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At least 253 records · Page 14Linked to original sources
PubMed1999

Screening of an intragenic second-site suppressor of purine-cytosine permease from Saccharomyces cerevisiae. Possible role of Ser272 in the base translocation process.

The purine-cytosine permease from Saccharomyces cerevisiae mediates the active transport through the plasma membrane of adenine, hypoxanthine, guanine and cytosine using the proton electrochemical potential difference as an energy source. Analysis of the activity of strains mutated in a hydrophilic segment (371-377) of the polypeptidic chain has shown the involvement of this segment in the maintenance of the active three-dimensional structure of the carrier. In an attempt to identify permease domains that could interact functionally and/or physically with this segment, we looked for second-site mutations that could suppress the effects of amino acid changes in this region. This paper describes a positive screen that has allowed the isolation of one suppressor from a permease mutant displaying the N374I change (fcy2-20 allele), a substitution that induces a dramatic decrease in the affinity of the carrier for adenine, cytosine and hypoxanthine. The second-site mutation corresponds to the replacement of the Ser272 residue by Leu. Its suppressive effect is shown to be a partial restoration of the binding of cytosine and hypoxanthine to the permease. To test whether this second-site mutation is specific for the fcy2-20 allele, two double mutants were constructed (Fcy2pT213I, S272L and Fcy2pS272L, N377G). Results obtained with these two double mutants showed that the suppressive effect of S272 L replacement was not specific for the original N374I change. To understand the general effect of this amino acid replacement for the three distinct double mutants, a strain overexpressing Fcy2pS272I, was constructed. Kinetic analysis of this strain showed that, by itself, the S272 L change induced an improvement in the base-binding step that could account for its global suppressive effect. Moreover, S272 L induced a decrease in the turnover of the permease, thus showing the involvement of S272 in the translocation process. Taking into account the topological model of the permease proposed here, this Ser residue is probably located in a transmembrane amphipathic alpha-helix (TM5). The location and the observed decrease in the turnover of the carrier observed with the S272 L change lead us to propose that S272 could be part of a hydrophilic pore involved in the translocation of the base and/or the proton.

Adenine↗
PubMed2004

The triplet state of cytosine and its derivatives: electron impact and quantum chemical study.

The excitation of the lowest electronic states and vibrational excitation of cytosine (C) have been studied using electron energy loss spectroscopy (EELS, 0-100 eV) with angular analysis. The singlet states have been found to be in good agreement with UV-VIS absorption results on sublimed films, slightly blueshifted by about 0.1 eV. The EEL spectra recorded at residual energy below 2 eV show clear shoulders at energy losses of 3.50 and 4.25 eV (+/-0.1 eV). They are assigned to the lowest triplet electronic states of cytosine. Energies and molecular structures of the lowest-lying triplet state of C and its methylated and halogenated 5-X-C, 6-X-C, and 5-X, 6-X-C substituted derivatives (X=CH3, F, Cl, and Br) have been studied using quantum chemical calculations with both molecular orbital and density functional methods, in conjunction with the 6-311++G(d,p), 6-311++G(3df,2p), and aug-cc-pVTZ basis sets. The triplet-singlet energy gap obtained using coupled-cluster theory [CCSD(T)] and density functional theory (DFT) methods agrees well with those derived from EELS study. The first C's vertical triplet state is located at 3.6 eV, in good agreement with experiment. The weak band observed at 4.25 eV is tentatively assigned to the second C's vertical triplet excitation. For the substituted cytosines considered, the vertical triplet state is consistently centered at 3.0-3.2 eV above the corresponding singlet ground state but about 1.0 eV below the first excited singlet state. Geometrical relaxation involving out-of-plane distortions of hydrogen atoms leads to a stabilization of 0.6-1.0 eV in favor of the equilibrium triplet. The lowest-lying adiabatic triplet states are located at 2.3-3.0 eV. Halogen substitution at both C(5) and C(6) positions tends to reduce the triplet-singlet separations whereas methylation tends to enlarge it. The vibrational modes of triplet cytosine and the ionization energies of substituted derivatives were also evaluated.

