Methylation of DNA by 3H-14C-methyl-labelled N-methyl-N-nitrosourea--evidence for transfer of the intact methyl group

The following products have been isolated from methyl-labelled N-methyl-N-nitrosourea (MNUA) and DNA after reaction at pH 8: 1-, 3-, 7-methyladenines, 3-methyldeoxycytidine, 3-, 7- and O⁶-methylguanines, 3- and O⁴-methylthymidines. Comparison of C³H₃ and ¹⁴CH₃ labelling showed that the ratio ${}^3\text{H}{}^{14}\text{C}$ in the products was the same and equal to that in the original reagents, in accord with the concept that the methyl group is transferred intact, and not via diazomethane. In some cases, most notably with O⁶-methyldeoxyguanosine, the ³H-labelled products were found to elute from Dowex 50 (NH₄⁺ form) slightly ahead of ¹⁴C-labelled or unlabelled products.     

Chronic or acute administration of various dialkylnitrosamines enhances the removal of O6-methylguanine from rat liver DNA in vivo

The effect of pretreatment of rats with various symmetrical dialkylnitrosamines on the repair of O⁶-methylguanine produced in liver DNA by a low dose of [¹⁴C]dimethylnitrosamine (DMN) has been examined. DMN, diethylnitrosamine (DEN), dipropylnitrosamine (DPN) or dibutylnitrosamine (DBN) were administered to rats for 14 consecutive weekdays at a daily dose of 5% of the LD₅₀. Animals were given [¹⁴C]DMN 24 h after the last dose and were killed 6 h later. DNA was extracted from the liver and analysed for methylpurine content after mild acid hydrolysis and Sephadex G-10 chromatography. While the amounts of 3-methyladenine and 7-methylguanine were only slightly different from controls, the amounts of O⁶-methylguanine in the DNA of the dialkylnitrosamine pretreated rats were about 30% of those in control rats, indicating a considerable increase in the capacity to repair this base. Liver ribosomal RNA from control and dialkylnitrosamine pretreated rats contained closely similar amounts of O⁶-methylguanine suggesting that the induced enzyme system does not act on this base in ribosomal RNA in vivo. Pretreatment with these dialkylnitrosamines also enhanced the repair of O⁶-methylguanine in liver DNA when they were given as a single dose (50% of the LD₅₀) either 3 or 7 days before the [¹⁴C]DMN. In addition, single low doses of DMN or DEN (5% of the LD₅₀) given either 1 or 6 days before [¹⁴C]DMN increased O⁶-methylguanine repair and the magnitude of the effect after DEN was similar to that produced by the other pretreatment schedules. The possible mechanism(s) of the induction of O⁶-methylguanine repair and its relation to hepatotoxicity, DNA alkylation, carcinogenesis and the adaptive response in Escherichia coli are discussed.     

The bacterial alkyltransferase-like (eATL) protein protects mammalian cells against methylating agent-induced toxicity

In both pro- and eukaryotes, the mutagenic and toxic DNA adduct O⁶-methylguanine (O⁶MeG) is subject to repair by alkyltransferase proteins via methyl group transfer. In addition, in prokaryotes, there are proteins with sequence homology to alkyltransferases, collectively designated as alkyltransferase-like (ATL) proteins, which bind to O⁶-alkylguanine adducts and mediate resistance to alkylating agents. Whether such proteins might enable similar protection in higher eukaryotes is unknown. Here we expressed the ATL protein of Escherichia coli (eATL) in mammalian cells and addressed the question whether it is able to protect them against the cytotoxic effects of alkylating agents. The Chinese hamster cell line CHO-9, the nucleotide excision repair (NER) deficient derivative 43-3B and the DNA mismatch repair (MMR) impaired derivative Tk22-C1 were transfected with eATL cloned in an expression plasmid and the sensitivity to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was determined in reproductive survival, DNA double-strand break (DSB) and apoptosis assays. The results indicate that eATL expression is tolerated in mammalian cells and conferes protection against killing by MNNG in both wild-type and 43-3B cells, but not in the MMR-impaired cell line. The protection effect was dependent on the expression level of eATL and was completely ablated in cells co-expressing the human O⁶-methylguanine-DNA methyltransferase (MGMT). eATL did not protect against cytotoxicity induced by the chloroethylating agent lomustine, suggesting that O⁶-chloroethylguanine adducts are not target of eATL. To investigate the mechanism of protection, we determined O⁶MeG levels in DNA after MNNG treatment and found that eATL did not cause removal of the adduct. However, eATL expression resulted in a significantly lower level of DSBs in MNNG-treated cells, and this was concomitant with attenuation of G2 blockage and a lower level of apoptosis. The results suggest that eATL confers protection against methylating agents by masking O⁶MeG/thymine mispaired adducts, preventing them from becoming a substrate for mismatch repair-mediated DSB formation and cell death.     

