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.     

High mobility group proteins in ram spermatids

The four major high mobility group proteins HMG 1, 2, 14 and 17, HMG 19B and histone H1(0) were identified in the ram testis by their extraction and solubility characteristics and by their electrophoretic mobilities. HMG 14 and 17 were isolated by chromatography and amino acid analysis revealed that they were similar to their calf thymus analogues. A protein, named 2R and co-extracted with HMG 14, was also purified and analysed. Electrophoretic analyses of the proteins extracted by 0.75 M perchloric acid (PCA) or by 0.35 M NaCl from round and non-round spermatids, separated by centrifugal elutriation, showed that the four major HMG proteins disappear from nuclei in the oldest round spermatids, at the time the nuclear content of protein 2R and histone H1(0) increases in spermatids. Ubiquitin and HMG 19B were present in the round and elongating spermatids, but not in elongated spermatids which contained only protamine. The relation was considered between several protein changes and genetic inactivation and structural reorganization of the spermatid chromatin.     

Distribution of high mobility group proteins in different tissues of rats during aging

The distribution of high mobility group (HMG) proteins has been studied in the liver, brain, kidney, lung, spleen, testis, thymus, and heart of young (19 weeks) and old (118 weeks) rats. These proteins were extracted with perchloric acid, fractionated by CM-Sephadex column chromatography, and analysed by acetic acid-urea polyacrylamide slab gel electrophoresis. As compared with that in young rats, the level of total HMG proteins in the old increased in liver and lung, decreased in thymus, heart, brain, and kidney, and remained unchanged in spleen and testis. In particular, the levels of HMG 1 and 2 were maximum in the thymus of young rats and dropped drastically in the old. However, the amount of HMG 17 was high in the spleen of both young and old rats, though it was comparatively higher in the former. Such age-dependent variation in the level of HMG proteins of different tissues denotes indirectly differences in the functional state of chromatin, and in growth and activity of cells, during aging.     

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.     

High-mobility group chromosomal proteins of wheat

Four proteins have been extracted from purified chromatin of wheat embryos with 0.35 M NaCl. These proteins are soluble in 2% (w/v) trichloroacetic acid and thus meet the original operational requirements to be classified as "high-mobility group" (HMG) chromosomal proteins. The proteins have been characterized by one- and two-dimensional electrophoresis, amino acid analysis, and peptide mapping. Three of the proteins (HMGb, c, and d) share the mammalian HMG characteristic of being rich in both acidic and basic amino acid residues. Unlike their putative mammalian counterparts, these plant HMG proteins contain less than 7 mol % proline. The fourth wheat protein (HMGa) is rich in both proline and in basic amino acid residues. This wheat protein, however, contains only about half the proportion of acidic residues found in mammalian HMG proteins--a characteristic also found in the trout testis HMG protein, H6. Comparative peptide maps show that none of the wheat HMG proteins are degradation products of other HMG proteins or the H1 histones. The peptide maps have not, however, been useful in establishing homologies with mammalian HMG proteins. Wheat HMG proteins are released from DNase I-treated nuclei and co-isolate with micrococcal nuclease-sensitive chromatin fractions. Similar observations concerning the HMG proteins of vertebrate animals have been considered consistent with a role for these proteins as structural components of actively transcribed chromatin.     

Characterization of high mobility group protein levels during spermatogenesis in the rat

