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PubMed1989

Cytosine methylation and the fate of CpG dinucleotides in vertebrate genomes.

The dinucleotide CpG is a "hotspot" for mutation in the human genome as a result of (1) the modification of the 5' cytosine by cellular DNA methyltransferases and (2) the consequent high frequency of spontaneous deamination of 5-methyl cytosine (5mC) to thymidine. DNA methylation thus contributes significantly, albeit indirectly, to the incidence of human genetic disease. We have attempted to estimate for the first time the in vivo rate of deamination of 5mC from the measured rate of 5mC deamination in vitro and the known error frequency of the cellular G/T mismatch-repair system. The accuracy and utility of this estimate (md) was then assessed by comparison with clinical data, and an improved estimate of md (1.66 X 10(-16) s-1) was derived. Comparison of the CpG mutation rates exhibited by globin gene and pseudogene sequences from human, chimpanzee and macaque provided further estimates of md, all of which were consistent with the first. Use of this value in a mathematical model then permitted the estimation of the length of time required to produce the level of "CpG suppression" currently found in the "bulk DNA" of vertebrate genomes. This time span, approximately 450 million years, corresponds closely to the estimated time since the emergence and adaptive radiation of the vertebrates and thus coincides with the probable advent of heavily methylated genomes. An accurate estimate of the 5mC deamination rate is important not only for clinical medicine but also for studies of gene evolution. Our data suggest both that patterns of vertebrate gene methylation may be comparatively stable over relatively long periods of evolutionary time, and that the rate of CpG deamination can, under certain limited conditions, serve as a "molecular clock".

Animals↗
PubMed1987

Asymmetric cytosine deamination revealed by spontaneous mutational specificity in an Ung- strain of Escherichia coli.

A collection of 164 spontaneous lacI- mutations were recovered from a uracil-DNA glycosylase deficient (Ung-) strain of Escherichia coli and analyzed by DNA sequencing. As predicted by genetic studies, G:C----A:T transitions predominated among base substitution events. However, DNA sequence analysis indicated that these events did not occur at random. Of the 31 G:C----A:T transitions recovered, 24 involved cytosine residues located in the nontranscribed strand of the gene and 15 of the 31 transitions occurred at cytosines located on the 3' side of 3 or more A:T base pairs. These differentials likely reflect the more single-stranded character of the non-transcribed strand of the gene and of regions rich in A:T base pairs. In addition, mutation at the frameshift hotspot was altered in the Ung- strain, suggesting a role for DNA repair in the formation of structural intermediates that potentiate these events. Also, the analysis of non-hotspot frameshifts, deletions and duplications showed that many involved local DNA sequence. Specifically, several of the frameshift, deletion and duplication mutations occurred near the sequence 5'-CTGG-3'. Thus, DNA sequence analysis of mutational specificity in an Ung- strain has provided evidence that gene expression, DNA repair and DNA context can all potentially influence the classes and frequencies of spontaneous mutation.

Base Composition↗
PubMed1975

A directed alteration of ribonuclease specificity. Hydrolysis of polyribonucleotides containing modified cytosine bases.

The action of ribonucleases on poly and oligoribonucleotides containing cytosine bases modified by methoxyamine and bisulphite was examined. Resistance of phosphodiester bonds in (Cp)nXp (where n greater than or equal to 1 and X stands for A, G or U) to T2 RNase hydrolysis was observed if substrates were modified chemically. The phenomenon formed the basis for isolation of (Cp)nXp blocks as an additional tool in sequence investigations. After modification of cytosine pancreatic RNase was unable to hydrolyse (Cp)nUp blocks. Therefore the specificity of pyrimidyl RNase may be narrowed to 'uridyl RNase'.

Cytosine↗
PubMedDate not supplied

Implantable enzyme capsules for cancer chemotherapy from bakers' yeast cytosine deaminase immobilized on epoxy-acrylic resin and urethane prepolymer.

