Illegitimate integration of single-stranded DNA in Saccharomyces cerevisiae

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

Development of a homologous transformation system for Aspergillus parasiticus with the gene encoding nitrate reductase

Summary The nitrate reductase structural gene (niaD) and an niaD mutant strain were isolated from Aspergillus parasiticus and used to develop a homologous transformation system. A transformation frequency of 110 to 120 transformants per microgram linear DNA was obtained with the 10.9 kb plasmid pSL82, which contained the niaD gene of A. parasiticus. Plasmid pSL82 was also capable of complementing Aspergillus nidulans FGSC A691, a niaD mutant, though at lower frequencies. Southern hybridization analyses of A. parasiticus niaD transformants showed that the niaD gene of pSL82 had integrated into the fungal genome. In addition, vector (bacterial plasmid) sequences were also present in one of the niaD transformants.Authors with primary and equal contribution in the research project     

Illegitimate integration of single-stranded DNA in Saccharomyces cerevisiae

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

Expression of a Thermomonospora fusca Cellulase Gene in Streptomyces lividans and Bacillus subtilis

A cellulase gene from Thermomonospora fusca coding for endocellulase E₅ was introduced into Streptomyces lividans by using shuttle plasmids that can replicate in either S. lividans or Escherichia coli. Plasmid DNA isolated from E. coli was used to transform S. lividans, selecting for thiostrepton resistance. The transformants expressed and excreted the endocellulase, but the ability to produce the endocellulase was unstable. This instability was shown to result from deletion of the endocellulase gene from the plasmid. Plasmid DNA prepared from a culture in which plasmid modification had occurred was used to transform E. coli, selecting for Amp⁺ cells, and all of the transformants were cellulase positive, showing that pBR322 and T. fusca DNA were deleted together. When a plasmid was constructed containing only T. fusca DNA in plasmid pIJ702, the transformants were more stable, and the level of endocellulase activity produced in the culture supernatant after growth on 0.2% glucose was close to the level produced by T. fusca cultures grown on 0.2% cellulose. About 50% of the total protein in the culture supernatant of the S. lividans transformant was endocellulase E₅. The enzyme produced by the S. lividans transformant was identical to pure T. fusca E₅ in its electrophoretic mobility and was completely inhibited by antiserum to E₅. Shuttle plasmids containing the E₅ gene that could replicate in Bacillus subtilis and E. coli were also constructed and used to transform B. subtilis. Again there was extensive deletion of the plasmid DNA during transformation and growth in B. subtilis. There was no evidence of E₅ activity, even in those B. subtilis transformants that retained the E₅ gene.     

Construction of Double-Copy Glucose Isomerase Gene Engineering Strain of Streptomyces diastaticus by Homologous Recombination

The plasmid pUT for homologous recombination was constructed by the insertion of the 1.1-kb thiostrepton resistance (tsr ^R) gene into the E. coli plasmid pUB1-GI1. Plasmid pUTK was produced through ligating the cleaved plasmid pUT by KpnI. After pUT and pUTK were introduced into Streptomyces diastaticus No.7 strain M1033 (SM33) by protoplast transformation, a series of tsr^R transformants were obtained, further based on enzyme assays. These results for polymerase chain reaction (PCR), DNA sequencing, restriction enzyme digestion, and recovery of cloned fragments from the transformant chromosome demonstrated the plasmid pUT and pUTK had integrated into the SM33 chromosome in three different patterns of single cross-over by homologous recombination. This directly results in double-copy GI gene in the transformant chromosome, of which one is wild-type GI gene, the other mutant GI (GIG138P, GI1) gene. Among the strains of the three kinds of recombinant patterns, one transformant was chosen and named K1, T2, and T3, respectively. The further identification of the three recombinant strains by PCR, DNA sequencing, restriction enzyme digestion, and Southern hybridization also proved there is a double-copy GI gene within their chromosome. Enzyme activity assay and thermostability analysis indicated that all three engineering strains expressed not only wild-type enzyme but also mutant GI. Received: 9 July 2001 / Accepted: 8 August 2001     

Genetical and Molecular Analyses of qa-2 Transformants in NEUROSPORA CRASSA

Neurospora crassa qa-2+ transformants from five different donor DNA clones were analyzed by genetical and molecular techniques. None of the 32 transformants have the qa-2+ DNA replacing the qa-2- gene in linkage group VII. In one transformant, the qa-2+ DNA was inserted adjacent to the qa-2- gene. Thirty-one transformants have the qa-2+ inserts at sites not linked, or not closely linked, to the qa-2 locus in LG VII. Plasmid sequences were integrated along with the qa-2+ gene in 28 transformants. In the unlinked duplication-type transformants, catabolic dehydroquinase (the qa-2+ gene product) was induced at 5-100% of the wild-type-induced enzyme activity, with 24 transformants in the 5-80% range. The reduced levels of enzyme activity may be due to "position effects" of sequences adjacent to the integration site either in the N. crassa genomic DNA or in the flanking plasmid (pBR322 or pBR325) sequences. Unexpected gene conversion-like events, in which a qa-2+ gene was changed to qa-2-, were observed in tetrads from intercrosses between unlinked duplication-type transformants and in selfings of such transformants.     

