Methylation Of Poly Research Articles

Methylation of poly(acrylic acid), prepared using RAFT polymerization, with trimethylsilyldiazomethane: A metamorphosis of the thiocarbonyl group to a thiol-end group

The Reversible Addition Fragmentation chain Transfer (RAFT) polymerizations of acrylic acid were performed in 1,4-Dioxane solution using three different chain transfer agent (CTA), and 4,4′-azobis(4-cyanopentanoic acid) (ACPA) as initiator. The molar ratios of monomer, CTA and initiator were kept similar for each experiment and all polymerization exhibited high conversion (>90%) at 8 h. Well-defined polyacrylic acids (PAAs) were obtained. Their molecular weights increased linearly with monomer conversion and the dispersity of the molecular weight distribution extended between 1.1 and 1.3. Nuclear magnetic resonance (NMR) spectroscopic studies showed that the PAA prepared are actually random copolymers of AA and the dimer of AA (H2C=CHCO2CH2CH2CO2H), which forms spontaneously from AA, even at room temperature. PAA samples were methylated by addition of trimethylsilyldiazomethane (TMSCHN2 a methylating agent) using a mixture of water/tetrahydrofuran (THF) or methanol/THF. Samples were submitted to different analyses before and after the methylation process in the order to examine the effect of the methylating agent. PAA were characterized by size exclusion chromatography (SEC) in aqueous phase and also in organic phase following methylation. Methylation is accompanied by variable amounts of chain cleavage in the case of PAA prepared using a symmetrical, trithiocarbonate RAFT chain transfer agent. Less extensive cleavage is seen on treatment of poly(methyl acrylate) (PMA), prepared from the trithiocarbonate RAFT reagent with TMSCHN2. Fourier transform infrared (FT-IR), UV-Vis, 1D and 2D NMR spectroscopic studies confirmed that TMSCHN2 reacts with the thiocarbonyl group, though all of the products of this reaction could not be identified. End group analysis using Ellman's reagent demonstrates that some of the polymer chains have a thiol end group, as does a thiol-ene reaction using 9-vinylcarbazole.

Quaternary ammonium ion dendrimers from methylation of poly(propylene imine)s

The poly(propylene imine) dendrimers DAB-dendr-(NH2)8, DAB-dendr-(NH2)32, and DAB-dendr-(NH2)64 were fully converted with iodomethane to quaternary ammonium ions at both chain ends and branch points and, with less iodomethane, were partially converted to quaternary ammonium ions mainly at end groups. Amidation of the primary amine ends followed by treatment with iodomethane gave the first dendrimers with quaternary ammonium ions only at branch points. After an exchange of iodide counterions for chloride, all of the quaternary ammonium ion dendrimers slightly increased the rate of decarboxylation of 6-nitrobenzisoxazole-3-carboxylate ion in an aqueous solution. Similar quaternary ammonium ion dendrimers with more hydrophobic interiors or more hydrophobic chains on the ends were much more active catalysts for the decarboxylation. © 2001 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 39: 821–832, 2001

Quaternary ammonium ion dendrimers from methylation of poly(propylene imine)s

The poly(propylene imine) dendrimers DAB-dendr-(NH2)8, DAB-dendr-(NH2)32, and DAB-dendr-(NH2)64 were fully converted with iodomethane to quaternary ammonium ions at both chain ends and branch points and, with less iodomethane, were partially converted to quaternary ammonium ions mainly at end groups. Amidation of the primary amine ends followed by treatment with iodomethane gave the first dendrimers with quaternary ammonium ions only at branch points. After an exchange of iodide counterions for chloride, all of the quaternary ammonium ion dendrimers slightly increased the rate of decarboxylation of 6-nitrobenzisoxazole-3-carboxylate ion in an aqueous solution. Similar quaternary ammonium ion dendrimers with more hydrophobic interiors or more hydrophobic chains on the ends were much more active catalysts for the decarboxylation. © 2001 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 39: 821–832, 2001

Influence of pre-existing methylation on the de novo activity of eukaryotic DNA methyltransferase.

Aberrant de novo methylation of CpG island DNA sequences has been observed in cultured cell lines or upon malignant transformation, but the mechanisms underlying this phenomenon are poorly understood. Using eukaryotic DNA (cytosine-5)-methyltransferase (of both human and murine origin), we have studied the in vitro methylation pattern of three CpG islands. Such sequences are intrinsically poor substrates of the enzyme, yet are efficiently methylated when a small amount of 5-methylcytosine is randomly introduced by the M.SssI prokaryotic DNA (cytosine-5)-methyltransferase prior to in vitro methylation by the eukaryotic enzyme. A stimulation was also found with several other double-stranded DNA substrates, either natural or of synthetic origin, such as poly(dG-dC).poly(dG-dC). An A + T-rich plasmid, pHb beta 1S, showed an initial stimulation, followed by a severe inhibition of the activity of DNA (cytosine-5)-methyltransferase. Methylation of poly(dI-dC).poly(dI-dC) was instead inhibited by pre-existing 5-methylcytosines. The extent of stimulation observed with poly(dG-dC).poly(dG-dC) depends on both the number and the distribution of the 5-methylcytosine residues, which probably must not be too closely spaced for the stimulatory effect to be exerted. The activity of the M.SssI prokaryotic DNA methyltransferase was not stimulated, but was inhibited by pre-methylation on either poly(dG-dC).poly(dG-dC) or poly(dI-dC).poly(dI-dC). The prokaryotic and eukaryotic DNA methyltransferases also differed in sensitivity to poly(dG-m5dC).poly(dG-m5dC), which is highly inhibitory for eukaryotic enzymes and almost ineffective on prokaryotic enzymes.

