Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “CYTOSINE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources
PubMed1997

Characterization of the Saccharomyces cerevisiae cytosine transporter using energizable plasma membrane vesicles.

The purine-cytosine permease is a carrier localized in the plasma membrane of the yeast Saccharomyces cerevisiae. The energetics of cytosine transport catalyzed by this permease has been studied in an artificial system obtained by fusion between proteoliposomes containing beef heart cytochrome c oxidase and plasma membrane-enriched fractions of a S. cerevisiae strain overexpressing the permease. Upon addition of an energy donor, a proton-motive force (inside alkaline and negative) is created in this system and promotes cytosine accumulation. By using different phospholipids, it is shown that cytosine uptake is dependent on the phospholipids surrounding the carrier. It was demonstrated that the purine-cytosine permease is able to catalyze a secondary active transport of cytosine. By using nigericin and valinomycin, the DeltapH component of the proton-motive force is shown to be the only force driving nucleobase accumulation. Moreover, transport measurements done at two pH values have shown that alkalinization of intravesicular pH leads to a significant increase in cytosine uptake rate. Finally, no specific role of K+ ions on cytosine transport could be demonstrated in this system.

Animals↗
PubMed1975

Characterization of cytosine permeation in Saccharomyces cerevisiae.

Cytosine permeation in Saccharomyces cerevisiae has been studied. Cytosine uptake is mediated by a permease which is also responsible for purines transport. The Km for the transport of various substrates of this permease have been determined. By means of appropriate selective techniques, mutants with altered Km and mutants lacking the permease have been selected. Cytosine transport is active and is inhibited by 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, and by N-ethylmaleimide, a reagent of--SH group. Internal labeled cytosine is chased by addition of unlabeled cytosine in the medium. These results support the hypothesis of a carrier-mediated transport, with reduced internal affinity, allowing the release and accumulation of cytosine in the inner compartment. The efflux of cytosine from cytosine permease-less cells has also been studied and shows first order kinetics. A diffusion coefficient of 5.7 per 10- minus 8 cm per S- minus 1 has been evaluated for this efflux.

Adenine↗
PubMed1998

Electron paramagnetic resonance and electron nuclear double resonance studies of X-irradiated crystals of cytosine hydrochloride. Part I: free radical formation at 10 K after high radiation doses.

Anhydrous single crystals of cytosine hydrochloride (protonated at N3) have been X-irradiated at 10 K and studied using K-band EPR, ENDOR and FSE spectroscopy. At least seven radicals were present at 10 K after X irradiation with a dose of about 150 kGy. Two different protonation states of the one-electron reduced cytosine cation were observed: an amino-protonated species (R1) and the pristine one-electron reduced species (R2) with zero net charge. Apparently three deprotonated versions of the one-electron oxidized cytosine cation were formed: the amino-deprotonated cation (R3), an N3-deprotonated cation (R4) and an N1-deprotonated cation (R5). Finally, two products formed by net hydrogen addition to the cytosine base were observed: a C5 hydrogen-addition radical (R6) and a C6 hydrogen-addition radical (R7). The crystalline lattice of cytosine hydrochloride is characterized in part by a cytosine base initially protonated at the N3-position, thus forming a cytosine base cation, and in part by an extended network of hydrogen bonding involving the chlorine anions. Proton transfer properties of pristine one-electron oxidation and reduction base products in this lattice are discussed and are suggested as explanations of the unusual multitude of positions for deprotonation of the one-electron oxidized species as well as for the two protonation states of the reduction product observed. The magnetic parameters for the amino-protonated species R1 agree well with those extracted from previous studies of cytosine derivatives in frozen solutions and in various glasses.

Crystallography↗
PubMed1999

Mechanism of inhibition of DNA (cytosine C5)-methyltransferases by oligodeoxyribonucleotides containing 5,6-dihydro-5-azacytosine.