Activation of AMP-activated protein kinase by MAPO1 and FLCN induces apoptosis triggered by alkylated base mismatch in DNA

O⁶-Methylguanine produced in DNA by the action of simple alkylating agents, such as N-methyl-N-nitrosourea (MNU), causes base-mispairing during DNA replication, thus leading to mutations and cancer. To prevent such outcomes, the cells carrying O⁶-methylguanine undergo apoptosis in a mismatch repair protein-dependent manner. We previously identified MAPO1 as one of the components required for the induction of apoptosis triggered by O⁶-methylguanine. MAPO1, also known as FNIP2 and FNIPL, forms a complex with AMP-activated protein kinase (AMPK) and folliculin (FLCN), which is encoded by the BHD tumor suppressor gene. We describe here the involvement of the AMPK–MAPO1–FLCN complex in the signaling pathway of apoptosis induced by O⁶-methylguanine. By the introduction of siRNAs specific for these genes, the transition of cells to a population with sub-G₁ DNA content following MNU treatment was significantly suppressed. After MNU exposure, phosphorylation of AMPKα occurred in an MLH1-dependent manner, and this activation of AMPK was not observed in cells in which the expression of either the Mapo1 or the Flcn gene was downregulated. When cells were treated with AICA-ribose (AICAR), a specific activator of AMPK, activation of AMPK was also observed in a MAPO1- and FLCN-dependent manner, thus leading to cell death which was accompanied by the depolarization of the mitochondrial membrane, a hallmark of the apoptosis induction. It is therefore likely that MAPO1, in its association with FLCN, may regulate the activation of AMPK to control the induction of apoptosis triggered by O⁶-methylguanine.     

The Molecular Weight and Other Biophysical Properties of Bromegrass Mosaic Virus

Data are presented which show that bromegrass mosaic virus has a particularly low molecular weight and nucleic acid content. A molecular weight of 4.6 × 10⁶ was calculated from the sedimentation coefficient, S°_20,w = 86.2S, the diffusion coefficient, D_20,w = 1.55 × 10^-7 cm²/sec., and an assumed partial specific volume, [UNK] = 0.708 ml/gm. The virus has a ribonucleic acid content of 1.0 × 10⁶ atomic mass units. Electrophoresis experiments showed that the virus is stable in 0.10 ionic strength buffers in the pH range 3-6. Breakdown of the virus was observed outside this pH range. Some characteristics of the breakdown products are described.     

Improved automated solid-phase microsequencing of peptides using DABITC

The methylated purines O⁶-methyl- and 7-methylguanine were isolated from mouse liver DNA hydrolysates by means of a column cleanup employing a Sep Pak C-18 reverse-phase cartridge. The purine bases were eluted from the cartridge with methanol, evaporated to dryness, and then dissolved in mobile phase for liquid chromatographic analysis by normalphase chromatography. The system consisted of a LiChrosorb Si 60 column with a watersaturated mobile phase of 20% methanol in chloroform containing 0.001% H₃PO₄. The two methylated bases eluted before adenine or guanine. For extremely low-level (<300 pmol) quantitation, the peaks corresponding to O⁶-methyl- and 7-methylguanine were collected and then analyzed by reverse-phase chromotography with a LiChrosorb RP-18 column and a mobile phase of 5% methanol in pH 7 phosphate buffer (for 7-methylguanine) or 9.5% methanol/buffer (for O⁶-methylguanine). Comparisons were made with fluorescence detection and with scintillation counting (in animal studies where [¹⁴C]dimethylnitrosamine was used). Minimum detectable levels at 254 nm were about 3 ng (3:1 signal to noise ratio) for each of the title compounds. As low as 10 pmol/mg of each could be detected in DNA hydrolysates. Recoveries of O⁶-methyl- and 7-methylguanine from DNA spiked at 750 pmol/mg were greater than 80%.     

