Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.4.1 (cytosine deaminase)
747 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The enzyme activity for cytosine deaminase, which converts cytosine to uracil in bacterial, is usually undetected in higher plants and animals. The enzyme also catalyzes conversion of non-toxic 5-fluorocytosine (5-FC) to 5- fluorouracil (5-FU), a toxic compound for plant growth. The gene encoding cytosine deaminase (codA) from Escherichia coli was fused to cauliflower mosaic virus (CaMV) 35S promoter (P35S), and cloned into a binary vector pLABR101. The resulting plasmid pLABR102 contained two marker genes for plants: a positive marker gene, bialaphos resistance (bar) gene driven by the promoter from nopaline synthase gene (Pnos) and a negative one, P35S-codA. The binary vector pLABR102 was transformed into Arabidopsis thaliana via Agrobacterium-mediated transformation. In transgenic progenies (T3) of the second (T2) generation heterozygous for a single T-DNA insertion, a 3:1 segregation ratio was observed on both bialaphos (resistance to sensitive) and 5-FC (sensitive to unaffected). From T2 plants homozygous for the T-DNA insert, on the other hand, no segregation was detected: all the T3 seedlings were resistant to bialaphos and sensitive to 5-FC. PCR and Northern analyses showed that the 5-FC sensitivity in transgenic descendants was caused by the integration and expression of the chimeric codA gene in the Arabidopsis genome. The results indicated that cytosine deaminase from E. coli is functional and useful for negative selection in Arabidopsis, and that sensitivity to 5-FC as well as the positive bialaphos resistance are dominant traits in Arabidopsis.
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PMID:A conditional negative selection for Arabidopsis expressing a bacterial cytosine deaminase gene. 763 43

The Escherichia coli codBA operon encodes cytosine permease (CodB) and cytosine deaminase (CodA). CodB mediates uptake of exogenously supplied cytosine, and CodA catalyses the hydrolytic deamination of cytosine to uracil and ammonia. The hydropathic profile of CodB indicates that it is an integral cytoplasmic membrane protein possessing several transmembrane-spanning domains. The membrane topology of CodB was investigated by using gene fusions containing varying lengths of the amino-terminus of CodB fused to either alkaline phosphatase (AP) or beta-galactosidase (BG). The AP activities expressed by the CodB-AP fusions are consistent with a topological model in which the amino- and the carboxy-termini of CodB are located in the cytoplasm, and in which CodB possesses 12 membrane-spanning segments. The enzyme activities of most of the CodB-BG fusions support the model. However, the results obtained with some of the CodB-BG fusions illustrate the limitations of using BG as a reporter protein in studies of membrane protein topology.
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PMID:Membrane topology analysis of the Escherichia coli cytosine permease. 853 18

Our laboratory and others have shown alternative splicing of up to ten exons at a discrete extracellular site to be primarily responsible for the generation of CD44 variant (CD44v) isoforms. Based on clear differences in the expression of these CD44v isoforms between normal and malignant tissues, we believe that elucidation of the mechanisms underlying the regulation of CD44 alternative splicing may provide a new gene therapeutic targeting approach based on CD44 pre-mRNA processing in vivo. This strategy incorporates utilization of CD44 alternative splicing control elements into a chimeric enzyme/prodrug therapy (CEPT), a novel modification of the virus-directed enzyme/prodrug therapy (VDEPT) approach for the treatment of brain metastases from tumors of systemic origin. As initial steps towards the development of a gene therapeutic approach based on targeting tumor cell expression of specific CD44v alternatively spliced isoforms, we have: (1) developed a novel in vivo assay system that allows the rapid analyses of potentially therapeutic CD44 alternative splicing minigene constructs; and (2) cloned the E. coli cytosine deaminase (CD) gene and fused its enzymatically active domain to alternatively spliced CD44 exons (CD44/CD). Deamination of cytosine by this CD44/CD chimeric fusion protein is demonstrated in E. coli cell lysates to be equal to that of wild type cytosine deaminase.
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PMID:Gene therapeutic approach to primary and metastatic brain tumors: I. CD44 variant pre-RNA alternative splicing as a CEPT control element. 875 Jan 90

