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 tissue specificity of the high-mobility-group non-histone chromosomal proteins from calf.

The high-mobility-group (HMG) non-histone chromosomal proteins from calf thymus, liver, spleen and kidney were extracted, and fractionated by CM-Sephadex chromatography and trichloroacetic acid precipitation. The isolated proteins HMG 1, HMG 2 and HMG 17 from the tissues were compared by polyacrylamide-gel electrophoresis, isoelectric focusing and amino acid analysis. The results show that the three proteins are very similar in the tissues studied, implying a lack of tissue specificity.     

Studies on the degradation of HeLa non-histone proteins

The hydrolysis of HeLa non-histone nuclear proteins over 24 h has been monitored in dilute alkali at 4, 15 and 25°C using the standard ninhydrin estimation, dansylation and various electrophoresis techniques. Under conditions (up to 0.2 N NaOH, 4°C) that do not release a significant quantity of ninhydrin-positive material or new N-terminal end group considerable breakdown was observed by two-dimensional electrophoresis analysis. The number of stained spots decreased from approx. 140 to 25–30. No internal protease activity could be found.Labelling studies (¹⁴C-labelled amino acids) showed that much of the hydrolysed material was extracted from the gel during normal staining and destaining procedures. Peptides could be extracted from alkali-hydrolysed non-histone protein with acid/ethanol and could be further separated by thin-layer chromatography on silica gel G. Short-term labelling of HeLa cells (¹⁴C-labelled amino acids for up to 60 min) revealed that these peptides probably have a high rate of turnover. [¹⁴C] Glucosamine studies also indicated the presence of considerable carbohydrate material in the low molecular weight products of this alkaline hydrolysis. Various standard proteins and histones were unaffected by hydrolysis in up to 0.2 N NaOH (4°C, 24 h) as judged by gel electrophoresis.Seven different phosphate-splitting enzymes and an esterase had no effect on the non-histone protein electrophoresis patterns but a preparation of phospholipase C which had no protease activity towards eight standard proteins did produce considerable breakdown in HeLa non-histone proteins similar to that produced by 0.2 N NaOH at 4°C.     

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.     

Complexes of viroids with histones and other proteins.

Complexes of potato spindle tuber viroid (PSTV) with nuclear proteins have been studied by in vitro reconstitution of the complexes and by isolation and characterization of in vivo complexes under non-dissociating conditions. For in vitro reconstitution, nuclear proteins were separated by SDS-gel-electrophoresis, renatured and blotted onto nitrocellulose filters, and incubated with viroid. The viroid-protein complexes were crosslinked covalently, and the viroid containing protein bands were detected by northern hybridization with a radioactive cDNA probe. The histones, a 41,000 dalton protein and to a small extent a 31,000 dalton protein were found in complexes with viroids. Raising the strength to 0.4 M NaCl destroys the complexes with the 41,000 dalton proteins but not those with the histones. From nucleoli, which are known to obtain the majority of viroids under non-dissociating conditions (Schumacher et al., (1983) EMBO J. 2, 1549-1555), a nucleosomal fraction was prepared. Viroids were found predominantly in this nucleosomal fraction. They are bound in a complex of 12-15 svedberg units.     

Distribution of 7-methylguanine and of replication sites in the different kinetic classes of DNA from rats treated with dimethylnitrosamine.

The distribution of 7-methylguanine in the families of repetitive and unique sequences of rat liver chromatin DNA has been studied using the technique of DNA-DNA reassociation. Rats were injected with di[14C]methylnitrosamine and chromatin DNA was prepared 3 h later. The distribution of 7-methylguanine was found to be random between these classes of DNA. We have also studied chromatin DNA from rats treated with unlabelled DMN plus [3H]thymidine in this way, in order to find if DMN affects DNA synthesis within any one kinetic class. Our results suggest that there is no difference in the extent of synthesis between these classes.     

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.