For trial use in the local chemotherapy of cancer by a combination of cytosine deaminase (EC 3.5.4.1) and 5-fluorocytosine (J. Biotechnol., (1985), 2, 13-21), 40 U of partially purified cytosine deaminase was obtained from 500 g of commercial compressed bakers' yeast. The enzyme, which is unstable, was immobilized to stabilize it by the use of commercial epoxy-acrylic beads (Eupergit C). The immobilized enzyme was made into enzyme capsules with cellulose tubing for dialysis to encapsulate it or urethane polymer to entrap it, which materials are biocompatible. The activity of the intact cellulose capsules thus made was 0.4% that of the immobilized enzyme inside. The enzyme capsules also were stable. Ten days after the cellulose capsules were implanted in rats, 25% of the starting activity remained. When the polyurethane capsules were tested in vitro for 9 mo for thermostability at 37 degrees C, the activity decreased rapidly (with a half-life of 28 d) during the first 4 mo, and then slowly (half-life, about 100 d) during the next 5 mo. A calculation to transform the biphasic decline into a sum of the exponential decline of two components of enzymic activities with different strengths and half-lives showed that the larger half-life was 5 mo.

Antineoplastic Agents↗
PubMed1997

Methylation of cytosines in nonconventional methylation acceptor sites can contribute to reduced gene expression.

Epigenetic silencing of gene expression is often correlated with extensive DNA methylation at cytosine residues in the promoter and the coding region of silenced genes. Increasing evidence indicates that, in such cases, DNA methylation can also occur in sequence contexts other than CG and CNG, resulting in genomic regions with almost complete modification of cytosines. Whether this nonconventional methylation at CNN sites also contributes to gene repression is not known. We constructed genes with a promoter and a coding region devoid of the conventional methylation acceptor sites CG and CNG in addition to constructs with the corresponding wild-type sequences containing these sites. We generated unmethylated and completely methylated DNA by the polymerase chain reaction and performed expression assays in plant protoplasts. Quantification of transcript levels by RNase protection assay demonstrated that DNA methylation at positions other than CG or CNG sites contributes to the reduction in gene expression.

Amino Acid Sequence↗
PubMed1998

Cytosine methylation at CG and CNG sites is not a prerequisite for the initiation of transcriptional gene silencing in plants, but it is required for its maintenance.

Transgenes integrated into plant chromosomes, and/or endogenous plant genes, may be subjected to epigenetic silencing at the transcriptional or post-transcriptional level. Transcriptional inactivation is correlated with hypermethylation of CG/CNG sites at the silent loci. It is not known whether local hypermethylation is part of the inactivation process, or just an outcome of the silent state. To address this issue, we generated transgenic tobacco lines containing a selectable marker gene controlled by a derivative of the 35S promoter of the cauliflower mosaic virus (CaMV) devoid of CG and CNG methylation acceptor sites. Silencing was triggered by crossing to the silencer locus of tobacco line 271. This line contains inactive and methylated copies of the 35S promoter and is able to silence homologous promoter copies at ectopic chromosomal positions. The mutated promoter lacking CG/CNG methylation acceptor sites was as susceptible to Trans-silencing as the unmodified 35S promoter control. Thus, methylation at CG and CNG sites is not a prerequisite for the initiation of epigenetic gene inactivation. Interestingly, while methylation of the remaining cytosines is usually only slightly affected by silencing, it was significantly increased in the absence of CG/CNG sequences. Since this sequence preference is the same as that of known methyltransferases, this may imply that silencing is accompanied or directly followed by recruitment of methyltransferase, which, in the absence of cytosines in the optimal sequence context, modifies other C residues in the affected area. However, silencing without CG/CNG methylation was immediately relieved in the absence of the silencer. Thus, CG/CNG methylation is probably essential for the maintenance of previously established epigenetic states.

Aminobutyrates↗
PubMed2006

Chemical carcinogens in non-enzymatic cytosine deamination: 3-isocyanatoacrylonitrile.

