LEU2 directed expression of β-galactosidase activity and phleomycin resistance in Yarrowia lipolytica

Summary The nucleotide sequence of a 968 by DNA fragment spanning the promoter and the 5 upstream sequence of the LEU2 coding sequence of the yeast Yarrowia lipolytica has been determined. A LEU2:lacZ gene fusion has been constructed and expressed in transformed yeast cells, showing that as few as 232 by of the LEU2 promotor were sufficient to direct gene expression. In order to develop new markers for transformation of this yeast, the LEU2 initiation codon was destroyed by in vitro mutagenesis and replaced by a cloning site. A gene confering phleomycin resistance in E. coli was attached to the LEU2 promoter and shown to be efficiently expressed in yeast: direct selection of phleomycin resistant transformants was possible.     

A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae

Summary A homologous transformation system for Aspergillus oryzae is described. The system is based on an A. oryzae strain deficient in orotidine-5-phosphate decarboxylase (pyrG) and the vector pA04-2, which contains a functional A. oryzae pyrG gene as selection marker. Transformation of the A. oryzae pyrG mutant with circular PA04-2 resulted in the appearance of Pyr⁺ transformants at a frequency of up to 20 per g of DNA, whereas with linear pA04-2 up to 200 transformants per g DNA were obtained. In 75 % of the Pyr⁺ transformants recombination events had occurred at the pyrG locus, most of which (90%) resulted in insertion of one or two copies of the vector and the others (10%) in a replacement of the mutant allele by the wild-type allele. Vector pA04-2 is also capable of transforming a corresponding mutant of Aspergillus niger. This transformation system was used to introduce into A. oryzae the heterologous and non-selectable bacterial genes lacZ, encoding -galactosidase, and uidA, encoding -glucuronidase. Using the Aspergillus nidulans gpdA promoter to drive bacterial gene expression in A. oryzae, relatively high levels of activity, as well as protein per se, as judged by western blot analyses, were obtained.     

Transformation of Neurospora crassa with the trp-1 gene and the effect of host strain upon the fate of the transforming DNA

Summary Neurospora trp-1 ⁺ transformants, obtained by transforming a trp-1 inl strain with plasmid DNA containing the wild type trp1 ⁺ gene, were characterized by genetic and Southern blot analyses. The transforming trp-1 gene integrated at or near the resident site in all of the trp-1 ⁺ transformants obtained with circular DNA or DNA cut within the trp-1 coding region. The frequency of homologous integration decreased substantially when the donor DNA was cleaved outside the trp-1 coding region. The transformants were very stable mitotically and, in general, also showed meiotic stability. Analysis of trp-1 ⁺ transformants obtained with another recipient strain, trp-1 ⁺ ga-2 aro-9 inl, showed that homologous integration of donor DNA occurred in only 20% of the transformants, whether circular or linear DNA was used. Thus, the host strain employed for transformation appears to be a major factor in determining the fate of transforming DNA. Southern blot analysis of transformants showed that integration of the transforming DNA at the homologous site occurred by double crossover or gene conversion events rather than by insertion of the entire plasmid DNA. Multiple and apparently non functional integration events were observed in some transformants.     

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     

High frequency FLP-independent homologous DNA recombination of 2 μ plasmid in the yeast Saccharomyces cerevisiae

Summary The purpose of this work is to identify and quantitate in vivo 2 plasmid FLP-independent recombination in yeast, using a nonselective assay system for rapid detection of phenotypic expression of the recombination events. A tester plasmid was constructed such that in vivo recombination between 2 direct repeat sequences produces the resolution of the plasmid into two circular DNA molecules. This recombinational event is detected as a phenotypic shift from red to white colonies, due to the mitotic loss of the plasmid portion containing the yeast ADE8 gene in a recipient ade1 ade2 ade8 genetic background. In the absence of the 2 FLP recombinase and/or its target DNA sequence, recombination is not abolished but rather continues at a high frequency of about 17%. This suggests that the FLP-independent events are mediated by the chromosomally-encoded general homologous recombination system. We therefore conclude that the totality of 2 DNA recombination events occurring in FLP⁺ cells is the contribution of both FLP-mediated and FLP-independent events.     

