High mobility group (HMG) proteins in the tammar wallaby Macropus eugenii: quantitative variations between tissues and testis-specific co-extracted proteins

1. Tammar wallaby (Macropus eugenii, Marsupialia) proteins with similar electrophoretic mobilities to calf non-histone chromosomal proteins HMG 1, 2, 14 and 17 are perchloric acid extracted from whole tissues (liver, kidney, spleen, brain and testis) and purified liver nuclei (using PCA or 0.35 M NaCl). 2. Tammar and calf HMG 1 have similar amino acid compositions. 3. Two testis-specific basic proteins co-extracting with HMG-like proteins from both tammar and red kangaroo (Megaleia rufa) are found in whole testis, purified testis nuclei, but not epididymis. 4. Tammar HMG 2 separates into two components on both acid urea and SDS gels. The larger, more basic protein, HMG 2b, is relatively abundant in proliferating tissues (testis, spleen).     

Analysis of the HMGI nuclear proteins in mouse neoplastic cells induced by different procedures

Four malignant tumors induced in mouse by different experimental procedures were compared as regards their high-mobility-group (HMG) proteins. All tumors showed the complete set of three HMG proteins which we call HMGI-C, I-D, and I-E. The presence of the three HMGI proteins is a characteristic of the transformed phenotype regardless of whether the tumor was chemically, virally, or spontaneously derived. However, the level of expression of the HMGI proteins is not constant in the four tumors. Using reverse-phase HPLC, the individual HMGI proteins were isolated from the spontaneously derived tumor (Lewis lung carcinoma) and shown by amino acid analysis to be similar to those previously obtained from a tumor grown in nude mice by inoculation of in vitro-transformed cells.     

HMG proteins released from the chromatin following incubation of mammalian nuclei with ethidium bromide

The low-molecular-mass high-mobility group (HMG) chromosomal proteins, namely HMG-14, HMG-17, and HMG-I, which have been found in several proliferating tissues, are released following incubation of nuclei isolated from young rat thymus and from human placenta in a low ionic strength medium containing the intercalating agent ethidium bromide. The amount of HMG proteins released is drug concentration-dependent, but at very high concentrations (20-40 mM) other low- and high-molecular-mass proteins, and even histones, are released. These results suggest a very weak interaction of the HMG proteins with DNA, so that they can be easily detached from the chromatin as a consequence of the interaction of DNA with the intercalating agent.     

Differential expression of the chromosomal high mobility group proteins 14 and 17 during the onset of differentiation in mammalian osteoblasts and promyelocytic leukemia cells

The expression of chromosomal proteins HMG 14 and HMG 17 during proliferation and differentiation into the osteoblast and monocyte phenotypes was studied. Cellular levels of HMG 14 and HMG 1 7 mRNA were assayed in primary cultures of calvarial-derived rat osteoblasts under conditions that (1) support complete expression of the mature osteocytic phenotype and development of a bone tissue-like organization; and (2) where development of osteocytic phenotypic properties are both delayed and reduced in extent of expression. HMG 14 and HMG 17 are preferentially expressed in proliferating osteoblasts and decline to basal levels post-proliferatively at the onset of extracellular matrix mineralization. In contrast, under conditions that are not conducive to extracellular matrix mineralization, HMG 14 is maximally expressed following the downregulation of proliferation. Consistent with previous reports by Bustin and co-workers [Crippa et al., 1990], HMG 14 and HMG 17 are expressed in proliferating HL-60 promyelocytic leukemia cells and downregulated post-proliferatively following phorbol ester-induced monocytic differentiation. However, differentiation into the monocyte phenotype is accompanied by reinitiation of HMG 17 gene expression. The results indicate that the levels of HMG 14 and HMG 17 mRNA are selectively down-regulated during differentiation.     

The isolation, characterization and partial sequences of the chicken erythrocyte non-histone chromosomal proteins HMG14 and HMG17. Comparison with the homologous calf thymus proteins.

Non-histone chromosomal proteins HMG14 and HMG17 were isolated from chicken erythrocyte nuclei. The proteins were characterized by amino acid analysis and by N-terminal sequence analyses. Comparison with the corresponding data for the calf thymus proteins shows that 11% of the residues in HMG14 protein and 5% of the residues in HMG17 protein differ between the two species. Proteins HMG14 and HMG17 therefore do not appear to exhibit the evolutionary stability shown by the nucleosome core histones. Detailed evidence for the amino acid sequence data has been deposited as Supplementary Publication SUP 50101 (4 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. 4. (1978) 169, 5.     