Uracil has long been known as the main product of nitrosative cytosine deamination in aqueous solution. Recent mechanistic studies of cytosinediazonium ion suggest that the cation formed by its dediazoniation can ring-open to N-protonated (Z,s-cis)-3-isocyanatoacrylonitrile 7. Stereochemical preferences are discussed of the 3-isocyanatoacrylonitriles (Z,s-cis)-10, (E,s-cis)-11, (Z,s-trans)-12, and (E,s-trans)-13. The electronic structures of 7 and 10-13 have been analyzed and a rationale is provided for the thermodynamic preference for (Z,s-cis)-10. It is shown that s-cis/s-trans-interconversion occurs via C-N rotation-inversion paths with barriers below 3 kcal mol(-1). The proton affinities of 3-isocyanatoacrylonitrile 10 and water are nearly identical and, thus, 3-isocyanatoacrylonitriles can and should be formed in aqueous media from 7 along with 3-aminoacrylonitriles 9. The results highlight the relevance of the chemistry of 3-isocyanatoacrylonitriles for the understanding of the chemical toxicology of nitrosation of the nucleobase cytosine.

Acrylonitrile↗
PubMed2025

An advanced cytosine base editor enabled the generation of cattle with a stop codon in the β-lactoglobulin gene.

β-Lactoglobulin (BLG) is an allergen present in milk that can induce an acute immune response in certain individuals. The successful use of cytosine base editors (CBEs) can introduce stop codons into premature mRNA, thereby generating animals with disrupted genes that negatively regulate target traits. In this study, we employed a CBE system to target the major milk allergen BLG in bovine embryos, mammary epithelial cells, and live cattle. First, the precise single-base editing of the BLG gene in bovine embryos was achieved by designing an effective sgRNA to induce a c.61C > T substitution in the coding region, converting codon 21Gln (p.21Gln) to a premature stop codon. Sanger sequencing revealed an editing efficiency of 83.3% (20 out of 24 embryos), including two homozygous edits. Second, a bovine mammary epithelial cell line harboring BLG edits was constructed using the same CBE system. Sequencing showed that the designed sgRNA1 enabled the simultaneous conversion of three consecutive cytosines (c.59-61CCC > TTT) to thymines. At position c.61, single-cell clones exhibited monoallelic or biallelic editing (BLGc.61C > T), with monoallelic edits at positions c.59 and c.60 (CC > TT). Gene expression analysis confirmed that the BLGc.61C > T mutation effectively suppressed BLG expression at both the mRNA and protein levels, even in monoallelically edited cells. Finally, we successfully generated a heterozygous BLGc.61C > T single-base-edited dairy cow that despite its heterozygosity, showed significantly reduced BLG expression in the mammary epithelial cells and milk. Collectively, this study demonstrates the feasibility of using CBEs to disrupt BLG expression in dairy cows and provides a foundation for application in generating hypoallergenic dairy products.

Animals↗
PubMed1990

Participation of the peroxisomal beta-oxidation system in the chain-shortening of PCA16, a metabolite of the cytosine arabinoside prodrug, YNKO1, in rat liver.

When PCA16, a metabolite of the cytosine arabinoside prodrug YNKO1, was incubated with isolated rat hepatocytes, time-dependent H2O2 generation was found. When the hepatocytes obtained from clofibrate-treated rat liver were used as an enzyme source, PCA16-dependent production of H2O2 was increased by around 6-fold. The activity of peroxisomal beta-oxidation for PCA16 assayed by H2O2 generation was 3-fold higher than that for palmitic acid, whereas the activity of mitochondrial beta-oxidation for PCA16 assayed by ketone body production was much less than that for palmitic acid. A subcellular distribution study revealed that the distribution of the activities of beta-oxidation and fatty acyl-CoA oxidase for PCA16-CoA coincided with those of cyanide-insensitive palmitoyl-CoA-dependent beta-oxidation and catalase, a marker enzyme of peroxisomes. The profile of the cofactor requirement for beta-oxidation of PCA16-CoA in isolated peroxisomes was similar to that for palmitoyl-CoA oxidation, and the reaction was not inhibited by KCN. The formation of CoA derivative prior to beta-oxidation reaction was essential. HPLC analysis of metabolites after incubation of PCA16-CoA with isolated peroxisomes demonstrated the production of four metabolites, two of which were identified as PCA14 and PCA12 by fast atomic bombardment-mass spectrometry. These results indicate that peroxisomal beta-oxidation participates in the shortening of the alkyl-side chain of PCA16 and plays an important role in the formation of antileukemic cytosine arabinoside from YNKO1.

