Heterologous gene expression of the glyphosate resistance marker and its application in yeast transformation

Summary The E. coli aroA gene was inserted between yeast promoter and terminator sequences in different shuttle expression plasmids and found to confer enhanced EPSP synthase activity as well as resistance to glyphosate toxicity. Subsequently, a transformation system using these newly constructed vectors in yeast was characterized. The efficiency of the glyphosate resistance marker for transformation and selection with plasmid pHR6/20-1 in S. cerevisiae laboratory strain SHY2 was found to be relatively high when compared with selection for LEU2 prototrophy. The fate of the recombinant plasmid pHR6/20-1 in the transformants, the preservation of the aroA E. coli DNA fragment in yeast, mitotic stability, EPSP synthase activity, and growth on glyphosate-containing medium have been investigated. As this plasmid also allows direct selection for glyphosate resistant transformants on rich media, the glyphosate resistance marker was used for transforming both S. cerevisiae laboratory strain SHY2 and brewer's yeast strains S. cerevisiae var. uvarum BHS5 and BHS2. In all cases, the vector pHR6/20-1 was maintained as an autonomously replicating plasmid. The resistance marker is, therefore, suitable for transforming genetically unlabeled S. cerevisiae laboratory, wild, and industrial yeast strains.Abbreviations EPSP 5-enolpyruvylshikimate 3-phosphate     

Highly-efficient electrotransformation of the yeast Hansenula polymorpha

A highly-efficient method for transformation of the methylotrophic yeast Hansenula polymorpha has been developed. Routinely, transformation frequencies of up to 1.7×10⁶/g plasmid DNA were obtained by applying an electric pulse of the exponential decay type of 7.5 kV/cm to a highly-concentrated cell mixture during 5 ms. Efficient transformation was dependent on: (1) pretreatment of the cells with the reducing agent dithiotreitol, (2) the use of sucrose as an osmotic stabilizer in an ionic electroporation buffer, and (3) the use of cells grown to the mid-logarithmic phase. Important parameters for optimizing the transformation frequencies were field strength, pulse duration, and cell concentration during the electric pulse. In contrast to electrotransformation protocols described for Saccharomyces cerevisiae and Candida maltosa, transformation frequencies (transformants per g DNA) for H. polymorpha remained high when large amounts (up to 10g) of plasmid DNA were added. This feature renders this procedure pre-eminently advantageous for gene cloning experiments when high numbers of transformants are needed.     

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.     

Cloning of a Candida utilis gene which complements leu2 mutation in Saccharomyces cerevisiae

Summary DNA fragments containing the LEU2 gene of Candida utilis have been isolated, utilizing the genome library (constructed in YRp12) of this organism. Two recombinant plasmids pZR84 and pZR32, containing the cloned LEU2 gene, were 4.24 kb and 10.4 kb, respectively, and were shown to complement leu2 mutation in Saccharomyces cerevisiae and leuB mutation in Escherichia coli. The cloned fragment in pZR84 contained one restriction site each for EcoRI and PvuII, and two for HindIII, but none for SalI, BamHI or Pstl. This cloned fragment hybridized with the total DNA from C. utilis and from Leu⁺ transformants of S. cerevisiae, but not with that from untransformed S. cerevisiae. Subcloning analyses showed that a 2.34 kb BamHI HindIII fragment of the cloned C. utilis sequence contains the region essential for the expression of the LEU2 gene.Journal Article No. 11669 from the Michigan Agricultural Experiment Station     

Optimisation of a host/vector system for heterologous gene expression by Hansenula polymorpha

Summary For the methylotrophic yeast, Hansenula polymorpha, expression vectors with different origins of replication have been constructed in order to analyse their influence on transformation and integration efficiency. The constructed plasmids are identical except for their origin of replication, which involve, respectively, that of the Saccharomyces cerevisiae 2-m plasmid and a H. polymorpha ARS sequence (HARS2). A plasmid with no origin of replication served as a control. The plasmids also contained the -galactosidase expression cassette, consisting of the Cyamopsis tetragonoloba -galactosidase gene, the H. polymorpha methanol oxidase promoter and terminator, and the S. cerevisiae invertase signal sequence. The transformation frequencies of the expression vectors containing the 2-m and the HARS2 origins of replication, and no origin of replication, were 2,50 and 15 per g of DNA respectively, which demonstrates the negative effect of the 2-m sequence on the transformation frequency. Autonomously replicating plasmids could be isolated from the transformants obtained with the plasmid containing either the 2-m or the HARS2 sequence. Integration of the 2-m based plasmid into the H. polymorpha genome could not be established using a standard procedure. This is in contrast with transformants containing a plasmid bearing the HARS2 sequence or else with no origin of replication, which shows that the 2-m sequence negatively influences the integration of the expression vector into the H. polymorpha genome. Integration of expression plasmids occurred in 50% of the analysed integrants on the homologous methanol oxidase locus, and tandem integration was favoured. The level of specific mRNA, and the expression of the -galactosidase protein by these integrants, was proportional to the number of integrated copies of the expression plasmid in the H. polymorpha genome.     

