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.     

Transient expression of a chloramphenicol acetyltransferase gene following transfection of Physarum polycephalum myxamoebae

Summary A plasmid was constructed containing a replication origin sequence from the Physarum ribosomal DNA molecule, and a bacterial chloramphenicol acetyltransferase (CAT) gene linked to a putative promoter of the long terminal repeat (LTR) of the Physarum HpaII-repeat element. The plasmid was transfected into Physarum myxamoebae either by electroporation or CaCl₂ treatment. In both cases significant transient levels of CAT gene expression were detected. Results were compared with those obtained with plasmids in which CAT gene expression was driven by eukaryotic virus promoters.     

Transformation of Rhizopus niveus using a bacterial blasticidin S resistance gene as a dominant selectable marker

Summary Rhizopus niveus has been transformed to blasticidin S resistance by vectors containing the bacterial blasticidin S resistance gene under the control of a Rhizopus promoter. Southern analysis of the total DNA from transformants indicated that the introduced DNA was rearranged, and that one of the transformants harbored extrachromosomal plasmids with rearranged DNA. Using this transformation system, the introduction of pUBSR101, a plasmid carrying the Escherichia coli lacZ gene fused to the promoter and the N-terminal region of the R. niveus aspartic proteinase-II (RNAP-II) gene, resulted in an increase of -galactosidase activity in the cell extract, indicating expression of the lacZ fusion gene in R. niveus. This is the first report of a transformation system for filamentous fungi using the blasticidin S resistance gene as a dominant selectable marker.     

Transformations of Penicillium islandicum and Penicillium frequentans that produce anthraquinone-related compounds

Wild-type strains of Penicillium islandicum and Penicillium frequentans, which produce anthraquinone and related compounds, were transformed to benomyl and hygromycin B resistance. Plasmids pSV50 and pBT6, with benomyl-resistant -tublin genes, and plasmids pAN7-1 and pDH25, with a bacterial hygromycin phosphotransferase gene under the control of Aspergillus nidulans sequences, were used respectively. Transformation frequencies with these plasmids were 10–20 transformants per g of DNA per 4-8×10⁷ viable protoplasts. Intergration of plasmid DNAs into chromosomal DNAs was confirmed by Southern-blot analysis. Copy numbers and sites of integration varied among transformants. The integrated plasmid DNAs conferring a drug-resistant phenotype were mitotically stable with or without selection. The demonstration of such transformation systems is the essential first step in the application of recombinant DNA technology to study the biosynthetic genes of anthraquinone and related compounds in P. islandicum and P. frequentans.     

A novel class of vector for yeast transformation

Summary We have constructed a set of hybrid yeast Escherichia coli vectors which utilise the site specific recombination function of the Saccharomyces cerevisiae 2 m plasmid to completely eliminate the bacterial moiety upon introduction into yeast. A number of these plasmids have been shown to exhibit high inheritable stability in both laboratory and industrial strains during non-selective growth. These plasmids are beneficial for the genetic modification of industrial yeast, particularly those used in the production of food and beverages, and are of benefit in the study of plasmid maintenance and heterologous gene expression.     

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

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

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

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

Cell type specific and inducible promoters for vectors in gene therapy as an approach for cell targeting

Gene therapy is used to correct genetic defects or to deliver new therapeutic functions to the target cells. Viral vectors are employed mainly as a gene delivery system. A great variety of viral expression systems have been developed and assessed for their ability to transfer genes into somatic cells. In particular, retroviral and adenoviral mediated gene transfer have been extensively studied and improved. Preclinical and clinical studies covering a large range of genetic disorders are currently underway to solve basic issues dealing with gene transfer efficiencies, regulation of gene expression, and potential risks of the use of viral vectors. The majority of clinical gene therapy trials that employ viral vectors perform ex vivo gene transfer into target cells. The main issue in potential clinical application of gene therapy is the need for increased gene transfer efficiency and target specificity associated with regulated gene expression at therapeutically relevant levels in vivo. Gene regulatory elements, such as promoters and enhancers, possess cell type specific activities and can be activated by certain induction factors (e.g., hormones, growth factors, cytokines, cytostatics, irradiation, heat shock) via responsive elements. A controlled and restricted expression of these genes can be achieved using such regulatory elements as internal promoters to drive the expression of therapeutic genes in viral vector constructs. In addition to high level and efficient gene expression, minimizing or excluding inappropriate gene expression in surrounding nontarget cells is of great importance for numerous gene therapeutic approaches. This contribution furnishes insight into the field of cell type specific promoter and enhancer systems which have been used for targeted and inducible expression of therapeutic genes in certain genetic disorders, viral infections, and malignancies. We also discuss promoters that represent attractive candidates for the construction of viral vectors.Abbreviations ADA Adenosine deaminase - AFP -Fetoprotein - AIDS Acquired immunodeficiency syndrome - CAT Chloramphenicol acetyltransferase - CD Cytosine deaminase - CEA Carcinoembryonic antigen - DMD Duchenne muscular dystrophy - 5-FC 5-Fluorocytosine - HIV Human immunodeficiency virus - LAD Leukocyte adherence deficiency - LCR Locus control region - LTR Long terminal repeats - MCK Muscle creatinine kinase - MLV Moloney murine leukemia virus - MMTV Mouse mammary tumor virus - PEPCK Phosphoenolpyruvate carboxykinase - PSA Prostate-specific antigen - SCLC Small cell lung cancer cells - SLPI Secretory leukoprotease inhibitor - SPA/B/C Human surfactant protein A/B/C - TAR Trans-activation-responsive - TNF Tumor necrosis factor-     

