DNA integration into recipient yeast chromosomes by trans-kingdom conjugation between Escherichia coli and Saccharomyces cerevisiae

Summary IncQ-derived conjugative shuttle vectors, which carried the yeast gene URA3 and/or the yeast autonomously replicating sequence (ARS1), were constructed. Both the ars-plus plasmid pAY205 and the ars-less plasmid pAY201 were successfully transmitted from E. coli to S. cerevisiae by the action of mob and tra. In this trans-kingdom conjugation, plasmid pAY205 could replicate and be retained in transconjugants. Plasmid pAY201 caused the formation of micro-colonies of abortive transconjugants due to its transient expression and rapid disappearance. Nevertheless, one per about 10³ colonies caused by transmitted pAY201 plasmids were uncurable by integration into the homologous region of a yeast chromosome. Analyses by restriction enzyme mapping and Southern hybridization indicate that this integration is primarily caused by a double crossover during conjugation and not by a single reciprocal recombination.     

The host range of the pKD1-derived plasmids in yeast

Summary pKDI is a 2-like circular plasmid found in the yeast Kluyveromyces drosophilarum that can also stably replicate in Kluyveromyces lactis. We have found a short intergenic region in this genome that appears to be functionally neutral; that is, the introduction of foreign sequences into the single EcoRI restriction site located near one of the inverted repeats did not affect the high stability of the natural plasmid. By introducing a G418 resistance gene at this site, we constructed an autonomous recombinant plasmid. Since this vector did not require cir ⁺ hosts for its stable maintenance, it could be used to examine the transformation host range of pKD1 among all the species belonging to the genus Kluyveromyces. Both species closely related to K. drosophilarum as well as a few other species that are very different in chromosomal GC % could be transformed to yield highly stable transformant clones.     

FLP-FRT mediated intrachromosomal recombination on a tandemly duplicated YEp integrant at the ILV2 locus of chromosome XIII in Saccharomyces cerevisiae

Summary A YEp chimaeric plasmid carrying SMR1 and URA3 genetic markers was integrated into chromosome XIII at the ilv2-1 locus in a [cir°] background. The 1.5 kb BglII deletion of ilv2-1 allowed the clear identification of an integrant structure which consisted of a direct tandem duplication (TD) of the chimaeric plasmid. Within the integrant structure, a single copy of the plasmid sequence was flanked by a direct duplication of the 2m site-specific recombinase (FLP) recognition target (FRT). Isogenic [cir°] and [cir ⁺] diploids formed by crossing the [cir°] TD strain to complementary haploids were analyzed for plasmid marker loss and chromosomal DNA alterations in the presence and absence of selection pressure for the URA3 and SMR1 plasmid borne markers. [cir°] diploids showed no plasmid marker loss and maintained the TD structure. In the absence of selection pressure, the [cir ⁺] diploid underwent FLP-FRT mediated unequal interchromatid recombination, resulting in the breakage-fusion-bridge cycle and homozygotization of chromosome XIII (Rank et al. 1988). Maintenance of selection pressure for the centromere distal plasmid URA3 marker selected against FLP-FRT interchromatid recombinants so that the effects of site specific recombinase on intrachromatid recombination could be evaluated. Intrachromatid recombination at the directly duplicated FRT sites of the TD structure resulted in the loss of a diagnostic internal fragment. These results show that in the presence of FLP, FRT sites separated by up to 13.3 kb of chromosomal DNA function as substrates for intra and interchromatid recombination.     

Integrative and replicative genetic transformation of Aureobasidium pullulans

A hybrid selectable marker for transformation was constructed by placing the promoter (TEF1p) from the gene encoding the Aureobasidium pullulans translation elongation factor 1- (TEF1) adjacent to the 5 end of the Escherichia coli hygromycin B phosphotransferase gene (HPT). Plasmids containing this hybrid gene (TEF1p/HPT) transformed A. pullulans strain R106 to a hygromycin B-resistant (HmB^R) phenotype. A PCR-generated DNA fragment consisting of the TEF1p/HPT resistance marker flanked by 41 bp of homologous DNA has also been shown to transform A. pullulans to HmB^R. Linearized plasmid DNA consistently produced more transformants than circular plasmid DNA. Analyses of 23 HmB^R transformants revealed integration of the plasmid in only eight of these transformants. In two transformants, integration into the largest chromosome (VIII) resulted in an alteration of the molecular karyotype. In four other transformants, integration occurred in chromosome VI (the chromosome containing TEF1) but only one was the result of homologous recombination with the genomic copy of the TEF1 promoter. The remainder of the transformants contained replicative plasmids that could be visualized on an agarose gel by ethidium bromide staining. These plasmids were generally 7–8 kb in size. One transformant appeared to contain four plasmids ranging in size from 4 to 8 kb, suggesting rearrangement of the transforming DNA. One plasmid obtained from a HmB^R A. pullulans transformant was able to transform E. coli to ampicillin resistance. However, after recovery from E. coli, this plasmid (approximately 4 kb) was unable to transform A. pullulans to HmB^R.     