Formation and subsequent removal of O6-methylguanine from deoxyribonucleic acid in rat liver and kidney after small doses of dimethylnitrosamine

1. The amounts of 7-methylguanine and O⁶-methylguanine present in the DNA of liver and kidney of rats 4h and 24h after administration of low doses of dimethylnitrosamine were measured. 2. O⁶-Methylguanine was rapidly removed from liver DNA so that less than 15% of the expected amount (on the basis of 7-methylguanine found) was present within 4h after doses of 0.25mg/kg body wt. or less. Within 24h of administration of dimethylnitrosamine at doses of 1mg/kg or below, more than 85% of the expected amount of O⁶-methylguanine was removed. Removal was most efficient (defined in terms of the percentage of the O⁶-methylguanine formed that was subsequently lost within 24h) after doses of 0.25–0.5mg/kg body wt. At doses greater or less than this the removal was less efficient, even though the absolute amount of O⁶-methylguanine lost during 24h increased with the dose of dimethylnitrosamine over the entire range of doses from 0.001 to 20mg/kg body wt. 3. Alkylation of kidney DNA after intraperitoneal injections of 1–50μg of dimethylnitrosamine/kg body wt. occurred at about one-tenth the extent of alkylation of liver DNA. Removal of O⁶-methylguanine from the DNA also took place in the kidney, but was slower than in the liver. 4. After oral administration of these doses of dimethylnitrosamine, the alkylation of kidney DNA was much less than after intraperitoneal administration and represented only 1–2% of that found in the liver. 5. Alkylation of liver and kidney DNA was readily detectable when measured 24h after the final injection in rats that received daily injections of 1μg of [³H]dimethylnitrosamine/kg for 2 or 3 weeks. After 3 weeks, O⁶-methylguanine contents in the liver DNA were about 1% of the 7-methylguanine contents. The amount of 7-methylguanine in the liver DNA was 10 times that in the kidney DNA, but liver O⁶-methylguanine contents were only twice those in the kidney. 6. Extracts able to catalyse the removal of O⁶-methylguanine from alkylated DNA in vitro were isolated from liver and kidney. These extracts did not lead to the loss of 7-methylguanine from DNA. 7. The possible relevance of the formation and removal of O⁶-methylguanine in DNA to the risk of tumour induction by exposure to low concentrations of dimethylnitrosamine is discussed.     

Fingerprinting nonradioactive ribonucleic acid with the aid of polynucleotide phosphorylase

We describe a method for obtaining radioactive fingerprints from nonradioactive ribonucleic acid. Fragments derived by T1 ribonuclease digestion of RNA are dephosphorylated with bacterial alkaline phosphatase. When these fragments are used as primers for the reaction of primer dependent polynucleotide phosphorylase with [α-³²P]GDP in the presence of T1 ribonuclease the 3′-hydroxyl group of each fragment becomes phosphorylated. The degree of phosphorylation is reasonably uniform. The method has been applied to T1 ribonuclease digests of Escherichia coli tRNA^Met_f; the oligonucleotides were further analyzed by spleen phosphodiesterase digestion. In a similar manner fingerprints of pancreatic ribonuclease digests of RNA can be obtained, when [α-³²P]UDP, polynucleotide phosphorylase and pancreatic ribonuclease are used.     

The alkylation of cell macromolecules in barley embryos with mutagenic N-methyl (14C)-N-Nitrosourea

In barley embryos, treated with N-methyl (¹⁴C)-N-nitrosourea, the alkylation of nucleic acids was much higher than that of proteins and lipids. At a concentration inhibiting the growth of M₁ seedlings to 50%, 0.27% of DNA-guanine was methylated to N-7-methylguanine. In barley DNA, alkylatedin vitro, the presence of 3-methyladenine and 0-6-methylguanine was chromatographically detected.     

