Characterization of the Saccharomyces cerevisiae FCY1 gene encoding cytosine deaminase and its homologue FCA1 of Candida albicans

By functional complementation of a fcy1 null mutant of Saccharomyces cerevisiae, we have cloned and characterized the FCY1 gene, encoding cytosine deaminase in Saccharomyces cerevisiae, and its homologue FCA1, encoding cytosine deaminase in Candida albicans. Disruption of FCY1 resulted in high resistance to 5-fluorocytosine (10⁻² M) and in total loss of cytosine deaminase activity. By contrast the transformation by FCY1 or FCA1 of the haploid FCY1-disrupted host strain restored sensitivity to 5-fluorocytosine and allowed growth on cytosine, as a source of pyrimidine, or ammonium. FCA1 as opposed to FCY1 contains an intron. FCA1 and FCY1 encode respectively 150- and 158- residue proteins of 60% identity. Both Fcy1p and Fca1p share common motifs with cytidine and CMP deaminases, but homology with cytosine deaminase of E. coli could not be detected. Received: 21 August / 12 September 1996     

Salvage of pyrimidine nucleosides by Trichomonas vaginalis

Trichomonas vaginalis is incapable of de novo pyrimidine biosynthesis because it cannot incorporate bicarbonate, aspartate or orotate into its pyrimidine nucleotides or nucleic acids. The organism can salvage exogenous cytidine greater than uridine greater than uracil and thymidine, and incorporate them into the nucleotide pool. A portion of cytidine is converted to CMP, CDP and CTP by cytidine phosphotransferase and nucleotide kinases. Some cytidine and most of uracil are, however, converted first to uridine by cytidine deaminase and uridine phosphorylase respectively; uridine is then incorporated into UMP, UDP and UTP by uridine phosphotransferase and nucleotide kinases. The two phosphotransferases, found mainly in the non-sedimentable fraction of T. vaginalis, provide the main avenue of pyrimidine salvage. No significant levels of pyrimidine phosphoribosyl transferase or nucleoside kinases can be detected in the extract. T. vaginalis has no appreciable dihydrofolate reductase or thymidylate synthetase; it grows normally in millimolar concentrations of methotrexate, pyrimethamine, or trimethoprim, and cannot incorporate labels from exogenous uracil or uridine into DNA. It has an enzyme thymidine phosphotransferase in the sedimentable fraction which converts thymidine to TMP. Thymidine salvage in T. vaginalis is thus totally isolated from the rest of the pyrimidine salvage.     

Pyrimidine base and nucleoside metabolism in Pseudomonas cepacia

Pyrimidine base and nucleoside metabolismin Pseudomonas cepacia was investigated. It was found that this pseudominad utilized uracila, cytosine or cytidine as a sole nitrogen source while prymidine bases and nucelosides supported little bacterial growth as sole carbon sources. Low nuceloside hydrolase and cytosine deminase activitiers were detected in P. cepacia. Synthesis of both enzymes appeared to be subject to repression by ammonium ions but repression of cytosine deaminase synthesis was more severe. Synthesis of nuceloside hydrolase or cytosine deminase was induced if the cells were grown on the substrate cytidine or cytosine, respectively, as a sole nitrogen source.     

The URH1 uridine ribohydrolase of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, uridine ribohydrolase activity is important for recycling, via the salvage pathway, pyrimidine deoxy- and ribonucleosides into uracil required for the growth of strains lacking the de novo pyrimidine synthesis pathway. We have shown that not only uridine and cytidine, but also 5-fluorouridine, 5-fluorocytidine and deoxycytidine are substrates for this enzyme. We identified, cloned and characterized the corresponding URH1 gene and its physiological function was determined by the measurement of metabolic fluxes in several mutants impaired in the pyrimidine salvage pathway. Sequence comparison revealed strong homology between Urh1p and the inosine/uridine-preferring nucleosidase and inosine/adenosine/guanosine nucleoside hydrolase proteins from the parasitic organisms Crithidia fasciculata and Trypanosoma brucei brucei. Moreover, the Asp and His residues in the putative active site were conserved. Site-directed mutagenesis demonstrated that the conserved His residue is involved in catalysis. These results allow us to speculate that the structure and catalytic mechanism of Urh1p are similar to the inosine/uridine nucleoside hydrolase from C. fasciculata.     