Plasmid cloning and expression of the E. coli polA + gene in S. cerevisiae

Summary The E. coli polA ⁺ gene has been subcloned from a specialised transducing phage onto a low copy number plasmid. Plasmid-encoded DNA polymerase I was synthesised at 2 to 3 times the wild-type E. coli level, and was biochemically indistinguishable from chromosomally-encoded protein. It was able to counteract the radio sensitivity of polA1, polAex1, polAex2 and polA12 mutants, but no complementation of polA107 mutants occurred, even though the plasmid polA⁺ gene was expressed. S. cerevisiae ars-1 or 2 replicative sequences were introduced into the polA⁺ plasmid. Transformation of yeast with these constructs increased total DNA polymerase levels 2–20 times, depending upon assay conditions. The additional activity was discriminated from yeast DNA polymerases by its ability to use low concentrations of substrate, by its resistance to chemical inhibition, and by co-electrophoresis with pure DNA polymerase I and its proteolytic fragments. The polA⁺ gene was expressed in yeast without the aid of yeast promotor sequences. However, deletion of cloned DNA more than 99 base pairs in front of the structural gene prevented expression in yeast but not in E. coli, indicating that the two organisms use different sequences for expression of the plasmid polA⁺ gene.     

Recovery of recombinant plasmids from Pleurotus ostreatus transformants

Summary A transformation system employing selectable resistance to hygromycin B has been developed for the mushroom-forming fungus, Pleurotus ostreatus. Vector pAN7-1, a commonly used non-replicative vector for integrative transformation in fungi, yielded 5–46 resistant colonies per g of DNA per 10⁷ viable protoplasts. Southern blot analysis of certain transformants revealed unexpected replicative plasmids containing pAN7-1 sequences, but modified for size, methylation and restriction enzyme pattern when compared to the initial transforming vector. Two such replicative derivatives of pAN7-1 have been rescued from P. ostreatus by cloning into Escherichia coli. Rescued plasmids have been used to probe DNA from untransformed P. ostreatus in an effort to identify fungal sequences that recombined in vivo with pAN7-1 to form replicative plasmids. Such replicative sequences have been localized in high molecular weight (chromosomal) DNA of wild-type P. ostreatus. Transformation has been obtained for P. ostreatus using a rescued plasmid, thereby confirming the role of this recombinant plasmid as a shuttle vector.     

Transformation of the yeast Kluyveromyces lactis by new vectors derived from the 1.6 μm circular plasmid pKD1

Summary The circular plasmid pKD1 (or 1.6 m DNA) has recently been isolated from Kluyveromyces drosophilarum. This plasmid appears to have a functional organization analogous to that of the 2 DNA of Saccharomyces cerevisiae, although the respective nucleotide sequences show little homology. pKD1 can be transferred to Kluyveromyces lactis where it is replicated stably. Using recombinant molecules derived from pKDl, a practical transformation system has been developed for Kluyveromyces lactis, with an efficiency and stability comparable to the 2 -based Saccharomyces cerevisiae transformation system.     