Genetic transformation of Clostridium botulinum hall a by electroporation

Summary A method was developed for the introduction of plasmids into Clostridium botulinum by electroporation. A 4.4 kb plasmid vector, pGK12, which contains genes for resistance to erythromycin (Em^r) and chloramphenicol (Cm^r) was electroporated into C. botulinum type A (Hall A). The highest transformation efficiency was obtained using midlog phase cells, 10% PEG 8000 as the electroporation solution, and 2.5 kV field strength. The transformation efficiency was highest (10³ transformants/g of DNA) when 1 g of plasmid DNA and 4 × 10⁸ CFU/ml of recipient cells were used. Plasmid DNA recovered from the transformants was indistinguishable from that introduced on the basis of restriction enzyme digestion and agarose gel electrophoresis.     

Isolation of physarum DNA segments that support autonomous replication in yeast

Summary Hybrid plasmids containing the bacterial resistance-transfer factor pBR322 and the yeast leu2 ⁺gene have been used to isolate DNA fragments of Physarum that are capable of initiating DNA replication in a yeast host. Five of forty hybrid plasmids containing Physarum sequences transform leu2 ^-yeast to Leu⁺ at high frequency. The resulting Leu⁺ transformants are characterized by phenotypic instability. Supercoiled plasmid molecules containing pBR322 sequences can be detected in the transformed yeast, indicating that the transforming DNA replicates autonomously. Plasmid DNA isolated from Leu⁺ yeast can transform leuB bacteria. The hybrid plasmid recovered from the Leu⁺ bacterial transformants is identical to the original plasmid, indicating structural integrity is maintained during passage through the yeast host. These hybrid plasmids containing Physarum sequences have the same characteristics as those containing autonomously replicating yeast chromosomal sequences. As the temporal sequence of DNA replication is particularly accessible to study in Physarum plasmodia, the functional significance of these segments should be amenable to study.     

嗜碱细菌NTT36嗜碱基因的亚克隆及其在大肠杆菌和农杆菌中的表达

将大肠杆菌ＨＢ１０１嗜碱转化子中质粒ｐＧＣＡ所携带的嗜碱基因亚克隆至双元载体ｐＢＩ１２１质粒中,构建了植物表达载体ｐＬＧＣ重组质粒。用其转化大肠杆菌ＨＢ１０１获得了能在碱性和卡那霉素抗性平板上生长的转化子,再通过三亲交配法将亚克隆质粒ｐＬＧＣ转化进农杆菌ＬＢＡ４４０４,又获得能在碱性平板和卡那霉素及利福平双抗平板上生长的转化子,Ｓｏｕｔｈｅｒｎ杂交结果表明ＨＢ１０１转化子亚克隆质粒ｐＬＧＣ是由来自于嗜碱芽孢杆菌ＮＴＴ３６染色体ＤＮＡ和双元载体ｐＢＩ１２１组成,且农杆菌ＬＢＡ４４０４转化子含有来自大肠杆菌亚克隆转化子的ｐＬＧＣ质粒。     

Genetic analysis of Saccharomyces cerevisiae transformed by plasmid containing a supressor transfer ribonucleic acid gene.

The behavior in Saccharomyces cerevisiae of plasmid pYTE1, which contains yeast tyrosine-inserting ochre suppressor SUP4.o, a 4-kilobase EcoRI fragment of yeast 2muDNA, and the bacterial plasmid pBR322, has been studied. Selection of yeast transformants was by suppression of multiple ochre mutations. About 10(3) to 10(4) transformants per microgram of pYTE1 dfeoxyribonucleic acid were obtained. The majority of transformants contained both an integrated copy of the SUP4.o gene plus pBR322 deoxyribonucleic acid sequences and autonomously replicating forms of the plasmid. The integrated copy was extremely stable mitotically and meiotically, but the associated nonintegrated copies were lost at meiosis. The chromosomally integrated pBR322 sequences were linked to the SUP4.o gene. The integration site was at the SUP4+ locus. In transformants with only nonintegrated copies of pYTE1, the expression of suppression was reduced, and the plasmid was unstable in mitosis. Plasmid deoxyribonucleic acid preparations from both types of transformant could be used to retransform yeast cells. Plasmid pYTE1 has restriction enzyme sites useful for the high frequency and stable transformation of other genes into yeasts. The potential uses of this plasmid for transformation of other organisms is discussed.     

