Methylation and ethylation of uridylic acid and thymidylic acid. Reactivity of the ring and phosphate as a function of pH and alkyl group.

At pH 6.8 in aqueous solution (4 hr, 22 degrees), all methylating agents tested, i.e., dimethyl sulfate, methyl methanesulfonate, and methylnitrosourea, react with both the N-3 of the ring and the phosphate of UMP and dTMP. Although the extent of reaction varies from 17 to 76%, the ratio of phosphate/ring methylation is approximately 4. Both the 3-methyl nucleotides and methyl ester of 3-methyl nucleotides are identified, as well as the methyl esters of unmodified UMP and dTMP. At pH 8.2 the extent of total methylation is similar but reactivity of the N-3 is increased and that of the phosphate decreased so that the phosphate/ring ratio is approximately 1. At pH 6 almost all reaction is with the phosphate group. Uridine, under the same conditions, is methylated at pH 6.8 to form 15% 3-methyluridine and, at pH 8.2, the N-3 of uridine and thymidine is methylated to about 50%. Neither uridine nor UMP forms detectable ribose methyl products at any of these pH's. The comparable ethylating agents (diethyl sulfate, ethyl methanesulfonate, and ethylnitrosourea) are less reactive and the total ethylation of UMP or dTMP is about 1/5 that of methylation. There is little ethylation of the N-3 but the phosphate is alkylated to a relatively high extent so that the phosphate/base ratio at pH 6.8 is 10-23, and at pH 8.2 the ratio is 5-8. The fact that ethylating agents have a greater affinity than methylating agents for alkylating phosphates is proposed as the basis for the previously reported analytical data in which ethylating agents, acting on DNA or RNA at neutrality, form more phosphotriesters than the analogous methylating agents.     

Modified nucleosides of Bacillus subtilis transfer ribonucleic acids.

An analysis of the kinds and amounts of minor nucleosides of transfer ribonucleic acids (tRNA's) from Bacillus subtilis 168 trpC2 is presented. Identification and quantitation were accomplished using ion exclusion chromatography, thin-layer and paper chromatography, and ultraviolet absorption properties. Nucleosides and their amount in moles per 80 residues are as follows: guanosine (25.7), cytidine (22.0), adenosine (15.2), uridine (13.1), 5-methyluridine (0.98), pseudouridine (1.54), 1-methyladenosine (0.15), N6-methyladenosine (0.01), 7-methyladenosine (0.10), 2-methyladenosine (0.03), 7-methylguanosine (0.20), N2-methylguanosine (0.14), 1-methylguanosine (0.14), a methylated pyrimidine (0.17), a methylated derivative of N6-(delta 2-isopentenyl)adenosine (0.02), ribose methylated nucleosides (0.02), 4-thiouridine (0.12), 2-thio-5-(N-methylaminomethyl) (0.09), and an unknown thionucleoside (0.12). Although the composition is similar to that of Escherichia coli in the proportion of major nucleosides, the content of pseudouridine and 5-methyluridine, and the degree of base and ribose methylation, the composition is more similar to that of the tRNA's of yeast and higher organisms in its lower degree of thiolation, the presence of significant amounts of 1-methyladenosine, and the low levels of 2-methyladenosine and 6-methyladenosine. Therefore, the nucleoside composition of B. subtilis presents some different aspects from those usually given as characteristic for bacterial tRNA's. It is not known whether these differences are due to variation between bacterial species in general or related to the process of differentiation.     

Composition and Characterization of tRNA from Methanococcus vannielii.

Purified bulk tRNA from Methanococcus vanielii (carbon source, formate) showed variation in the modified nucleoside pattern reported for Escherichia coli as analyzed by both ion-exchange and thin-layer chromatography. Ribothymidine and 7-methylguanosine were absent; 1-methyladenosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, thiolated nucleosides, pseudouridine, dihydrouridine, and O2'-methylcytidine were quantitated. In vitro methylation by M. Vannielii extracts with S-adenosylmethionine and undermethylated E. coli tRNA revealed active tRNA methyltransferases for formation of methylated residues found in native M. vannielii tRNA, but none for the formation of 7-methylguanosine or ribothymidine. The native M. vannielii tRNA became methylated in the 7-methylguanosine position by E. Coli extracts, but ribothymidine was not formed. Both M. vannielii and E. coli tRNA methyltransferases produced unidentified methylated residues in tRNA's lacking or deficient in ribothymidine.     