A key step in the predicted mechanism of enzymatic transfer of methyl groups from S-adenosyl-l-methionine (AdoMet) to cytosine residues in DNA is the transient formation of a dihydrocytosine intermediate covalently linked to cysteine in the active site of a DNA (cytosine C5)-methyltransferase (DNA C5-MTase). Crystallographic analysis of complexes formed by HhaI methyltransferase (M.HhaI), AdoMet and a target oligodeoxyribonucleotide containing 5-fluorocytosine confirmed the existence of this dihydrocytosine intermediate. Based on the premise that 5,6-dihydro-5-azacytosine (DZCyt), a cytosine analog with an sp3-hybridized carbon (CH2) at position 6 and an NH group at position 5, could mimic the non-aromatic character of the cytosine ring in this transition state, we synthesized a series of synthetic substrates for DNA C5-MTase containing DZCyt. Substitution of DZCyt for target cytosines in C-G dinucleotides of single-stranded or double-stranded oligodeoxyribonucleotide substrates led to complete inhibition of methylation by murine DNA C5-MTase. Substitution of DZCyt for the target cytosine in G-C-G-C sites in double-stranded oligodeoxyribonucleotides had a similar effect on methylation by M. HhaI. Oligodeoxyribonucleotides containing DZCyt formed a tight but reversible complex with M.HhaI, and were consistently more potent as inhibitors of DNA methylation than oligodeoxyribonucleotides identical in sequence containing 5-fluorocytosine. Crystallographic analysis of a ternary complex involving M.HhaI, S-adenosyl-l-homocysteine and a double-stranded 13-mer oligodeoxyribonucleotide containing DZCyt at the target position showed that the analog is flipped out of the DNA helix in the same manner as cytosine, 5-methylcytosine, and 5-fluorocytosine. However, no formation of a covalent bond was detected between the sulfur atom of the catalytic site nucleophile, cysteine 81, and the pyrimidine C6 carbon. These results indicate that DZCyt can occupy the active site of M.HhaI as a transition state mimic and, because of the high degree of affinity of its interaction with the enzyme, it can act as a potent inhibitor of methylation.

Animals↗
PubMed1999

On the substrate specificity of DNA methyltransferases. adenine-N6 DNA methyltransferases also modify cytosine residues at position N4.

Methylation of DNA is important in many organisms and essential in mammals. Nucleobases can be methylated at the adenine-N6, cytosine-N4, or cytosine-C5 atoms by specific DNA methyltransferases. We show here that the M.EcoRV, M.EcoRI, and Escherichia coli dam methyltransferases as well as the N- and C-terminal domains of the M. FokI enzyme, which were formerly all classified as adenine-N6 DNA methyltransferases, also methylate cytosine residues at position N4. Kinetic analyses demonstrate that the rate of methylation of cytosine residues by M.EcoRV and the M.FokI enzymes is reduced by only 1-2 orders of magnitude in relation to methylation of adenines. This result shows that although these enzymes methylate DNA in a sequence specific manner, they have a low substrate specificity with respect to the target base. This unexpected finding has implications on the mechanism of adenine-N6 DNA methyltransferases. Sequence comparisons suggest that adenine-N6 and cytosine-N4 methyltransferases have changed their reaction specificity at least twice during evolution, a model that becomes much more likely given the partial functional overlap of both enzyme types. In contrast, methylation of adenine residues by the cytosine-N4 methyltransferase M.BamHI was not detectable. On the basis of our results, we suggest that adenine-N6 and cytosine-N4 methyltransferases should be grouped into one enzyme family.

Adenine↗
PubMed2002

Optimization of baculovirus-mediated expression and purification of hexahistidine-tagged murine DNA (cytosine-C5)-methyltransferase-1 in Spodoptera frugiperda 9 cells.

Enzymatic DNA methylation of carbon 5 of cytosines is an epigenetic modification that plays a role in regulating gene expression, differentiation, and tumorigenesis. DNA (cytosine-C5)-methyltransferase-1 is the enzyme responsible for maintaining established methylation patterns during replication in mammalian cells. It is composed of a large ( approximately 1100 amino acids (a.a.)) amino-terminal region containing many putative regulatory domains and a smaller ( approximately 500 a.a.) carboxy-terminal region containing conserved, catalytic domains. In this study, murine DNA (cytosine C5)-methyltransferase-1, fused to an amino-terminal hexahistidine tag, was expressed by infecting Spodoptera frugiperda cells for 46 h with a recombinant baculovirus carrying the DNA (cytosine-C5)-methyltransferase-1 cDNA. A total of 3 x 10(8) infected S. frugiperda cells yielded approximately 1 mg of full-length, hexahistidine-tagged DNA (cytosine-C5)-methyltransferase-1, which was purified approximately 450-fold from RNase-treated S. frugiperda cell extracts by nickel affinity chromatography. The characterization of hexahistidine-tagged DNA (cytosine-C5)-methyltransferase-1 through DNA methylation and inhibitor-binding assays indicated that the purified enzyme had at least a 30-fold higher catalytic efficiency with hemimethylated double-stranded oligodeoxyribonucleotide substrates than unmethylated substrates and was most active with small oligodeoxyribonucleotide substrates with a capacity for forming stem-loop structures. The expression and purification procedures reported here differ significantly from the original reports of baculovirus-mediated hexahistidine-tagged DNA (cytosine-C5)-methyltransferase-1 expression and purification by nickel affinity chromatography and provide a consistent yield of active enzyme.