FLP recombinase induction of the breakage-fusion-bridge cycle and gene conversion in Saccharomyces cerevisiae

Summary A YEp chimaeric plasmid containing URA3 and SMR1 [sulfometuron methyl resistant (SM^R) allele of ILV2] as selectable markers, and the 2 m site-specific recombination FLP recognition target (FRT), was integrated at the ilv2-1 site in chromosome XIII in a cir°] haploid. Southern analysis defined two integrant structures. Structure I had URA3 distal and SMR1 proximal to FRT whereas in structure II both markers were distal to FRT. Selectable markers were stably inherited in [cir°] haploids and [cir°] diploids heterozygous for the integrant and ILV2. Approximately 14% of heterozygous [cir ⁺] diploid cells exhibited homozygotization for the distal (500 kb) ade4 marker in trans. In [cir ⁺] diploids FLP-FRT recombination resulted in the simultaneous loss of both structure II markers, whereas the structure I distal URA3 marker loss always preceded the variable loss of the proximal SMR1 marker. URA^– cells continued to segregate for loss of SMR1 until stable URA^– SM^R or URA^–SM^S cells were produced. Gene conversion was identified in stable URA^–SM^R cells that were homozygous SMR1/SMR1 but contained wild type ILV2 restriction endonuclease sites. These observations support a model based on concerted FLP-FRT action resulting from the secondary integration of native 2 m DNA followed by unequal sister chromatid exchange (USCE) within inverted FRTs. The resultant chromatid bridge resulted in a double-stand break. Fusion of the broken ends of sister chromatids generated a breakage-fusion-bridge cycle (BFBC). Repeated rounds of the BFBC resulted in proximal marker loss and the generation of additional double-strand breaks. Recombinogenic properties of the double-strand break initiated events leading to homozygotization and gene conversion.     

The byp1-3 allele of the Saccharomyces cerevisiae GGS1/TPS1 gene and its multi-copy suppressor tRNAGLN (CAG): Ggs1/Tps1 protein levels restraining growth on fermentable sugars and trehalose accumulation

Byp1-3 is an amber nonsense allele of the Sacchromyces cerevisiae GGS1/TPS1 gene which encodes the small subunit of the trehalose synthase complex. Mutations in this gene confer an inability to grow on glucose or fructose but the phenotype of byp1-3 mutants is leaky in a strain-dependent manner. Overexpression of the isolated byp1-3 allele suppressed the growth defect of a ggs1/tps1 mutant. Expression of an in-vitro-generated mutant allele of GGS1/TPS1 that lacks all the coding sequences downstream from the byp1-3 mutation led to the production of a shortened protein that did not complement the ggs1/tps1 mutant. We have isolated, as an allele-specific multi-copy suppressor of the growth defect of the byp1-3 mutant on fructose, the gene for tRNA^GLN (CAG). Thus the leaky phenotype of byp1-3 mutants is due to a low level of read through of the internal nonsense codon by tRNA^GLN (CAG). Using overexpression of the isolated byp1-3 allele, as well as of the tRNA^GLN (CAG) gene, we were able to demonstrate that as little as about 10% of the normal Ggs1/Tps1 protein level is sufficient for slow growth on fructose. We also show a correlation between the level of Ggs1/Tps1, the ability to accumulate trehalose in stationary phase and the ability to grow on fermentable sugars. Sequence analysis of the cloned tRNA^GLN (CAG) gene showed that it is located 700 bp upstream of URA10. However, we found considerable differences to the reported sequence of URA10, in particular in the non-coding region.Communicated by K. Wolf     

Construction of a marker gene cassette which is repeatedly usable for gene disruption in yeast

A disruption cassette has been constructed containing the LEU2 gene flanked by directly repeated sitespecific recombination sites of the yeast plasmid, pSB3, which resembles the 2 m DNA of Saccharomyces cerevisiae. A disruption constructed by inserting this DNA fragment acquires a Leu⁺ phenotype, which can be easily removed by expressing the FLP-PSB3 gene encoding the site-specific recombinase of pSB3. A test was made using a Schizosaccharomyces pombe host. The ura4 ⁺ gene of S. pombe was replaced with the ura4::LEU2 gene constructed by inserting the disruption cassette into the ura4 ⁺ gene. Then, the FLP-pSB3 gene driven by the nmt1 ⁺ promoter was introduced into this disruptant. Upon de-repression of the nmt1 promoter by removing thiamine from the medium, the rate of appearance of Leu^- was increased. As expected the ura4 ⁺ locus underwent a structural change. Thus, the FLP-pSB3 protein and its target site can function adequately in S. pombe.     

