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.     

Phosphorylation of high-mobility-group chromatin proteins by protein kinase C from rat brain

Chromosomal high-mobility-group (HMG) proteins have been examined as substrates for calcium/phospholipid-dependent protein kinase C. Protein kinase C from rat brain phosphorylated efficiently both HMG 14 and HMG 17 derived from calf thymus and the reactions were calcium/phospholipid-dependent. About 1 mol of ³²P was incorporated per mol of HMG 14 and HMG 17. Phosphopeptide mapping suggested that the same major site was phosphorylated in both proteins at serine. The apparent K_m values for HMG 14 and HMG 17 were about 5 μM. HMG 14, HMG 17 and the five histone H1 subtypes prepared from rat thymus, liver and spleen were phosphorylated by the kinase. HMG 14 and HMG 17 from transformed human lymphoblasts (Wi-L2) were also phosphorylated in a calcium/phospholipid-dependent manner. HMG 1 and HMG 2 from the tissues examined were found to be poor substrates for the kinase.     

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.     

Changes in quantities of high-mobility-group protein 1 in oviduct cellular fractions after oestrogen stimulation.

Antiserum against chick oviduct high-mobility-group protein 1 (HMG 1) has been induced in the rabbit. With this antiserum, immunobiochemical techniques have been used to probe the quantitative change of HMG 1 in the cellular fractions of chick oviduct before or after oestrogen stimulation. HMG 1 is detectable in the cytosol, microsomal and nuclear fraction of the chick oviduct cell. After administration of oestrogen to young chicks in vivo for 5 days, the quantity of HMG 1 is increased 4-fold in the cytosol, 3.5-fold in the microsomal fraction and 1.6-fold in the nuclear fraction. The finding of large amounts of HMG 1 in cytoplasm of oviduct cell is not likely due to its leakage from the nucleus. We anticipate that HMG 1 is synthesized in the cytoplasm and then transported into the nucleus. The synthesis and transportation of HMG proteins is probably regulated by oestrogen.     

Analysis of putative high-mobility-group (HMG) proteins in neuronal and glial nuclei from rabbit brain

Putative high-mobility-group (HMG) proteins 1, 2, and 17 were detected in neuronal and glial nuclei isolated from the cerebral hemisphere of rabbit brain. Although divergent chromatin structures are present in these two populations of brain nuclei (i.e., neuronal nuclei exhibit a short DNA repeat length), no differences were apparent in the electrophoretic mobilities of putative HMG proteins 1, 2, and 17 on SDS gels.     

Detection of monoclonal antibody to high-mobility-group protein 17 from chick oviduct.

Total chromosomal HMG (high-mobility-group) proteins have been isolated from oestrogen-stimulated chick oviduct. The antibodies against these proteins were induced in mice and subsequently their spleen cells were fused with myeloma cells to form hybridomas. A highly purified HMG protein, 17, was used to select for the hybridomas that produce antibody against HMG protein 17. The hybridomas were cultured and injected into mice to produce ascites. The antibody against HMG protein 17 in the IgG (immunoglobulin G) fraction of the ascites fluid was obtained by Protein A-Sepharose column chromatography. We have devised a solid-phase radioimmunoassay and enzyme-linked serological assay for the detection and characterization of this antibody directed against HMG protein 17. This anti-(HMG protein 17) IgG interacted only with HMG protein 17, but not with other chromosomal proteins, e.g. histone H1, "95K protein' (a chick oviduct-specific chromosomal protein) and HMG proteins 1, 2 and 14. The monospecific nature of this anti-(HMG protein 17) IgG fraction is confirmed.     