Animals↗
PubMed1975

On the nature of the cytosine-methylated sequence in DNA of Bacillus brevis var. G.-B.

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of [Me-3H]methionine, practically all the radioactivity incorporated into DNA is found to exist in 5-methylcytosine and N6-methyladenine. The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of 5-methylcytosine in R-m5 C-R and R-m5 C-T-R oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring 5-methylcytosine to be identified and shows that 5-methylcytosine localizes in G-m5 C-A and G-m5 C-Tr fragments. B. brevis S DNA methylase modifying cytosine residues recognizes the GCA/TGC degenerate nucleotide sequence which is a part of the following complementary structure with a two-fold rotational axis of symmetry: (5')...N'-G-C-T-G-C-N... (3') (3')...N-C-G-A-C-G-N'... (5') (Methylated cytosine residues are askerisked). Cytosine-modifying DNA methylase activity is isolated from B. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence DNA in bacterial cells can be undermethylated. This enzyme methylates cytosine residues in native and denatured DNA in the same nucleotide sequences. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA. DNA methylases of different variants of B. brevis (R, S, P+, P-)) methylate cytosine residues in the same nucleotide sequences. It means that specificity or methylation of DNA cytosine residues in the cells of different variants of B. brevis is the same.

Bacillus↗
PubMed1979

The secondary structure of bacteriophage DNA in situ. VIII. The reaction of sodium bisulphite with intraphage cytosine as a probe for studying the DNA-protein interaction.

To obtain data on the viral nucleoprotein a study has been made of the reaction of sodium bisulphite with cytosine in the intraphage DNA of the phage Sd. The CHlO4 hydrolysates of the bisulphite-modified phage Sd have demonstrated a decrease of 18% in the cytosine content and the presence of the products with the properties of cytosyl-amino acids (the main amino acid responsible for the DNA-protein interaction involving cytosine is lysine). But when prior to hydrolysis the modified phage was disintegrated under mild conditions in 0.1--1 M NaCl solution or Tris-HCl buffer (pH 7), neither the decrease in the cytosine content nor cytosyl-amino acids have been found. An exception is the heating of the phage at 70 degrees C in a medium containing 0.05 M phosphate buffer (pH 7.9--8.5), when an 18% decrease in the cytosine content and subsequent appearance of cytosyl-amino acids have also been observed. The presence of cytosyl-amino acids which are the nucleotide-protein cross-links is confirmed by the results of viscometry, equilibrium centrifugation in cesium sulphate gradient and determinations of the survival percentage. It is suggested that the reaction between bisulphite and cytosine in the phage Sd stops at the stage of the intermediate product C5-C6-dihydro-C6-sulphopyrimidine whose amino group is shielded by interaction with protein (product VII). This product can exist only under in situ conditions: with disintegration of nucleoprotein (destruction of phage particles or ejection of the DNA) in phosphate-free media the product VII reverts into the initial cytosine. Under the conditions of acid hydrolysis or destruction of phage in the presence of phosphate ions product VII undergoes transamination with cleavage of SO3 and restoration of the C5-C6 double bond producing cytosyl-amino acids. The factors determining the stability of the product VII are discussed.

Bacteriophages↗
PubMed1982

Purification and some properties of cytosine deaminase from Salmonella typhimurium.

Cytosine deaminase (EC 3.5.4.1) from Salmonella typhimurium has been purified 419-fold to apparent homogeneity. SDS polyacrylamide gel electrophoresis indicated that the final cytosine deaminase preparation was homogeneous. The molecular weight of cytosine deaminase was determined to be approx. 230000 containing four identical subunits with each subunit having a molecular weight 54000. Cytosine was deaminase has a pH optimum of 7.30 to 7.50 and a temperature optimum of 45 to 50 degrees C. Cytosine was deaminated specifically; 5-fluorocytosine was deaminated to a lesser extent. The Km and V values for cytosine were 0.74 mM and 47.16 mumole/min, respectively. As effectors of enzyme activity, PPi stimulated the deamination while metal ions and orotidine monophosphate inhibited it. The physical characteristics of cytosine deaminase lend credence to its proposed salvage role in pyrimidine metabolism as indicated previously by physiological studies (West, T.P. and O'Donovan, G.A., J. Bacteriol. (1982) 149, 1171-1174).