γ-Irradiation of Thymine Dimers in Aqueous Solution

Thymine dimers were irradiated in aqueous solution with ⁶⁰Co γ-rays in N₂ or O₂. Thymine and unidentified non-UV-absorbing products appeared. The thymine was identified by spectrophotometry, chromatography, and ability to support the growth of Escherichia coli 15 T^-. Residual dimer was determined by a UV-reversibility assay. The G-values for dimer breakage were approximately equal in N₂ and O₂. At low γ-doses, about two thymines were produced per dimer broken in N₂, whereas only about one thymine appeared per dimer broken in O₂. For dimer irradiated in frozen solution, the yield of thymine was at least 100 times less than in liquid.     

3'-Exonuclease resistance of DNA oligodeoxynucleotides containing O6-[4-oxo-4-(3-pyridyl)butyl]guanine

Tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a chemical carcinogen thought to be involved in the initiation of lung cancer in smokers. NNK is metabolically activated to methylating and pyridyloxobutylating species that form promutagenic adducts with DNA nucleobases, e.g. O⁶-[4-oxo-4-(3-pyridyl)butyl]guanine (O⁶-POB-dG). O⁶-POB-dG is a strongly mispairing DNA lesion capable of inducing both G→A and G→T base changes, suggesting its importance in NNK mutagenesis and carcinogenesis. Our earlier investigations have identified the ability of O⁶-POB-dG to hinder DNA digestion by snake venom phosphodiesterase (SVPDE), a 3′-exonuclease commonly used for DNA ladder sequencing and as a model enzyme to test nuclease sensitivity of anti-sense oligonucleotide drugs. We now extend our investigation to three other enzymes possessing 3′-exonuclease activity: bacteriophage T4 DNA polymerase, Escherichia coli DNA polymerase I, and E.coli exonuclease III. Our results indicate that, unlike SVPDE, 3′-exonuclease activities of these three enzymes are not blocked by O⁶-POB-dG lesion. Conformational analysis and molecular dynamics simulations of DNA containing O⁶-POB-dG suggest that the observed resistance of the O⁶-POB-dG lesion to SVPDE-catalyzed hydrolysis may result from the structural changes in the DNA strand induced by the O⁶-POB group, including C3′-endo sugar puckering and the loss of stacking interaction between the pyridyloxobutylated guanine and its flanking bases. In contrast, O⁶-methylguanine lesion used as a control does not induce similar structural changes in DNA and does not prevent its digestion by SVPDE.     

Mutagenic specificity of N′-methyl-N′-nitro-N-nitrosoguanidine in the gpt gene on a chromosome of Chinese hamster ovary cells and of Escherichia coli cells

Summary DNA base sequence changes induced by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis have been determined for the Escherichia coli gpt gene stably incorporated in a chromosome of Chinese hamster ovary cells and in the chromosome of both growing and starving E. coli cells, instead of on a plasmid as in most previous studies. In the three cases, nearly all mutations were G: C to A: T transitions, with a 2-to 4-fold higher mutation rate, compared to other sites, at guanines flanked on the 5 side by another guanine. Mutagenic hot spots in these experiments were less prominent than in published results for MNNG mutagenesis of gpt and of other genes. A suggested explanation involves repair of O⁶meG. At low levels of mutagenic products, most are repaired and even small differences in the repair rates leads to large differences in the relative amounts of residual O⁶meG at various sites; in contrast, at high levels of mutagenic products there is little effect of repair on the distribution.Abbreviations MNNG N-methyl-N-nitro-N-nitrosoguanidine - MNU N-methyl-N-nitrosourea - O⁶meG O⁶-methylguanine - N7meG N7-methylguanine - CHO Chinese hamster ovary     