Cytosine↗
PubMed2002

Cytosine substituted calix[4]pyrroles: neutral receptors for 5'-guanosine monophosphate.

The synthesis and characterization of two cytosine-substituted calix[4]pyrrole conjugates, bearing the appended cytosine attached at either a beta- or meso-pyrrolic position, is described. These systems were tested as nucleotide-selective carriers and as active components of nucleotide-sensing ion-selective electrodes at pH 6.6. Studies of carrier selectivity were made using a Pressman-type model membrane system consisting of an initial pH 6.0 aqueous phase, an intervening dichloromethane barrier containing the calix[4]pyrrole conjugate, and a receiving basic aqueous phase. Good selectivity for the Watson-Crick complementary nucleotide, 5'-guanosine monophosphate (5'-GMP), was seen in the case of the meso-linked conjugate with the relative rates of through-membrane transport being 7.7:4.1:1 for 5'-GMP, 5'-AMP, and 5'-CMP, respectively. By contrast, the beta-substituted conjugate, while showing a selectivity for 5'-GMP that was enhanced relative to unsubstituted calix[4]pyrrole, was found to transport 5'-CMP roughly 4.5 times more quickly than 5'-GMP. Higher selectivities were also found for 5'-CMP when both the beta- and meso-substituted conjugates were incorporated into polyvinyl chloride membranes and tested as ion selective electrodes at pH 6.6, whereas near-equal selectivities were observed for 5'-CMP and 5'-GMP in the case of unsubstituted calix[4]pyrroles. These seemingly disparate results are consistent with a picture wherein the meso-substituted cytosine calix[4]pyrrole conjugate, but not its beta-linked congener, is capable of acting as a ditopic receptor, binding concurrently both the phosphate anion and nucleobase portions of 5'-GMP to the calixpyrrole core and cytosine "tails" of the molecule, respectively, with the effect of this binding being most apparent under the conditions of the transport experiments.

Calixarenes↗
PubMed1976

A gene of bacteriophage T4 whose product prevents true late transcription on cytosine-containing T4 DNA.

T-even coliphages have 5-hydroxymethylcytosine in their DNA instead of cytosine. In some T4 mutants, the replicated DNA contains cytosine, but then no late gene products are made. We show that the inability to make late gene products with cytosine-containing T4 DNA is due to a T4 gene products. This gene product, while probably nonessential under normal conditions, interacts with an essential part of the transcription apparatus. Mutations in this gene allow viable T4 particles to be made whose DNA has been substituted almost 100% with cytosine.

Coliphages↗
PubMed1985

Endonuclease-resistant apyrimidinic sites formed by neocarzinostatin at cytosine residues in DNA: evidence for a possible role in mutagenesis.

When defined-sequence DNA from the lacl region of plasmid pMC1 was treated with the nonprotein chromophore of neocarzinostatin in the presence of various thiols, the predominant lesions were direct strand breaks, occurring primarily at thymine and adenine residues. In the presence of glutathione, however, alkali-dependent strand breaks, occurring at certain cytosine residues, were also detected but were virtually absent when other thiols were used. Chromophore-induced release of free cytosine base from [3H]cytosine-labeled DNA was 2- to 3-fold greater with glutathione than with the other thiols. These results suggest that the alkali-dependent strand break is some form of apyrimidinic site. These sites were substrates for endonuclease IV of Escherichia coli, although a 5-fold greater concentration of enzyme was required for their cleavage than was required for cleavage of apurinic sites in depurinated DNA. These sites were also less sensitive to E. coli endonuclease VI (exonuclease III) by a factor of at least 5 and less sensitive to E. coli endonuclease III by a factor of at least 10. These and other results suggest that these sites are chemically different from normal apurinic/apyrimidine sites. When chromophore-induced apyrimidinic sites were quantitated as alkali-dependent breaks at 11 specific sites in the lacl gene, a correlation was found between occurrences of these lesions and the reported frequencies of G-C to A X T transitions at the same sites. All occurrences of the trinucleotide sequence A-G-C, including the ochre 21 mutational hot spot, were particularly prominent sites. The selective formation of endonuclease-resistant apyrimidinic sites at specific cytosine residues may explain the high frequency of G X C to A X T transitions in the mutational spectrum of neocarzinostatin.