Occurrence of 1-methyladenosine and absence of ribothymidine in transfer ribonucleic acid of Mycobacterium smegmatis.

The minor base composition of Mycobacterium smegmatis tRNA has been studied. Thin-layer chromatographic patterns of a ribonuclease T2 digest of mycobacterial tRNA indicated the presence of appreciable amounts of 1-methyladenosine (which is commonly present only in eucaryotic tRNA), dihydrouridine, and 7-methylguanosine. Ribothymidine was absent. The S-adenosylmethionine-dependent tRNA methylases of M. smegmatis catalyzed the formation of 1-methyladenosine when Escherichia coli tRNA was used as acceptor. Similarly, E. coli extracts methylated the tRNA of M. smegmatis, forming ribothymidine.     

Methylated nucleotides block 5' terminus of HeLa cell messenger RNA.

Polyadenylylated [poly(A)+] mRNA from HeLa cells that were labeled with [3H-methyl]-methionine and 14C-uridine was isolated by poly(U)-Sepharose chromatography. The presence of approximately two methyl groups per 1000 nucleotides of poly(A)+ RNA was calculated from the 3H/14C ratios and known degrees of methylation of 18S and 28S ribosomal RNAs. All four 2'-O-methylribonucleosides, but only two base-methylated derivatives, 7-methylguanosine (7MeG) and 6-methyladenosine (6MeA), were identified. 6MeA was the major component accounting for approximately 50% of the total methyl-labeled ribonucleosides. 7MeG, comprising about 10% of the total, was present exclusively at the 5' terminus of the poly(A)+ RNA and could be removed by periodate oxidation and beta elimination. Evidence for a 5' to 5' linkage of 7MeG to adjacent 2'-O-methylribonucleosides through at least two and probably three phosphates to give structures of the type 7MeG5'ppp5pNMep- and 7MeG5'ppp5'NMepNmep- was presented. The previous finding of similar sequences of methylated nucleotides in mRNA synthesized in vitro by enzymes associated with virus cores indicates that blocked 5' termini may be a characteristic feature of mRNAs that function in eucaryotic cells.     

The interaction of aquated cis-[(NH3)2Pt(II)]with nucleic acid constituents. II. Modified nucleosides.

The binding interaction of aquated cis-(NH3)2Pt(II) with 1-methylguanosine (1-MeGuo), 7-methylguanosine (7-MeGuo), 1-methyladenosine (1-MeAdo+) and protonated adenosine has been studied using UV difference spectroscopy. The magnitude of the binding constants for the 1 : 1 interaction of cis-(NH3)2Pt(II) with 1-MeDuo and 1-MeAdo are log K = 3.5 and 3.7, respectively. These data are presented and compared to other cis-(NH3)2Pt(II)-nucleoside interactions.     

Reverse-phase high-performance liquid chromatography of chemically modified DNA

A reverse-phase high-performance liquid chromatographic method has been developed to determine the sites of reaction and the product distribution of modified salmon sperm DNA. The DNA was reacted with methyl methanesulfonate in neutral solution, and then degraded into deoxyribonucleosides by snake venom phosphodiesterase and alkaline phosphatase. Four products were identified and quantitated: 7-methyldeoxyguanosine (37.1%), 7-methylguanine (7.3%), 3-methyldeoxycytidine (28.8%), and 1-methyldeoxyadenosine (26.8%). This method provides a rapid procedure for analysis of chemically or biochemically modified nucleic acids.     

Wheat germ cytoplasmic ribosomes. Localization of 7-methylguanosine and 6-methyladenosine by electron microscopy of immune complexes

Nucleoside analysis of the RNA from the small subunit of wheat germ cytoplasmic ribosomes shows 1 mol each of N7-methylguanosine and N6-methyladenosine/mol of RNA. Antibodies directed against each methylated nucleoside were used to localize these residues within the subunit by electron microscopy of immune complexes. Antibodies to 7-methylguanosine bound 40 S subunits at a single site, at or slightly above the division between the upper and lower segments of the particle and on the surface furthest from the platform (or large lobe) of the subunit. This site is essentially equivalent to that previously seen with Escherichia coli and chloroplast 30 S subunits (Trempe, M. R., Ohgi, K., and Glitz, D. G. (1982) J. Biol. Chem. 257, 9822-9829). Antibodies to N6-monomethyladenosine were induced in rabbits with a nucleoside-albumin conjugate and shown to be specific for the modified nucleoside. Electron microscopy of antibody-subunit complexes placed the methyladenosine residue in a position that is essentially indistinguishable from that of 7-methylguanosine.     

