A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality

A novel method to efficiently generate unmarked in-frame gene deletions in Rhodococcus equi was developed, exploiting the cytotoxic effect of 5-fluorocytosine (5-FC) by the action of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) enzymes. The opportunistic, intracellular pathogen R. equi is resistant to high concentrations of 5-FC. Introduction of Escherichia coli genes encoding CD and UPRT conferred conditional lethality to R. equi cells incubated with 5-FC. To exemplify the use of the codA::upp cassette as counter-selectable marker, an unmarked in-frame gene deletion mutant of R. equi was constructed. The supA and supB genes, part of a putative cholesterol catabolic gene cluster, were efficiently deleted from the R. equi wild-type genome. Phenotypic analysis of the generated ΔsupAB mutant confirmed that supAB are essential for growth of R. equi on cholesterol. Macrophage survival assays revealed that the ΔsupAB mutant is able to survive and proliferate in macrophages comparable to wild type. Thus, cholesterol metabolism does not appear to be essential for macrophage survival of R. equi. The CD-UPRT based 5-FC counter-selection may become a useful asset in the generation of unmarked in-frame gene deletions in other actinobacteria as well, as actinobacteria generally appear to be 5-FC resistant and 5-FU sensitive.     

Importance of codB for new codA-based markerless gene deletion in Gluconobacter strains

For the detailed molecular analysis, genomic modification, and application of acetic acid bacteria such as Gluconobacter in biotechnological processes, a simple markerless deletion system is essential. The available methods have either low efficiencies or their applicability is restricted to strains containing an upp mutation. We now developed a method based on counterselection by cytosine deaminase, encoded by the codA gene from Escherichia coli, in the presence of the fluorinated pyrimidine analogue 5-fluorocytosine (FC). The codA-encoded enzyme converts nontoxic FC to toxic 5-fluorouracil, which is channeled into the metabolism by the uracil phosphoribosyltransferase, encoded by the chromosomal upp gene of Gluconobacter. We found that the presence of E. coli codB, encoding a cytosine permease, was needed for a high efficiency of gene deletion. The system is applicable in wild-type strains because no preceding deletions are required. Based on the fact that a codA gene is absent and an upp gene is present in almost all acetic acid bacteria sequenced so far, the method should also be applicable for other genera of the Acetobacteraceae.     

Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PP_i). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PP_i product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.     

Cytosine deaminase as a substrate-dependent negative selectable marker in Brassica napus

The enzyme cytosine deaminase, encoded by the codA gene, catalyzes the deamination of the non- toxic compound 5-fluorocytosine (5-FC) to the highly toxic compound 5-fluorouracil (5-FU). Cytosine deaminase activity is not found in higher plants and Brassica napus seedlings are unaffected by the presence of 5-FC in the growth medium. In codA-transformed B. napus seedlings, expression of cytosine deaminase results in a reduction of root and hypocotyl lengths, and a severe suppression of true leaf development. This phenotype is dependent on the presence of the 5-FC substrate and no effects are seen in plants grown in the absence of the substrate or in sibling plants lacking the transgene. The codA transformants have been assessed over three generations of growth and in each generation the transgene is stably inherited and confers the same 5-FC-sensitive phenotype. Transfer of 5-FC-sensitive seedlings to soil results in the restoration of normal growth in up to 100% of the seedlings. These results indicate that codA is a versatile dominant marker gene that can be used effectively in B. napus for substrate-dependent negative selection. Received: 24 June 1999 / Accepted: 22 July 1999     

Functional characterization of a gene encoding a dual domain for uridine kinase and uracil phosphoribosyltransferase in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Uridine kinase (UK) and uracil phosphoribosyltransferase (UPRT) are enzymes catalyzing the formation of uridine 5′-monophosphate (UMP) from uridine and adenine 5′-triphosphate (ATP) and from uracil and phosphoribosyl-α-1-pyrophosphate (PRPP), respectively, in the pyrimidine salvage pathway. Here, we report the characterization and functional analysis of a gene AtUK/UPRT1 from Arabidopsis thaliana. Sequencing of an expressed sequence tag clone of this gene revealed that it contains a full-length open reading frame of 1461 nucleotides and encodes a protein with a molecular mass of approximately 53 kDa. The sequence analysis revealed that the N-terminal region of AtUK/UPRT1 contains a UK domain and the C-terminal region consists of a UPRT domain. Expression of AtUK/UPRT1 in upp and upp-udk mutants of Escherichia coli supplied with 5-fluorouracil (5-FU) and 5-fluorouridine (5-FD) led to growth inhibition. Identical results were obtained with 5-FD and 5-FU treatments when the UK and UPRT domains were separated by the introduction of translation initiation and stop codons prior to complementation into the upp-udk and upp mutants. These results suggest that the AtUK/UPRT1 product can use uracil and uridine as substrates for the production of UMP. We also investigated the function of AtUK/UPRT1 in an Arabidopsis mutant. The wild-type Arabidopsis plants showed drastic growth retardation when they were treated with 5-FU and 5-FD while the growth of atuk/uprt1 mutant plants was not significantly affected. These findings confirm that AtUK/UPRT1 has a dual role in coding for both uridine kinase and uracil phosphoribosyltransferase that form UMP through the pyrimidine salvage pathway in Arabidopsis.     