Transformation of Aspergillus niger using the homologous orotidine-5′-phosphate-decarboxylase gene

Summary A homologous transformation system for the filamentous fungus Aspergillus niger has been developed, based on the orotidine-5-phosphate-decarboxylase gene. A. niger Pyr^– mutants have been selected from 5-fluoroorotic acid resistant mutants. These mutants were found to comprise two complementation groups, pyrA and pyrB. The A. niger OMP-decarboxylase gene was isolated from a gene library by heterologous hybridization with the Neurospora crassa pyr4 gene. The cloned gene is capable to transform A. nidulans pyrG mutants at high frequencies. Transformation of A. niger pyrA mutants occurs with moderate frequencies (about 50 transformants/g DNA) whereas the pyrB mutants cannot be complemented with the cloned OMP-decarboxylase gene. Analysis of the DNA of the A. niger PyrA⁺ transformants showed that transformation resulted in integration of the vector DNA into the genome by homologous recombination. Both gene replacements and integration of one or more copies of the complete vector have been observed.     

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.     

Genetic and molecular characterization of argB+ transformants of Aspergillus nidulans

Summary Thirty-three argB^– to argB⁺ transformants of Aspergillus nidulans have been subjected to genetic and molecular analysis. Two showed high levels of mitotic instability although it is suggested that this is a consequence of heterokaryosis rather than instability of the transformation event. Most transformants resulted from the integration of the transforming DNA in tandem with the chromosomal argB locus. The maximum number of inserted sequences was two, to generate three copies of the argB locus. The other main transformant type showed replacement of the argB^– mutation by the wild-type allele present on the transforming plasmid. Transformants were also recovered in which the transforming DNA had integrated into non-homologous chromosomal regions. Selfed or hybrid cleistothetica from all transformants, except the gene replacement types gave arginine requiring recombinants. Most transformants showed low levels of meiotic instability. Others displayed varying levels which in some cases differed between selfed and hybrid cleistotheticia. There was some correlation between meiotic instability and the nature of the transformation event. Diploid parasexual and aneuploid analysis located the integrated DNA in each transformant to chromosome III. Two transformants were isolated as heterozygous diploids. A third diploid was isolated as a stable mitotic segregant from one of the mitotically unstable transformants.     

Expression of a Bacillus α-amylase gene in yeast

Summary A recombinant plasmid, pSR11.3, containing the -amylase gene (AMY) of Bacillus amyloliquefaciens was characterized and expressed in Bacillus subtilis. A 2.3 kilobase BamHI-BglII fragment carrying AMY was cloned into pBR322 (pEL322) and in both orientations into a multi-copy Escherichia coli-yeast shuttle vector YEp13 (pAM13) and expressed in E. coli HB101 and various Saccharomyces strains. We report on the successful secretion of an active bacterial enzyme in yeast without using yeast promoter and secretory signals. Enzyme production in B. subtilis 1A297(pSR11.3), E. coli HB101(pEL322) and Saccharomyces JM277315B(pAM13) transformants was measured as 125, 22 and 123 U/ml, respectively. The molecular weight of the purified -amylase secreted by B. subtilis 1A297(pSR11.3) and Saccharomyces JM2773-15B(pAM13) was estimated to be 55 kDa. The pH and temperature optima for the -amylase activities of the transformants were 6.5 to 8.0 and 50 to 65 °C, respectively. Amylose hydrolysis profiles of the -amylases secreted by B. subtilis 1A297(pSR11.3) and Saccharomyces JM2773-15B(pAM13) indicate effective meso-thermostable hydrolytic enzymes with maltotriose and maltose, respectively, as major end products.     

Pathogenicity and growth of Metarhizium anisopliae stably transformed to benomyl resistance

Summary The insect pathogenic hyphomycete Metarhizium anisopliae was transformed to benomyl resistance using pBENA3, a plasmid containing the benA3 allele from Aspergillus nidulans. The transformation rate was 9 transformants/50 g DNA/2×10⁶ viable protoplasts. Southern hybridization analyses indicated that the plasmid integrated by nonhomologous recombination at multiple loci. The sites of integration differed among transformants. There was no evidence for autonomous plasmid replication in the transformants. Transformants grew at benomyl concentrations up to 10 times that which inhibits wild type, and they were mitotically stable on either selective or non-selective medium or insect tissue. The transformants were pathogenic to the hornworm, Manduca sexta, producing both appressoria and the cuticle-degrading enzyme, chymoelastase, in the presence of 50 g/ml of benomyl. These studies demonstrate the potential of using transgenic strains of entomopathogenic fungi along with other components of pest control such as fungicides.     

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.     

Transformation ofGaeumannomyces graminis to benomyl resistance

Summary Gaeumannomyces graminis var.graminis andtritici were transformed to benomyl resistance using pBT3, a plasmid encoding fungicide-resistant -tubulin. Either circular or linear plasmid DNA producedG. graminis var.graminis transformants in which plasmid DNA was integrated into the fungal genome. There was no evidence for autonomous plasmid replication in any of the transformants examined. 4/11 linear DNA transformants had a single plasmid copy, whereas 8/9 circular DNA transformants had multiple copies of the plasmid. Integration of transforming DNA occurred by nonhomologous recombination in all (20/20) of these transformants.     