Cations↗
PubMed1999

Prebiotic cytosine synthesis: a critical analysis and implications for the origin of life.

A number of theories propose that RNA, or an RNA-like substance, played a role in the origin of life. Usually, such hypotheses presume that the Watson-Crick bases were readily available on prebiotic Earth, for spontaneous incorporation into a replicator. Cytosine, however, has not been reported in analyses of meteorites nor is it among the products of electric spark discharge experiments. The reported prebiotic syntheses of cytosine involve the reaction of cyanoacetylene (or its hydrolysis product, cyanoacetaldehyde), with cyanate, cyanogen, or urea. These substances undergo side reactions with common nucleophiles that appear to proceed more rapidly than cytosine formation. To favor cytosine formation, reactant concentrations are required that are implausible in a natural setting. Furthermore, cytosine is consumed by deamination (the half-life for deamination at 25 degrees C is approximately 340 yr) and other reactions. No reactions have been described thus far that would produce cytosine, even in a specialized local setting, at a rate sufficient to compensate for its decomposition. On the basis of this evidence, it appears quite unlikely that cytosine played a role in the origin of life. Theories that involve replicators that function without the Watson-Crick pairs, or no replicator at all, remain as viable alternatives.

Cytosine↗
PubMed2001

Extrahelical cytosine bases in DNA duplexes containing d[GCC](n).d[GCC](n) repeats: detection by a mechlorethamine crosslinking reaction.

The cytosine-cytosine (C-C) pair is one of the least stable DNA mismatch pairs. The bases of the C-C mismatch are only weakly hydrogen bonded, and previous work has shown that, in certain sequence contexts, they can become unstacked from the core helix, and adopt an 'extrahelical' location. Here, using DNA duplexes with d[GCC](n).d[GCC](n) fragments containing C-C mismatches in a 1,4 bp relationship, we show that cytosine bases of different formal mismatch pairs can be crosslinked by mechlorethamine. For example, in the duplex d[CTCTCGCCGCCGCCGTATC].d[GATACGCCGCCGCCGAGAG], where underlined cytosine bases are present as the formal C-C mismatch pairs C(7)-C(32), C(10)-C(29) and C(13)-C(26), we show that two mechlorethamine crosslinks form between C(13) and C(29) and between C(10) and C(32), in addition to crosslinks at C(7)-C(32), C(10)-C(29) and C(13)-C(26) (we have reported previously the crosslinking of formal C-C pairs by mechlorethamine). We interpret the formation of the C(13)-C(29) and C(10)-C(32) crosslinks as evidence of an extrahelical location of the crosslinkable cytosines. Such extrahelical cytosine bases have been observed previously for a single C-C mismatch pair (in the so-called E-motif conformation). In the E-motif, the extrahelical cytosines are folded back towards the 5'-end of the duplex, consistent with our crosslinking data, and also consistent with the absence of C(7)-C(29) and C(10)-C(26) crosslinks in the current work. Hence, our data provide evidence for an extended E-motif DNA (eE-DNA) conformation in short d[GCC](n).d[GCC](n) repeat fragments, and raise the possibility that such structures might occur in much longer d[GCC](n).d[GCC](n) repeat tracts.

Base Pair Mismatch↗
PubMed2006

Sequence-dependent enhancement of hydrolytic deamination of cytosines in DNA by the restriction enzyme PspGI.

Hydrolytic deamination of cytosines in DNA creates uracil and, if unrepaired, these lesions result in C to T mutations. We have suggested previously that a possible way in which cells may prevent or reduce this chemical reaction is through the binding of proteins to DNA. We use a genetic reversion assay to show that a restriction enzyme, PspGI, protects cytosines within its cognate site, 5'-CCWGG (W is A or T), against deamination under conditions where no DNA cleavage can occur. It decreases the rate of cytosine deamination to uracil by 7-fold. However, the same protein dramatically increases the rate of deaminations within the site 5'-CCSGG (S is C or G) by approximately 15-fold. Furthermore, a similar increase in cytosine deaminations is also seen with a catalytically inactive mutant of the enzyme showing that endonucleolytic ability of the protein is dispensable for its mutagenic action. The sequences of the mutants generated in the presence of PspGI show that only one of the cytosines in CCSGG is predominantly converted to thymine. Our results are consistent with PspGI 'sensitizing' the cytosine in the central base pair in CCSGG for deamination. Remarkably, PspGI sensitizes this base for damage despite its inability to form stable complexes at CCSGG sites. These results can be explained if the enzyme has a transient interaction with this sequence during which it flips the central cytosine out of the helix. This prediction was validated by modeling the structure of PspGI-DNA complex based on the structure of the related enzyme Ecl18kI which is known to cause base-flipping.

