Transformation of commercial baker's yeast strains by electroporation

Summary We developed an electroporation protocol for transformation which was particularly optimized for commercial baker's yeast strains. The protocol is based on the standard BIORAD GENE PULSER/PULSE CONTROLLER machine. It works efficiently both for the introduction of standard multicopy plasmids (ARS and 2m based) and for integrative transformation. In particular we were able to transform genuine prototrophic baker's yeast strains with a 2m-based multicopy plasmid, carrying the dominant sulfometuron methyl resistance marker. For plasmids requiring the introduction of more than one copy for complementation, the transformation frequency was considerably lower. This suggests that transformation by the electroporation method introduces on average only one or a few copies of the transforming plasmid per cell.     

High frequency excision of Ty elements during transformation of yeast.

Yeast (Saccharomyces cerevisiae) transposons (Ty elements) are excised from up to 20% of supercoiled plasmids during transformation of yeast cells. The excision occurs by homologous recombination across the direct terminal repeats (deltas) of the Ty element, leaving behind a single delta in the transforming plasmid. Only the initial transforming plasmid is susceptible to excision, and no high frequency excision is observed in plasmids that have become established in transformed cells or in plasmids that are resident in cells undergoing transformation. High frequency excision from plasmids during yeast transformation is not specific for Ty elements and can be observed with other segments of plasmid DNA bounded by direct repeats. The frequency of Ty excision from supercoiled plasmids is greatly reduced when the host yeast cells contain the rad52 mutation, a defect in double-strand DNA repair. When linear or ligated-linear plasmid DNAs containing a Ty element are used for transformation, few or no excision plasmids are found among the transformant colonies. These results suggest that when a yeast cell is transformed with a supercoiled plasmid, the plasmid DNA is highly susceptible to homologous recombination for a short period of time.     

Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells.

We have examined the fate of plasmids containing a segment of a mouse rDNA repeat after they were introduced by transfection into cultured mouse cells. In addition to the rDNA segment, the plasmids contained the thymidine kinase gene from herpes simplex virus 1 to allow for selection of the plasmid after transfection into thymidine kinase-deficient mouse cells. Thus far, no cases of homologous recombination between transfected plasmid DNAs and host cell sequences have been documented. We reasoned that the high repetition frequency of the rRNA genes in the mouse genome (200 copies per diploid cell) might create a favorable situation for obtaining homologous recombination events between the plasmids containing rDNA and host cell rDNA sequences. The plasmids were introduced into cells in both the presence and the absence of carrier DNA and both as covalently closed circles and linear molecules. The sites of plasmid integration in the genomes of various cell lines were examined by DNA restriction digests and hybridization, molecular cloning, and nuclear fractionation. In the seven cell lines examined, there was no evidence that the plasmids had integrated into the rRNA gene clusters of the cell. Thus, the apparent absence of site-specific integration of cloned DNAs introduced into mammalian cells does not appear to be due simply to the small target presented by most host cell sequences.     

Genetic transformation of the filamentous yeast, Trichosporon cutaneum, using dominant selection markers

An efficient transformation system for the filamentous yeast, Trichosporon cutaneum, has been developed. Transformation was obtained with plasmids carrying either the Escherichia coli hygromycin B phosphotransferase-encoding gene (hph) or the Streptoalloteichus hindustanus phleomycin-resistance gene (ble), as dominant selection markers. Expression of both resistance-conferring genes was controlled by the gpd promoter and the trpC terminator, from Aspergillus nidulans. The transformation frequency was up to 500 colonies/micrograms of transforming DNA, using the ble gene, and up to 100 colonies/micrograms of transforming DNA, using the hph gene. Co-transformation frequencies using unselected DNA varied between 50 and 65%. The transforming DNA was found to consist of multiple tandem plasmid copies of high Mr. This polymeric structure, in nonselective media, was mitotically unstable, possibly indicating that it existed in an episomal state.     