Antibiotics, Antineoplastic↗
PubMed1995

Stable loop in the crystal structure of the intercalated four-stranded cytosine-rich metazoan telomere.

In most metazoans, the telomeric cytosine-rich strand repeating sequence is d(TAACCC). The crystal structure of this sequence was solved to 1.9-A resolution. Four strands associate via the cytosine-containing parts to form a four-stranded intercalated structure held together by C.C+ hydrogen bonds. The base-paired strands are parallel to each other, and the two duplexes are intercalated into each other in opposite orientations. One TAA end forms a highly stabilized loop with the 5' thymine Hoogsteen-base-paired to the third adenine. The 5' end of this loop is in close proximity to the 3' end of one of the other intercalated cytosine strands. Instead of being entirely in a DNA duplex, this structure suggests the possibility of an alternative conformation for the cytosine-rich telomere strands.

Animals↗
PubMed2001

Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage.

Ribozymes of hepatitis delta virus have been proposed to use an active-site cytosine as an acid-base catalyst in the self-cleavage reaction. In this study, we have examined the role of cytosine in more detail with the antigenomic ribozyme. Evidence that proton transfer in the rate-determining step involved cytosine 76 (C76) was obtained from examining cleavage activity of the wild-type and imidazole buffer-rescued C76-deleted (C76 Delta) ribozymes in D(2)O and H(2)O. In both reactions, a similar kinetic isotope effect and shift in the apparent pKa indicate that the buffer is functionally substituting for the side chain in proton transfer. Proton inventory of the wild-type reaction supported a mechanism of a single proton transfer at the transition state. This proton transfer step was further characterized by exogenous base rescue of a C76 Delta mutant with cytosine and imidazole analogues. For the imidazole analogues that rescued activity, the apparent pKa of the rescue reaction, measured under k(cat)/K(M) conditions, correlated with the pKa of the base. From these data a Brønsted coefficient (beta) of 0.51 was determined for the base-rescued reaction of C76 Delta. This value is consistent with that expected for proton transfer in the transition state. Together, these data provide strong support for a mechanism where an RNA side chain participates directly in general acid or general base catalysis of the wild-type ribozyme to facilitate RNA cleavage.

Base Sequence↗
PubMed1995

Investigation of the influence of cytosine methylation on DNA flexibility.

To test the influence of pyrimidine methyl groups on DNA flexibility and helix repeat, two sets of 14 mixed sequence DNA molecules, spanning a range of lengths from 158 to 180 base pairs, were cyclized with T4 DNA ligase. The two sets differed only in that the Cyt-5 positions of all cytosines (80-90 cytosine residues per molecule) were fully methylated in the members of one set. Determination of the molar cyclization factors, persistence lengths, helix repeats, and torsional elastic constants revealed no significant differences between the two sets. These results imply that, at least for mixed sequence DNA, the biological consequences of cytosine methylation are likely to derive from either local structural distortions in the helix, which do not propagate as altered twist, or from direct protein-methyl cytosine interactions.

Animals↗
PubMed2003

In vitro deamination of cytosine to uracil in single-stranded DNA by apolipoprotein B editing complex catalytic subunit 1 (APOBEC1).