Animals↗
PubMed1986

F.t.-i.r. and laser-Raman spectra of cytosine and cytidine.

Fourier-transform infrared (F.t.-i.r.) and laser-Raman spectra of cytosine and cytidine in the solid state have been recorded and assignments of the frequencies made. Comparison of the observed frequencies for cytosine with those for cytidine permits identification of the bands characteristic of the sugar on the one hand, and of the pyrimidine base on the other.

Cytidine↗
PubMed1981

The isolation and characterization of 3-(2-carboxyethyl)cytosine following in vitro reaction of beta-propiolactone with calf thymus DNA.

The new adduct 3-(2-carboxyethyl)cytosine (3-CEC) was isolated following in vitro reaction of the carcinogen beta-propiolactone (BPL) with calf thymus DNA. The structure of 3-CEC was confirmed by synthesis from BPL and dCyd. Reaction of BPL with cCyd (pH 7.0-7.5, 37 degrees C) gave 3-(2-carboxyethyl)deoxycytidine (3-CEdCyd) (9% yield) and 3,N4-bis(2-carboxyethyl)deoxycytidine (3,N4-BCEdCyd) (0.6% yield). 3-CEdCyd and 3,N4-BCEdCyd were hydrolyzed (1.5 N HCl, 100 degrees C, 2 h) to 3-CEC and 3,N4-bis(2-carboxyethyl)cytosine (3,N4-BCEC), respectively. The structure of 3-CEC was assigned on the basis of UV and NMR spectra and the electron impact (EI) mass spectra of 3-CEC and a tri-trimethylsilyl (TMS) derivative of 3 CEC as well as deuterated (d27) tri-TMS derivative of 3-CEC. The structure of 3,N4-BCEC was assigned on the basis of UV spectra and the EI mass spectra of a tri-TMS derivative. Ei and isobutane chemical ionization mass spectra of 3-methylcytosine (3-MeCyt) and a di-TMs derivative of 3-MeCyt were obtained and were helpful in deducing the structures of 3-CEC and 3,N4-BCEC. This is the first report of the alkylation by BPL of an exocyclic atom on a base in DNA. Compound 3,N4-BCEC was not detected in BPL-reacted calf thymus DNA. The relative amounts of 1-(2-carboxyethyl)-adenine (1-CEA), 7-(2-carboxyethyl)guanine (7-CEG), 3-(2-carboxyethyl)-thymine (3-CET) and 3-CEC isolated from BPL-reacted DNA following perchloric acid hydrolysis were 0.23, 1.00, 0.39 and 0.41 respectively, when the alkylation reaction was conducted in phosphate buffer at 0-5 degrees C and pH 7.5 and 0.10, 1.00, 0.29 and 0.28 respectively when the reaction was conducted in H2O at 37b degrees C and pH 7.0-7.5.

Animals↗
PubMed1982

1H-NMR lifetimes of cytosine interactions with the DNA melting probe, methylmercury.

Studies on monomeric cytosine were undertaken to establish a kinetic foundation for the progressive melting of DNA by the mutagen, methylmercury. The reversible displacement of protons by methylmercury at the amino group of cytosine is slow on the 1H-NMR time scale at 100 and 360 MHz. Exchange coupled resonances are produced, not only for all protons of the free- and mercurated amino species, but for the rotational isomers of the latter. These spectra provide for assignment of all exchange-coupled resonances, selection of resonances providing mercuration rates from line shape and measurement of pH-dependent reciprocal lifetimes of the free-amino species (less than or equal to 6 s-1 at pH 3 and 15 s-1 at pH 4). Evidence is presented for the existence of an amino-mercurated species of cytidine thus far not reported (formation constant, 10.