Genetic analysis of transformation in a microconidiating strain of Neurospora crassa

Summary We have characterized Neurospora crassa transformants obtained with plasmid pDV1001 bearing the cloned catabolic dehydroquinase (qa-2 ⁺) gene (Hughes et al. 1983) and fluffy 268 host strain producing only uninucleate microconidia allowing to isolate individual transformation products. The percentage of transformed nuclei in the mycelium and their stability were determined by genetic analysis of microconidia produced on selective or non-selective medium. About half of the transformants originating from mycelial spheroplasts were apparently homokaryotic. Catabolic dehydroquinase activity was in agreement with the proportion of transformed nuclei. The DNAs from four transformants analyzed by Southern hybridization showed restriction fragments expected for integration of pDV1001 into genomic DNA by non-homologous recombination. No plasmids could be rescued from the undigested DNAs of the transformants by transformation of E. coli. One transformant, 8268-6, was unstable and generated a high proportion of segregants. Plasmid pDV1001 sequences were absent in their DNA. Colonies originating from microconidia of strain fl268-6 on selective plates often lost the transformed character. These results suggest that instability in this transformant is due to the loss of integrated plasmid sequences during vegetative growth.     

Transformation ofNeurospora crassa with circular and linear DNA and analysis of the fate of the transforming DNA

Summary We have conducted a detailed study of 108qa-2 ⁺ Neurospora transformants which were obtained by use of circular plasmid DNAs and various linear DNAs. Parallel genetic and molecular analyses have revealed that three classes of transformants can be identified: linked transformants, in which theqa-2 gene has integrated at the resident locus, unlinked transformants, where integration has occurred at other genomic sites, and a third class designated non-transmissible which fail to pass theqa-2 gene through a cross. The non-transmissible class comprises the majority of transformants and may identify those which harbor autonomously replicating plasmids. Evidence is presented which suggests that a 1.2 kB BamHI-Bg1IIqa-2 ⁺ DNA fragment might possess anars sequence. Transformation with linear plasmid DNAs and DNA fragments carrying theqa-2 gene resulted in a demonstrable increase in transformation frequency beyond that achieved with circular plasmid DNAs, but did not permit precise targeting to the resident locus. Southern analysis showed that linked transformants have only the normal residentqa-2 band whereas the unlinked transformants always possess the resident band plus at least one additional band. Multiple integration events appear to be common and include cases where only a portion of the transforming DNA has been integrated.     

Terminal segment of Kluyveromyces lactis linear DNA plasmid pGKL2 supports autonomou sreplication of hybrid plasmids in Saccharomyces cerevisiae

Summary By use of linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis we have constructed hybrid plasmids carrying a LEU2 gene of Saccharomyces cerevisiae as a selectable marker. The replication properties of hybrid plasmids in yeasts were investigated. We demonstrated that the insertion of a LEU2 gene into pGKL2 resulted in circularization of the hybrid plasmids and pGKL2 segment supported autonomous replication of the plasmids. Moreover, the hybrid plasmids propagated autonomously, independently of the presence of the natural pGKL2 plasmid.     

<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.     

Transformation of the mycoparasite Parasitella simplex to neomycin resistance

Summary The facultatively parasitic zygomycete Parasitella simplex was transformed to neomycin resistance by a vector, which had been developed primarily for transformation of its host Absidia glauca. This plasmid, pAmNEF21, contained the bacterial resistance gene for neomycin (NPTII) under the control of the promoter region from the gene for elongation factor 1 (tef) isolated from A. glauca. Both flanking regions of the marker gene contain parts of the structural tef gene. DNA isolated from two Parasitella transformants was re-transformed in E. coli and the resulting plasmids, pAt21 and pAt35, were analyzed. The restriction map and Southern blot analysis show that both plasmids are rearranged. They had lost the structural tef information and were found to contain new DNA fragments, which were identical in both cases. Southern blot analysis of the transformants indicates that the rearranged plasmids are present in the fungal transformants and that the changes are not the result of re-transformation in E. coli. Plasmids were only recovered after growth under selective conditions. Southern blot analysis and re-transformation with undigested transformant DNA shows that the plasmids are replicated autonomously.     