High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression

Yeast vectors suitable for high-level expression of heterologous proteins should combine a high copy number with a high mitotic stability under non-selective conditions. Since high stability can best be assured by integration of the vector into chromosomal DNA we have set out to design a vector that is able to integrate into the yeast genome in a large number of copies. The rDNA locus appeared to be an attractive target for such multiple integration since it encompasses 100-200 tandemly repeated units. Plasmids containing several kb of rDNA for targeted homologous recombination, as well as the deficient LEU2-d selection marker were constructed and, after transformation into yeast, tested for both copy number and stability. One of these plasmids, designated pMIRY2 (for multiple integration into ribosomal DNA in yeast), was found to be present in 100-200 copies per cell by restriction analysis. The pMIRY2 transformants retained 80-100% of the plasmid copies over a period of 70 generations of growth in batch culture under non-selective conditions. To explore the potential of pMIRY2 as an expression vector we have inserted the homologous genes for phosphoglycerate kinase (PGK) and Mn2+-dependent superoxide dismutase (SOD) as well as the heterologous genes for thaumatin from Thaumatococcus danielli (under the GAPDH promoter), into this plasmid and analyzed the yield of the various proteins. Under optimized conditions the level of PGK in cells transformed with pMIRY2-PGK was about 50% of total soluble protein. The yield of thaumatin in the pMIRY2-thaumatin transformants exceeded by about a factor of 100 the level of thaumatin observed in transformants carrying only a single thaumatin gene integrated at the TRP1 locus in chromosome IV.     

Mechanism of high-copy-number integration of pMIRY-type vectors into the ribosomal DNA of Saccharomyces cerevisiae

Targeted integration of the yeast plasmid pMIRY2 into the ribosomal DNA (rDNA) of Saccharomyces cerevisiae by homologous recombination results in transformants carrying 100-200 copies of the plasmid per cell which are stably maintained over a large number of generations [Lopes et al., Gene 79 (1989) 199-206]. These properties make pMIRY2 an attractive vector for high-level production of (heterologous) proteins by yeast cells. We have investigated the mechanism underlying high-copy-number (hcn) integration of pMIRY-type plasmids and show that either targeting to a location outside the rDNA locus or use of the wild-type LEU2, instead of the deficient LEU2d gene, as selection marker reduces the copy number to the low value characteristic of standard integrating (YIp-type) yeast plasmids. Further experiments demonstrate that the hcn of pMIRY-type plasmids is achieved by amplification of a small number of copies initially integrated into the rDNA locus. Amplification depends upon the strong selection pressure created by the extremely low expression of the deficient LEU2d gene, but not on the presence of this gene per se. The hcn integration also occurs when either the TRP1 or URA3 gene is used as the selection marker, provided expression of the marker gene is severely curtailed, e.g., by removal of most of its 5'-flanking region.     

Microprojectile-mediated transient and integrative transformation of rice embryogenic suspension cells: effects of osmotic cell conditioning and of the physical configuration of plasmid DNA

Microprojectile-mediated transient and integrative transformation frequencies in rice (Oryza sativa cv. Taipei 309) embryogenic suspension cells were studied as a function of various parameters. Mannitol at concentrations of 0.5 and 0.6 m was best for osmotic preconditioning of the cells for transient, but not for integrative transformation, for which sucrose yielded the best and most reliable results. Denaturation of the transforming plasmid DNA prior to bombardment improved transient and integrative transformation frequencies two to three fold. Delivery of double-stranded plasmids in linear form had no effect on transient transformation when compared to supercoiled plasmid DNA, but led to an overall two fold increase in integrative transformation frequency. This shows that optimized protocols for generating transgenic plants should not be based exclusively on transient gene expression assays. Received: 29 September 1997 / Revision received: 27 February 1998 / Accepted: 2 April 1998     

Integrative transformation system for the metabolic engineering of the sphingoid base-producing yeast Pichia ciferrii