Cytidine deamination and resistance to retroviral infection: towards a structural understanding of the APOBEC proteins

The human apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G, or hA3G) protein, provides cells with an intracellular antiretroviral activity that is associated with the hypermutation of viral DNA through cytidine deamination. Indeed, hA3G belongs to a family of vertebrate proteins that contain one or two copies of a signature sequence motif unique to cytidine deaminases (CTDAs). We have constructed secondary structure models of the APOBEC proteins through a combination of structure prediction and subsequent alignment with nucleotide CTDAs whose structures have been solved to high resolution. Secondary structure elements common to all CTDAs are predicted, in addition to structural features that are apparently unique to the APOBEC family of proteins. Most notably, a putative looped-out helix abuts an amino acid that modulates the susceptibility of A3G proteins to the antagonistic action of the human and simian immunodeficiency virus (HIV and SIV) Vif proteins. Using the structure models as a guide, we reflect on mutagenesis studies of the APOBEC1 (A1), hA3G and activation induced deaminase (AID) proteins, with emphasis on the determinants of cytidine deamination and antiviral activities.     

Molecular Modelling of the Biosynthesis of the Rna-Editing Enzyme Apobec-1, Responsible for Generating the Alternative forms of Apolipoprotein B

We discovered in 1987 that the shorter form of apolipoprotein B (B48) synthesized in the intestine is due to the action, previously unrecognized in mammalian cells, of an mRNA-editing process, and more recently we demonstrated that this was due to a specific enzyme (APOBEC-1) with cytidine deaminase activity. We show here, by sequence alignment, molecular modelling and mutagenesis, that APOBEC-1 is a cytidine deaminase, responsible for editing apoB mRNA, and that is related in crystal structure to the cytidine deaminase of Escherichia coli (ECCDA). The two enzymes are both homodimers with composite active sites formed with loops from each monomer. In the sequence of APOBEC-1, three gaps compared with ECCDA match the size and contour of the minimal RNA substrate. We propose a model in which the asymmetric binding of one active site to the substrate cytidine which is positioned by the downstream binding of the product uridine and that this helps to target the other active site for deamination.     

Cytosine deaminase versus thymidine kinase: a comparison of the antitumor activity

Abstract. The efficacy of single and combination suicide gene therapy was evaluated using a Herpes simplex virus thymidine kinase/ganciclovir system and Escherichia coli cytosine deaminase/5-fluorocytosine system on the rat prostate tumor cell line R3327 AT-1. The wild-type R3327 AT-1 cell line was transfected with a bifunctional fusion gene CDglyTK, which had the advantage that the resulting R3327 AT-1/CDglyTK cell line has the same amount of cytosine deaminase and thymidine kinase molecules. The percentage of viable R3327 AT-1/CDglyTK cells after 96 h incubation with 0.1 µg/ml ganciclovir or 10 µg/ml 5-fluorocytosine were 85% and 52% of controls, respectively. The cell viability when both suicide genes systems were activated was 43%. For in vivo analysis, Copenhagen rats were injected subcutaneously with R3327 AT-1 or R3327 AT-1/CDglyTK cells and treated with 30 mg/kg ganciclovir, 500 mg/kg 5-fluorocytosine, or both prodrugs together. A survival of 83% with the thymidine kinase/ganciclovir and 57% with the CD/5-FC could be observed. Only co-administration of thymidine kinase- and cytosine deaminase-specific prodrugs resulted in a 100% recurrence-free survival of the Copenhagen rats with a Dunning R3327 AT-1/CDglyTK prostate tumor and showed an additive cytotoxic effect. Calculation of the degree of activation and the potential of activation can be used to predict the success of a suicide gene therapy. In our case, the cytosine deaminase/5-fluorocytosine system had a low degree of activation (value 40), which is also found in the low response to 5- fluorocytosine in vivo (57% tumor free).     