Conversion of homothallic yeast to heterothallism through HO gene disruption

A simple method was developed for the conversion of homothallic Saccharomyces cerevisiae yeast strains to heterothallism through HO gene disruption. An integrative ho::neo disrupted allele was constructed by cloning a dominant selectable marker, the bacterial neo gene, within the HO gene. Transformation of a homothallic diploid S. cerevisiae strain with plasmid DNA containing the ho::neo allele yielded G418-resistant yeast transformants in which one of the HO alleles was replaced by the disrupted ho::neo allele. Meiotic tetrad analysis of four-spored asci from these G418-resistant transformants gave rise to haploid heterothallic and diploid homothallic tetrad progeny. The presence of the ho::neo and HO alleles in the heterothallic and homothallic progeny was confirmed by Souther-blot analysis.     

Effects of the CDC2 gene on adaptive mutation in the yeast Saccharomyces cerevisiae

We have studied the influence of a temperature-sensitive cdc2-1 mutation in DNA polymerase on the selection-induced mutation occurring at the LYS-2 locus in the yeast Saccharomyces cerevisiae. It was found that in cells plated on synthetic complete medium lacking only lysine, the numbers of Lys⁺ revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number. When cdc2-1 mutant cells, after selective plating, were incubated at the restrictive temperature of 37°C for 5 h daily for 7 days, the frequency of an adaptive reversion of lys ^- Lys⁺ was significantly higher than the frequency in cells incubated only at the permissive temperature, or in wild-type cells incubated either at 23°C or 37°C. Therefore, when the proof-reading activity of DNA polymerase is impaired under restrictive conditions, the frequency of adaptive mutations is markedly enhanced.     

Secretion of Trichoderma reesei β-glucosidase by Saccharomyces cerevisiae

An intronless form of thebgl1 gene encoding an extracellular-glucosidase fromTrichoderma reesei was expressed in the yeast Saccharomyces cerevisiae under the control of the yeast GAL 1 promoter. Transformation of a yeast strain with this vector resulted in transformants that produce and secrete active-glucosidase into the growth medium. Additionally, active recombinant-glucosidase protein was shown to be localized predominantly in the periplasmic space by using ap-nitrophenyl-D-glycoside hydrolysis assay against fractionated yeast cells. The apparent size of the recombinant enzyme was 10–15 kDa larger than that of the native form. Treatment of the recombinant-glucosidase with endoglycosidase-H indicated the apparent increase in size was due to N-linked glycosylation.     

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.     

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.     

Integration of vectors by homologous recombination in the plant pathogen Glomerella cingulata

An homologous transformation system has been developed for the plant pathogenic fungus Glomerella cingulata (Colletotrichum gloeosporioides). A transformation vector containing the G. cingulata gpdA [romoter fused to the hygromycin phosphotransferase gene was constructed. Southern analyses indicated that this vector integrated at single sites in most transformants. A novel method of PCR amplification across the recombination junction point indicated that the integration event occurred by homologous recombination in more than 95% of the transformants. Deletion studies demonstrated that 505 bp (the minimum length of homologous promoter DNA analysed which was still capable of promoter function) was sufficient to target integration events. Homologous integration of the vector resulted in duplication of the gdpA promoter region. When transformants were grown without selective pressure, a high incidence of vector excision by recombination between the duplicated regions was evident. The significance of these recombination characteristics is discussed with reference to the feasibility of performing gene disruption experiments.     

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.     

Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha

Summary For the transformation of the yeast Hansenula polymorpha we have constructed a set of hybrid plasmids carrying the LEU2 gene of Saccharomyces cerevisiae as a selective marker and fragments of mitochondrial DNA of Candida utilis and H. polymorpha or chromosomal DNA fragments of H. polymorpha as replicator sequences. The replication properties of chimeric plasmids in the yeast H. polymorpha were investigated. We showed that for plasmids propagated autonomously in this yeast the plasmid monomers could be detected in the transformants only during the immediate time after the transformation event. Further growth under selective conditions led to the selection of polymeric forms of plasmid DNA as it was clearly shown for transformants carrying cosmid pL2 with mtDNA fragment of C. utilis. Such transformants carrying polymerized plasmids showed a remarkably increased stability of the transformed phenotype. Cosmid pL2 was able to shuttle between Escherichia coli, S. cerevisiae and H. polymorpha, whereas plasmids with DNA fragments from H. polymorpha did not transform S. cerevisiae effectively.