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.     

Introduction of nonselectible 2μ plasmid into [cir°] cells of the yeast S. cerevisiae by DNA transformation and in vivo site-specific resolution

Summary The 2 DNA plasmid of the yeast Saccharomyces cerevisiae does not confer any known selectable phenotype to the host cell carrying it. Selection of cells transformed with purified 2 DNA therefore cannot be achieved, and the intracellular presence of 2 can only be assessed by molecular analysis of the DNA complement. In addition, 2 alone does not replicate in bacterial hosts, thus rendering its amplification by conventional methods impossible. We have isolated a shuttle plasmid, pBH-2L, generated by in vivo sites-pecific recombination between the endogenous 2 DNA plasmid and pRL, a pBR322 derivative containing the yeast LEU2 gene and one 2 repeat sequence associated with the origin of replication. This new shuttle plasmid has the property, when transformed into yeast, of undergoing site-specific recombinational resolution between its two direct repeat sequences. This releases 2 plasmid and pRL as individual molecules. The latter can undergo progressive mitotic loss during growth in nonselective medium, ultimately leaving leucine auxotrophic transformants that contain only 2 DNA plasmid. This system can be utilized to introduce 2 DNA alone into cells lacking it, thereby providing a novel means to study the biology and the molecular genetics of the plasmid and its potential practical applications as a vector.     

A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific integration system for Aspergillus awamori

A homologous gene transfer system for Aspergillus awamori for site-specific integration is described, based on two components. First, a defined A. awamori pyrG mutant strain constructed by a selection strategy for gene-replacement in fungi. Second, a vector with a homologous pyrG selection marker containing a defined mutation at a site different from that of the mutations in the pyrG gene of the defined mutant strain. Defined mutation in the A. awamori pyrG gene, isolated from a genomic library by heterologous hybridisation with the A. niger pyrG gene as a probe, were introduced by specifically altering sequences at restriction sites in the coding region of the gene. After transformation of the A. awamori wild-type strain with vectors containing these mutated pyrG genes, and selection for 5-fluoro-orotic acid resistance (5-FOA^R), on the average 60% of the 5-FOA^R colonies originated from replacement of the wild-type pyrG gene by the mutated pyrG allele. After transformation of a mutant strain, carrying a mutation near the 5 end of the pyrG gene with vectors containing a mutation near the 3 end of the pyrG gene, 35% of the resulting transformants contained one copy of the vector at the pyrG locus.     

Liver-directed gene transfer and application to therapy

The liver is an important and attractive target for the development of gene therapy strategies. Many genetic diseases are manifested in the liver, and both infectious and malignant diseases affect this organ. Retroviral and adenoviral vectors have been shown to infect hepatocytes with varying efficiently in vitro and in vivo. The presence of unique receptors at the cellular membrane of hepatocytes has stimulated the development of transfer strategies based on receptor targeting of vectors. The results of a first clinical trial for gene therapy in the liver based on ex vivo gene delivery has shown both the feasibility and the limits of current technology. This review discusses both existing vectors and strategies and prospective developments towards liver-directed gene therapy of genetic and malignant diseases.Abbreviations AFP -Fetoprotein - EBV Epstein-Barr virus - HBV Hepatitis B virus - LDL Low-density lipoprotein     