The isolation and nucleotide sequence of the pyruvate decarboxylase gene from Kluyveromyces marxianus

We have determined the nucleotide sequence of a 2360-basepair (bp) region of the Kluyveromyces marxianus genome containing the structural gene for the enzyme pyruvate decarboxylase (PDC). Comparison of the deduced amino-acid sequence of this gene to that of the Saccharomyces cerevisiae PDC genes reveals extensive homology including a motif common to thiamin diphosphate-dependent enzymes.     

The use of electrophoretic karyotypes in the classification of yeasts: Kluyveromyces marxianus and K. lactis

Summary The relationship between varieties marxianus and lactis of Kluyveromyces marxianus was investigated. Strains of these varieties readily form hybrids, but their classification in one species has recently been contested. Enzyme patterns and the GC contents of their DNAs differed significantly. Moreover, DNA-DNA reassociation was less than 15%. Since the generally accepted definition of a species is based on gene exchange, we used two approaches for directly detecting genetic recombination in crosses between representatives of both species. First, multiple marked strains were crossed, and the offspring from the resulting hybrids analyzed. Secondly, the fate of individual chromosomes in identical crosses was followed by comparing the karyotypes of the parent strains, the hybrids formed between them, and the descendants of these hybrids. Karyotypes were obtained by orthogonal-field-altemation gel electrophoresis (OFAGE). Gene exchange was not detected with either method. We therefore concluded that the formation of hybrids, even when they produced viable offspring, was not sufficient to include var. lactis and var. marxianus in one species.     

Behaviour of recombinant plasmids in Aspergillus nidulans: structure and stability

Summary A pyrG ^– Aspergillus strain was transformed with plasmid pDJB-1, derived from pBR325 by insertion of the Neurospora crassa pyr4 gene (orotidine 5-phosphate carboxylase), giving mitotically unstable transformants. Aspergillus DNA which acted as an autonomously replicating sequence (ARS) in yeast was inserted into pDJB-1 and the resulting construct, pDJB12.1, gave mitotically stable transformants when introduced into Aspergillus. Transformants obtained with pDJB-1 and pDJB12.1 gave few pyr ^– progeny in crosses to a pyrG ⁺ strain. Southern hybridisation analysis of pyr ⁺ transformants obtained with pDJB-1 revealed restriction fragments expected for integrated plasmid but transformants obtained with pDJB12-1 showed only bands derived from free plasmid. pDJB-1 and derivatives of pDJB12.1 could be recovered from transformants. These derivatives could not be explained by straightforward excision of integrated pDJB12.1 sequences but could result from recombination between plasmid molecules. Hybridisation of undigested transformant DNAs showed that the transforming DNA was present in a high molecular weight form. These results suggest: (1) pDJB12.1 derivatives and possibly pDJB-1 can replicate autonomously in Aspergillus; (2) A. nidulans DNA acting as an ARS in yeast enhances replication and/or segregation of transforming plasmids in Aspergillus; and (3) recombinant plasmids may undergo rearrangements when introduced into Aspergillus.Abbreviations PABA para-amino benzoic acid - EDTA disodium salt of ethylene diamine tetra-acetic acid - SDS sodium dodecyl sulphate - DTT dithiothreitol - UV ultra violet - SSC standard saline citrate; 0.15 M sodium chloride, 0.015 M trisodium citrate pH 7. - ARS('s) autonomously replicating sequence(s) - kb kilobase pairs     

Recombinational properties of the Saccharomyces cerevisiae FLP gene expressed in Escherichia coli

Summary The FLP gene from the 2-m DNA of Saccharomyces cerevisiae is shown to be functionally expressed in Escherichia coli leading to site-specific intramolecular as well as intermolecular recombination between IR sequences. The expression was achieved under control of a low expression as well as a high expression E. coli promoter. The FLP gene was found to complement in trans a Flp^– plasmid and promote its interconversion.By the use of a low Flp expression plasmid, it could be shown that the rate of interconversion of a Flp^– plasmid by complementation in trans, was lower than that of a Flp⁺ plasmid, suggesting that in addition to the IR sequences another cis-acting function exists.Expression of the FLP gene fused to the lac promoter in an in vitro system yielded two polypeptides with apparent molecular weights of 44,000 and 37,000. The 37,000 dalton polypeptide can also be produced from Flp^– plasmids and is generated from a translation start within the FLP gene. The 44,000 dalton polypeptide is considered to represent the FLP gene product.     

