1,453 base pairs of yeast chromosomal DNA. TRP1 RI Circle contains both the TRP1 gene and a sequence called ARS1. This plasmid is found in 100 to 200 copies per cell and is relatively stable during both mitotic and meiotic cell cycles. Replication of TRP1 RI Circle requires the products of the same genes (CDC28, CDC4, CDC7, and CDC8) required for replication of chromosomaL DNA. Like chromosomal DNA, its replication does not occur in cells arrested in the B1 phase of the cell cycle by incubation with the yeast pheromone alpha-factor. In addition, TRP1 RI Circle DNA is organized into nucleosomes whose size and spacing are indistinguishable from that of bulk yeast chromatin. These results indicate that TRP1 RI Circle has the replicative and structural properties expected for an origin of replication from yeast chromosomal DNA. Thus, this plasmid is a suitable model for further studies of yeast DNA replication in both cells and cell-free extracts.     

Invertase gene (SUC2) of Saccharomyces cerevisiae as a dominant marker for transformation of Pichia pastoris

A two-step method for the selection of transformants of prototrophic industrial strains of the methylotrophic yeast Pichia pastoris has been developed. This method is based on our observation that P. pastoris cannot use sucrose as the sole carbon source (Suc-) and that introduction of the invertase gene (SUC2) of Saccharomyces cerevisiae renders P. pastoris Suc+. P. pastoris was transformed with a plasmid which contains the SUC2 gene of S. cerevisiae and an autonomously replicating sequence PARS1 from P. pastoris. The transformants were initially allowed to regenerate on medium containing dextrose and the regenerated cells were pooled and plated on sucrose medium to screen for Suc+ transformants. It was shown that the Suc+ transformants of P. pastoris with the autonomously replicating plasmid were highly unstable with respect to the plasmid maintenance, even when grown on sucrose as the sole carbon and energy source. This high instability was attributed to an efficient cross-feeding by Suc- segregants on glucose and fructose generated due to hydrolysis of sucrose by the invertase enzyme secreted by Suc+ cells. Spontaneous integration of the plasmid DNA resulting in a stable Suc+ phenotype was also observed. However, stable Suc+ transformants were obtained more readily by integration of SUC2 into P. pastoris genome following transformation with a linearized plasmid with the ends homologous to P. pastoris HIS4 locus. All such integrants were completely stable for Suc+ phenotype after 20 generations of growth in a nonselective medium.     

Nonhomologous end joining and homologous recombination DNA repair pathways in integration mutagenesis in the xylose-fermenting yeast Pichia stipitis

Pichia stipitis integrates linear homologous DNA fragments mainly ectopically. High rates of randomly occurring integration allow tagging mutagenesis with high efficiency using simply PCR amplificates of suitable selection markers from the P. stipitis genome. Linearization of an autonomously replicating vector caused a distinct increase of the transformation efficiency compared with the circular molecule. Cotransformation of a restriction endonuclease further enhanced the transformation efficiency. This effect was also observed with integrative vector DNA. In most cases vector integration in chromosomal targets did not depend on microhomologies, indicating that restriction-enzyme-mediated integration (REMI) does not play an essential role in P. stipitis. Small deletions were observed at the ends of the integrated vectors and in the target sites. Disruption of the PsKU80 gene increased the frequency of homologous integration considerably but resulted in a remarkable decrease of the transformation efficiency. These results suggest that in P. stipitis the nonhomologous end joining (NHEJ) pathway obviously predominates the homologous recombination pathway of double-strand break repair.     

Autonomously replicating fragments of mitochondrial DNA from Penicillium urticae in Saccharomyces cerevisiae

Eight fragments which cover the whole range of the mitochondrial genome of Penicillium urticae were subcloned into the yeast integration vector YIp5. Transformation of Saccharomyces cerevisiae with the constructed plasmids by the alkali cation method indicated that six plasmids are able to replicate in yeast. Both closed and open circular forms of the plasmids were detected in the DNA extracts from transformants. Distribution of the autonomously replicating sequence in the mitochondrial genome was similar to that in P. chrysogenum except for one small region.     

Transformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors

Summary A high frequency transformation system for the methylotrophic yeast Hansenula polymorpha has been developed. This system depends on complementation of isolated uracil auxotrophs by the URA3 gene of Saccharomyces cerevisiae. Maintenance of the uracil prototrophy is based on integration of plasmid YIp5 at random sites within the H. polymorpha genome and on autonomously replicating plasmids containing ARS1 of S. cerevisiae or related sequences cloned from the host DNA. The sequence of one autonomously replicating sequence (HARS1) from H. polymorpha has been determined showing an AT-rich region of 9 bp with some similarity to the consensus sequence of known eukaryotic replication origins. Mitotic loss of autonomously replicating sequences is high; selection for stable uracil prototrophs yields multiple tandem arrangement of the transformed DNA with no detectable loss of the phenotype on non-selective medium. These features offer the possibility for extensive gene expression in H. polymorpha.