In vivo inhibition of Novikoff cytoplasmic messenger RNA methylation by S-tubercidinylhomocysteine.

The analogue S-tubercidinylhomocysteine (STH) has been used to study the methylation of mRNA in vivo. Partial inhibition of cytoplasmic poly(A)-RNA methylation was observed using a level of inhibitor which still permitted cell growth. Characterization of the partially methylated mRNA indicated the presence of cap structures lacking 2'-O-methylnucleosides, m7GpppN', which are normally not found in mammalian mRNA. Inhibition of additional methylated sites in mRNA at the second 2'-O-methynucleoside, and at internal N6-methyladenosine was also observed Methylation of 7-methylguanosine was not affected under the conditions used in these experiments. The methylnucleoside composition of cap structures differed in STH-inhibited and uninhibited cells. These results indicate that a completely methylated cap is not required for transport of mRNA into the cytoplasm. Furthermore, it may now be possible to assess in vivo the sequential nature of mRNA methylation and its potential role in mRNA processing.     

Free radical oxidation of coriolic acid (13-(S)-hydroxy-9Z,11E-octadecadienoic acid)

The reaction of (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (1a), one of the major peroxidation products of linoleic acid and an important physiological mediator, with the Fenton reagent (Fe(2+)/EDTA/H(2)O(2)) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with >80% substrate consumption after 4h to give a defined pattern of products, the major of which were isolated as methyl esters and were subjected to complete spectral characterization. The less polar product was identified as (9Z,11E)-13-oxo-9,11-octadecadienoate (2) methyl ester (40% yield). Based on 2D NMR analysis the other two major products were formulated as (11E)-9,10-epoxy-13-hydroxy-11-octadecenoate (3) methyl ester (15% yield) and (10E)-9-hydroxy-13-oxo-10-octadecenoate (4) methyl ester (10% yield). Mechanistic experiments, including deuterium labeling, were consistent with a free radical oxidation pathway involving as the primary event H-atom abstraction at C-13, as inferred from loss of the original S configuration in the reaction products. Overall, these results provide the first insight into the products formed by oxidation of 1a with the Fenton reagent, and hint at novel formation pathways of the hydroxyepoxide 3 and hydroxyketone 4 of potential (patho)physiological relevance in settings of oxidative stress.     

Free radical oxidation of 15-(S)-hydroxyeicosatetraenoic acid with the Fenton reagent: characterization of an epoxy-alcohol and cytotoxic 4-hydroxy-2E-nonenal from the heptatrienyl radical pathway

The oxidation of (5Z,8Z,11Z,13E,15S)-15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-(S)-HETE, 1a) with the Fenton reagent (Fe2+/EDTA/H2O2) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with 75% substrate consumption after 1 h to give a mixture of products, one of which was identified as (2E,4S)-4-hydroxy-2-nonenal (3a, 18% yield). Methylation of the mixture with diazomethane allowed isolation of another main product which could be identified as methyl (5Z,8Z,13E)-11,12-trans-epoxy-15-hydroxy-5,8,13-eicosatrienoate (2a methyl ester, 8% yield). A similar oxidation carried out on (15-(2)H)-15-HETE (1b) indicated complete retention of the label in 2b methyl ester and 3b, consistent with an oxidation pathway involving as the primary event H-atom abstraction at C-10. Overall, these results support the recently proposed role of 1a as a potential precursor of the cytotoxic gamma-hydroxyalkenal 3a and disclose a hitherto unrecognized interconnection between 1a and the epoxy-alcohol 2a, previously implicated only in the metabolic transformations of the 15-hydroperoxy derivative of arachidonic acid.     