构建携带胞嘧啶脱氨酶基因骨髓间充质干细胞及其对胶质瘤细胞的体外抑制作用

背景：转染胞嘧啶脱氨酶基因的骨髓间充质干细胞能有效地将化疗前药5-氟尿嘧啶转化成具有细胞毒性的化疗药物5-氟尿嘧啶,并在体外对胶质瘤细胞有显著的生长抑制作用。 目的：探讨以骨髓间充质干细胞为基因治疗载体表达外源基因胞嘧啶脱氨酶基因对胶质瘤C6细胞增殖的影响。 方法：分离、培养小鼠间充质干细胞,构建胞嘧啶脱氨酶基因与GFP联合的慢病毒载体,通过慢病毒包装法将胞嘧啶脱氨酶基因及GFP转染至小鼠骨髓间充质干细胞,获得稳定表达胞嘧啶脱氨酶基因及GFP的骨髓间充质干细胞,使其与胶质瘤C6细胞共培养,在培养液中加入5-氟胞嘧啶后应用流式细胞仪检测胞嘧啶脱氨酶基因对胶质瘤细胞的增殖影响。 结果与结论：慢病毒介导的胞嘧啶脱氨酶基因及GFP基因成功转染小鼠骨髓间充质干细胞形成C57BL/6 mMSC-codA/eGFP细胞,C57BL/6 mMSC-codA/eGFP在5-氟胞嘧啶的作用下可引起胶质瘤C6细胞的明显凋亡,在5-氟胞嘧啶浓度为1×10⁶ μg/ L条件下C6胶质瘤细胞凋亡率为60%(P < 0.05)。提示,C57BL/6 mMSC-codA/eGFP可将5-氟胞嘧啶转化成5-氟尿嘧啶并对C6胶质瘤细胞生长有显著的限制作用甚至是致死效应。     

Functional analysis of the two cytidine deaminase domains in APOBEC3G

Human APOBEC3G (hA3G), a cytidine deaminase with two cytidine deaminase domains (CDs), has been identified as an anti-HIV-1 host factor. Although the two CDs of hA3G have been extensively characterized, there is still debate on the role of the CDs in the biological function of hA3G. In this work, we constructed three hA3G mutants CD1-1, CD2-2 and CD2-1, which contain duplicate CD1 domain, duplicate CD2 domain and position switched CD domain respectively, and investigated the effect of CD domain replacement or switch upon virion encapsidation, Vif-mediated degradation, deamination and antiviral activity of hA3G. The results showed that the two CD domains were functionally equivalent in virion encapsidation and the interaction with HIV-1 Vif of hA3G, whereas CD domain switch or replacement greatly affected the sensitivity to Vif induced degradation, editing and antiviral activity of hA3G. Although the CD2 domain was shown to possess the deamination activity, CD2-2 incorporated efficiently into HIV-1 was unable to mutate viral cDNA, suggesting that CD1 also involved in the enzymatic function. Interestingly, CD2-1 retained considerable deamination activity with a different sequence preference. Taken together, our results suggest that CD domain may play a structural role in virion encapsidation and Vif-mediated degradation of hA3G, and coordination of the two CD domains is required for its editing and antiviral activity.     

