Studies on liver chromatin from rats treated with dimethylnitrosamine.

Shortly after injecting a single low dose of N,N-dimethylnitrosamine into rats, the DNA, RNA and histones are methylated, the level in the DNA greatly exceeding that in the histones. The composition of the chromatin and the electrophoretic profiles of the histone and non-histone proteins are not detectably different from those obtained from control animals. Electric birefringence studies suggest that methylation may result in both interparticle cross-linking and some localised loosening of the DNA-protein complex complex.     

Studies on the high-mobility-group non-histone proteins from hen oviduct.

Nuclear high-mobility-group (HMG) proteins were isolated from hen oviduct. These were proteins HMG-1, -2, -3, -14 and -17, which are equivalent to the classification of calf thymus HMG proteins. Hen oviduct proteins HMG-1 and -2 were individually isolated by HCIO4.extraction and CM-Sephadex chromatographic separation. Their mol.wts. were determined as 28 000 and 27 000, respectively. The proteins have a high content of acidic and basic amino acids. The association of proteins HMG-1 and -2 with the genome of hen oviduct nuclei was probed by a limited digestion with nucleases. Hen oviduct nuclei were incubated with deoxyribonuclease I or micrococcal nuclease until 10% of the DNA was digested. The nuclear suspension was centrifuged and the contents of proteins HMG-1 and -2 in the supernatant and sediment fractions were analysed by polyacrylamide-gel electrophoresis. HMG proteins were found to be preferentially released by micrococcal-nuclease digestion rather than by deoxyribonuclease I.     

Metabolism of histones and non-histone proteins in liver cell nuclei and chromatin from rats of various ages

The metabolism of various classes of histones and nonhistone proteins in intact nuclei and in liver chromatin of albino Wistar rats aged 1, 3, 12 and 24 months, was studied. It was shown that in the course of postnatal development the metabolism of nonhistone proteins extracted with 0.14 M NaCl in murine liver is increased. Later in ontogenesis, the incorporation of labeled precursors into proteins HMG 14 and HMG 17 decreases; the specific radioactivity of proteins HMG 1 + 2 is higher in 3- and 24-month-old animals. The intensity of metabolism of nonhistone proteins and histones is higher within the composition of the chromatin complex than in the intact nucleus at all stages of postnatal development. Among other histone proteins, histones H1 are characterized by the highest level specific radioactivity in rats of all age groups.     

Replication of hepatic DNA in rats treated with dimethylnitrosamine.

Replication of DNA containing unrepaired lesions such as depurinated sites, single-strand breaks or methylated bases such as O-6 and N-7 methylguanine was studied in the rat liver. Rat liver DNA was damaged by administering 10 mug dimethylnitrosamine (DMN)/g body wt i.p. 4 h prior to partial hepatectomy. The analysis of DNA on alkaline sucrose gradient revealed considerable damage to the parental strand at the time of and 48 h subsequent to partial hepatectomy. During this time interval, the synthesis of new strands was studied using labeled thymidine. In the control liver, radioactivity in DNA appeared as small fragments at 15 and 30 min following the administration of labeled thymidine which became bigger within 4 h. In the carcinogen-treated livers, the newly made DNA remained as small fragments for longer periods of time. Sometime between 4 and 24 h these became bigger in size than the parental damaged template DNA. Thus, with a delay, the newly made strands became eventually bigger, in spite of the fact that the parental template DNA strand was damaged. Such replication of DNA with unrepaired lesions (miscoding and/or non-coding) offers a mechanism by which the original damage to DNA caused by the carcinogen can be permanently imprinted on the newly made cell, a phenomenon that could account for the initiation of carcinogenesis under certain circumstances.     

Hepatocyte chromosomal non-histone proteins in developing rats.

Rat hepatocytes taken a different stages of the perinatal period were partially purified. On sodium dodecylsulphate acrylamide electrophoresis chromosomal non-histone proteins showed important variations in complexity during development. Chromosomal phosvitin kinase strongly increased during the last days of fetal life; it strongly decreased just after birth and increased again for a short time, while the cytosol phosvitin kinase increased more significantly after birth. Chromosomal non-histone proteins prepared at varoius stages were incubated with [gamma-32P]ATP and resolved on polyacrylamide gel. The incorporation was very low in sample taken at the 15th say of the fetal life. A dramatic increase was observed at the 17th day. This incorporation strongly decreased in the samples taken thereafter and it was negligible in proteins from adult rats. The variations in protein kinase and in 32P incorporation into non-histone proteins were correlated with the pattern of appearance of enzymes in this period of life, with cell growth and with the hormone-induced maturation.     