Simultaneous measurement of CYP1A2 activity, regioselectivity, and coupling: Implications for environmental sensitivity of enzyme-substrate binding

The cytochrome P450 (CYP) reaction mechanism often yields a broad array of coupled and uncoupled products from a single substrate. While it is well known that reaction conditions can drastically affect the rate of P450 catalysis, their effects on regioselectivity and coupling are not well characterized. To investigate such effects, the CYP1A2 oxidation of 7-ethoxymethoxy-3-cyanocoumarin (EOMCC) was examined as a function of buffer type, buffer concentration, pH, and temperature. A high-throughput, optical method was developed to simultaneously measure the rate of substrate depletion, NADPH depletion, and generation of the O-dealkylated product. Increasing the phosphate buffer concentration and temperature increased both the NADPH and EOMCC depletion rates by 6-fold, whereas coupling was constant at 7.9% and the regioselectivity of O-dealkylation to other coupled pathways was constant at 21.7%. Varying the buffer type and pH increased NADPH depletion by 2.5-fold and EOMCC depletion by 3.5-fold; however, neither coupling nor regioselectivity was constant, with variations of 14.4% and 21.6%, respectively. Because the enzyme–substrate binding interaction is a primary determinant of both coupling and regioselectivity, it is reasonable to conclude that ionic strength, as varied by the buffer concentration, and temperature alter the rate without affecting binding while buffer type and pH alter both.     

Persistence of methylated bases in ribonucleic acid of syrian golden hamster liver after administration of dimethylnitrosamine.

1. Syrian golden hamster liver ribosomal RNA was isolated up to 96 h after administration of [14C]dimethylnitrosamine at 25 mg/kg or 2.5 mg/kg body weight. 2. The chemical alkyation products, 7-methylguanine, 3-methylcytosine, O6-methylguanosine and 1-methyladenosine, were measured after acidic or enzymic hydrolysis of the RNA to bases or mononucleosides followed by ion-exchange chromatography. 3. Between 7 and 96 h, the relative amounts of alkylation products did not change with time even though the absolute amounts fell by approx. 80% and 51% after the high and low doses respectively. 4. The results suggest that base specific excision repair does not exist for RNA alkylation products in this experimental system.     

Partial purification and characterization of a human 3-methyladenine-DNA glycosylase.

A DNA glycosylase was purified about 30-fold from cultured human lymphoblasts (CCRF-CEM line) and was found to cleave 3-methyladenine from DNA alkylated with methyl methanesulfonate. The enzyme did not promote the release of 1-methyladenine, 7-methyladenine, or 7-methylguanine from DNA nor did it act on denatured methylated DNA. It produced apurinic sites in DNA alkylated with N-methyl-N-nitrosourea and ethyl methane-sulfonate as well as methyl methanesulfonate but not in untreated DNA or in DNA alkylated with nitrogen mustard or irradiated with ultraviolet light or X-rays. The glycosylase was free of detectable endonuclease activity in experiments with untreated DNA or DNA exposed to ultraviolet light; low levels of endonuclease activity, obtained when X-irradiated, alkylated, or depurinated DNA was the substrate, were attributed to contaminant apurinic endonuclease activity. This 3-methyladenine-DNA glycosylase has an estimated molecular weight of 34,000, is not dependent on divalent metal ions, and shows optimal activity at pH 7.5--8.5.     

An in vivo giemsa chromosome banding technique.

An in vivo chromosome banding technique has been developed. Swiss albino mice were injected with the DNA alkylating agents ethyl methanesulfonate, methyl methanesulfonate, or methyl ethanesulfonate 12, 24, 48 or 72 hours prior to cell harvesting. After harvesting, the cells were fixed with 3:1 methanol-acetic acid and slides were prepared by air drying. The slides were stained 2 1/2 minutes in 3% Giemsa in pH 6.8 Sorensen's buffer. All three alkylating agents induced chromosome bands similar to the Giemsa bands induced by other banding techniques which involve postfixation treatments.     

Mutagenic and inactivating effects of methyl alkylaminosulfonates on Escherichia coli.

Methyl alkylamino methanesulfonates are mutagenic agents as shown by treating several strains of E. coli at pH 7. Methyl methylaminosulfonate (CH3-NH-SO3-CH3) was more efficient than methyl ethylaminosulfonate (C2H5-NH-SO3-CH3) which itself was more efficient than methyl isopropylaminosulfonate (C3H7-NH-SO3-CH3). Methyl methylaminosulfonate seemed to be at least as effective as methyl methanesulfonate (CH3-SO3-CH3). Methyl methylaminosulfonate produced a yield of up to 1% of auxotrophic mutants. All three new mutagens appeared to react according to the same mechanism by ester fission and methylation of nucleophilic groups as is known for methyl methanesulfonate. The reaction mechanism seems to be of the SN2 type.     