The binding sites for large and small high-mobility-group (HMG) proteins. Studies on HMG-nucleosome interactions in vitro

Studies in vitro of binding high-mobility-group (HMG) proteins to nucleosomal particles that differ in their DNA contents reflect several aspects pertinent to their function in vivo. Two molecules of HMG 14 or 17 are accommodated by particles with 140 or 180 base pairs of DNA whereas HMG 1 or 2 are only bound by the larger specimens irrespective of the presence of HMG 14/17. It is concluded that one molecule of HMG 1 or 2 binds to the 40 base pairs of linker DNA whereas the HMG 14 or 17 molecules associate with the nucleosomal core. At physiological ionic strength, HMG 14 binding is cooperative, probably by triggering a conformational change in the nucleosomal particle. The phenomenon has been studied by two independent techniques. Besides the common gel-electrophoretic system, a centrifugation assay is described, which permits the derivation of a Hill coefficient nH = 1.3 and dissociation constants in the range of 30-90 nM at 0.15 M NaCl, pH 6.8.     

Comparison of the high-mobility-group chromosomal proteins in rainbow-trout (Salmo gairdnerii) liver and testis.

Chromatography and characterization of the proteins extracted by 5% (w/v) HClO4 from rainbow-trout (Salmo gairdnerii) liver and testis show that the two tissues present a characteristically different spectrum of high-mobility-group (HMG) proteins. A variant subfraction of HMG C is found in liver, but is not detectable in testis, where even the main fraction of HMG C is present in only very low quantity. A protein, F, which appears to be related to protein H6 has similarly been isolated only from liver and not from testis. Quantification of the HMG proteins in total 5%-HClO4 extracts of trout liver and testis nuclei shows that, in relation to DNA, levels of HMG T1 and T2, and D are more than 2-fold, and C, 20-fold higher in liver than in testis. However, these differences do not result merely from the sequential withdrawal of HMG proteins at the same time that histones are replaced by protamines in the developing spermatid, since in testis, at some stages of maturation, levels of H6 are almost 2-fold higher than in liver. The implications of these findings for the function of HMG proteins are discussed.     

Adenosine diphosphate ribosylation of chicken-erythrocyte histones H1, H5 and high-mobility-group proteins by purified calf-thymus poly(adenosinediphosphate-ribose) polymerase

Poly(ADP-ribosylation) of histones H1, H5 and non-histone chromosomal high-mobility-group proteins HMG 1, 2, 14 and 17 from chicken erythrocytes by purified calf thymus poly(ADP-ribose) polymerase was studied using acid/urea/Triton gel electrophoresis and autoradiography. With histone H1, besides ADP-ribosylated H1 supporting short chains of polymer, the appearance of H1 'dimer' was observed and this reaction was dependent on NAD concentration and incubation time. In addition, highly modified and/or aggregated species of histone H1 were observed. Histone H5 was slightly ADP-ribosylated at low NAD concentrations. At higher NAD concentrations or after longer incubations the formation of H5 'dimer' and of more modified forms of H5 could be observed. HMG 1 and HMG 2 were found to be ADP-ribosylated, the reaction being dependent on NAD concentration and time. Here again some discrete intermediates appeared. HMG 14 and HMG 17 were only slightly ADP-ribosylated under our experimental conditions. These results indicate that the purified DNA-independent poly(ADP-ribose) polymerase can catalyse the formation of H1 'dimer' as in nuclei and nucleosomes and that H5 and HMG proteins can also be ADP-ribosylated and produce well-defined higher complexes. These modifications of nuclear proteins may provide a means of localized conformational changes of the chromatin structure in vivo.     

Studies on the stability of the higher-order structure of rat liver chromatin containing high-mobility-group proteins

The stability of the higher-order structure of chromatin containing high-mobility-group (HMG) proteins has been studied in rat liver nuclei by mild micrococcal nuclease digestion at low temperature and fractionation by sucrose gradient centrifugation. Nuclei preparation and digestion, chromatin solubilization and analysis have been carried out in two ionic conditions, 140 mM and 40 mM monovalent cation concentration, avoiding drastic changes in ionic conditions and temperature during preparation and analysis. During the time course of digestion at 140 mM ionic strength a material stable at 80 S appears, whose DNA is cleaved at values around 12 nucleosomes. The distribution of HMG proteins in different chromatin fractions was analyzed by immunodot using antibodies elicited against proteins HMG-1, HMG-2, and HMG-14 and 17. It appears that these proteins have a distribution distinctly different from the bulk of chromatin. They are never found in the chromatin fragments that keep their internucleosomal interactions, indicating that these proteins tend to accumulate in points where the chromatin has a less stable structure.     