The distribution, quantitation, and synthesis of high mobility group (HMG) proteins during spermatogenesis in the rat have been determined. HMG1, -2, -14, and -17 were isolated from rat testes by Bio-Rex 70 chromatography combined with preparative gel electrophoresis. Amino acid analysis revealed that each rat testis HMG protein was similar to its calf thymus analogue. Tryptic peptide maps of somatic and testis HMG2 showed no differences and, therefore, failed to detect an HMG2 variant. Testis levels of HMG proteins, relative to DNA content, were equivalent to other tissues for HMG1 (13 micrograms/mg of DNA), HMG14 (3 micrograms/mg of DNA), and HMG17 (5 micrograms/mg of DNA). The testis was distinguished in that it contained a substantially higher level of HMG2 than any other rat tissue (32 micrograms/mg of DNA). HMG protein levels were determined from purified or enriched populations of testis cells representing the major stages of spermatogenesis; spermatogonia and early primary spermatocytes, pachytene spermatocytes, early spermatids, and late spermatids; and testicular somatic cells. High levels of HMG2 in the testis were due to pachytene spermatocytes and early spermatids (56 +/- 4 and 47 +/- 6 micrograms/mg of DNA, respectively). Mixtures of spermatogonia and early primary spermatocytes showed lower levels of HMG2 (12 +/- 3 micrograms/mg of DNA) similar to proliferating somatic tissues, whereas late spermatids had no detectable HMG proteins. The somatic cells of the testis, including isolated populations of Sertoli and Leydig cells, showed very low levels of HMG2 (2 micrograms/mg of DNA), similar to those in nonproliferating somatic tissues. HMG proteins were synthesized in spermatogonia and primary spermatocytes, but not in spermatids. Rat testis HMG2 exhibited two bands on acid-urea gels. A "slow" form comigrated with somatic cell HMG2, while the other "fast" band migrated ahead of the somatic form and appeared to be testis-specific. The "fast" form of HMG2 accounted for the large increase of HMG2 levels in rat testes. These results show that the very high level of HMG2 in testis is not associated with proliferative activity as previously hypothesized.     

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.     

Analysis of age-associated alteration in the synthesis of HMG nonhistone proteins of the rat liver

HMG proteins were extracted with 5% PCA or 0.35 M NaCl from whole tissue, nuclei or chromatin of the liver of young (19 weeks) and old (118 weeks) male rats. They were resolved on acetic acid-urea polyacrylamide gel. The electrophoretic patterns of the major HMG proteins 1, 2, 14 and 17 of both ages are similar. The in vitro synthesis of HMG 1 and 2 decreases, but that of HMG 14 and 17 increases considerably in the liver of old rats. The synthesis of different HMG proteins is modulated differentially by spermine, butyrate, dexamethasone and 3-aminobenzamide in the liver of young and old rats. These findings suggest that HMG proteins contribute to alterations in the organization of chromatin and expression of genes during aging.     

In vitro ADP-ribosylation of nuclear proteins in mouse tissues. Identification of an unusual group of acceptors in testis nuclei

1. Acceptor proteins for poly(ADP-ribose) have been identified in nuclei from mouse testis, liver, kidney and spleen. Purified nuclei were incubated in vitro with [14C]NAD, extracted sequentially with 5% HClO4 and 0.25 N-HCl and labelled proteins analysed on acetic acid/urea polyacrylamide gels pH 2.9. 2. Results show that: (a) in vitro there are significant differences between tissues in the extent of poly(ADP-ribosylation) of nuclear proteins; (b) in testis nuclei two tissue specific proteins are poly(ADP-ribosylated) to higher specific activity than histones; (c) there are significant differences between in vivo and in vitro studies on poly(ADP-ribosylation) of nuclear proteins in testis nuclei.     

The high mobility group of nuclear proteins as biomarkers of age and caloric restriction in rats.

The quantitative levels and phosphorylation states of the high mobility group (HMG) of proteins were investigated in bone marrow, brain, heart, kidney, liver, pancreas, spleen, testis and thymus of three groups of male Fischer 344 rats. Two groups of rats, young ad libitum (Y/AL - 1 1/2 mo.) and old ad libitum (O/AL - 28 mo.), had free access to rat chow, and a third group of old rats were maintained on a caloric restricted intake (O/CR - 28 mo.). The quantities of HMGs 1,2,14 and 17 were significantly reduced in O/AL rats compared with Y/AL rats in all tissues examined, and in many cases, the amount of HMGs of O/CR rats were increased by varying degrees from O/AL animals. In G2-phase nuclei of bone marrow, spleen and testis, phosphorylation of HMG proteins was reduced significantly in O/AL rats, but was enhanced in O/CR animals (especially HMG14). These levels of HMGs in O/CR animals, altered by age and diet dependent factors, reflect a condition which is more reminiscent of Y/AL than O/AL animals.     

