Study of the methylation and lack of deamination of deoxyribonucleic acid by N-methyl-N′-nitro-N-nitrosoguanidine

1. DNA labelled with (14)C in the purine residues was prepared by treating newborn rats with [(14)C]formate and killing them for preparation of nucleic acids at 11-17 months. This DNA was incubated with N-methyl-N'-nitro-N-nitrosoguanidine, and then analysed for products of methylation and deamination reactions. 2. Evidence was found for the formation of 7-methylguanine and a smaller amount of 3-methyladenine, and, after preliminary denaturation of the DNA, 1-methyladenine was detected. The presence of cysteine increased the extent of methylation. No evidence was found for the formation of xanthine or hypoxanthine, even at pH5.5.     

Influence of 0.5 ppm nitrogen dioxide exposure of mice on macrophage congregation in the lungs

Summary The lungs of 12 mice, half of which were exposed to continuous 0.5 ppm nitrogen dioxide for 3 weeks, were explanted in culture, and the instances of macrophage congregation were quantitated according to numbers of target cells involved, categories of congregation from three to 11 or more, numbers of macrophages participating in each category for the total cultures, and the influence of delaying explantation for 24 and 96 hr. A total of 9042 macrophages and 2140 epithelial and spindle target cells were counted in the outgrowths from 306 explants. The incidence of macrophage congregation (or numbers of target cells) was greater for the cultures from the NO₂-exposed animals, both with respect to total incidences between groups (p→0), and the 0-hr (p<0.001) and 24-hr (p<0.01) culture subgroups. In addition, the values for T₃ to T₆ macrophage congregation were individually and consistently greater for the exposed animal group. Postmortem interval stress at 96 hr appeared to result in large colonies, but they were reduced greatly in number. Also the incidence of macrophage congregation fell by 28% as compared to 0-hr and 24-hr subgroups. Supported by Grants NHLI No. HL 17412 and EPA No. R. 800881.     

Overproduction and purification of the M.HhaII methyltransferase from Haemophilus haemolyticus

The HhaII methyltransferase gene from Haemophilus haemolyticus was subcloned in an expression vector under control of the hybrid trp-lac promoter. Induction with isopropyl-beta-D-thiogalactopyranoside results in overproduction of the methyltransferase to about 3% of total cellular protein. The methyltransferase was purified to near electrophoretic homogeneity by phosphocellulose, DEAE, and gel chromatography. Its monomer Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 25 kDa, in good agreement with that predicted from the nucleotide sequence. Crystals of the methyltransferase were obtained in the presence of a two-fold molar excess of the duplex oligodeoxynucleotide substrate 5'd-GGACTCC.CCTGAGG.     

Stability of deoxyribonucleic acid methylated in the intact animal by administration of dimethylnitrosamine. Rate of breakdown in vivo and in vitro at different dosages

1. The effect of administration of various dosages of dimethylnitrosamine on the extent of methylation of liver and kidney nucleic acids in the intact rat was studied. Methylation of liver nucleic acids was linearly related to the dosage, but decreasing the dose produced relatively less lowering of the extent of alkylation of kidney nucleic acids. 2. The rates of disappearance of 7-methylguanine from DNA during the 2 days after administration of dimethylnitrosamine in the intact animal and on incubation under simulated physiological conditions in vitro were compared. At a high dosage this rate was greater in vivo than in vitro. At a low dosage the small difference between the two rates was not thought to be sufficient evidence for existence of a specific enzymic excision of the abnormal base.     

Effect of administration of the carcinogen dimethylnitrosamine on urinary 7-methylguanine

1. Evidence is presented for the excretion of 7-methylguanine in normal rat urine at a rate of approx. 65μg./day. Experiments with animals in which the nucleic acids had been prelabelled by treatment of the neonatal rats with [¹⁴C]-formate gave evidence that the methylated base originated in the nucleic acids of the rat. 2. Injection of [¹⁴C]dimethylnitrosamine leads to an increased excretion of 7-methylguanine, and the base becomes labelled in the methyl group. The disappearance of labelled 7-methylguanine formed in nucleic acids of rats treated with the carcinogen therefore does not take place by an N-demethylation reaction, but by liberation of the intact methylated base.     

The fate of 7[14C]-methylguanine after administration to the rat

1. To assess the significance of the methylation of nucleic acids known to be caused by certain carcinogens, the metabolic fate of 7[¹⁴C]-methylguanine was studied, with special reference to its possible incorporation into RNA and DNA. 2. The major part (approx. 95%) of the dose was excreted unchanged in the urine. A small amount of N-demethylation took place, as evidenced by the formation of radioactive adenine and guanine, and expiration of ¹⁴C-labelled carbon dioxide. No evidence was obtained for the direct incorporation of 7-methylguanine into systems synthesizing nucleic acids, i.e. RNA in liver, DNA in intestine or in the foetus.     

HLA class II associations with rheumatic heart disease among clinically homogeneous patients in children in Latvia

Genetic control of immune reactions has a major role in the development of rheumatic heart disease (RHD) and differs between patients with rheumatic fever (RF). Some authors think the risk of acquiring RHD is associated with the HLA class II DR and DQ loci, but other views exist, due to the various HLA-typing methods and ways of grouping cases. Our goal was to determine the relations between HLA class II alleles and risk of or protection from RF in patients with relatively homogeneous clinical manifestations. A total of 70 RF patients under the age of 18 years were surveyed in Latvia. HLA genotyping of DRB1*01 to DRB1*18 and DQB1*0201-202, *0301-305, *0401-402, *0501-504, and *0601-608 was performed using polymerase chain reaction sequence-specific primers. Data for a control group of 100 healthy individuals typed for HLA by the same method were available from the databank of the Immunology Institute of Latvia. Of the RF patients, 47 had RHD and 8 had Sydenham's chorea. We concluded that HLA class II DRB1*07-DQB1*0401-2 and DRB1*07-DQB1*0302 could be the risk alleles and HLA class II DRB1*06 and DQB1*0602-8, the protective ones. Patients with mitral valve regurgitation more often had DRB1*07 and DQB1*0401-2, and patients with multivalvular lesions more often had DRB1*07 and DQB1*0302. In Sydenham's chorea patients, the DQB1*0401-2 allele was more frequent. Genotyping control showed a high risk of RF and RHD in patients with DRB1*01-DQB1*0301-DRB1*07-DQB1*0302 and DRB1*15-DQB1*0302-DRB1*07-DQB1*0303.     

Detection of summer truffle (Tuber aestivum Vittad.) in ectomycorrhizae and in soil using specific primers

Tuber aestivum is becoming an important commodity of great economical value in some European countries. At the same time, it is a highly protected organism in other countries, where it needs careful treatment. A reliable method of detection in roots and soil is thus needed for assessment of geographic distribution, ecological studies and inoculation efficiency testing in man-made experiments. A PCR-based method of detection of T. aestivum using specific primers was therefore developed. A pair of PCR primers Tu1sekvF/Tu2sekvR selective for T. aestivum and some genotypes of Tuber mesentericum was designed on the basis of the known internal transcribed spacer T. aestivum sequences. TaiI restriction cleavage was then used to distinguish the two species. The selectivity of the designed primer pair was evaluated using DNA extracted from specimens of a further 13 Tuber spp. Subsequently, the selectivity and robustness to false-positive results with nontarget DNA of the designed primers was compared with two other primer pairs (UncI/UncII and BTAE-F/BTAEMB-R). The occurrence of T. aestivum in soil and ectomycorrhizae collected in its native habitat has been successfully detected using the designed primers and nested PCR. The method is reliable and thus suitable for detection of T. aestivum in the field.