HMG (high-mobility-group)-14/17-like proteins in calf thyroid. Thyrotropin-dependent phosphorylation and comparison with calf thymus proteins.

Two-dimensional polyacrylamide-gel electrophoresis of acid extracts of thyroid and thymus tissue, and of thyroid nuclei, revealed the presence of three HClO4-soluble nuclear proteins, PS.1, PS.2 and PS.3, whose electrophoretic mobilities closely resembled those of HMG (high-mobility-group) proteins 14 and 17. PS.1 co-migrated with HMG 14 on CM-Sephadex column chromatography. Like HMG 14, PS.2 and PS.3 were phosphorylated in calf thyroid slices; 32P-labelling of PS.3 was stimulated by thyrotropin. Thyrotropin also induced a rapid increase in the labelling of A5, an HMG-14/17-like protein found in whole calf thyroid and thymus tissue, but not in thyroid nuclei.     

The isolation and partial sequence of peptides produced by cyanogen bromide cleavage of calf thymus non-histone chromosomal high-mobility-group protein 2. Sequence homology with non-histone chromosomal high-mobility-group protein 1.

Peptides produced by CNBr cleavage of non-histone chromosomal protein HMG 2 (CNBr peptides) were isolated and characterized, and their partial sequences were determined. The present sequence data account for over half of the sequence of the protein HMG (high-mobility-group) 2 molecule, and, together with previously published results, provide interesting information on the charge distribution within the molecule. Comparison of the CNBr-peptide-sequence data for protein HMG 2 with the previously published data on the CNBr peptides from protein HMG 1 reveals extensive sequence homology between the two proteins. Detailed evidence for the amino acid-sequence data has been deposited as Supplementary Publication SUP 50095 (6 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1978) 169, 5.     

Phosphorylation of a nonhistone protein fraction which coextracts with the high-mobility-group proteins of chromatin

³²P-labelled chromatin proteins from rat liver and ventral prostate were fractionated according to the procedure designed to enrich high-mobility-group (HMG) nonhistone proteins. This fraction, however, reproducibly demonstrated small amounts of apparently basic nonhistone proteins other than HMG nonhistone proteins. These proteins appeared to be tissue specific and were highly labelled with ³²P. The ³²P-labelled phosphoproteins were soluble in trichloroacetic or perchloric acid, migrated in acid-urea polyacrylamide gels, and demonstrated pI values ranging from 6.8 to 7.5. The HMG proteins 1 and 2 showed no incorporation of radioactivity under these experimental conditions.     

Phosphorylation of high-mobility-group proteins by the calcium-phospholipid-dependent protein kinase and the cyclic AMP-dependent protein kinase

Purified lamb thymus high-mobility-group (HMG) proteins 1, 2, and 17 have been investigated as potential substrates for the Ca2+-phospholipid-dependent protein kinase and the cAMP-dependent protein kinase. HMG proteins 1, 2, and 17 are phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the reactions are totally Ca2+ and lipid dependent and are not inhibited by the inhibitor protein of the cAMP-dependent protein kinase. HMG 17 is phosphorylated predominantly in a single seryl residue, Ser 24 in the sequence Gln-Arg-Arg-Ser 24-Ala-Arg-Leu-Ser 28-Ala-Lys, with the second seryl moiety, Ser 28, modified to a markedly lesser degree. HMGs 1 and 2 are also phosphorylated in only seryl residues but with each there are multiple phosphorylation sites. HMG 17, but not HMG 1 or 2, is also phosphorylated by the cAMP-dependent protein kinase with the site phosphorylated being the minor of the two phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the Km for phosphorylation by the cAMP-dependent enzyme is 50-fold higher than that by the Ca2+-phospholipid-dependent enzyme. HMG 17 is an equally effective substrate for the Ca2+-phospholipid-dependent protein kinase either as the pure protein or bound to nucleosomes. Preliminary evidence has indicated that lamb thymus HMG 14 is also a substrate for the Ca2+-phospholipid-dependent enzyme. It is phosphorylated with a Km similar to that of HMG 17 (4-6 microM), and a comparison of tryptic peptides suggests that it is phosphorylated in a site that is homologous with Ser 24 of HMG 17 and distinct from the sites phosphorylated by the cAMP-dependent protein kinase.     