Base Sequence↗
PubMed2000

Cytosine deamination plays a primary role in the evolution of mammalian isochores.

DNA melting is rate-limiting for cytosine deamination, from which we infer that the rate of cytosine deamination should decline twofold for each 10% increase in GC content. Analysis of human DNA sequence data confirms that this is the case for 5-methylcytosine. Several lines of evidence further confirm that it is also the case for unmethylated cytosine and that cytosine deamination causes the majority of all C-->T and G-->A transitions in mammals. Thus, cytosine deamination and DNA base composition each affect the other, forming a positive feedback loop that facilitates divergent genetic drift to high or low GC content. Because a 10 degrees C increase in temperature in vitro increases the rate of cytosine deamination 5. 7-fold, cytosine deamination must be highly dependent on body temperature, which is consistent with the dramatic differences between the isochores of warm-blooded versus cold-blooded vertebrates. Because this process involves both DNA melting and positive feedback, it would be expected to spread progressively (in evolutionary time) down the length of the chromosome, which is consistent with the large size of isochores in modern mammals.

5-Methylcytosine↗
PubMed1990

Photoaffinity labelling of the purine-cytosine permease of Saccharomyces cerevisiae.

8-Azidoadenine was used as a photoaffinity reagent to characterize the purine-cytosine permease of Saccharomyces cerevisiae. It is a potent competitive inhibitor of cytosine uptake and irradiation of the cells incubated with the label induced the irreversible inactivation of cytosine uptake. Addition of excess cytosine prevented this labelling which was restricted to the outer face of the plasma membrane since it was not accumulated by the cells. In the strain with the amplified purine-cytosine permease gene the maximum cytosine uptake rate was increased 4-5-fold relative to wild type without a modification of the Michaelis constant of uptake (Kt); no uptake could be measured in the deleted strain. The relative amounts of specific labelling determined for the cells and for membrane preparations were 0, 1 and 4 for the null, the wild-type and the amplified strains, respectively. One major band specifically labelled by [3H]azidoadenine, corresponding to a polypeptide with an apparent molecular mass of 45 kDa, was observed in the wild type, amplified in the strain carrying the multicopy plasmid and not detected in the deleted strain. Therefore this polypeptide corresponds to the purine-cytosine permease.

Adenine↗
PubMed1969

Biological effects of substituting cytosine for 5-hydroxymethylcytosine in the deoxyribonucleic acid of bacteriophage T4.

Previous work from this laboratory has shown that the cytosine-containing T4 deoxyribonucleic acid (DNA) made by deoxycytidine triphosphatase (dCTPase) amber mutants is extensively degraded, and that nucleases controlled by genes 46 and 47 participate in this process. In this paper, we examine other consequences of a defective dCTPase. Included are studies of DNA synthesis and phage production, and of the control of both early and late protein synthesis after infection of Escherichia coli B with various T4 mutants defective in genes 56 (dCTPase), 42 (dCMP hydroxymethylase), 1 (deoxynucleotide kinase), 43 (DNA polymerase), 30 (polynucleotide ligase), 46 and 47 (DNA breakdown) or e(lysozyme). By varying the temperature of infection with a temperature-sensitive dCTPase mutant, we have been able to control intracellular dCTPase activity, and thus vary the cytosine content of the phage DNA. We have produced and characterized viable T4 phage in which cytosine replaces 20% of the 5-hydroxymethylcytosine (HMC) in the DNA. We present evidence which suggests that intact, cytosine-containing T4 DNA is much less efficient than is normal T4 DNA in directing the synthesis of tail-fiber antigen. Lysozyme production is much less affected by progressively decreasing dCTPase activity; however, complete substitution of cytosine is correlated with a depression of lysozyme synthesis greater than expected from the defective synthesis of DNA. Low but significant lysozyme synthesis is observed late after infection of E. coli B with T4 amber mutants defective in a number of genes controlling DNA synthesis. The "20% cytosine" T4 phage, once produced, can initiate an apparently normal infection at permissive temperatures; the synthesis of early enzymes, DNA, and phage does not appear to be impaired. Two roles for HMC in T4 DNA have been indicated previously: (i) involvement in host-controlled restriction of the phage, in which glucosylation of the hydroxymethyl group plays a crucial role (16, 29, 53, 58), and (ii) protection of vegetative DNA against phage-controlled nucleases, a protection not dependent on glucosylation (41, 66, 67). A third role is suggested by our present results: transcription of at least some late genes can occur only from HMC-containing DNA and not from cytosine-containing DNA.