The prodrug activation system formed by the E. coli codA gene encoding cytosine deaminase (CD) and 5-fluorocytosine (5-FC) developed for selective cancer chemotherapy suffers from a sensitivity limitation in many tumour cells. In an attempt to improve the CD/5-FC suicide association, we combined the E. coli upp gene encoding uracil phosphoribosyltransferase (UPRT) with codA gene to create the situation prevailing in E. coli, a bacterium very efficient in metabolising 5-FC. The constitutive expression of the two genes cloned on an E. coli-animal cell shuttle plasmid either in a linked or in a fused configuration was evaluated in E. coli strains selected and engineered to mimic the 5-FC metabolism encountered in mammalian cells. The simultaneous expression of codA and upp genes generated a cooperative effect resulting in a dramatic increase in 5-FC sensitivity of cells compared to the expression of codA alone. Furthermore, it was shown that the association of UPRT with CD facilitated the uptake of 5-FC, in the situation where the drug penetrates cells by passive diffusion as in mammalian cells, by directly channeling 5-fluorouracil, the product of CD, to 5-fluoroUMP, the product of UPRT.
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PMID:Concomitant expression of E. coli cytosine deaminase and uracil phosphoribosyltransferase improves the cytotoxicity of 5-fluorocytosine. 978 50

To improve genetic models available for the analysis of apicomplexan protozoan parasites, bacterial sequences encoding the 427 amino acid cytosine deaminase (CD) gene were fused, in-frame, to an engineered linker domain of the high level pyrimethamine resistant form of the parasite bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. Toxoplasma gondii was transformed with the plasmid containing the fused pyrimethamine resistant dihydrofolate reductase-cytosine deaminase-thymidylate synthase (DHFRm2m3-CD-TS) gene and parasites were selected in a high level of pyrimethamine. Transfected parasites that acquired resistance to pyrimethamine were cloned and evaluated for expression of the CD genetic marker. CD transgenic parasites acquired a high sensitivity to 5-fluorocytosine due to the intraparasitic conversion of this non-toxic prodrug to the cytotoxic compound 5-fluorouracil. Exogenously supplied cytosine or uracil rescued the growth of CD transgenic T. gondii parasites that were cultured in the presence of cytotoxic concentrations of 5-fluorouracil or 5-fluorocytosine. Bacterial CD fused to the pyrimethamine resistant DHFR-TS marker provides a novel genetic tool for new positive and negative genetic selection strategies in several protozoan parasites. An advantage of the CD genetic marker is that it is derived from a bacterial gene and can therefore be used in nearly any parasite genetic background for negative selection. This novel system should facilitate new approaches for the development of improved model genetic systems for the biological investigation of apicomplexan parasites.
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PMID:Stable transformation of Toxoplasma gondii based on a pyrimethamine resistant trifunctional dihydrofolate reductase-cytosine deaminase-thymidylate synthase gene that confers sensitivity to 5-fluorocytosine. 1002 12

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.
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PMID:Genetic selection scheme for isolation of signal transduction pathway mutants. 1476 78

We have previously constructed an antibody-avidin (Av) fusion protein, anti-transferrin receptor (TfR) IgG3-Av, which can deliver biotinylated molecules to cells expressing the TfR. We now describe the use of the fusion protein for antibody-directed enzyme prodrug therapy (ADEPT). The 67 amino acid carboxyl-terminal domain (P67) of human propionyl-CoA carboxylase alpha subunit can be metabolically biotinylated at a fixed lysine residue. We genetically fused P67 to the carboxyl terminus of the yeast enzyme FCU1, a derivative of cytosine deaminase that can convert the non-toxic prodrug 5-fluorocytosine to the cytotoxic agent 5-fluorouracil. When produced in Escherichia coli cells overexpressing a biotin protein ligase, the FCU1-P67 fusion protein was efficiently mono-biotinylated. In the presence of 5-fluorocytosine, the biotinylated fusion protein conjugated to anti-rat TfR IgG3-Av efficiently killed rat Y3-Ag1.2.3 myeloma cells in vitro, while the same protein conjugated to an irrelevant (anti-dansyl) antibody fused to Av showed no cytotoxic effect. Efficient tumor cell killing was also observed when E. coli purine nucleoside phosphorylase was similarly targeted to the tumor cells in the presence of the prodrug 2-fluoro-2'-deoxyadenosine. These results suggest that when combined with P67-based biotinylation, anti-TfR IgG3-Av could serve as a universal delivery vector for targeted chemotherapy of cancer.
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PMID:A human biotin acceptor domain allows site-specific conjugation of an enzyme to an antibody-avidin fusion protein for targeted drug delivery. 1574 88