We have developed an integrative transformation system for metabolic engineering of the tetraacetyl phytosphingosine (TAPS)-secreting yeast Pichia ciferrii. The system uses (i) a mutagenized ribosomal protein L41 gene of P. ciferrii as a dominant selection marker that confer resistance to the antibiotic cycloheximide and (ii) a ribosomal DNA (rDNA) fragment of P. ciferrii as a target for multicopy gene integration into the chromosome. A locus within the nontranscribed region located between 5S and 26S rDNAs was selected as the integration site. A maximum frequency of integrative transformation of approximately 1,350 transformants/ microg of DNA was observed. To improve the de novo synthesis of sphingolipid, the LCB2 gene, encoding a subunit of serine palmitoyltransferase, which catalyzes the first committed step of sphingolipid synthesis, was cloned from P. ciferrii and overexpressed under the control of the P. ciferrii glyceraldehyde-3-phosphate dehydrogenase promoter. After transformation of an LCB2 gene expression cassette, several transformants that contained approximately five to seven copies of transforming DNA in the chromosome and exhibited about 50-fold increase in LCB2 mRNA relative to the wild type were identified. These transformants were observed to produce approximately two times more TAPS than the wild type.     

Construction of hybrid plasmids containing the yeast replicator

In Saccharomyces cerevisiae strain 6-1G-P188 about 10 per cent of rRNA genes exist as extrachromosomal copies of rDNA repeating units. These extrachromosomal copies can be isolated as covalently closed molecules with lengths around 3mu. We have constructed a set of hybrid plasmids containing the bacterial vector pBR325, the LEU2 gene of yeast encoding beta-isopropylmalatedehydrogenase and various EcoRI restriction fragments of the 3mu DNA. We have tested the ability of our hybrid plasmids to transform LEU2 strain DC5 to leucine prototrophy. One of the plasmids Rcp21/11 transforms DC5 at the frequency comparable with that obtained with YEp13, containing the 2mu DNA replication origin. The 2400 bp EcoRI-B fragment of the 3mu DNA in Rcp21/11 carries a gene for 5S rRNA and two spacers. Our results on transformation experiments allow un to suggest that this EcoRI fragment also carries the 3mu DNA replication origin. Yeast transformants containing this plasmid are highly unstable but during the prolonged growth in selective conditions the stabilization of the LEU+ phenotype is observed being most likely a result of integration of Rcp21/11 into the yeast chromosome.     

Development of a high-frequency transforming vector for Aspergillus nidulans

The pyr4 gene of Neurospora crassa, which codes for orotidine-5'-phosphate decarboxylase, is capable of transforming an Aspergillus nidulans pyrG mutant by chromosomal integration, despite low homology between the transforming DNA and the recipient genome. Integration of pFB6, a plasmid carrying pyr4 and capable of replication in Escherichia coli, was not observed at the pyrG locus. The efficiency of transformation was considerably enhanced (50-100 fold) by inclusion in the transforming vector of a 3.5-kb A.nidulans chromosomal sequence, ans1. Although this sequence was isolated on the basis of replicating activity in Saccharomyces cerevisiae, there was no evidence for such activity in A.nidulans. Part of the ans1 fragment appears to be reiterated in the A.nidulans genome, though it is not yet clear whether this is directly responsible for the high transformation frequency. The efficiency of transformation of A.nidulans by plasmids bearing ans1, using an improved protocol, was approx. 5 X 10(3) stable transformants per microgram of plasmid DNA.     

Features of transformation of competent cells and Bacillus subtilis protoplasts by integrative vectors

The effect of structural peculiarities of DNAs from integrative plasmids on the transformation activity was studied. Monomeric forms of the plasmids can only transform B. subtilis competent cells, when plasmid selective marker is inserted into chromosomal fragment within the plasmid. Polymeric forms are needed for efficient transformation. Both single- and double-stranded DNAs of integrative plasmids transform no B.subtilis protoplasts, this being irrespective of plasmid structure.     