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.     

Comparison of the high mobility group proteins and their chromatin distribution in trout testis and liver

The trout testis contains two major high mobility group (HMG) proteins HMG-T and H6 which, although related to the four mammalian HMGs, exhibit distinct variation as evidenced by differences in electrophoretic mobility and amino acid sequence. Previous work using various endonucleases as probes has shown that HMG-T and H6 are located at specific sites in the testis chromatin. The differentiation of testis cells during spermatogenesis is characterized by a unique transition from a histone-packaged genome to one bound by a class of small molecular weight, highly basic proteins, the protamines. Questions arise as to whether any of the HMG variability may be unique to the process of spermatogenesis and whether the histone-protamine transition occurring in most testis cells affects the HMG protein distribution and/or the specificity of the probe. In an attempt to answer these questions, the distribution of the HMG proteins in the chromatin of trout liver, a tissue lacking protamine, has been studied and comparisons made with testis. Liver HMGs exhibit the same electrophoretic characteristics as the testis HMGs indicating that the variability when compared to mammalian HMGs is primarily phylogenetic in origin rather than tissue-specific. Furthermore, micrococcal nuclease digestion of liver nuclei and its effect on the subsequent HMG protein distribution during chromatin fractionation yields a pattern very similar to that for testis, suggesting that the interaction of the HMGs with the remaining testis nucleohistone is not significantly altered by the ongoing transition to nucleoprotamine. Finally, the HMGs represent an unusually high proportion of the total testis non-histone protein population; the implications of this are discussed.     

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.     

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.     

High mobility group nonhistone chromosomal proteins of the developing sea urchin embryo

The high mobility group or HMG proteins are nonhistone chromosomal proteins that have been found in relatively high amounts in nuclei of many tissues. A number of studies have shown that some of these proteins are preferentially associated with actively transcribed regions of the genome and may play a role in maintaining these regions in an active state. In this study, we undertook an investigation of the high mobility group proteins from the sea urchin, Stronglyocentrotus purpuratus. Initially the putative sea urchin HMGs were extracted from isolated nuclei of hatching blastula-stage embryos with 5% perchloric acid (PCA). The major proteins in this extract were characterized according to their electrophoretic mobility, amino acid composition, and association with isolated deoxyribonucleoprotein particles. The results indicate there is only one "major" sea urchin HMG protein, termed P2 in this paper. An estimate of the amount of P2 in relation to the inner histones, however, was low compared to what has been found for other HMG proteins. Of the other major 5% PCA-extractable proteins, one was identified as the cleavage stage H1. Another protein apparently resulted from H3 contamination in the 5% PCA extract, and the fourth major protein did not have all the characteristics of an HMG. In particular, it was not found associated with nucleosomal particles. The HMG proteins from other developmental stages were then examined. Five percent PCA extracts of nuclei from unfertilized eggs, 2-cell, 16-cell, hatching blastula, gastrula, and pluteus stages were analyzed on SDS- and acetic acid-urea gels. This analysis indicated that P2 exists in two different forms differing slightly in charge. The less basic form was found in the egg, 2-cell and 16-cell extracts. At the hatching blastula stage, both forms were present and by pluteus stage, the more basic form predominated. It appears that P2 is undergoing a developmental change from a less to more basic form. The presence of P2 in the 5% PCA extract of egg nuclei is proof that P2 does not initially appear sometime during embryogenesis but is already in the egg nucleus prior to fertilization.