Apolipoprotein B-editing complex catalytic subunit 1 (APOBEC1) is the catalytic component of an RNA-editing complex that deaminates C6666 --> U in apolipoprotein B RNA in gastrointestinal tissue, thereby generating a premature stop codon. Whereas RNA is the physiological substrate of APOBEC1, recent experiments have strongly indicated that, when expressed in bacteria, APOBEC1 and some of its homologues can deaminate cytosine in DNA. Indeed, genetic evidence demonstrates that the physiological function of activation-induced deaminase, a B lymphocyte-specific APOBEC1 homologue, is to perform targeted deamination of cytosine within the immunoglobulin locus, thereby triggering antibody gene diversification. However, biochemical evidence of in vitro DNA deamination by members of the APOBEC family is still needed. Here, we show that deamination of cytosine to uracil in DNA can be achieved in vitro using partially purified APOBEC1 from extracts of transformed Escherichia coli. Thus, APOBEC1 can deaminate cytosine in both RNA and DNA. Strikingly, its activity on DNA is specific for single-stranded DNA and exhibits dependence on local sequence context.

APOBEC-1 Deaminase↗
PubMed2005

The retroviral hypermutation specificity of APOBEC3F and APOBEC3G is governed by the C-terminal DNA cytosine deaminase domain.

The human proteins APOBEC3F and APOBEC3G restrict retroviral infection by deaminating cytosine residues in the first cDNA strand of a replicating virus. These proteins have two putative deaminase domains, and it is unclear whether one or both catalyze deamination, unlike their homologs, AID and APOBEC1, which are well characterized single domain deaminases. Here, we show that only the C-terminal cytosine deaminase domain of APOBEC3F and -3G governs retroviral hypermutation. A chimeric protein with the N-terminal cytosine deaminase domain from APOBEC3G and the C-terminal cytosine deaminase domain from APOBEC3F elicited a dinucleotide hypermutation preference nearly indistinguishable from that of APOBEC3F. This 5'-TC-->TT mutational specificity was confirmed in a heterologous Escherichia coli-based mutation assay, in which the 5'-CC-->CT dinucleotide hypermutation preference of APOBEC3G also mapped to the C-terminal deaminase domain. An N-terminal APOBEC3G deletion mutant displayed a preference indistinguishable from that of the full-length protein, and replacing the C-terminal deaminase domain of APOBEC3F with AID resulted in an AID-like mutational signature. Together, these data indicate that only the C-terminal domain of APOBEC3F and -3G dictates the retroviral minus strand 5'-TC and 5'-CC dinucleotide hypermutation preferences, respectively, leaving the N-terminal domain to perform other aspects of retroviral restriction.

APOBEC-3G Deaminase↗
PubMed2003

Molecular structure and properties of protonated and methylated derivatives of cytosine.

Ab initio techniques were used to determine the effects of protonation and methylation on cytosine's molecular geometry, molecular affinities, relative stability, and structural rigidity. The geometries of local minima were fully optimized by the gradient procedure at DFT and MP2 levels of theory with the medium size 6-31G(d,p) basis set. The results of energetic analysis indicate that N(3)-methyl-cytosine and C(5)-methyl-cytosine are the most stable derivatives for monocationic and neutral species, respectively. The structural rigidity of each species was assessed by an analysis of normal out-of-plane frequencies, the amplitudes, and by the contribution of internal coordinates to the potential energy distributions. The obtained evidence suggests that methylation increases the overall structural flexibility of cytosine and that all molecules in this study populate a non-planar conformation 50% of the time.

Cytosine↗
PubMed1984

Analysis of the variations in proviral cytosine methylation that accompany transformation and morphological reversion in a line of Rous sarcoma virus-infected Rat-1 cells.