Chemical Phenomena↗
PubMed1990

Induction of cytosine-rich poly(A)+ RNAs in Chlamydomonas reinhardtii by cell wall removal.

RNA accumulation in Chlamydomonas reinhardtii during the course of wall regeneration in protoplasts prepared by treatment of vegetative cells with autolysin was studied. In vitro translation of poly(A)+ RNA isolated from wall-regenerating cells indicated that cell wall removal induced the accumulation of several large RNA transcripts. Some of these induced RNAs were enriched following oligo(dG)-cellulose chromatography (referred to as cytosine-rich). The induced and cytosine-rich poly(A)+ RNAs potentially code for hydroxyproline-rich cell wall glycoproteins (HRGPs). Immunoprecipitation of in vitro translated products with an antiserum against a deglycosylated wall fraction (2BII) confirmed that these RNAs contained species involved in cell wall biosynthesis.

Blotting, Northern↗
PubMed1993

Cooperative face-to-face and edge-to-face aromatic interactions of tryptophan indole ring with N7-quarternized guanine and neutral cytosine bases.

In order to investigate the effect of cytosine base upon the stacking interaction of N7-quarternized guanine base with tryptophan indole ring, the X-ray crystal structure of a 1:1 complex of model compounds 1 and 2 was carried out. Contrary to the expectation of the interaction of both molecules in aqueous solution, the crystal structure showed the first example of the simultaneous recognition of the Trp indole ring by guanine and cytosine bases by the coupling of the face-to-face and edge-to-face aromatic interactions, respectively.

Cytosine↗
PubMed1995

Selective inhibition of cytosine-DNA methylases by polyamines.

We have advanced the hypothesis that polyamines affect DNA methylation and thus promote the expression of developmentally controlled genes. We demonstrate that the activity of cytosine-DNA methyltransferases HpaII, HhaI, HaeIII and SssI is inhibited by physiological concentrations of polyamines. On the other hand, activity of the adenine-DNA methyltransferase EcoRI, and restriction enzymes HpaII, HhaI, HaeIII and EcoRI, is insensitive to polyamine concentrations up to 40 mM. Our results indicate that the effect of polyamines on cytosine-DNA methyltransferases is rather selective and suggest a possible mode of action in vivo.

DNA↗
PubMed1996

Mammalian DNA cytosine-5 methyltransferase interacts with p23 protein.

In higher eukaryotic genomes, methylated cytosine residues (m(5)C) are distributed in heritable, cell-type-specific patterns, which are believed to be involved in the control of gene expression, developmental regulation and genomic imprinting. These methylation patterns are established and maintained by DNA cytosine-5 methyltransferase (MTase), a approximately 1500 amino acid enzyme containing a regulatory N-terminal domain and a catalytic C-terminal domain. The mechanism responsible for targeting MTase to particular genes is poorly understood and might possibly involve interactions with other proteins. In an effort to identify proteins that interact with the mammalian MTase, we used the yeast two-hybrid system with several different MTase domains as baits. Here we report an interaction between the C-terminal catalytic domain of the MTase and p23, a protein previously reported to associate with the progesterone receptor (PR) complex.

Animals↗
PubMed1990

Cloning and sequencing the cytosine methyltransferase gene M. CviJI from Chlorella virus IL-3A.

The Chlorella virus IL-3A gene encoding the DNA methyltransferase M.CviJI, which methylates the internal cytosine in (G/A)GC(T/C/G) sequences, was cloned and expressed in Escherichia coli. The region containing the M.CviJI gene was sequenced and a single open reading frame of 1101 bp was identified that could code for a polypeptide of 367 amino acids with a predicted molecular weight of 41,864. M.CviJI contained regions of amino acids which were similar to bacterial cytosine methyltransferases. Eighteen other Chlorella viruses, of 36 tested, contained DNA sequences which hybridized to the M.CviJI gene; DNA from some, but not all, of these 18 viruses also contained 5-methylcytosine in (G/A)GC(T/C/G) sequences.

Amino Acid Sequence↗