Methylation of DNAase-digestible DNA and of RNA in chromatin from rats treated with dimethylnitrosamine.

After injecting rats with di[14C]methylnitrosamine we have prepared liver chromatin and have examined firstly, the methylation level of the DNAase I-degradable fraction of the DNA and secondly, the level of methylation and the stability of methylated sites in chromatin RNA. Our results show that the level of 7-methylguanine in the degradable DNA is about 1.3 times that of whole DNA; therefore in the 20% or so of the DNA which is undegradable by DNAase I, the level must be very low or zero. Experiments using chromatin from rats injected with unlabelled dimethylnitrosamine plus [3H]thymidine show that the specific activity is similar in the DNAase I degradable and undegradable fractions, suggesting that there is no preferential repair in the latter region. In chromatin RNA, the level of 7-methylguanine is higher than that of whole DNA and decreases fairly rapidly within 30 h after dimethylnitrosamine treatment. Our results indicate that this decrease is due to some type of excision or repair process rather than to normal turnover.     

Studies on the association of the high mobility group non-histone chromatin proteins with isolated nucleosomes.

Nucleosomes have been isolated from rabbit thymus by sucrose gradient centrifugation, and their high mobility group (HMG) protein content analysed by electrophoresis on polyacrylamide gels. The results suggest that proteins HMG 14 and HMG 17 are associated with the core particle of the nucleosome, and that there are two or more sub-populations of both HMG 1 and HMG 2 molecules. One sub-population appears to be fairly tightly bound to the nucleosome, while another is rapidly released from the chromatin by digestion with micrococcal nuclease. The latter fraction may participate in a higher order folding of the nucleosomes.     

The erythroid cells of anaemic Xenopus laevis. II. Studies on nuclear non-histone proteins.

The mass ratio of nuclear non-histone protein: DNA in the immature circulating erythroid cells of phenylhydrazine-induced anaemic Xenopus is approximately threefold higher than in mature erythrocytes. This is largely due to the presence of increased amounts of low and intermediate molecular weight proteins in the nuclei of the immature cells. There are a few qualitative differences in the components of this class of proteins between the mature and immature cells, the most notable of which is the presence of a protein of molecular weight approximately 115000 in the former which is not detectable in the latter. These changes are discussed in relation to the changing synthetic capacities of cells and to certain generalizations about the function of the nuclear non-histone protein based on studies of other differentiating systems.     

Effect of cadmium and zinc-metallothionein on methemoglobin and nitric oxide in dimethylnitrosamine treated rats

Protective effects of metallothionein (MT) have been studied against dimethylnitosamine (DMN) toxicity in laboratory rats. MT was induced by feeding rats on repeated sublethal doses of cadmium and zinc. These rats were subsequently administered DMN. Methemoglobin and nitric oxides, the established markers of DMN toxicity, were estimated in the blood samples of MT protected rats. Preinduction of MT decreased methemoglobin and ameliorated the generation of nitric oxides. Antioxidative effects of MT may have manifested these results, however, an effect on N-nitrosation is also speculated.     

Interaction of histones with human serum proteins.

The authors describe the interactions of whole calf thymus histone and its fractions with the human serum proteins. Immunoelectrophoretic analysis revealed two types of interactions: 1) a decrease in the electrophoretic mobility of a whole, immunochemically uniform fraction, and 2) a decrease in the electrophoretic mobility of only some molecules of such a fraction. In this association, the arginine-rich histone fraction (F3) was found to be the most active. The findings are important for interpreting the results of biological treatments.     

Interaction of non-histone proteins HMG1 and HMG2 with core histones in nucleosomes and core particles revealed by chemical cross-linking

Chemical cross-linking was used to study the interaction of the non-histone chromosomal proteins HMG1 and HMG2 with core histones in H1,H5-depleted nucleosomes or core particles. Cross-linking with a 'zero-length' cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and with a longer (cleavable) cross-linker dimethyl-3,3'-dithiobispropionimidate revealed an interaction of HMG1 and HMG2 with (or proximity to) core histones in both types of particles. These results indicated that the presence of the 40-50-base-pairs-long segment of the 'linker' DNA in nucleosomes was not necessary for the establishment of mutual contacts of HMG1 and HMG2 proteins with core histones. Possible implications of the interaction of HMG1 and HMG2 proteins with histones for the structure and functioning of chromatin are discussed.