Integrative Transformation of Aspergillus oryzae with a Plasmid Containing the Aspergillus nidulans argB Gene

The transformation of Aspergillus oryzae has been achieved with a plasmid carrying the Aspergillus nidulans argB gene coding for ornithine carbamoyltransferase (OCTase). The frequency of transformation was relatively low (0.7 transformants/μg DNA) but the transformed phenotype was extremely stable for many generations without selective pressure.

Southern blot analysis revealed that transformation had occurred by integration of multiple tandem copies of plasmid DNA into the host genome through non-homologous recombination. There was no evidence of the existence of free plasmid in the transformants. The number of integrated copies of the plasmid ranged from 15 to 60. The specific activity of OCTase in the cell- free extract was proportional to the copy number of the plasmid, indicating that most of the integrated argB gene was expressed.     

Electrotransformation ofStreptococcus sanguis Challis

Plasmid DNA was introduced into noncompetent cells ofStreptococcus sanguis Challis by an electrotransformation technique. The procedure was simple and rapid, did not require elaborate pretreatment of cells, and yielded transformant colonies in 24 h. The maximum transformation efficiency attained was 2.1×10⁴ transformants per g of pVA736. Molecular rearrangements and deletions were not detected in plasmid DNA isolated from transformants.     

Reversible inactivation of a foreign gene, hph, during the asexual cycle in Neurospora crassa transformants

Summary A plasmid construct carrying the hygromycin phosphotransferase (hph) gene fused to the expression elements of the trpC gene of Aspergillus nidulans was used to obtain hygromycin B (Hyg)-resistant transformants of Neurospora crassa. The plasmid does not have any homology with the N. crassa genome. Here we demonstrate that most of the transformants arise from integration of the transforming DNA into only one of the nuclei present in the protoplasts. Furthermore, in most of the transformants the integrated transforming DNA is physically stable after growth of the transformants for about 25 nuclear divisions without Hyg selection, in spite of being present in multiple copies. In transformants carrying only a single insertion, phenotypic expression of the hph gene remains unaltered in conidial isolates obtained withoug Hyg selection. On the other hand, about 40% of transformants harbouring plasmid DNA integrated at more than one location yield conidial isolates showing reversible inactivation of the hph genes. Interestingly, the presence of methylated cytosine residues in the integrated DNA is strongly correlated with the number of plasmid copies. The hph genes are heavily methylated in transformants harbouring multiple copies but not in those harbouring only one copy of the plasmid. Phenotypic expression of the inactive hph genes can be restored by growing the transformants either under Hyg selection pressure or in the presence of 5-azacytidine. In the first case the hph genes are again inactivated when Hyg selection pressure is removed, while the activation of the hph gene by 5-azacytidine gives stable Hyg^r strains.Dedicated to Dr. T.A. Trautner on the occasion of his 60^th birthday     

Construction of Plasmid Vectors and Transformation of the Marine Yeast Debaryomyces hansenii

We have constructed two plasmid vectors (pMR95 and pMR96) with selectable markers for the marine yeast Debaryomyces hansenii. Plasmid pMR95 contains an autonomously replicating sequence previously isolated from Debaryomyces and a hygromycin B resistance gene from the plasmid pLG90 under the control of the isocytochrome C1 promoter and terminator sequences, while pMR96 has, in addition, the Saccharomyces URA3 gene. Transformation in Debaryomyces was accomplished by electroporation. Plasmid pMR95 was capable of transforming both Saccharomyces cerevisiae and D. hansenii to hygromycin resistance at low frequencies; pMR96 transformed both yeasts at low frequencies when selected for hygromycin B resistance and at very high efficiencies when selected for uracil prototrophy. The presence of the plasmids in the transformed yeast was confirmed by polymerase chain reaction. The plasmids could be recovered back in Escherichia coli when transformed with total DNA from the yeast transformants, indicating at least a partial autonomous existence of the plasmids in the marine yeast. To our knowledge this is the first successful attempt to transform D. hansenii. Received April 16, 1998; accepted June 30, 1998.     