Cloned mitochondrial DNA from the zygomycete Absidia glauca promotes autonomous replication in Saccharomyces cerevisiae

Summary We have cloned fragments from mitochondrial and chromosomal DNA of the zygomycete Absidia glauca in Saccharomyces cerevisiae using the ARS selection vector YIp5. Though it has not been possible to select ARS elements from chromosomal DNA, we succeeded in isolating two clones of mitochondrial origin that support autonomous replication in bakers' yeast. DNA from these plasmids has been shown to hybridize with mitochondrial DNA from both mating types. Generation times of the transformed yeast strain in selective medium are around 20 h. In liquid minimal medium only 6% of the cells contain the plasmid; in complete medium a mitotic stability of 50% has been determined.     

Transformation of Schizosaccharomyces pombe by non-homologous,unstable integration of plasmids in the genome

Summary In the fission yeast, Schizosaccharomyces pombe, transformation with recombinant plasmids always results in a high proportion of mitotically unstable transformants. This suggested that specialised (ARS) sequences might not be required for autonomous replication of plasmids in S. pombe, contrary to the situation in Saccharomyces cerevisiae. We have shown that specialised ARS sequences, analogous to those in S. cerevisiae, do exist in S. pombe, supporting the view that ARS elements are a general feature of eukaryotes. In addition, there is a further mechanism of plasmid maintenance which involves homologous and non-homologous integration into, and excision from the genome.     

Cloning of a DNA fragment from Cephalosporium acremonium which functions as an autonomous replication sequence in yeast

Summary A fragment of DNA which functions as an autonomous replication sequence in yeast was cloned from Cephalosporium acremonium. Mitochondrial DNA (mtDNA) was isolated from an industrial strain of C. acremonium (08G-250-21) highly developed for the production of the antibiotic, cephalosporin C. Size, 27 kb, and restriction pattern indicated this DNA was identical to mtDNA previously isolated (Minuth et al. 1982) from an ancestral strain (ATTC 14553) which produces very low amounts of cephalosporin C. A 1.9 kb Pst1 fragment of the Cephalosporium mtDNA was inserted into a Pst1 site of the yeast integrative plasmid, Ylp5, to produce a 7.5 kb plasmid, designated pPS1. The structure of pPS1 was verified by restriction analysis and hybridization.PS1 transformed Saccharomyces cerevisiae (DBY-746) to uracil prototrophy at a frequency of 272 transformants/g DNA. Transformation frequencies of 715 transformants/g DNA and zero were obtained for the replicative plasmid, YRp7, and the integrative plasmid YIp5, respectively. Southern hybridization and transformation of E. coli by DNA from yeast transformed by pPS1 verified that pPS1 replicates autonomously in yeast.The uracil-independent pPS1-yeast transformants were mitotically unstable. The average retention of pPS1 after three days growth in selective and non-selective medium was 4.5% and 0.4%, respectively, compared to retentions of 4.6% and 0.5% for YRp7. The properties of pPS1 were compared to those of a related plasmid, pCP2. pCP2 was constructed (Tudzynski et al. 1982) by inserting the C. acremonium 1.9 kb Pst1 fragment into the yeast integrative plasmid, pDAM1.     

Cloning by genetic complementation and restriction mapping of the yeast HEM1 gene coding for 5-aminolevulinate synthase

Summary We have cloned the structural gene HEM1 for 5-aminolevulinate (ALA) synthase from Saccharomyces cerevisiae by transformation and complementation of a yeast hem1–5 mutant which was previously shown to lack ALA synthase activity (Urban-Grimal and Labbe Bois 1981) and had no immunodetectable ALA synthase protein when tested with yeast ALA synthase antiserum. The gene was selected from a recombinant cosmid pool which contained wild-type yeast genomic DNA fragments of an average size of 40 kb. The cloned gene was identified by the restauration.of growth on a non fermentable carbon source without addition of exogenous ALA. Sub cloning of partial Sau3A digests and functional analysis by transformation allowed us to isolate three independent plasmids, each carrying a 6 kb yeast DNA fragment inserted in either orientation into the single BamHI site of the vector pHCG3 and able to complement hem1–5 mutation. Analysis of the three plasmids by restriction endonucleases showed that HEM1 is contained within a 2.9 kb fragment. The three corresponding yeast trans formants present a 1, 2.5 and 16 fold increase in ALA synthase activity as compared to the wild-type strain. The gene product immunodetected in the transformant yeast cells has identical size as the wild-type yeast ALA synthase and its amount correlates well with the increase in ALA synthase activity.     