Co-transformation with autonomous replicating and integrative plasmids in Penicillium chrysogenum is highly efficient and leads in some cases to rescue of the intact integrative plasmid

The efficiency of co-transformation in Penicillium chrysogenum Wisconsin 54-1255 pyrG(-) and the fate of the transforming DNA were studied using an integrative (pEF43) and an autonomous replicating plasmid (pAM9L). The results showed a co-transformation frequency of nearly 70% of all transformants tested. The total efficiency of transformation was shown to be dependent on the plasmid marker used as transformant selection (i.e., markers in the integrative or autonomous replicating vector). Analysis of the plasmids re-isolated from several co-transformants showed that different populations of plasmids co-exist in the fungal host. Interestingly, in all co-transformants studied, the integrative plasmid was found to be replicating autonomously without integrating into the host genome. In some cases, co-integrates were formed by recombination between autonomous replicating (pAM9L) and integrative (pEF43) plasmids. However, unexpectedly in some cases, the non-reorganised pEF43 integrative plasmid used in the co-transformation assays was rescued from some co-transformants.     

Two separate regions of the extrachromosomal ribosomal deoxyribonucleic acid of Tetrahymena thermophila enable autonomous replication of plasmids in Saccharomyces cerevisiae.

Plasmids containing the nontranscribed central and terminal, but not the coding, regions of the extrachromosomal ribosomal deoxyribonucleic acid (rDNA) of Tetrahymena thermophila are capable of autonomous replication in Saccharomyces cerevisiae. These plasmids transform S. cerevisiae at high frequency; transformants are unstable in the absence of selection, and plasmids identical to those used for transformation were isolated from the transformed yeast cells. One plasmid contains a 1.85-kilobase Tetrahymena DNA fragment which includes the origin of bidirectional replication of the extrachromosomal rDNA. The other region of Tetrahymena rDNA allowing autonomous replication of plasmids in S. cerevisiae is a 650-base pair, adenine plus thymine-rich segment from the rDNA terminus. Neither of these Tetrahymena fragments shares obvious sequence homology with the origin of replication of the S. cerevisiae 2-microns circle plasmid or with ars1, an S. cerevisiae chromosomal replicator.     

Recombination of plasmids into the Saccharomyces cerevisiae chromosome is reduced by small amounts of sequence heterogeneity.

As a model system for studying the properties of mitotic recombination in the yeast Saccharomyces cerevisiae, we have examined recombination between a recombinant plasmid (introduced into the S. cerevisiae cell by transformation) and homologous chromosomal loci. The recombinant plasmids used in these experiments contained S. cerevisiae rRNA genes. We found that the frequency of integrative recombination is sensitive to small amounts of sequence heterogeneity. In addition, the frequency and specificity of these recombination events are affected by the lengths of the interacting homologous DNA sequences.     

Nonintegrative transformation in the filamentous fungus Podospora anserina: stabilization of a linear vector by the chromosomal ends of Tetrahymena thermophila.

The effect of the chromosomal ends of Tetrahymena thermophila on the stability of linear transforming molecules in the filamentous fungus Podospora anserina was tested. A derivative of an integrative vector for this fungus has been constructed, so that after linearization, the ends of the plasmid are the telomeric sequences of T. thermophila. After transformation, this linear molecule was maintained as an extrachromosomal plasmid with no integrated copies in about 50% of the transformants. Under selective conditions, there was approximately one linear molecule per 5 to 10 nuclei, and these extrachromosomal molecules were rapidly lost under nonselective conditions. The circular plasmid carrying an inverted repeat of T. thermophila telomeres could be linearized and processed in vivo.     

Insertion of nonhomologous DNA into the yeast genome mediated by homologous recombination with a cotransforming plasmid

Bacterial plasmids containing no detectable homology with yeast DNA sequences were inserted into the yeast genome by cotransforming with a plasmid containing a yeast gene. Analysis of the yeast transformants confirmed that recombination events occurred between the prokaryotic sequences shared by the two plasmids and between the yeast sequences common to the cotransforming plasmid and to the genome. Multiple copies of the two plasmids, in both tandem and interspersed arrays, are inserted by this method. Populations of cells grown from individual transformants are heterogenous for the number of integrated sequences. The number of integrated bacterial sequences is greatly reduced after 100 generations of growth in the populations that initially contained large numbers of sequences, while it is stable in those populations that initially contained either a single or a small number of copies.     