Tetracycline-Regulated Suppression of Amber Codons in Mammalian Cells

As an approach to inducible suppression of nonsense mutations in mammalian cells, we described recently an amber suppression system in mammalian cells dependent on coexpression of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) along with the E. coli glutamine-inserting amber suppressor tRNA. Here, we report on tetracycline-regulated expression of the E. coli GlnRS gene and, thereby, tetracycline-regulated suppression of amber codons in mammalian HeLa and COS-1 cells. The E. coli GlnRS coding sequence attached to a minimal mammalian cell promoter was placed downstream of seven tandem tetracycline operator sequences. Cotransfection of HeLa cell lines expressing a tetracycline transactivator protein, carrying a tetracycline repressor domain linked to part of a herpesvirus VP16 activation domain, with the E. coli GlnRS gene and the E. coli glutamine-inserting amber suppressor tRNA gene resulted in suppression of the amber codon in a reporter chloramphenicol acetyltransferase gene. The tetracycline transactivator-mediated expression of E. coli GlnRS was essentially completely blocked in HeLa or COS-1 cells grown in the presence of tetracycline. Concomitantly, both aminoacylation of the suppressor tRNA and suppression of the amber codon were reduced significantly in the presence of tetracycline.     

Co-transformation using a negative selectable marker gene for the production of selectable marker gene-free transgenic plants

A negative selectable marker gene, codA, was successfully co-transformed with a GUS reporter gene to develop selectable marker gene-free transgenic plants. The pNC binary vector contained a T-DNA harboring the codA gene next to the nptII gene, while a second binary vector, pHG, contained a GUS reporter gene. Tobacco plants (Nicotiana tabacum cv. Samsun NN) were co-transformed via the mixture method with Agrobacterium tumefaciens LBA4404 strains harboring pNC and pHG, respectively. Seeds harvested from the co-transformants were sown on germination media containing 5-fluorocytosine (5-FC). Analysis of the progeny by GUS staining and PCR amplification revealed that all of the 5-FC-resistant R₁ plants were codA free, and that the codA gene segregated independently of the GUS gene. Because codA-free seedlings developed normally on 5-FC-containing medium, we suggest that co-transformation with negatively selectable markers is a viable method for the production of easily distinguished, selectable marker gene-free transgenic plants.     

A new idea for simple and rapid monitoring of gene expression: requirement of nucleotide sequences encoding an N-terminal HA tag in the T7 promoter-driven expression in E. coli

Mammalian expression vectors are used to overexpress genes of interest in mammalian cells. High temperature requirement protein A1 (HtrA1), used as a specific target, was expressed from the pHA-M-HtrA1 plasmid in HEK293T cells, inducing cell death. Expression of HtrA1 was driven by the pHA-M-HtrA1 mammalian expression vector in E. coli resulting in growth suppression of E. coli in an HtrA1 serine protease-dependent manner. By using various combinations of promoters, target genes and N-terminal tags, the T7 promoter and N-terminal HA tag in the mammalian expression vector were shown to be responsible for expression of target genes in E. coli. Thus the pHA-M-HtrA1 plasmid can be used as a novel, rapid pre-test system for expression and cytotoxicity of the specific target gene in E. coli before assessing its functions in mammalian cells.     

Genetic Selection Scheme for Isolation of Signal Transduction Pathway Mutants

Genetic characterization of a signal transduction pathway requires the isolation of mutations in the pathway. Characterization of these mutated genes and their loci enumerates the components of the pathway and leads to an understanding of the role of each gene locus in the pathway under study. We have designed and developed a strategy based on resistance to the chemical flucytosine for the identification of mutations in a given pathway. In this study, the Escherichia coli codA gene, which encodes the enzyme cytosine deaminase, was fused to the light-intensity-regulated gene promoter psbDII. Cytosine deaminase converts 5′-fluorocytosine to the toxic product 5-fluorouracil. Wild-type cells containing an intact signal transduction pathway that regulates the psbDII promoter will die in the presence of this chemical. Cells that carry mutations in the pathway that inactivate the psbDII promoter will not express the codA gene and, consequently, will live on 5′-fluorocytosine, allowing the isolation and subsequent characterization of mutations in this signaling pathway. Utilizing this selection method, we have successfully isolated and characterized mutations in the psbDII pathway. This selection scheme can be used with a tissue-specific or phase-specific promoter fused to the codA gene to direct the timing of expression of codA to obtain mutants defective in temporal or cell-specific expression of a particular pathway. This scheme also allows the isolation of mutants even when a clearly identifiable phenotype is not available. The selection scheme presented here extends the molecular tools available for the genetic dissection of signal transduction pathways.     