Novel poly(aryl ether phthalazinium) ionomers and their properties

The synthesis of N-phenyl phthalazinium salts by condensation of a 1,2-diaroylbenzene with phenylhydrazine is described. This reaction was utilized to prepare novel poly(aryl ether phthalazinium) ionomers by the direct condensation of poly(aryl ether ketone)s with phenylhydrazine. The preparation of poly(aryl ether phthalazinium) ionomers by methylation of poly(aryl ether phthalazine)s is also described. Most of the ionomers are amorphous, thermally stable, and soluble in organic solvents. They can be cast into flexible and strong films. The glass transition temperature of the ionomers ranges from 297 to 363°C, an increase of 12 to 100°C over the corresponding neutral polymers. © 1996 John Wiley & Sons, Inc.

Induction of murine erythroleukemia cell differentiation is associated with methylation and differential stability of poly(A)+ RNA transcripts.

Murine erythroleukemia (MEL) cells exposed to DMSO were assessed for their ability to methylate poly(A)+ RNA and accumulate RNA transcripts of globin and nonglobin genes (c-myc, beta-actin and MER5). Cells were pulse-labeled with L-[methyl-3H]methionine, cytoplasmic RNA was isolated, selected for poly(A)+ RNA and analyzed by HPLC chromatography for methylated nucleosides. When MEL cells were exposed to inhibitors of RNA methylation (neplanocin A, 3-deazaneplanocin A and cycloleucine) and assessed for their ability to differentiate by DMSO, accumulate RNA transcripts, produce hemoglobin, methylate poly(A)+ and poly(A)- RNA and synthesize S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), we observed the following: (a) MEL cells treated with DMSO underwent hypermethylation in poly(A)+ RNA that preferentially occurred at the 5'-cap structures (7-methylguanosine and 2'-O-methylcytidine and 2'-O-methyluridine); (b) inducer-treated MEL cells exhibited a decrease in the intracellular level of SAH that led to a lower ratio of SAH/SAM, an event that favors methylation; and (c) treatment of MEL cells with inhibitors of RNA methylation suppressed methylation of poly(A)- and poly(A)+ RNA, reversed the ratio SAH/SAM seen in differentiated MEL cells and prevented differentiation to occur. Moreover, we observed that treatment of MEL cells with selective inhibitors of RNA methylation caused fragmentation of beta major globin and c-myc mRNAs, two RNA transcripts coded by developmentally regulated genes, while had no detectable effect on the structural integrity of poly(A)+ RNA transcripts transcribed by two housekeeping genes (beta-actin and MER5). These data indicate that induction of erythroid cell differentiation of MEL cells is associated with changes in methylation of poly(A)+ RNA and selective differential stability of RNA transcripts, two events that might be related to each other.

In vitro methylation of undermethylated yeast poly(A)-rich RNA using mRNA (guanine-7-)-methyltransferase purified from wheat germ or yeast

By crossing two strains of Saccharomyces cerevisiae deficient for each of the two methionine adenosyltransferase isoenzymes (ATP: L-methionine S-adenosyltransferase EC 2.5.1.6) respectively, we have constructed a strain strictly auxotrophic for S-adenosylmethionine and used it as a source of undermethylated mRNA suitable for in vitro transmethylation studies. RNA has been phenol-extracted from yeast cells shifted down to S-adenosylmethionine-free medium for 90 min and poly(A)-rich RNA has been prepared by oligo(dT)-cellulose chromatography. Upon incubation in vitro in the presence of methyl-labeled S-adenosylmethionine and mRNA (guanine-7-)-methyltransferase purified from wheat germ or yeast, undermethylated poly(A)-rich RNA became significantly labeled as compared to non-starved cells from the same strain, or from a wild-type control. Cap structures were resolved by paper chromatography afer T2 and P1 RNase digestion, and shown to be a mixture of m7G5'ppp5'G and m7G5'ppp5'A, irrespective of the enzyme source, in agreement with earlier in vivo studies in yeast mRNA capping and methylation.