Purification and some properties of human DNA-O6-methylguanine methyltransferase

DNA-O⁶-methylguanine methyltransferase was purified from the nuclear fraction of fresh human placenta using ammonium sulphate precipitation, gel filtration, affinity chromatography on DNA-cellulose and hydroxyapatite. The methyltransferase preparation was approximately 1–2% pure based on specific activity, and was free of nucleic acids. The protein reacts stoichiometrically with O⁶-methylguanine in DNA with apparent second-order kinetics. The human methyltransferase has a pH optimum of about 8.5, similar to that of the corresponding rat and mouse proteins. NaCl inhibits the reaction in a concentration-dependent fashion. The human protein, like the rodent andE. coli methyltransferases, needs no cofactor. While lmM MnCl₂, lmM spermidine, 5mM MgCl₂ and 10 mM EDTA individually do not significantly inhibit the initial rate of reaction, the protein is nearly completely inactive in 5 mM A1Cl₃ or FeCl₂ or 10 mM spermidine. The initial rate of reaction increases as a function of temperature at least up to 42°. The reaction is inhibited by DNA in a concentration-dependent manner, with single-stranded DNA being more inhibitory than duplex DNA.     

Direct assay for O6-methylguanine-acceptor protein in cell extracts

A direct in vitro assay for O⁶-methylguanine-acceptor protein in cell extracts that measures the transfer of radioactivity from labeled O⁶-methylguanine (O⁶MeGua) adducts in an exogenous DNA substrate to protein is described. The protein-bound radioactivity is released and separated from that remaining in the DNA by sequential digestion with protease K and aminopeptidase M, and appears in the alcohol-soluble fraction of the digest. Data obtained by the direct assay are similar to those obtained by an indirect assay that measures the amount of O⁶MeGua-acceptor protein as the loss of O⁶MeGua from the DNA. In addition to its accuracy, the direct assay is also simple and can measure the amount of O⁶MeGua-acceptor activity in cell extracts prepared from as few as 0.5–1.0 × 10⁶ mammalian cells.     

Transformation-specific cell killing by a cancer-associated galactosyltransferase acceptor and cellular binding

Cancer-associated galactosyltransferase acceptor (CAGA glycoprotein), a small glycoprotein purified from human malignant effusion that selectively kills transformed cells, was tritiated by reductive methylation in the presence of NaB³H₄. CAGA-glycoprotein-sensitive cells (baby-hamster kidney cells transformed by polyoma virus and chick-embryo fibroblasts infected with Ts68 temperature-sensitive mutant of Rous sarcoma virus grown at 37°C, the permissive temperature) bound 3–5-fold more ³H-labelled CAGA glycoprotein than did their CAGA-glycoprotein-resistant non-transformed counterparts. The Rous-sarcoma-virus-infected chick-embryo fibroblasts grown at non-permissive temperature (41°C) bound an intermediate amount of ³H-labelled CAGA glycoprotein; however, this intermediate amount appeared to be sufficient to induce inhibition of cell growth when the infected chick-embryo fibroblasts treated at 41°C were switched to 37°C. Binding of ³H-labelled CAGA glycoprotein was time- and temperature-dependent and was not inhibited by monosaccharide. Binding was completely inhibited by the oligosaccharide liberated by endoglucosaminidase H treatment or by exhaustive Pronase digestion of intact CAGA glycoprotein. However, the isolated oligosaccharide failed to demonstrate the growth-inhibition characteristics of the intact glycopeptide. Binding of ³H-labelled CAGA glycoprotein was unaffected by co-incubation with the peptide core released by endoglucosaminidase H treatment. ³H-labelled CAGA glycoprotein bound to intact cells could be removed by trypsin treatment up to 4h after addition of the glycoprotein but not thereafter. This time course paralleled the decreasing reversibility of growth inhibition. However, all ³H-labelled CAGA glycoprotein was found in the supernatant when cells were first disrupted by sonication followed by trypsin treatment for up to 12h. ³H-labelled CAGA glycoprotein linked to Sepharose 4B failed to cause growth inhibition in CAGA-glycoprotein-sensitive cells. These findings suggest that binding of CAGA glycoprotein occurs via its oligosaccharide moiety. Binding appears to be a necessary but not sufficient condition to induce cell killing. Growth inhibition appears to depend on internalization of the glycoprotein and the presence of a transformation-specific cell process.     