An In Vivo Giemsa Chromosome Banding Technique

An in vivo chromosome banding technique has been developed. Swiss albino mice were injected with the DNA alkylating agents ethyl methanesulfonate, methyl methanesulfonate, or methyl ethanesulfonate 12, 24, 48 or 72 hours prior to cell harvesting. After harvesting, the cells were fixed with 3:1 methanol-acetic acid and slides were prepared by air drying. The slides were stained 2¹/₂ minutes in 3% Giemsa in pH 6.8 Sorensen's buffer. All three alkylating agents induced chromosome bands similar to the Giemsa bands induced by other banding techniques which involve postfixation treatments.     

Comparative structural analysis of 1-methyladenosine, 7-methylguanosine, ethenoadenosine and their protonated salts IV: 1H, 13C, and 15N NMR studies at natural isotope abundance.

The 1H, 13C, and 15N NMR spectra of neutral and protonated forms of the nucleosides 1-methyladenosine (m1A), 7-methylguanosine (m7G) and ethenoadenosine (EA), as a model compound, have been analyzed in order to assign the site of protonation in m1A and m7G. Protonation of these nucleosides occurs in the pyrimidine ring of m1A and EA and in the imidazole ring of m7G, with the charge being distributed rather than localized. Structural differences for both m1A and m7G were observed in solution and compared with those existing in the crystal state of monomers as well as in tRNA where these nucleosides occur quite often. The protonated nucleoside structures in solution compared favorably in sugar pucker and glycosidic bond conformations with x-ray crystallographic data. Methyl group carbon chemical shifts of the protonated mononucleosides corresponded to those of the methyls of the respective nucleosides in native tRNA structures. Therefore, the tRNA methyl group carbon chemical shifts are indicative of fully protonated nucleosides in the native, three dimensional structure of the nucleic acid.     

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.     

The specificity of different classes of ethylating agents toward various sites in RNA.

The alkyl products of neutral in vitro ethylation of TMV-RNA by [14C]diethyl sulfate, [14C]ethyl methanesulfonate, and [14C]ethylnitrosourea have been determined and found to differ significantly depending on the ethylating agent. Diethyl sulfate and ethyl methanesulfonate ethylate the bases of TMV-RNA in the following order: 7-ethylguanine greater than 1-ethyladenine, 3-ethylcytidine greater than 7-ethyladenine, 3-ethyladenine, O6-ethylguanosine, 3-ethylguanine. Ethyl methanesulfonate was more specific for the 7 position of guanine, and other derivatives were found in lesser amounts than with diethyl sulfate. Neither reagent caused the formation of detectable amounts (smaller than 0.26 percent) of 1-ethylguanine, 1,7-diethylguanine, N2-ethylguanine, N6-ethyladenine, N4-ethylcytidine, or 3-ethyluridine. Identified ethyl bases account for over 85% of the total radioactivity of [14C]ethyl methanesulfonate and [14C]diethyl sulfate treated TMV-RNA. Phosphate alkylation accounts for about 13 and 1%, respectively, In contrast, [14C]ethylnitrosourea-treated TMV-RNA, while reacting to a similar extent (15-70 ethyl groups/6400 nucleotides), is found to cause considerably more phosphate alkylation. Upon either U4A RNase or acid hydrolysis up to 60% of the radioactivity is found as volatile ethyl groupw in the form of [14C]ethanol, and a further 15% appears to be primarily ethyl phosphate and nucleosides with ethylated phosphate. Of the remaining radioactivity, half is found as O6-ethylguanosine, the major identified ethyl nucleoside. Other ethyl bases found in ethylnitrosourea-treated TMV-RNA are 7-ethylguanine greater than 1-ethyladenine, 3-ethyladenine, 7-ethyladenine, 3-ethylcytidine, and 3-ethylguanine. It appears that ethylnitrosourea preferentially alkylates oxygens, and that formation of phosphotriesters is by far the predominant chemical event. Since the number of ethyl groups introduced into TMV-RNA by ethylnitrosourea is similar to the number of lethal events, one may conclude that phosphate alkylation leads to loss of infectivity. None of the three ethylating agents studied are strongly mutagenic on TMV-RNA or TMV. The role of phosphate alkylation in regard to in vivo mutagenesis and oncogenesis remains to be established. At present it appears possible that the extent of this reaction may correlate better with the oncogenic effectiveness of different ethylating agents, than the extent of any base reaction. Unfractionated HeLa cell RNA is ethylated primarily in acid labile manner even by diethyl sulfate and ethyl methanesulfonate, a fact that is attributed to its high content of low molecular weight trna rich in terminal phosphates which alkylate readily.