Functional domain organization of human APOBEC3G

Human APOBEC3 proteins exist in two forms containing either a single cytidine deaminase domain (CDA) or two CDAs. Strikingly, the proteins that are capable of effectively inhibiting the infectivity of Vif-deficient HIV-1 (HIV-1ΔVif), such as APOBEC3G (A3G), contain two CDAs. In contrast, single-domain APOBEC3 proteins such as APOBEC3A (A3A) are weak inhibitors of HIV-1ΔVif, even though A3A is an active cytidine deaminase and a potent inhibitor of retrotransposon mobility. Here, we demonstrate that the ability to bind to Gag and package into HIV-1 virions is entirely contained within the amino-terminal half of A3G. By changing three adjacent amino acids in A3A, to the sequence found in the N-terminal half of A3G, we were able to confer on A3A the ability to be efficiently incorporated into HIV-1 virions and to bind HIV-1 Gag. Nevertheless, this A3A mutant remained a weak inhibitor of HIV-1 infectivity, suggesting that segregation of the Gag-binding/virion incorporation and cytidine deaminase/virus-inhibition activities of APOBEC3 proteins into two tandem CDA regions promotes the efficient inhibition of retrovirus infectivity by APOBEC3 proteins.     

Cytidine deaminases: AIDing DNA demethylation?

The presence of 5-methylcytosine (5-mC) in DNA is a vital epigenetic mark in vertebrates. While the enzymes responsible for methylating DNA in vertebrates have been identified, the means by which this mark can be removed are still unclear. Recently, it has been shown that activation-induced cytidine deaminase (AID) contributes to the demethylation of DNA in certain systems. This enzyme has been intensely studied in its role as a key driver of antibody diversification in B cells, but recent observations from early development in zebrafish and mice as well as heterokaryons point to a role beyond immunology. This review takes stock of the reports linking AID and related deaminases to DNA demethylation, and describes the many important questions left to be answered in this field.     

The effect of lentinan on proliferative processes in parenchymal organs of rats--I. The effect on pyrimidine and nucleic acid syntheses.

The present study investigated the effect of Lentinan on the biochemical events associated with the pyrimidine and nucleic acid syntheses in the liver, kidney, thymus and spleen of rats. Lentinan was used at a dose of 4 mg/kg/day (twice) and in a single dose of 20 mg/kg. The following changes were observed. (1) The utilization of (14C)orotic acid for the synthesis of uridine components of liver acid-soluble extract and RNA uracil was activated after the administration of both doses of the drug. The specific activity of cytidine components of the acid-soluble extract and RNA were, on the other hand, not affected. The same holds true for the kidney. The ratio of the specific activity of cytidine:uridine components of the acid soluble extract as well as RNA decreased after the administration of both doses of the drug. The specific activity of DNA cytosine and thymine are slightly suppressed in the liver after the administration of a high dose of Lentinan; no effect was observed in the kidney. (2) The uptake of (14C)cytidine by the liver was not affected; the specific activity of DNA cytosine and thymine were increased after the administration of a high dose of Lentinan. (3) The uptake of (14C)thymidine by the liver was not affected; the specific activity of liver DNA thymine was increased after the administration of both doses of the drug. In the thymus an increase of specific activity of DNA thymine has also been observed. (4) Repeated doses of the drug (4 mg/kg for 6 consecutive days) increased the weight of the spleen. The specific activity of DNA thymine of the liver and spleen were significantly increased.     

Human uracil-DNA glycosylase deficiency associated with profoundly impaired immunoglobulin class-switch recombination

Activation-induced cytidine deaminase (AID) is a 'master molecule' in immunoglobulin (Ig) class-switch recombination (CSR) and somatic hypermutation (SHM) generation, AID deficiencies are associated with hyper-IgM phenotypes in humans and mice. We show here that recessive mutations of the gene encoding uracil-DNA glycosylase (UNG) are associated with profound impairment in CSR at a DNA precleavage step and with a partial disturbance of the SHM pattern in three patients with hyper-IgM syndrome. Together with the finding that nuclear UNG expression was induced in activated B cells, these data support a model of CSR and SHM in which AID deaminates cytosine into uracil in targeted DNA (immunoglobulin switch or variable regions), followed by uracil removal by UNG.     