Alkylation of transfer RNA by N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea

The alkylation of a number of purified tRNA preparations by reaction with the carcinogens, N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea was studied in order to investigate the role of nucleic acid structure on the distribution of alkylation products within the nucleotide sequence. The rate of alkylation was greatly increased by increasing the pH over the range 6 to 8 and the degree of alkylation (expressed as moles alkyl groups/mole tRNA) was directly proportional to the concentration of the nitrosamide added and independent of the amount of tRNA present. There was no significant difference in the degree of alkylation of any of the tRNA preparations tested. Reaction with N-ethyl-N-nitrosourea resulted in a degree of alkylation some 13 times less than that produced by reaction with a similar concentration of N-methyl-N-nitrosourea. The major product of the reaction was 7-alkylguanine amounting to about 80% of the total, but 3-methylcytosine, 6-O-methylguanine and 1-methyl-, 3-methyl-, and 7-methyladenine were also identified as products of the reaction of tRNA^fMet with N-methyl-N-nitrosourea.The possibility that preferential alkylation of certain residues within the polynucleotide sequence was produced by reaction with the nitrosamides was examined by degradation of the alkylated tRNA with pancreatic ribonuclease and separation of the oligonucleotide fragments by chromatography on DEAE cellulose. When tRNA^fMet which had been alkylated by reaction with N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea was analysed in this way, the distribution of 7-alkylguanine was, within the limits of experimental error, in agreement with that expected for a random reaction of the alkylating agent with all of the guanosine residues throughout the molecule. A similar result was seen when tRNA^Phe was examined. These results were obtained by alkylation under conditions where the native configuration of the tRNA was maintained and show that the tertiary structure of the nucleic acid does not impart any specificity to the reaction with the nitrosamide producing 7-alkylguanine but the possibility that such specificity does exist for the minor products of alkylation cannot be excluded.     

Effect of pretreatment with other dialkylnitrosamines on excision from hepatic DNA of O6-methylguanine produced by dimethylnitrosamine

Pretreatment with diethylnitrosamine or dipropylnitrosamine increased the amount of labelled O⁶-methylguanine found in liver DNA 4 and 24 h after injection of 10 μg/kg [³H] dimethylnitrosamine. Dibutylnitrosamine treatment had a similar, though smaller effect at 4 h but was ineffective when measurements were made 24 h after the dimethylnitrosamine was given. These pretreatments did not affect 7-methylguanine levels in the DNA showing that the metabolic conversion of dimethylnitrosamine into a methylating agent was not altered. Previous studies have shown that O⁶-methylguanine is rapidly removed from hepatic DNA after methylation to a small extent but removal is less efficient after higher amounts of methylation. Therefore, the most probable explanation for the present findings is that these longer dialkylnitrosamines produce a similar product in DNA which interferes with the loss of O⁶-methylguanine. This hypothesis was supported by experiments showing that diethylnitrosamine did give rise to O⁶-ethylguanine which was lost from the DNA at a rate comparable to the observed loss of O⁶-methylguanine in diethylnitrosamine pretreated rats. This method may, therefore, be of value for determination of whether other nitrosamines, not available in a radioactively labelled form, react with DNA at external oxygen atoms. The present results also suggest that different dialkylnitrosamines might have additive effects in prolonging damage to DNA which could be important in carcinogenesis.     

