Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.4.1 (cytosine deaminase)
 747 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

The genome sequence of Plasmodium falciparum, the causative agent of the most severe form of malaria in humans, rapidly approaches completion, but our ability to genetically manipulate this organism remains limited. Chromosomal integration has only been achieved following the prolonged maintenance of circularised episomal plasmids which selects for single crossover recombinants. It has not been possible to construct genetic deletions via double crossover recombination, presumably due to the low frequency of this event. We have used the Herpes simplex virus thymidine kinase gene and the Escherichia coli cytosine deaminase gene for negative selection of P. falciparum. Parasites were transformed with plasmids expressing the thymidine kinase and cytosine deaminase genes by positive selection for the human dihydrofolate reductase gene. Parasites expressing thymidine kinase are susceptible to the pro-drug ganciclovir while those expressing cytosine deaminase are sensitive to 5-fluorocytosine. Parental parasites were inherently resistant to these drugs. A significant 'bystander effect' was evident in cultures with either ganciclovir or 5-fluorocytosine. Positive and negative selection of the thymidine kinase transformants with both ganciclovir and WR99210 resulted in the selection of parasites containing a genetic deletion of the Pfrh3 gene, the first targeted double crossover deletions in P. falciparum. The use of negative selection for gene disruptions via double crossover recombination will dramatically improve our ability to analyse protein function and opens the possibility of using this strategy for a variety of gene deletion and modification experiments in the analysis of this important infectious agent.
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PMID:Negative selection of Plasmodium falciparum reveals targeted gene deletion by double crossover recombination. 1179 25

We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1-145) is similar to cytosine deaminase and the C-terminal domain (146-367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP(+)) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159-173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.
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PMID:The crystal structure of the bifunctional deaminase/reductase RibD of the riboflavin biosynthetic pathway in Escherichia coli: implications for the reductive mechanism. 1776 62

Protein-fragment complementation assays (PCAs) are a family of assays for detecting protein-protein interactions (PPIs) that have been developed to provide simple and direct ways to study PPIs in any living cell, multicellular organism or in vitro. PCAs can be used to detect PPI between proteins of any molecular weight and expressed at their endogenous levels. Proteins are expressed in their appropriate cellular compartments and can undergo any posttranslational modification or degradation that, barring effects of the PCA fragment fusion, they would normally undergo. Applications of PCAs in yeast have been limited until recently, simply because appropriate expression plasmids or cassettes had not been developed. However, we have now developed and reported on several PCAs in Saccharomyces cerevisiae that cover the gamut of applications one could envision for studying any aspect of PPIs. Here, we present detailed protocols for large-scale analysis of PPIs with the survival-selection dihydrofolate reductase (DHFR) reporter PCA and a new PCA based on a yeast cytosine deaminase reporter that allows for both survival and death selection. This PCA should prove a powerful way to dissect PPIs. We then present a method to study spatial localization and dynamics of PPIs based on fluorescent protein reporter PCAs and finally, two luciferase reporter PCAs that have proved useful for studies of dynamics of PPIs.
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PMID:A toolkit of protein-fragment complementation assays for studying and dissecting large-scale and dynamic protein-protein interactions in living cells. 2094 17

Protein-fragment Complementation Assays (PCAs) are a family of assays for detecting protein-protein interactions (PPIs) that have been developed to provide simple and direct ways to study PPIs in any living cell, multicellular organism, or in vitro. PCAs can be used to detect PPI between proteins of any molecular weight and expressed at their endogenous levels. Proteins are expressed in their appropriate cellular compartments and can undergo any posttranslational modification or degradation that, barring effects of the PCA fragment fusion, they would normally undergo. Assays can be performed in any cell type or model organism that can be transformed or transfected with gene expression DNA constructs. Here we focus on recent applications of PCA in the budding yeast, Saccharomyces cerevisiae, that cover the gamut of applications one could envision for studying any aspect of PPIs. We present detailed protocols for large-scale analysis of PPIs with the survival-selection dihydrofolate reductase (DHFR), reporter PCA, and a new PCA based on a yeast cytosine deaminase reporter that allows for both survival and death selection. This PCA should prove a powerful way to dissect PPIs. We then present methods to study spatial localization and dynamics of PPIs based on fluorescent protein reporter PCAs.
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PMID:Protein-fragment complementation assays for large-scale analysis, functional dissection and dynamic studies of protein-protein interactions in living cells. 2187 Feb 42

CRISPR/Cas9 has been successfully adapted for gene editing in malaria parasites including Plasmodium falciparum and Plasmodium yoelii. However, the reported methods were limited to editing one gene at a time. In practice, it is often desired to modify multiple genetic loci in a parasite genome. Here we describe a CRISPR/Cas9 mediated genome editing method that allows successive modification of more than one gene in the genome of P. yoelii using an improved single-vector system (pYCm) we developed previously. Drug resistant genes encoding human dihydrofolate reductase (hDHFR) and a yeast bifunctional protein (yFCU), with cytosine deaminase (CD) and uridyl phosphoribosyl transferase (UPRT) activities in the plasmid, allowed sequential positive (pyrimethamine, Pyr) and negative (5-fluorocytosine, 5FC) selections and generation of transgenic parasites free of the episomal plasmid after genetic modification. Using this system, we were able to efficiently tag a gene of interest (Pyp28) and subsequently disrupted two genes (Pyctrp and Pycdpk3) that are individually critical for ookinete motility. Disruption of the genes either eliminated (Pyctrp) or greatly reduced (Pycdpk3) ookinete forward motility in matrigel in vitro and completely blocked oocyst development in mosquito midgut. The method will greatly facilitate studies of parasite gene function, development, and disease pathogenesis.
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PMID:CRISPR/Cas9 mediated sequential editing of genes critical for ookinete motility in Plasmodium yoelii. 2803 75