An improved large scale fractionation of high mobility group non-histone chromatin proteins.

1. Methodology is presented for the large scale preparation and fractionation of high mobility group proteins from calf thymus chromatin. The total high mobility group protein from approx. 1 kg calf thymus tissue can be separated into five fractions by CM-Sephadex C25 ion-exchange chromatography. High mobility group proteins 1 and 2 comprise two fo the fractions. From a third fraction two more chromatin proteins, protein 3 and 17, can be isolated by trichloroacetic acid precipitation and CM-cellulose chromatography at pH 5.5. 2. The four proteins thus purified are lysine-rich proteins. Proteins 1 and 2 are additionally characterised by their high contents of acidic amino acids, as described previously (Goodwin, G. H. and Johns, E. W. (1973) Eur. J. Biochem. 40, 215-219). Proteins 3 and 17, having lower contents of acidic amino acids, are basic proteins similar to the histones. All four proteins exhibit single N-terminal amino acids; glycine is the N-terminal group of proteins 1, 2 and 3; protein 17 has a proline N-terminal amino acid. The proteins are not highly phosphorylated nor are they associated with appreciable quantities of nucleic acid.     

Immunochemical studies of high mobility group non-histone chromatin proteins HMG 1 and HMG 2

Sera were raised to non-histone chromatin proteins HMG 1 and HMG 2. Immunoperoxidase staining localised these proteins on chromosomes during mitosis and indicated a cell cycle-related variation in these proteins during interphase. Some species differences in HMG 1 and HMG 2 were also observed.     

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.     

Phosphorylation alters the affinity of high mobility group protein HMG 14 for single-stranded DNA

The effect of phosphorylation on the affinity of HMG 14 from calf thymus for single-stranded DNA (ssDNA) was studied, using a cyclic GMP-dependent protein kinase from bovine lung and a nuclear protein kinase II from rat liver. When phosphorylated by G-kinase, HMG 14 eluted at 0.27 M NaCl from the ssDNA-column, whereas the native protein eluted at 0.30 M salt concentration. In contrast, phosphorylation by nuclear protein kinase II did not alter dissociation of HMG 14 from ssDNA and the phosphoprotein consequently coeluted with the native HMG 14. Thus, addition of a negative charge by phosphorylation of the Ser-6 residue by G-kinase presumably weakens the interaction between the DNA-binding amino acids of HMG 14 and the negatively charged phosphate groups of DNA.     

Removal of degradation products from calf thymus high mobility group non-histone chromatin proteins by chromatography on immobilized double-stranded DNA

The substantial protease activity in calf thymus chromatin inevitably produces some degradation of high mobility group (HMG) non-histone proteins in NaCl extracts of calf thymus chromatin. We have found that proteins considered to be degradation products can be conveniently and cleanly separated from intact high mobility group proteins 1 and 2 by chromatography on double-stranded DNA-cellulose in 0.2 M NaCl/1 mM Tris-HCl (pH 7.5). Under those conditions, only the presumptive degradation products are retained by the column.     

Absence of metabolic turnover of N-methyl groups in non-histone and high mobility group chromosomal proteins

Turnover of N-methyl groups in non-histone chromosomal (NHC) and high mobility group (HMG) proteins from chinese hamster ovary cell nuclei was compared with that of the peptide backbone. Cells grown with tritiated amino-acids and methionine (Me-14C) were resuspended in unlabeled medium, and aliquots removed at 4 time points. Halflives were calculated from the decay of the respective specific activities by the method of least squares. Ratios of halflives for 14C and 3H calculated from the first order rate decay curves of the specific activities were shown to be close to unity.     

Distribution of high mobility group and other acid-soluble proteins in fractionated chromatin

Chromatin was fractionated by digestion with deoxyribonuclease II and precipitation with MgCl₂. The Mg²⁺-soluble fraction, known to be enriched in transcribed DNA sequences, was enriched also in high mobility group proteins 1 and 2 and contained almost all other acid-soluble nonhistone proteins.     

Acid-soluble, non-histone proteins in rat liver chromatin. Interaction with DNA

Some properties of nonhistone proteins of rat liver chromatin (Mr 40 +/- 1 and 41 +/- 1 KD) are described. These proteins are abundant in monomeric particles formed at the early steps of chromatin fragmentation by Ca2+,Mg2+-DNase. The proteins are not extracted from chromatin by 5% HClO4 and 1 M NaCl, but can be extracted by 0.4 n H2SO4 and 2 M NaCl. Study on proteins binding to DNA demonstrated that in 0.05 M NaCl these proteins are bound both to bovine satellite DNA and to the plasmid pBR 322 DNA.     

Effect of non-histone proteins on thermal transition of chromatin and of DNA.

The effect of chromatin non-histone protein on DNA and chromatin stability is investigated by differential thermal denaturation method. 1) Chromatin (rat liver) yields a multiphasic melting profile. The major part of the melting curve of this chromatin is situated at temperatures higher than pure DNA, with a distinct contribution due to nucleosomes melting. A minor part melts at temperatures lower than DNA which may be assigned to chromatin non-histone protein-DNA complex which destabilized DNA structure. 2) Heparin which extracts histones lowers the melting profile of chromatin and one observes also a contribution with a Tm lower that of pure DNA. In contrast, extraction on non-histone proteins by urea supresses the low Tm peak. 3) Reconstitution of chromatin non-histone protein-DNA complexes confirms the existence of a fraction of chromatin non-histone protein which lowers the melting temperature when compared to pure DNA. It is concluded that chromatin non-histone proteins contain different fractions of proteins which are causing stabilizing and destabilizing effect on DNA structure.     

Immunochemical study of chromatin non-histone proteins

Summary Rabbit antibodies against rat thymus and liver chromatin are obtained. Antithymus IgG are found to interact only with homologous chromatin, while antiliver IgG interact with both liver and thymus chromatin. After preincubation of antiliver IgG with thymus chromatin the antibodies interact with homologous chromatin only. Thus chromatin of both organs contains tissue-specific proteins. Antiliver and antithymus IgG are used to investigate a distribution of tissue-specific and tissue-non-specific immunogenic proteins in thymocyte nuclei. It is shown that these proteins are practically lacking in nuclear membranes, matrix, nuclear sap, nucleous chromatin and do not participate in attaching DNA to the nuclear matrix.Abbreviations NHP non-histone chromatin proteins - IgG immunoglobulins G - PMSF PhMeSO₃     

Lectin activity of chromatin non-histone proteins

Non-histone proteins from chromatin of sea urchin embryos were found to possess the ability to agglutinate erythrocytes.