Studies on the conformational properties of the high-mobility-group chromosomal protein HMG 17 and its interaction with DNA

The conformation of the non-histone chromatin protein, HMG 17, has been studied using circular dichroism, infrared and nuclear magnetic resonance spectroscopies, and by small-angle scattering. The results show that in free solution this protein has little or no secondary or tertiary structure in contrast to the other high-mobility-group proteins, HMG 1 and 2, which exhibit highly ordered structures. Protein HMG 17 binds to calf thymus DNA in an ionic-dependent manner, precipitating the DNA at high protein/DNA ratio. The nuclear magnetic resonance data suggest that the principle DNA-binding segment of HMG 17 is that between about residues 15 and 40.     

Fractionation by high-performance liquid chromatography of the low-molecular-mass high-mobility-group (HMG) chromosomal proteins present in proliferating rat cells and an investigation of the HMG proteins present in virus transformed cells

The low-molecular-mass high-mobility-group (HMG) proteins from young rat thymus nuclei were fractionated by high-performance liquid chromatography. Two proteins analogous to calf HMG14 and HMG17 were found together with a third major component HMGI similar to that found in HeLa cells [Lund et al. (1983) FEBS Lett. 152, 163-167]. HMGI has as amino acid composition similar to but distinct from HMG14 and HMG17. The three proteins form a family of proteins with HMG14 having an amino acid composition intermediate between HMG17 and HMGI. HMGI is present in proliferating fibroblasts and embryos but is present in very low levels in rat liver, a non-dividing tissue, supporting the notion that HMGI is required for proliferating cells. Fibroblasts transformed with avian sarcoma virus have high levels of HMGI and an additional band HMGI' but the presence of HMGI and HMGI' is not dependent on a functional src gene product.     

Comparison of the NH2-Terminal sequences of chick Type I preprocollagen chains synthesized in an nRNA-dependent reticulocyte lysate

The histone variants and high-mobility-group (HMG) proteins of a transcribing fraction of chromatin, described in the preceding paper of this journal, have been analysed qualitatively and quantitatively by a combination of one-dimensional and two-dimensional gel electrophoresis. The stoichiometry of the four core histones (all variants included) in this fraction is equimolar and is not detectably different from that in the nontranscribing fraction or in total chromatin. The molar ratio of histone H1 to the core histones is markedly lower, by approximately 72%, than that in the nontranscribing fraction. A minor histone variant identified as M1 (an H2A variant) is detected only in the transcribing fraction, while variant H3.1 is found only in the non-transcribing fraction. Proteins A24, HMG1 and HMG2 are essentially absent from the transcribing fraction; HMG14 is found uniquely in this fraction, while HMG17 occurs at a relatively lower level.     

A study on the amount of high-mobility-group chromatin proteins in T-cells at different stages of differentiation

The amounts of high-mobility-group proteins (HMG) 1 and 2 in different mouse T-cell populations are studied. It is shown that the quantity of HMG 1 and 2 is different in functionally distinct T-cells. The level of these proteins in thymus cells is higher than in cortisone-resistant thymocytes and peripheral T-cells; it increases in the cytotoxic cells generated in mixed lymphocyte culture. The quantity of HMG is negligible in memory T-cells and increases when the latter cells are stimulated again. The differences found in the levels of HMG 1 and 2 could be related to the rate of cell proliferation and to the changes in chromatin structure at each functional stage of differentiating T-cells.