Cytosine deaminase as a negative selective marker for Arabidopsis

Cytosine deaminase (CD), produced by prokaryotes but not by higher eukaryotes including plants, deaminates cytosine to uracil. The enzyme likewise converts 5-fluorocytosine (5FC), which by itself is not toxic, to 5-fluorouracil (5FU), which is toxic. The Escherichia coli codA-coding sequence encoding CD, together with appropriate regulatory elements, was introduced into Arabidopsis. Neither untransformed controls, nor transgenic plants expressing no CD mRNA, were sensitive to 5FC. Conversely, for most transgenic plants expressing CD mRNA, in the presence of 5FC calli and seedlings failed to proliferate, and seeds failed to germinate. A few transgenic plants with many codA copies expressed less CD mRNA and remained insensitive to 5FC, which likely reflected epigenetic repeat-induced gene silencing. Thus 5FC, presumably through conversion by the enzyme to 5FU, can be used to select against plants that express CD.     

Transformation of tomato with a bacterial codA gene enhances tolerance to salt and water stresses

Genetically engineered tomato (Lycopersicon esculentum) with the ability to synthesize glycinebetaine was generated by introducing the codA gene encoding choline oxidase from Arthrobacter globiformis. Integration of the codA gene in transgenic tomato plants was verified by PCR analysis and DNA blot hybridization. Transgenic expression of gene was verified by RT-PCR analysis and RNA blot hybridization. The codA-transgenic plants showed higher tolerance to salt stress during seed germination, and subsequent growth of young seedlings than wild-type plants. The codA transgene enhanced the salt tolerance of whole plants and leaves. Mature leaves of codA-transgenic plants revealed higher levels of relative water content, chlorophyll content, and proline content than those of wild-type plants under salt and water stresses. Results from the current study suggest that the expression of the codA gene in transgenic tomato plants induces the synthesis of glycinebetaine and improves the tolerance of plants to salt and water stresses.     

Suicide gene therapy with an adenovirus expressing the fusion gene CD::UPRT in human glioblastomas: different sensitivities correlate with p53 status

BACKGROUND: Several gene therapy strategies have been designed for cancer treatment. Intra-tumoral injection of adenoviruses expressing pro-drug-converting enzymes is one such strategy. Although the efficacy of these therapies was tested in animal models, little work has been devoted to the determination of critical variables for success. In this work, we aimed at better understanding variables that affect the cytosine deaminase::uracil phosphoribosyl transferase (CD::UPRT)-based strategy in two human glioblastomas. METHODS: We have constructed two adenoviruses expressing either CD or the fusion protein CD::UPRT. We have tested their anti-tumor activity in combination with 5-fluorocytosine (5-FC) in the glioblastoma cell lines U87 and U251, which are p53-wt and p53-deficient, respectively. Anti-tumor activity has also been investigated in spheroid structures. RESULTS: The superiority of CD::UPRT over CD was confirmed in both glioblastoma cells. We found that the pro-drug concentration required for anti-tumor activity was 9-fold higher in U251 than in U87, while multiplicity of infection (MOI) as low as 6 was sufficient to achieve 50% killing. Bystander activity was observed with as few as 2 and 6% transduced cells for U87 and U251, respectively. Differences in sensitivity between U87 and U251 were not due to differences in transduction, transgene expression, or intercellular transport, but were related to 5-FU sensitivity and p53 status. Also, it is noteworthy that, in contrast to U87, U251 spheroids barely responded to the treatment, whereas their monolayer counterparts were very sensitive. CONCLUSIONS: Our study has shown that p53 status is important for CD::UPRT/5-FC treatment. Moreover, this study demonstrated that the three-dimensional spheroid model is a more stringent in vitro model for suicide gene therapy evaluation than are monolayer cultures.     