Study of the stability of hybrid plasmids replicating in Saccharomyces cerevisiae due to DNA fragments from polyoma virus

Hybrid plasmid pSP97 carrying the entire genome of polyoma virus (PY), inserted into bacterial vector psV3, transforms yeast cells with the frequency 1 x 10(-2). Plasmid pSP97 is capable of autonomous replication in S. cerevisiae, while its structure remains unaltered, the stability of hybrid plasmid in transformants is 44%--100%. Plasmid pSP155 consisting of Ori-containing DNA segment from polyoma, pBR322 and yeast gene arg4, transforms yeast cells with the frequency 5 x 10(-3), the stability of plasmid in transformants is 23%--29%. Two types of plasmids were isolated from transformants: one was identical to SP155, while the another differed structurally and phenotypically from SP155. Plasmids pSP113 and pSP114, in addition to pBR322 and yeast gene arg4, contain a viral DNA segment that encodes genes from small and middle T-antigens. These plasmids transform yeast cells with low frequency (2 x 10(-4), 3 x 10(-5)), the stability of plasmids in yeast transformants is 100%. However, hybrid plasmids identical to pSP113 were isolated from transformants. Structural rearrangements have been observed in pSP114, which carries the arg4 gene in reversed orientation compared to pSP113.     

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.     

Construction of a hybrid plasmid capable of replication in Amycolatopsis mediterranei

A new plasmid, pA387, has been isolated from "Amycolatopsis sp." (DSM 43387). This plasmid could be isolated from liquid culture as well as mycelium from agar plates by a modified procedure. Plasmid pA387 is about 29.6 kb and can be cured at low frequency by protoplasting and ethidium bromide and heat treatment. Hybridization experiments showed that this plasmid is present in free form and does not integrate into the chromosome. A hybrid plasmid was constructed by cloning a 5.1-kb fragment of pA387 into the Escherichia coli vector pDM10. This hybrid plasmid, termed pRL1, could be transformed into Amycolatopsis mediterranei and A. orientalis by electroporation. A transformation frequency of 2.2 x 10(3) transformants per micrograms of DNA at 12.5 kV/cm and a pulse duration of 10.8 ms was obtained in A. mediterranei, whereas 1.1 x 10(5) transformants per microgram of DNA were obtained at a field strength of 7.5 kV/cm and a pulse duration of 7.6 ms in A. orientalis. Plasmid pRL1 is the first hybrid plasmid which could be used successfully for the transformation of A. mediterranei. The plasmid has a rather high copy number, is genetically stable, and can be easily reisolated from A. mediterranei. Plasmid pRL1 will be useful for further construction of a shuttle vector for E. coli and A. mediterranei and becomes the basis for the development of gene cloning techniques in Amycolatopsis spp.     

Optimum Conditions for Electric Pulse-mediated Gene Transfer to Bacillus subtilis Cells

It was found that plasmid DNA (pUB 110) can be introduced into not only protoplasts but also intact cells of Bacillus subtilis by electric field pulses. The transformation of, B. subtilis using protoplasts results in an efficiency of 2.5 × 10⁴ transformants per μg of DNA, with a single pulse of 50 jisec with an initial electric field strength of 7kV/cm. Even transformation of intact B. subtilis cells results in a maximum efficiency of 1.5 × 10³ transformants per μg DNA, with a single pulse of 400 μsec with an initial electric field strength of 16kV/cm. The cell survival of protoplasts and intact cells was approximately 100% and 30%, respectively, under the conditions found to be optimal for the transformation process. Plasmid DNA isolated from pUB 110 containing transformants was indistinguishable from authentic preparations of pBU 110 on gel electrophoretic analysis.     

Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae.

Summary A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu^–) was transformed by pRC2312 to Leu^– at a frequency of 1.41 × 10⁵ colonies per g DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura⁺ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 × 10³ per g DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per g DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15±3 per haploid genome in S. cerevisiae and 2–3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7–12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade^– strains of both C. albicans and S. cerevisiae to Ade⁺. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.     

Development of a genetic transformation system for Histoplasma capsulatum: complementation of uracil auxotrophy

Summary We have developed a simple and efficient transformation system for the dimorphic fungus Histoplasma capsulatum. Mutants of H. capsulatum defective in orotidine-5-monophosphate pyrophosphorylase were transformed to prototrophy by the cloned URA5 gene of the filamentous fungus Podospora anserina. Abortive and mitotically stable transformants were obtained. The stable transformants had integrated copies of the plasmid, some in tandem head-to-tail orientation. Free plasmid identical to the transforming plasmid was present in some of the transformants. We obtained a transformation efficiency of up to 30 transformants/g DNA for plasmid pPAura5-1 (9.2 kb). pPW2001, a smaller plasmid (4.7 kb) derived from pPAura5-1, transformed H. capsulatum more efficiently (up to 155 transformants/gm DNA).