Improving live attenuated bacterial carriers for vaccination and therapy

Live attenuated bacteria are well established as vaccines. Thus, their use as carriers for prophylactic and therapeutic macromolecules is a logical consequence. Here we describe several experimental applications of bacteria to carry heterologous macromolecules into the murine host. First, Listeria monocytogenes are described that are able to transfer eukaryotic expression plasmids into host cells for gene therapy. High multiplicities of infection are still required for efficient gene transfer and we point out some of the bottlenecks that counteract a more efficient transfer and application in vivo. Then, we describe Salmonella enterica serovar Typhimurium (S. typhimurium) as an expression plasmid transfer vehicle for oral DNA vaccination of mice. We demonstrate that the stabilization of the plasmid transformants results in an improved immune response. Stabilization was achieved by replacing the origin of replication of the original high-copy-number plasmid by a low-copy-number origin. Finally, we describe Salmonella carriers for the improved expression of heterologous proteins. We introduce a system in which the plasmid is carried as a single copy during cultivation but is amplified several fold upon infection of the host. Using the same in vivo inducible promoter for both protein expression and plasmid amplification, a substantial increase in antigen expression in vivo can be achieved. A modification of this approach is the introduction of inducible gene expression in vivo with a low-molecular-weight compound. Using P_BAD promoter and l-arabinose as inducer we were able to deliberately activate genes in the bacterial carrier. No background activity could be observed with P_BAD such that an inducible suicide gene could be introduced. This is adding an important safety feature to such live attenuated carrier bacteria.     

Extrachromosomal genetics of Cephalosporium acremonium

Summary In order to facilitate strain improvement by concerted breeding in the cephalosporin producing imperfect filamentous fungus Cephalo sporium acremonium, it is attempted to develop a eukaryotic vector for molecular cloning based on mitochondrial (mt) DNA.Fragments of mtDNA from C. acremonium were inserted into a yeast/bacterial hybrid plasmid (pDAM1) lacking a eukaryotic replicon. Six hybrid plasmids were obtained each containing a different mt fragment which together comprise about 60% of the total mtDNA. One of these recombinant plasmid vectors (pCP2) showed a high replication efficiency which is comparable to that of vectors containing yeast 2 m DNA. This plasmid therefore fulfills the requirements for practical application.     

Comparison of expression of the endo-β-1,3-1,4-glucanase gene from Bacillus subtilis in Saccharomyces cerevisiae from the CYC1 and ADH1 promoters

Summary The endo--1,3-1,4-glucanase gene from B. subtilis was placed under yeast promoter control in a number of different yeast expression vectors. The hybrid plasmids were transformed into S. cerevisiae where they directed the synthesis of varying amounts of active enzyme. The presence of B. subtilis DNA sequences 5 to the initiation codon for the B. subtilis -glucanase gene reduced expression of the gene in yeast. A 1,000-fold increase in the yield of -glucanase was obtained using the ADH1 promoter compared with the CYC1 promoter.     

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.     

Expression of exogenous genes in<Emphasis Type="Italic"> Trypanosoma cruzi</Emphasis>: improving vectors and electroporation protocols

To improve transfection efficiency in Trypanosoma cruzi, we developed a new electroporation protocol and expression vectors which use luciferase and green and red fluorescent proteins as reporter genes. In transient transfections, the electroporation conditions reported here resulted in luciferase expression 100 times higher than the levels obtained with previously described protocols. To verify whether sequences containing different trans-splicing signals influence reporter gene expression, we compared DNA fragments corresponding to 5 untranslated plus intergenic (5 UTR plus Ig) regions from GAPDH, TcP2, - and -tubulin and amastin genes. Vectors containing sequences derived from the first four genes presented similar efficiencies and resulted in luciferase expression in transiently transfected epimastigotes that was up to 10 times higher than that for a control vector. In contrast, the amastin 5 UTR plus Ig resulted in lower levels of reporter gene expression. We also constructed a vector containing an expression cassette designed to be targeted to the tubulin locus of the parasite.     

Curing of Saccharomyces cerevisiae 2-μm DNA by transformation

Summary A general procedure for the curing of 2-m in Saccharomyces cerevisiae is described. The method is based on the displacement of endogenous 2-m DNA by the recombinant plasmid pMP78-1, which carries the yeast leu2 gene and the 2 -m DNA replicon, but cannot be maintained stably in a yeast cell without endogenous 2-m DNA. After transformation with pMP78-1 cells are grown selectively to displace 2-m DNA. During the non-selective growth which follows, plasmid pMP78-1 is lost and up to 100% of the cells completely lack plasmids. In conjunction with a kanamycin resistance marker, as present in plasmid pMP81, this method should be applicable to cure any wild-type yeast strain. The stability of recombinant plasmids in cir ⁺ and cir ⁰ strains has been compared.     

Expression of a yeast gene can be blocked by insertion of short yeast DNA fragments between a UAS and the TATA box

We have constructed a plasmid, pOV10, which facilitates the introduction of putative upstream activating sequences (UAS) or upstream repressing sequences (URS) from yeast genes into plasmids containing CYC1-lacZ fusions. We have observed that the insertion of yeast sequences from 155 to 195 bp between the UAS and the TATA box of a CYC1-lacZ fusion gene can block -galactosidase expression. It is suggested that this block is related to the formation of nucleosomes on the DNA.