High-level production and purification of biologically active proteins from bacterial and mammalian cells using the tandem pGFLEX expression system

Because of the complexities involved in the regulation of gene expression in Escherichia coli and mammalian cells, it is considered general practice to use different vectors for heterologous expression of recombinant proteins in these host systems. However, we have developed and report a shuttle vector system, pGFLEX, that provides high-level expression of recombinant glutathione S-transferase (GST) fusion proteins in E. coli and mammalian cells. pGFLEX contains the cytomegaloma virus (CMV) immediate-early promoter in tandem with the E. coli lacZpo system. The sequences involved in gene expression have been appropriately modified to enable high-level production of fusion proteins in either cell type. The pGFLEX expression system allows production of target proteins fused to either the N or C terminus of the GST π protein and provides rapid purification of target proteins as either GST fusions or native proteins after cleavage with thrombin. The utility of this vector in identifying and purifying a component of a multi-protein complex is demonstrated with cyclin A. The pGFLEX expression system provides a singular and widely applicable tool for laboratory or industrial production of biologically active recombinant proteins in E. coli and mammalian cells.     

DNA modification and functional delivery into human cells using Escherichia coli DH10B

The availability of almost the complete human genome as cloned BAC libraries represents a valuable resource for functional genomic analysis, which, however, has been somewhat limited by the ability to modify and transfer this DNA into mammalian cells intact. Here we report a novel comprehensive Escherichia coli-based vector system for the modification, propagation and delivery of large human genomic BAC clones into mammalian cells. The GET recombination inducible homologous recombination system was used in the BAC host strain E.coli DH10B to precisely insert an EGFPneo cassette into the vector portion of a ~200 kb human BAC clone, providing a relatively simple method to directly convert available BAC clones into suitable vectors for mammalian cells. GET recombination was also used for the targeted deletion of the asd gene from the E.coli chromosome, resulting in defective cell wall synthesis and diaminopimelic acid auxotrophy. Transfer of the Yersinia pseudotuberculosis invasin gene into E.coli DH10B asd^– rendered it competent to invade HeLa cells and deliver DNA, as judged by transient expression of green fluorescent protein and stable neomycin-resistant colonies. The efficiency of DNA transfer and survival of HeLa cells has been optimized for incubation time and multiplicity of infection of invasive E.coli with HeLa cells. This combination of E.coli-based homologous recombination and invasion technologies using BAC host strain E.coli DH10B will greatly improve the utility of the available BAC libraries from the human and other genomes for gene expression and functional genomic studies.     

Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor tRNA genes

We describe the generation of mammalian cell lines carrying amber suppressor genes. Nonsense mutants in the herpes simplex virus thymidine kinase (HSV tk) gene, the Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco-gpt) gene and the aminoglycoside 3′ phosphotransferase gene of the Tn5 transposon (NPT-II) were isolated and characterized. Each gene was engineered with the appropriate control signals to allow expression in both E. coli and mammalian cells. Expression in E. coli made possible the use of well developed bacterial and phage genetic manipulations to isolate and characterize the nonsense mutants. Once characterized, the nonsense mutants were transferred into mammalian cells by microinjection and used, in turn, to select for amber suppressor genes. Xenopus laevis amber suppressor genes, prepared by site-specific mutagenesis of a normal X. laevis tRNA gene, were microinjected into the above cell lines and selected for the expression of one or more of the amber mutant gene products. The resulting cell lines, containing functional amber suppressor genes, are stable and exhibit normal growth rates.     

In Vitro and In Vivo Effect of 5-FC Combined Gene Therapy with TNF-α and CD Suicide Gene on Human Laryngeal Carcinoma Cell Line Hep-2

This study was aimed to investigate the effect of combined cancer gene therapy with exogenous tumor necrosis factor-alpha (TNF-α) and cytosine deaminase (CD) suicide gene on laryngeal carcinoma cell line Hep-2 in vitro and in vivo. Transfection of the recombinant eukaryotic vectors of pcDNA3.1 (+) containing TNF-α and/or CD into Hep-2 cells resulted in expression of TNF-α and/or CD gene in vitro. The significant increase in apoptotic Hep-2 cells and decrease of Hep-2 cell proliferation were observed using 5-FC treatment combined with TNF-a expression by CD/5-FC suicide system. Moreover, bystander effect was also observed in the TNF-α and CD gene co-expression group. Laryngeal squamous cell carcinoma (LSCC) mice model was established by using BALB/c mice which different transfected Hep-2 cells with pcDNA3.1 (+) containing TNF-α and/or CD were applied subcutaneously. So these mice are divided into four groups, namely, Hep-2/TIC group; Hep-2/CD group; Hep-2/TNF-α group; Hep-2/0 group. At day 29 after cell inoculation, volume of grafted tumor had significant difference between each two of them (P<0.05). These results showed that the products of combined CD and TNF-α genes inhibited the growth of transplanted LSCC in mice model. So by our observed parameters and many others results, we hypothesized that 5-FC combined gene therapy with TNF-αand CD suicide gene should be an effective treatment on Laryngeal carcinoma.