Autoradiographic study of nucleic acid precursor incorporation in nerve cells of the mouse brain

Following intravenous administration of five tritiated nucleic acid bases and five nucleosides in mice the intensity of labelling of the cerebral cortex cells and the liver cells show the following pattern. Cytidine and uridine cause intense labelling; uracil, adenine, adenosine, guanosine, guanine, thymine and cytosine only a weak labelling. The choroid plexus is labelled intensely by cytidine and uridine, moderately by guanosine and adenosine, weakly by other substances.In vitro experiments and intracerebral injection with uridine, uracil, adenine and adenosine give the same labelling pattern as intravenous injection. Inhalation of CO₂ does not alter the labelling of nerve cells apart from that due to the uridine group, where a weaker labelling of the nerve cells is observed.It is concluded that, in the case of the nucleic acid precursors used here, metabolic specificities are of greater significance for the labelling pattern of the nerve cells than the transport specificities of the blood-brain barrier.     

Stoichiometry of the antiviral protein APOBEC3G in HIV-1 virions

A host cytidine deaminase, APOBEC3G (A3G), inhibits replication of human immunodeficiency virus type 1 (HIV-1) by incorporating into virions in the absence of the virally encoded Vif protein (Deltavif virions), at least in part by causing G-to-A hypermutation. To gain insight into the antiretroviral function of A3G, we determined the quantities of A3G molecules that are incorporated in Deltavif virions. We combined three experimental approaches-reversed-phase high-pressure liquid chromatography (HPLC), scintillation proximity assay (SPA), and quantitative immunoblotting-to determine the molar ratio of A3G to HIV-1 capsid protein in Deltavif virions. Our studies revealed that the amount of the A3G incorporated into Deltavif virions was proportional to the level of its expression in the viral producing cells, and the ratio of the A3G to Gag in the Deltavif virions produced from activated human peripheral blood mononuclear cells (PBMC) was approximately 1:439. Based on previous estimates of the stoichiometry of HIV-1 Gag in virions (1400-5000), we conclude that approximately 7 (+/-4) molecules of A3G are incorporated into Deltavif virions produced from human PBMCs. These results indicate that virion incorporation of only a few molecules of A3G is sufficient to inhibit HIV-1 replication.     

Aedes albopictus cells resistant to adenosine because of a defect in nucleoside transport

By growing Aedes albopictusmosquito cells in media containing increasing concentrations of adenosine and subsequently plating low numbers of cells in the presence of EHNA (an inhibitor of adenosine deaminase), three clones were obtained which were resistant to adenosine. The adenosine-resistant clones contained levels of adenosine and thymidine kinase similar to those in the parental cells, but were unable to incorporate labeled nucleosides (adenosine, uridine, thymidine, or guanosine) into TCA-precipitable material. The inability to incorporate nucleosides was also reflected in an enhanced resistance to several nucleoside analogs such as 5-fluorodeoxyuridine and tubercidin but not to the unribosylated base, 5-fluorouracil. Direct measurements over short time intervals indicated that the primary defect in these cells was at the level of nucleoside transport.     

Complementary function of the two catalytic domains of APOBEC3G

The HIV-1 viral accessory protein Vif prevents the encapsidation of the antiviral cellular cytidine deaminases APOBEC3F and APOBEC3G by inducing their proteasomal degradation. In the absence of Vif, APOBEC3G is encapsidated and blocks virus replication by deaminating cytosines of the viral cDNA. APOBEC3G encapsidation has been recently shown to depend on the viral nucleocapsid protein; however, the role of RNA remains unclear. Using APOBEC3G deletion and point mutants, we mapped the encapsidation determinant to the Zn(2+) coordination residues of the N-terminal catalytic domain (CD1). Notably, these residues were also required for RNA binding. Mutations in the two aromatic residues of CD1 but not CD2, which are conserved in cytidine deaminase core domains and are required for RNA binding, prevented encapsidation into HIV-1, HTLV-I and MLV. The Zn(2+) coordination residues of the C-terminal catalytic domain (CD2) were not required for encapsidation but were essential for cytidine deaminase activity and the antiviral effect. These findings suggest a model in which CD1 mediates encapsidation and RNA binding while CD2 mediates cytidine deaminase activity. Interestingly, HTLV-I was relatively resistant to the antiviral effects of encapsidated APOBEC3G.