Cells of the A11 lineage of Rat-1 contain a single complete Rous sarcoma provirus. Variation in the activity of this provirus accompanies fluctuations in the lineage between normal and transformed phenotypes. Increased proviral cytosine methylation of the doublet CpG in the tetranucleotide CCGG correlates with transcriptional inactivity and this pattern of cytosine hypermethylation is stable, even when the cells are transformed by another virus. However, transformation can also be induced by 5-azacytidine (but not by other mutagens) and in these transformants reduced proviral cytosine methylation is accompanied by increased proviral transcription. Differences in CCGG methylation between normal and transformed cells are found mainly in the 3' half of the provirus; sites near and within the src gene are heavily methylated only when the provirus is transcriptionally inactive. On the other hand, both transformed and normal A11 derivatives show little, if any, cytosine methylation of CCGG sequences in and flanking the 5' portion of the provirus.

5-Methylcytosine↗
PubMed1992

Substitutions of a cysteine conserved among DNA cytosine methylases result in a variety of phenotypes.

The proposed mechanism for DNA (cytosine-5)-methyltransferases envisions a key role for a cysteine residue. It is expected to form a covalent link with carbon 6 of the target cytosine, activating the normally inactive carbon 5 for methyl transfer. There is a single conserved cysteine among all DNA (cytosine-5)-methyltransferases making it the candidate nucleophile. We have changed this cysteine to other amino acids for the EcoRII methylase; which methylates the second cytosine in the sequence 5'-CCWGG-3'. Mutants were tested for their methyl transferring ability and for their ability to form covalent complexes with DNA. The latter property was tested indirectly with the use of a genetic assay involving sensitivity of cells to 5-azacytidine. Replacement of the conserved cysteine with glycine, valine, tryptophan or serine led to an apparent loss of methyl transferring ability. Interestingly, cells carrying the mutant with serine did show sensitivity to 5-azacytidine, suggesting the ability to link to DNA. Unexpectedly, substitution of the cysteine with glycine results in the inhibition of cell growth and the mutant allele can be maintained in the cells only when it is poorly expressed. These results suggest that the conserved cysteine in the EcoRII methylase is essential for methylase action and it may play more than one role in it.

Azacitidine↗
PubMed1994

High sensitivity mapping of methylated cytosines.

An understanding of DNA methylation and its potential role in gene control during development, aging and cancer has been hampered by a lack of sensitive methods which can resolve exact methylation patterns from only small quantities of DNA. We have now developed a genomic sequencing technique which is capable of detecting every methylated cytosine on both strands of any target sequence, using DNA isolated from fewer than 100 cells. In this method, sodium bisulphite is used to convert cytosine residues to uracil residues in single-stranded DNA, under conditions whereby 5-methylcytosine remains non-reactive. The converted DNA is amplified with specific primers and sequenced. All the cytosine residues remaining in the sequence represent previously methylated cytosines in the genome. The work described has defined procedures that maximise the efficiency of denaturation, bisulphite conversion and amplification, to permit methylation mapping of single genes from small amounts of genomic DNA, readily available from germ cells and early developmental stages.

5-Methylcytosine↗
PubMed2003

De novo cytosine methylation in the differentiating macronucleus of the stichotrichous ciliate Stylonychia lemnae.

Dramatic DNA reorganization and elimination processes occur during macronuclear differentiation in ciliates. In this study we analyzed whether cytosine methylation of specific sequences plays a functional role during DNA rearrangement. Three classes of sequences, macronuclear-destined sequences (MDSs, pCE7), members from a large family of transposon-like elements and micronuclear-specific sequences (pLJ01), differing in their structure and future destiny during nuclear differentiation, were studied in the micronucleus, the developing macronucleus and, when present, in the mature macronucleus. While the MDSs become processed to a 1.1 and 1.3 kb gene-sized macronuclear DNA molecule, the family of transposon-like elements represented by MaA81 becomes removed late in the course of polytene chromosome formation. The micronuclear-specific sequence pLJ01 is eliminated together with bulk micronuclear DNA during degradation of polytene chromosomes. No methylated cytosine could be detected in the vegetative macronucleus and no difference in methylation pattern was observed either between micronucleus and developing macronucleus in MDSs or in a micronuclear-specific sequence. However, a significant percentage of the cytosines contained in the transposon-like element becomes methylated de novo in the course of macronuclear differentiation. This is the first demonstration that cytosine methylation in specific sequences occurs during macronuclear differentiation and may provide a first step towards understanding epigenetic factors involved in DNA processing.

