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)

Pyrimidine salvage pathways are vital for all bacteria in that they share in the synthesis of RNA with the biosynthetic pathway in pyrimidine prototrophs, while supplying all pyrimidine requirements in pyrimidine auxotrophs. Salvage enzymes that constitute the pyrimidine salvage pathways were studied in 13 members of Pseudomonas and former pseudomonads. Because it has been established that all Pseudomonas lack the enzyme uridine/cytidine kinase (Udk) and all contain uracil phosphoribosyl transferase (Upp), these two enzymes were not included in this experimental work. The enzymes assayed were: cytosine deaminase [Cod: cytosine + H2O --> uracil + NH3], cytidine deaminase [Cdd: cytidine + H2O --> uridine + NH3], uridine phosphorylase [Udp: uridine + Pi <--> uracil + ribose - 1 - P], nucleoside hydrolase [Nuh: purine/pyrimidine nucleoside + H2O --> purine/pyrimidine base + ribose], uridine hydrolase [Udh: uridine/cytidine + H2O --> uracil/cytosine + ribose]. The assay work generated five different Pyrimidine Salvage Groups (PSG) designated PSG1 - PSG5 based on the presence or absence of the five enzymes. These enzymes were assayed using reverse phase high-performance liquid chromatography techniques routinely carried out in our laboratory. Escherichia coli was included as a standard, which contains all seven of the above enzymes.
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PMID:Pathways of pyrimidine salvage in Pseudomonas and former Pseudomonas: detection of recycling enzymes using high-performance liquid chromatography. 1796 97

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.
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PMID:Importance of codB for new codA-based markerless gene deletion in Gluconobacter strains. 2395 75

Here, we present a detailed protocol for studying in yeast cells the contingent interaction between a substrate and its multisubunit enzyme complex by using a death selection technique known as the optimized yeast cytosine deaminase protein-fragment complementation assay (OyCD PCA). In yeast, the enzyme cytosine deaminase (encoded by FCY1) is involved in pyrimidine metabolism. The PCA is based on an engineered form of yeast cytosine deaminase optimized by directed evolution for maximum activity (OyCD), which acts as a reporter converting the pro-drug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a toxic compound that kills the cell. Cells that have OyCD PCA activity convert 5-FC to 5-FU and die. Using this assay, it is possible to assess how regulatory subunits of an enzyme contribute to the overall interaction between the catalytic subunit and the potential substrates. Furthermore, OyCD PCA can be used to dissect different functions of mutant forms of a protein as a mutant can disrupt interaction with one partner, while retaining interaction with others. As it is scalable to a medium- or high-throughput format, OyCD PCA can be used to study hundreds to thousands of pairwise protein-protein interactions in different deletion strains. In addition, OyCD PCA vectors (pAG413GAL1-ccdB-OyCD-F[1] and pAG415GAL1-ccdB-OyCD-F[2]) have been designed to be compatible with the proprietary Gateway technology. It is therefore easy to generate fusion genes with the OyCD reporter fragments. As an example, we will focus on the yeast cyclin-dependent protein kinase 1 (Cdk1, encoded by CDC28), its regulatory cyclin subunits, and its substrates or binding partners.
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PMID:Dissecting the Contingent Interactions of Protein Complexes with the Optimized Yeast Cytosine Deaminase Protein-Fragment Complementation Assay. 2780 54

Programmable nucleases are cutting edge genetic technology which edits targeted DNA sequences through generation of site-specific double-strand DNA breaks (DSBs). To improve the efficiency and precision of genetic modification, scientists have developed a single-base editing system (base editor) through combining of CRISPR/Cas9 system with cytosine deaminase. Compared with Cas9 system, this base editor can convert cytosine to thymine (C > T) at specific site more efficiently without inducing DSBs to avoid generation of indels. However, the base editor can only generate transition of pyrimidine but could not modify purines. Recently, Nature published a novel base editing system to convert adenine to guanine (ABEs, adenine base editors) through fusion of Cas9 nickase to a modified deaminase which is evolved through screening of random library based on tRNA adenine deaminase from E. coli. Here, we summarize the development of single-base editing tools and the latest research progress, especially the optimization process of ABEs, as well as the potential directions of the base editors.
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PMID:The "new favorite" of gene editing technology-single base editors. 2925 82

Cell type-specific transcription is a key determinant of cell fate and function. An ongoing challenge in biology is to develop robust and stringent biochemical methods to explore gene expression with cell type specificity. This challenge has become even greater as researchers attempt to apply high-throughput RNA analysis methods under in vivo conditions. TU-tagging and EC-tagging are in vivo biosynthetic RNA tagging techniques that allow spatial and temporal specificity in RNA purification. Spatial specificity is achieved through targeted expression of pyrimidine salvage enzymes (uracil phosphoribosyltransferase and cytosine deaminase) and temporal specificity is achieved by controlling exposure to bioorthogonal substrates of these enzymes (4-thiouracil and 5-ethynylcytosine). Tagged RNAs can be purified from total RNA extracted from an animal or tissue and used in transcriptome profiling analyses. In addition to identifying cell type-specific mRNA profiles, these techniques are applicable to noncoding RNAs and can be used to measure RNA transcription and decay. Potential applications of TU-tagging and EC-tagging also include fluorescent RNA imaging and selective definition of RNA-protein interactions. TU-tagging and EC-tagging hold great promise for supporting research at the intersection of RNA biology and developmental biology. This article is categorized under: Technologies > Analysis of the Transcriptome.
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PMID:Uncovering cell type-specific complexities of gene expression and RNA metabolism by TU-tagging and EC-tagging. 2936 22


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