A major limitation in cancer gene therapy, specifically gene-dependent enzyme prodrug therapy (GDEPT), is inefficient gene delivery and expression. The suicide gene cytosine deaminase (CD) and its substrate, 5-fluorocytosine (5-FC), have been extensively explored due to the inherent 'bystander' effect achieved through diffusion of the toxic metabolite 5-fluorouracil (5-FU). In this study, we aimed to enhance this 'bystander' effect by fusing the Saccharomyces cerevisiae CD to the HSV-1 tegument protein vp22, a novel translocating protein. Two constructs were created: one with vp22 fused to CD (vp22CD) and a second wherein a truncated vp22, lacking the necessary residues for trafficking, fused to CD (delvp22CD). The generated 9L stable lines exhibited similar growth rates, enzyme expression, CD activity, and sensitivity to 5-FC and 5-FU. However, mixed population colony formation assays demonstrated greater bystander effect with the vp22CD fusion as compared to delvp22CD. This enhancement was maintained in vivo where 9L tumors expressing 20 or 50% vp22CD exhibited increased growth delay compared to the respective delvp22CD tumors. Moreover, adenoviral transduction of established wild-type 9L tumors showed increased growth delay with vp22CD (Ad-EF_vp22CD) as compared to equivalent CD (Ad-EF_CD) transduced tumors. Finally, confirming the increased efficacy, (19)F magnetic resonance spectroscopy (MRS) of vp22CD-expressing tumors demonstrated increased 5-FU levels as compared to tumors expressing the nontranslocating CD. These results together demonstrated that fusion of vp22 to CD resulted in CD translocation, which in turn amplified conversion of 5-FC to 5-FU in vivo and enhanced the therapeutic benefit of this GDEPT strategy.
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PMID:Fusion of the HSV-1 tegument protein vp22 to cytosine deaminase confers enhanced bystander effect and increased therapeutic benefit. 1616 81

Endostatin, an angiogenesis inhibitor tested in multiple clinical trials, selectively targets neovascular endothelial cells, suppressing tumor growth. To enhance the therapeutic efficacy of endostatin, we fused endostatin with cytosine deaminase, which converts a prodrug 5-flucytosine into a cytotoxic 5-fluorouracil. This therapeutic strategy was developed based on the observation that the endostatin-green fluorescence protein gene and endostatin-luciferase gene selectively target to endothelial cells in vitro and to the tumor site in vivo, respectively. When we used the endostatin-cytosine deaminase fusion protein to treat s.c. grafted tumors or experimental metastasis tumors, our results showed that endostatin-cytosine deaminase treatment provided stronger tumor growth suppression and increased mean survival time of the mice compared with the treatments of endostatin alone, cytosine deaminase alone, or endostatin plus cytosine deaminase. The endostatin-cytosine deaminase protein significantly inhibited the growth of endothelial cells and preferentially induced tumor cell apoptosis. This endostatin-cytosine deaminase fusion approach opens an avenue for cancer-targeting therapy.
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PMID:Endostatin-cytosine deaminase fusion protein suppresses tumor growth by targeting neovascular endothelial cells. 1639 52

To further develop gene therapy for patients with glioblastomas, an experimental gene therapy protocol was established comprising a series of imaging parameters for (i) noninvasive assessment of viable target tissue followed by (ii) targeted application of herpes simplex virus type 1 (HSV-1) amplicon vectors and (iii) quantification of treatment effects by imaging. We show that viable target tissue amenable for application of gene therapy vectors can be identified by multitracer positron emission tomography (PET) using 2-(18)F-fluoro-2-deoxy-D-glucose, methyl-(11)C-L-methionine, or 3'-deoxy-3'-(18)F-fluoro-L-thymidine ([(18)F]FLT). Targeted application of HSV-1 amplicon vectors containing two therapeutic genes with synergistic antitumor activity (Escherichia coli cytosine deaminase, cd, and mutated HSV-1 thymidine kinase, tk39, fused to green fluorescent protein gene, gfp) leads to an overall response rate of 68%, with 18% complete responses and 50% partial responses. Most importantly, we show that the "tissue dose" of HSV-1 amplicon vector-mediated gene expression can be noninvasively assessed by 9-[4-(18)F-fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]FHBG) PET. Therapeutic effects could be monitored by PET with significant differences in [(18)F]FLT accumulation in all positive control tumors and 72% in vivo transduced tumors (P = 0.01) as early as 4 days after prodrug therapy. For all stably and in vivo transduced tumors, cdIREStk39gfp gene expression as measured by [(18)F]FHBG-PET correlated with therapeutic efficiency as measured by [(18)F]FLT-PET. These data indicate that imaging-guided vector application with determination of tissue dose of vector-mediated gene expression and correlation to induced therapeutic effect using multimodal imaging is feasible. This strategy will help in the development of safe and efficient gene therapy protocols for clinical application.
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PMID:Imaging-guided gene therapy of experimental gliomas. 1730 12


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