The CDC8 gene product is required for transformation with episomal and integrative plasmids in Saccharomyces cerevisiae

Summary The product of the yeast CDC8 gene (thymidylate kinase), which is required for chromosomal, mitochondrial and 2 plasmid replication, also participates in plasmid transformation processes in S. cerevisiae. The thermosensitive cdc8-1 mutant strain was transformed with episomal pDQ9 and integrative pDQ9-1 plasmids both of which carry the CDC8 gene. The results suggest that thymidylate kinase is essential for the expression of genes carried on transforming episomal plasmid DNA (probably through its replication) and is also essential for homologous recombination between chromosomal and linearized integrative plasmid 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.     

Behaviour of a replicating mitochondrial DNA sequence fromAspergillus amstelodami inSaccharomyces cerevisiae andAspergillus nidulans

Summary An amplified sequence of mitochondrial DNA from a ragged (rgd) mutant ofAspergillus amstelodami has been shown to exist in multimeric circular form, suggesting that it is excised from the genome and can exist independently of it. This sequence has replicative (ARS) activity inSaccharomyces cerevisiae, and a subfragment responsible for this activity has been identified and sequenced. A homologous sequence fromAspergillus nidulans mtDNA also has ARS activity inS. cerevisiae. BothA. amstelodami andA. nidulans ARS elements have been incorporated into the integrative transformation vector pDJBI and the derived vectors used to transformA. nidulans. Inclusion of theA. nidulans ARS element enhanced the transformation frequency 5-fold relative to pDJBI. No increase in transformation frequency was evident with the ARS element fromA. amstelodami. The stability of transformants was variable but in comparison to pDJBI, ARS-containing plasmids were mitotically unstable inA. nidulans. Although plasmid DNAs could be rescued inEscherichia coli from undigested transformant DNA, no freely replicating plasmids were detected by Southern hybridisation.     

Transformation of the phytopathogenic fungus Septoria nodorum to hygromycin B resistance

Summary The phytopathogenic fungus Septoria nodorum has been transformed using a plasmid (pAN7-1) containing the Escherichia coli hygromycin phosphotransferase gene (hph). Large, stable hygromycin-resistant transformant colonies appeared at frequencies between 2 and 25 per g DNA when wheat-adapted and barley-adapted wild type strains were used as recipients. These transformants grew at hygromycin concentrations up to ten times that which inhibits the wild types. A second type of colony also developed on transformation plates. These appeared at higher frequencies, grew less vigorously and could not be subcultured in the presence of hygromycin. They are believed to be abortive transformants. Southern hybridization analyses indicated that transformation takes place via the integration of plasmid DNA into the fungal chromosomal DNA. Multiple integrations occur producing tandemly iterated arrays of plasmid molecules. Some transformants arose as heterokaryons. These could be resolved by propagation through a single spore and transformants purified in this way remained mitotically stable. All of 1,025 transformants tested were unchanged in pathogenicity. Reisolates from leaves retained their hygromycin-resistance, indicating that transformants remain stable during growth in plant tissue. Cotransformation of an unselected plasmid (p3SR2) carrying the Aspergillus nidulans amdS gene occurred at a high frequency.     

Cosmids carrying Aspergillus terreus DNA can integrate into Saccharomyces cerevisiae chromosome XII via recombination between yeast and foreign DNAs

Summary A genome clonotheque consisting of 25- to 40-kb Sau3A1 fragments of Aspergillus terreus DNA was constructed in the episomal cosmid vector pES33 containing the yeastARG4 gene. From the 475 transformants of cir° yeast strain ESH-0, 23 stable Arg ⁺ transformants were independently selected. Genetic and Southern analysis of these stable transformants showed that 39% arose as a result of recombination between cloned A. terreus DNA sequences and yeast chromosome XII. The recombination events most likely occurred in the regions of homology within the rDNA clusters of A. terreus and Saccharomyces cerevisiae.     

Transformation of Botrytis cinerea with the hygromycin B resistance gene,hph

A transformation method has been developed for the phytopathogenic fungus Botrytis cinerea. Protoplasts were transformed with pAN7-1 plasmid carrying the Escherichia coli hygromycin phosphotransferase gene (hph), confering hygromycin B resistance, downstream from an Aspergillus nidulans promoter. Molecular analysis showed that transformation resulted in an integration of the plasmid into different regions of the B. cinerea genome and occurred through non-homologous recombination. The frequency was 2–10 transformants per g of DNA. Transformants expressed phosphotransferase activity confirming that the hph gene conferred the hygromycin-resistance phenotype. All transformants analysed so far proved to be stable after several subcultures without any selective pressure.