High-efficiency electrotransformation of the yeast Schwanniomyces occidentalis

A method has been developed for introducing heterologous DNA rapidly and efficiently by electroper-meabilization into the yeast Schwanniomyces occidentalis. A transformation efficiency as high as 2×10⁵ transformants/g of plasmid DNA was obtained with a squarewave electric pulse of 2.17 kV/cm during 18 ms. Small quantities of DNA (5 ng) can be used to transform 3×10⁸ cells. The main parameters which have been optimized are: presence of adenine in the culture medium, pretreatment of the cells with dithiothreitol during the exponential growth phase of the cells, amount of cells treated, and pulse-field strength and duration. Competent cells can be stored to allow electrotransformation whenever needed.     

Cloning and biochemical characterization of LYS5 gene of Saccharomyces cerevisiae

Summary In Saccharomyces cerevisiae, the functions of two unlinked genes (LYS2 and LYS5) are required for the synthesis of the lysine biosynthetic enzyme, -aminoadipate reductase. The LYS5 gene of S. cerevisiae was cloned by functional complementation of a lys5 mutant, X4004-3A, using a YEp24 plasmid library. The cloned LYS5 gene was contained within a 7.5 kb DNA insert of the recombinant plasmid pSC5. Cloning of LYS5 gene was confirmed by second cycle transformation of a lys5 mutant with the pSC5 plasmid, growth response studies, and plasmid loss experiments with Lys5 ⁺ transformants. Analysis of restriction digests of the pSC5 plasmid revealed 3 EcoRI, 5 PvuII, 1 PstI, 1 BglII and 2 HpaI sites in the 7.5 kb insert. A 3.9 kb internal pSC5 fragment hybridized only to the plasmid pSC5, but no homology was observed with LYS2 DNA or the YEp24 vector. The pSC5 transformed Lys5 ⁺ cells and the wild-type strain exhibited same level of -aminoadipate reductase activity, whereas lys5 mutant and plasmid-cured transformed strain exhibited none. Lys2 ⁺ transformants consistently had five times greater -aminoadipate reductase activity when compared with the wildtype and the Lys5 ⁺ transformant. The -aminoadipate reductase activity was repressed in lysine-grown wildtype and Lys5 ⁺ transformed cells but not in Lys2 ⁺ transformed cells. A Lys2 ⁺ and Lys5 ⁺ double transformant exhibited higher a-aminoadipate reductase activity than lys2 ⁺ or lys5 ⁺ transformant.     

Co-transformation with autonomously-replicating helper plasmids facilitates gene cloning from an Aspergillus nidulans gene library

Autonomously-replicating, marker-less helper plasmids were added to transformations of Aspergillus nidulans with plasmids which normally transform by chromosomal integration. This resulted in as much as a 200-fold increase in transformation efficiency. Recovery of autonomously-replicating plasmid co-integrates indicated that co-transformation involves recombination between integrating and helper plasmids, which occurs at a high frequency. Increasing DNA sequence-homology between pairs of plasmids used in simultaneous transformations enhanced co-transformation efficiency. Using helper plasmids and an A. nidulans gene library in a normally-integrating vector, the genes adC and adD were cloned as part of such a co-integrate. In effect, the addition of helper plasmid converts an integrating into an autonomously-replicating gene library in vivo.     

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.     

Homologous transformation of the edible basidiomycete Agrocybe aegerita with the URA1 gene: characterization of integrative events and of rearranged free plasmids in transformants

The URA1 gene, encoding dihydroorotate dehydrogenase of the pyrimidine pathway, cloned into pUC18 (pUra1-1) was used to develop an homologous transformation system for the cultivated mushroom Agrocybe aegerita. Protoplasts of a ura1 auxotrophic strain were transformed by electroporation with efficiencies ranging from 1 to 26 transformants per g of DNA. The phenotype of the stable Ura+transformants suggested a strong nuclear heterogeneity further confirmed by Southern-blot analysis. All transformants acquired extrachromosomal forms derived from pUra1-1. Integration of pUra1-1 into chromosomal DNA occurred for some transformants. Plasmids containing the integrant of pUC18 recombined to different parts of the URA1 gene were rescued from A. aegerita transformants through transformation of E. coli. Their molecular analysis indicated that they represent products of the continuous excision of primary-integrated vector sequences rather than ARS-dependent autoreplicative forms.