Plasmid transformation of Streptococcus sanguis (Challis) occurs by circular and linear molecules

Summary Transformation of Streptococcus sanguis (Challis) by antibiotic resistance plasmids has shown that (a) competente developed with identical kinetics for chromosomal and plasmid DNA; (b) dependence of transformant yield on plasmid DNA concentration was second order; (c) open circular plasmid DNA transformed Challis, although at reduced frequency; (d) linearization of plasmid DNA by restriction enzymes cutting at unique sites inactivated the transforming capacity; (e) transforming activity was restored when linear plasmid molecules generated by different restriction enzymes were mixed; (f) restoration of transforming activity depended on the distance between the linearizing cuts, i.e. on the presence of sufficiently long overlapping homologous sequences; (g) when linear deletion mutants were mixed with linear parental plasmids the smaller plasmid was restored with significantly higher frequency.Based on these data, a model for plasmid transformation of Challis is proposed according to which circular plasmid is linearized during binding and uptake. One DNA strand enters the cell and restoration of circular plasmids inside the cell occurs by annealing of complementary single strands from two different donor molecules. Implications of this model for recombinant DNA experiments in streptococci are discussed.     

Insertion of a genetic marker into the ribosomal DNA of yeast

Plasmid pBR322 carrying the yeast LEU2+ gene transforms leu⁻ yeast into LEU+ at a low frequency by integration at homologous chromosomal DNA. When one-half of the yeast rDNA repeat unit (BglII-A) is inserted into the plasmid, the frequency of yeast transformation increases 100- to 200-fold, in proportion to the increased amount of homologous repetitive rDNA available for integration. When the other half of the repeat unit (BglII-B) is inserted into the plasmid, the transformation frequency increases by a factor of 10⁴, and the transformants are very unstable. It is likely that this fragment of rDNA contains a yeast origin of replication. This plasmid is a useful vector for cloning fragments of yeast DNA in yeast. We have used the LEU2+ gene, inserted into the rDNA locus, as a genetic marker for mapping the rDNA, in a procedure analogous to the use of antibiotic resistance transposons in the mapping of bacterial genes. Yeast ribosomal DNA is on chromosome XII between asp5 and ura4 as determined by mitotic linkage. Genetic analysis of markers inserted at the rDNA locus should be a useful tool for studying the conservation of sequence homology and the conservation of copy number of repeated genes.     

Efficient Transformation of the Astaxanthin-Producing Yeast Phaffia Rhodozyma

An efficient transformation system for the astaxanthin-producing yeast Phaffia rhodozyma was developed based on electroporation that routinely yields approximately 1000 transformants per g of plasmid DNA. The high transformation efficiency depends on vector integration in the ribosomal DNA (rDNA) and the presence of the homologous glycolytic glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and terminator to drive the expression of the transposon Tn5 encoded kanamycin resistance gene (Km^R) as a selective marker. Using this system stable transformants were obtained, carrying multiple plasmid copies. Plasmid copy number could be markedly increased by deletion of the gpd terminator from the transforming plasmid.     

Isolation and characterization of an autonomously replicating sequence from Ustilago maydis.

DNA fragments that function as autonomously replicating sequences (ARSs) have been isolated from Ustilago maydis. When inserted into an integrative transforming vector, the fragments increased the frequency of U. maydis transformation several-thousandfold. ARS-containing plasmids were transmitted in U. maydis as extrachromosomal elements through replication. They were maintained at a level of about 25 copies per cell but were mitotically unstable. One ARS characterized in detail, which we called UARS1, was localized to a 1.7-kilobase fragment. UARS1 contained a cluster of active sequences. This element could be reduced further into three separate subfragments, each of which retained ARS activity. The smallest one was 383 base pairs (bp) long. Although not active itself in yeast, this small fragment contained seven 8-bp direct repeats, two contiguous 30-bp direct repeats, and five 11-bp units in both orientations with sequences similar but not identical to the consensus sequence found to be crucial for ARS activity in Saccharomyces cerevisiae.