On the phosphorylation of low molecular mass HMG (high mobility group) proteins in Ehrlich ascites cells

This paper shows that the low molecular mass HMG proteins 14 and 17 do not seem to be phosphorylated in Ehrlich ascites cells whereas two other small HMG proteins designated HMG I and Y are. Amino acid analysis and peptide mapping of all four proteins demonstrated that HMG I and Y were not phosphorylated modifications of HMG 14 or 17.     

Differences between HMG1 proteins isolated from normal and tumour cells

The properties of the non-histone chromosomal high-mobility-group 1 (HMG1) proteins from rat liver and Guerin ascites tumour cells (GAT cells) were compared and showed the following differences: (1) five spots were missing in the peptide map of HMG1 from GAT cells in comparison with that of HMG1 from rat liver; (2) HMG1 from GAT cells was about 5-times more poly(ADP)-ribosylated; (3) HMG1 from GAT cells which was found acetylated in vivo and incorporated [14C]acetate in vitro, whereas no incorporation of the label was detected in HMG1 from rat liver; (4) HMG1 from GAT cells exhibited pronounced ability to form oligomers at physiological ionic strength, while HMG1 from rat liver was predominantly in monomeric form. This property of HMG1 from GAT cells was lost upon deacetylation.     

A novel, highly phosphorylated protein, of the high-mobility group type, present in a variety of proliferating and non-proliferating mammalian cells

The present work describes a perchloric-acid-soluble high-mobility-group (HMG)-like protein present in HeLa and Ehrlich ascites cells, rat and calf liver. The protein is designated P1 and has, depending on the source, a molecular mass 48-53 kDa and an amino acid composition which, like the HMG proteins, is characterized by a high content of acidic and basic residues and of proline. The protein contains about 10 mol serine/100 mol amino acid residues, is highly phosphorylated and has, in contrast to the known HMG proteins, an acidic isoelectric point of 5.0. An estimate suggests that protein P1 in HeLa interphase cells contains 25-30 residues of phosphate. Like HMG 1 and 2 it is distributed between the nucleus and the cytoplasm. In HeLa metaphase cells P1 is further modified, resulting in an increase in apparent molecular mass from 53 kDa to 56 kDa.     

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.     

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.     

Persistence of chromosomal proteins HMG-14/-17 in myotubes following differentiation-dependent reduction of HMG mRNA.

The expression of chromosomal proteins HMG-14 and HMG-17 during cellular differentiation was studied in cultured mouse myoblasts. During myogenesis the level of both HMG-14 and HMG-17 mRNA decreased to less than 20% of that found in myoblasts. The down-regulation of HMG-14/-17 mRNA occurred simultaneously with activation of muscle-specific actin mRNA and was not linked to DNA synthesis, indicating that it is a differentiation-, rather than a cell cycle-related event. Incorporation of radiolabeled lysine into HMG proteins was similar to that into the major histone fractions in that it was significant in myoblasts and undetectable in myotubes. The decrease in mRNA and protein synthesis did not affect the cellular levels of HMG protein. These results indicate that the regulation of HMG-14/-17 mRNA levels is different from that of the histones and is linked to differentiation rather than to DNA synthesis.     

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.     

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.     

On the presence of the chromosomal proteins HMG I and HMG Y in rat organs

Using antiserum raised against HMG I, we have shown that HMG I and HMG Y are present in perchloric acid extracts of kidney, lung, heart, brain, liver and intestine in the rat, suggesting that the expression of these proteins may not be dependent upon proliferative activity. The results also show that the ratio between HMG I and HMG Y varies between different organs.     

High mobility group-like proteins of the insect Plodia interpunctella

Nuclei from Plodia interpunctella larvae contain four major proteins, which are extracted by 5% perchloric acid and 0.35 M NaCl. The proteins have been designated PL1, PL2, PL3, and PL4. The amino acid analyses of these proteins show that they have high proportions of acidic and basic amino acid residues, a property characteristic of the high mobility group (HMG) proteins isolated from vertebrate tissues. Immunological characterization of these proteins clearly shows that PL1, PL2, and PL4 are more closely related to HMG1 dipteran proteins, while PL3 is more closely related to HMGI dipteran proteins. The possible relatedness of these proteins to HMG proteins is discussed.