Photorespiration and Internal CO(2) Accumulation in Chara corallina as Inferred from the Influence of DIC and O(2) on Photosynthesis

An O₂ electrode system with a specially designed chamber for `whorl' cell complexes of Chara corallina was used to study the combined effects of inorganic carbon and O₂ concentrations on photosynthetic O₂ evolution. At pH = 5.5 and 20% O₂, cells grown in HCO₃⁻ medium (low CO₂, pH ≥ 9.0) exhibited a higher affinity for external CO₂ (K_½(CO₂) = 40 ± 6 micromolar) than the cells grown for at least 24 hours in high-CO₂ medium (pH = 6.5), (K_½(CO₂) = 94 ± 16 micromolar). With O₂ ≤ 2% in contrast, both types of cells showed a high apparent affinity (K_½(CO₂) = 50 − 52 micromolar). A Warburg effect was detectable only in the low affinity cells previously cultivated in high-CO₂ medium (pH = 6.5). The high-pH, HCO₃⁻-grown cells, when exposed to low pH (5.5) conditions, exhibited a response indicating an ability to fix CO₂ which exceeded the CO₂ externally supplied, and the reverse situation has been observed in high-CO₂-grown cells. At pH 8.2, the apparent photosynthetic affinity for external HCO₃⁻ (K_½[HCO₃⁻]) was 0.6 ± 0.2 millimolar, at 20% O₂. But under low O₂ concentrations (≤2%), surprisingly, an inhibition of net O₂ evolution was elicited, which was maximal at low HCO₃⁻ concentrations. These results indicate that: (a) photorespiration occurs in this alga and can be revealed by cultivation in high-CO₂ medium, (b) Chara cells are able to accumulate CO₂ internally by means of a process apparently independent of the plasmalemma HCO₃⁻ transport system, (c) molecular oxygen appears to be required for photosynthetic utilization of exogenous HCO₃⁻: pseudocyclic electron flow, sustained by O₂ photoreduction, may produce the additional ATP needed for the HCO₃⁻ transport.     

Excision of O6-methylguanine from DNA of various mouse tissues following a single injection of N-methyl-N-nitrosourea

The persistence of O⁶-methylguanine produced by a single dose of N-methyl-N-nitrosourea (MNU) was determined in DNA of various murine tissues and compared with the location of tumours induced by MNU and related alkylating carcinogens in this species. A/J and C3HeB/FeJ mice received a single intravenous injection of MNU (10 mg/kg) and were killed at different time intervals ranging from 4 h to 7 days.The rate of loss of O⁶-methylguanine from brain DNA was considerably slower than from liver DNA; tumours have been found in both organs after administration of MNU and other alkylnitrosoureas. There was no difference in the rate of excision from cerebral DNA of A/J and C3HeB/FeJ mice, although these strains differ significantly in their susceptibility to the neurooncogenic effect of MNU and related carcinogens. Excision of O⁶-methylguanine from hepatic DNA was significantly slower in A/J than in C3HeB/FeJ mice; both strains have been found to develop hepatic carcinomas following MNU administration. Seven days after the injection of ³H-MNU, O⁶-methylguanine concentrations were highest in brain and lung DNA, lowest in the liver, and intermediate in kidney, spleen, small intestine and stomach. The lung is a principal target organ for tumour induction by MNU and other carcinogens in mice; however, neural tumours are usually induced at a low incidence.The results obtained do not contradict the hypothesis that O⁶-alkylation of guanine in DNA is a critical event in the initiation of tumour induction by alkylating agents. However, the location of tumours produced in mice does not seem to depend solely on the formation and persistence of O⁶-alkylguanine in DNA.     

Methylation of DNA in rat liver and intestine by dimethylnitrosamine and N-methylnitrosourea

A chromatographic procedure for improved separation of deoxyribonucleosides and methylated deoxyribonucleosides is described. DNA was isolated from liver and small intestine of rats treated with [¹⁴C]dimethylnitrosamine ([¹⁴C]DMN) or $\text{N-[}{}^3\text{H}\text{]}\text{methyl}\text{-N-}\text{nitrosourea}$ ([³H]MNU), and the purified DNA was hydrolyzed enzymatically. The deoxyribonucleosides were chromatographed on an Aminex A-6 cation exchange column at 37°C with 0.4 M ammonium formate, pH 4.5, as eluant. In addition to showing the presence of the expected alkylated products, N⁷-methyldeoxyguanosine (determined as N⁷-methylguanine) and O⁶-methyldeoxyguanosine, several other minor methylated products were found in liver and intestinal DNA of rats treated with DMN or MNU. Two of these products are believed to be N³-methylthymidine and O⁴-methylthymidine.