Enzymatic methylation of DNA and poly(dG-dC)·poly(dG-dC) modified by 4-acetoxyaminoquinoline-1-oxide, the ultimate carcinogen of 4-nitroquinoline-1-oxide

Abstract Both the initial velocity and the overall methylation of Ac-4HAQO modified DNA by a calf brain DNA (cytosine-5-)-methyltransferase are increased as compared to native DNA. The affinity of the modified DNA for the enzyme decreases as a function of the extent of the modification. Heat-denatured, single-stranded DNA shows exactly the opposite results; the more it is modified, the less it is methylated. The poly(dG—dC) · poly(dG—dC) modified by 4NQO is as well methylated as the non-modified one. The carcinogen may induce a tertiary structure favouring the ‘walking’ of the enzyme along the DNA. The hypermethylation caused by this carcinogen could have a significance in gene activity and cellular differentiation.

Misincorporation in DNA synthesis after modification of template or polymerase by MNNG, MMS and UV radiation

The synthetic DNA polymers, poly(dG-dC), poly(dC), poly(dA-dT), poly(dA) and poly(dT), were treated with N-methyl- N′-nitro- N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS) and UV irradiation. The modified polymers were use as templates to examine the incorporation of non-complementary nucleotides by E. coli DNA polymerase I. Methylation of poly(dG-dC) by MNNG predominantly induced the misincorporation of dTMP, whereas methylation by MMS induced that of dAMP. Treatment of poly(dT) with MNNG caused the misincorporation of dGMP to a considerable extent, but MMS did not enhance the error on poly(dT). The misincorporation of dAMP on poly(dC) and that of dGMP on poly(dA) were also increased by these chemicals. UV irradiation of poly(dT) and poly(dC) induced the error of dGMP and dAMP, respectively. These data on MNNG and MMS in vitro were in fair agreement with the directions of mutation in vivo. But the predominant induction of transitions by UV in vitro did not agree with the UV-induced transversions in E. coli. This inconsistency suggested the participation of other factors than direct mispairing in UV-induced transversion. Modification of DNA polymerase I by MNNG changed the ratio of polymerase to 3′→5∼ exonuclease activity altering the fidelity of this enzyme, whereas MMS and UV-irradiation did not alter the fidelity of the enzyme.

Age-dependent gene induction in quail oviduct. XI. Alterations on the post-transcriptional level (analytical aspect)

For the elucidation of the age-dependent reduction of avidin synthesis in the oviduct of quails studies on the level of post-transcriptional modification of mRNA were performed. Incorporation of [ 3H]adenosine into mRNA is drastically reduced in senescent animals compared to adult animals. Double labeling of mRNA with [ 3H]adenosine and [ 3H]uridine revealed that in the presence of actinomycin D the incorporation ratio adenosine/uridine into mRNA increases to higher values in the case of adult animals compared with the ratio determined for mRNA from senescent animals. These data support the assumption that in senescent animals the length of the poly(A) sequence on mRNA might be shorter. Because of the low efficiency of label incorporation into poly(A) in whole animals, a method for directly introducing a radioactive label in vitro into the isolated poly(A) was developed for the estimation of the length of the poly(A) segments. A randomly labeled product with no apparent degradation is achieved by methylation of poly(A) with [ 3H]dimethyl sulphate. Therefore the method is a powerful tool for the detection and size determination of small amounts of poly(A). Size determination of poly(A) from oviducts of adult and senescent quails by the methylation method revealed that in the case of senescent animals an increase in shorter poly(A) tracts can be detected. These results support the assumption that the age-dependent reduction of avidin synthesis is also caused by changes in the control mechanisms on the post-transcriptional level.

Physical studies on synthetic DANs containing 5-methylcytosine

In synthetic DNAs in which cytosine or 5-methylcytosine is base-paired with guanine or hypoxanthine, the physical properties depend both on base composition and base sequence. The absorption spectra of poly(dI) · poly(m 5dC) and poly(dI-m 5dC) · poly(dI-m 5dC) closely resemble the spectra of an equimolar mixture of nucleotides; the maxima near 250 nm corresponds to dIMP absorption and the shoulder at 278 nm corresponds to 5-methyldeoxyCMP absorption. In contrast, when dIMP is replaced by dGMP, the maxima remain near 250 nm, but there is no shoulder; the pyrimidine absorption appears to be blue-shifted by approx. 28 nm. The fluorescence of 5-methyldeoxyCMP is unperturbed when incorporated into randomcoil poly(m 5dC) and is blue-shifted by approx. 8 nm when incorporated into poly(dI-m 5dC) · poly(dI-m 5dC). Incorporation of 5-methyldeoxyCMP into poly(dG-m 5dC) · poly(dG-m 5dC) reduces the fluorescence yield several-fold and blue-shifts the emission by approx. 50 nm. Methylation of pyrimidines raises the melting temperature of both poly(dR-dY) · poly(dR-dY) and Poly(dR) · poly(dY) and reverses the order of melting: methylation (and also bromination) gives the homopolymer pair a higher melting temperature than the alternating copolymer. Methylation also invariably reduces the buoyant density of the synthetic DNAs in neutral and alkaline CsCl, but the decrement is not constant. Methylation of poly(dI-dC) also recudes its buoyant density in neutral and alkaline Cs 2SO 4. Methylation of poly(dG-dC) increases its buoyant density in neutral Cs 2SO 4 and reduces it in alkaline Cs 2SO 4.