Animals↗
PubMed2004

Random mutagenesis and selection of Escherichia coli cytosine deaminase for cancer gene therapy.

Cytosine deaminase (CD) is currently being used as a suicide gene for cancer gene therapy. The premise of this therapy is the preferential deamination of 5-fluorocytosine (5FC) to 5-fluorouracil by cancer cells expressing cytosine deaminase. However, a lack of efficient gene transfer to tumors combined with inefficient 5FC turnover currently limits the clinical applications of this gene therapy approach. We have used random mutagenesis to create novel bacterial cytosine deaminases that demonstrate an increased preference for 5FC over cytosine. Among the 15 mutants isolated, one conferred sensitivity to Escherichia coli in a negative selection system at a concentration of 5FC that was 10-fold lower than a sublethal dose for wild-type CD. Evaluation of individual substitutions found in this double mutant (Q102R, D314G) demonstrated that the substitution at residue D314 was solely responsible for the observed increase in sensitivity to 5FC. Additional mutagenesis at D314 resulted in the identification of two more substitutions with the ability to confer enhanced 5FC sensitivity to E.coli. Structure determinations of the three CD variants in the presence and absence of a transition state 5FC analogue provide insights to the determinants of substrate binding specificity at the 5' position of the pyrimidine ring. CD mutant D314A is a promising candidate for further gene therapy studies.

Amino Acid Sequence↗
PubMed2006

Comparative isoschizomer profiling of cytosine methylation: the HELP assay.

The distribution of cytosine methylation in 6.2 Mb of the mouse genome was tested using cohybridization of genomic representations from a methylation-sensitive restriction enzyme and its methylation-insensitive isoschizomer. This assay, termed HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR), allows both intragenomic profiling and intergenomic comparisons of cytosine methylation. The intragenomic profile shows most of the genome to be contiguous methylated sequence with occasional clusters of hypomethylated loci, usually but not exclusively at promoters and CpG islands. Intergenomic comparison found marked differences in cytosine methylation between spermatogenic and brain cells, identifying 223 new candidate tissue-specific differentially methylated regions (T-DMRs). Bisulfite pyrosequencing confirmed the four candidates tested to be T-DMRs, while quantitative RT-PCR for two genes with T-DMRs located at their promoters showed the HELP data to be correlated with gene activity at these loci. The HELP assay is robust, quantitative, and accurate and is providing new insights into the distribution and dynamic nature of cytosine methylation in the genome.

Animals↗
PubMed2005

Cytosine-containing hybrid dipeptides: N-[2-(4-amino-2-oxo-1,2-dihydropyrimidin-1-yl)propionyl]-L-phenylalanine N-[2-(4-amino-2-oxo-1,2-dihydropyrimidin-1-yl)propionyl]-L-serine monohydrate and N-[2-(4-amino-2-oxo-1,2-dihydropyrimidin-1-yl)propionyl]-L-lysine.

The title compounds are cytosine-incorporating hybrid dipeptides showing affinities for 9-ethyl-7-methylguanine (7mG). Four molecules of the L-phenylalanine (L-Phe) derivative, C16H14N4O4, are present in the asymmetric unit, with similar folded conformations but with slightly different torsion angles involving the L-Phe group. The L-serine (L-Ser) derivative crystallizes as a monohydrate, C10H14N4O5.H2O, the two independent molecules having extended conformations, whereas the two independent molecules of the L-lysine (L-Lys) system in the final compound, C13H21N5O4, are folded. The cytosine-cytosine base pair (pyrimidine NN interactions) was observed only for the L-lysine derivative. Conformational comparisons with previous structures of cytosine hybrid dipeptides may show the relationships between side-chain position and binding phases for 7mG.

Cadmium↗