Bacteriophages↗
PubMed2006

Endostatin-cytosine deaminase fusion protein suppresses tumor growth by targeting neovascular endothelial cells.

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.

Adenocarcinoma↗
PubMed2000

Adenovirus mediated cytosine deaminase gene transduction and 5-fluorocytosine therapy sensitizes mouse prostate cancer cells to irradiation.

PURPOSE: We assess the ability of adenovirus mediated expression of the Escherichia coli cytosine deaminase gene in conjunction with the prodrug 5-fluorocytosine to result in radiation sensitization in the mouse prostate cancer cell line RM-1 in vitro. MATERIALS AND METHODS: To document cytotoxicity of gene therapy, RM-1 cells were exposed to escalating doses of adenovirus mediated cytosine deaminase and a fixed dose of 5-fluorocytosine or phosphate buffered saline. Viable cells as determined by exclusion of trypan blue were counted the following day. Cytosine deaminase expressing RM-1 cells were then irradiated as single cell suspensions at various doses of radiation in a cesium source (4.4 Gy. per minute) and randomized to receive 5-fluorocytosine therapy at different times in relation to the external radiation therapy. End points were determined in a clonogenic assay by counting colonies with greater than 50 cells 7 days after replating. RESULTS: Use of adenovirus mediated cytosine deaminase plus 5-fluorocytosine demonstrated viral dose dependent killing of RM-1 cells to a maximum of 85%, while either therapy alone was nontoxic. Neither adenovirus mediated cytosine deaminase infection nor 5-fluorocytosine alone influenced external radiation therapy killing. However, after controlling for death due to gene therapy alone, the combination of adenovirus mediated cytosine deaminase plus 5-fluorocytosine and external radiation therapy resulted in synergistic activity to approximately 2 logs of cell kill at low doses of radiation (p = 0.001). While altering the chronology of prodrug exposure in relation to external radiation therapy maintained synergy in all scenarios tested, starting 5-fluorocytosine 24 hours before external radiation therapy resulted in the most profound killing (p = 0.04), which indicates the importance of maintaining prodrug therapy during external radiation therapy. CONCLUSIONS: The combination of adenovirus mediated cytosine deaminase plus 5-fluorocytosine and radiation therapy resulted in radiation sensitization with clinically relevant doses of radiation suggesting a potential usefulness of this treatment in patients with prostate cancer.

Adenoviridae↗
PubMedDate not supplied

[Specificity of methylation of cytosine risidues in DNA of Bacillus brevis var. G-B].

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of (methyl-3H)-methionine practically the total radioactivity included into DNA is found to exist in 5-methylcytosine (MC) and 6N-methyladenine (MA). The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of MC in Pur-MC-Pur and Pur-MC-T-Pur oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring with MC to be revealed and shows that MC localizes in G-MC-A and G-MC-T-Pu fragments. Bac. brevis S DNA-methylase modifying cytosine residues recognizes the GCAT GC degenerative nucleotide sequence which is a part of the following complementary structure with rotational symmetry: (5') ... N'--G--MC--T--G--C--N ... (3') (3') ... N--C--G--A--MC--G--N' ... (5') Cytosine modifying DNA-methylase activity is isolated from Bac. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence, DNA in bacterial cells can be partially undermethylated. This enzyme methylates cytosine residues in native and deneaturated DNA in the same nucleotide sequences. As compared to the native DNA, the denaturated DNA is indicative of a decrease in the level of methylation of adenine, rather than cytosine residues. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA (calf thymus, Pseudomonas aeruginosa etc.). DNA-methylases of different variants of Bac. brevis (R, S, P+, P-) methylate cytosine residues in the same nucleotide sequences. It means that specificity of methylation of DNA cytosine residues in the cells of different variants of Bac. brevis is the same.

Bacillus↗