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.     

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.     

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).     

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.     

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.     

Release of ribonucleoprotein during digestion of rat testis chromatin with deoxyribonuclease II (3.1.4.6)

The composition of rat testis chromatin proteins in fractions produced by limited DNase II digestion followed by differential precipitation with MgCl2 has been studied. Over 50% of the acid-soluble proteins in the soluble chromatin fraction appeared to be quite similar to proteins which are associated with ribonucleoprotein (RNP) particles in HeLa cells. Although the ratios of the testis RNP protein components differed from those of HeLa RNP particles, the three major polypeptides were most similar to the HeLa components designated A2, B2, and C1. The soluble chromatin fraction was also enriched in the high mobility group proteins HMG1 and HMG2.     

Isolation and sequence of cDNA clones coding for a member of the family of high mobility group proteins (HMG-T) in trout and analysis of HMG-T-mRNA''s in trout tissues.

A specific oligonucleotide has been used to isolate a cDNA prepared from the mRNA for a trout High Mobility Group (HMG) protein closely related to trout HMG-T and bovine HMG 1 and 2 proteins. The sequence isolated more closely resembles bovine HMG-1 than the previously sequenced HMG-T protein in regions corresponding to the N terminal half of the protein. Northern blot analysis at low stringency indicated that 2 related sequences are expressed in total trout testis mRNA. Southern blots of total trout DNA indicate that several different forms of the homologous sequence are present in the trout genome and an estimate of copy number by dot-blot shows 4 HMG-T genes per trout sperm DNA equivalent. Analysis of mRNA from several trout tissues, including testis, liver and kidney indicates that expression of genes for histones and the larger HMG proteins in trout is not closely coupled.     

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.     

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.     

Identification of a single-stranded DNA binding protein from rat liver with high mobility group protein 1

The rat liver single-stranded DNA binding protein, S25 and HD25, isolated by differential DNA cellulose affinity chromatography was compared to the high mobility group proteins, HMG1 and HMG2, isolated from rat liver chromatin by the technique of Goodwin et al. (Goodwin, G. H., Sanders, C., and Johns, E. W. (1973) Eur. J. Biochem. 38, 14-19). Analysis of their amino acid composition, electrophoretic mobility, and tryptic peptide map reveal the identity of the single-stranded DNA binding protein with HMG1 protein, implying that the rat liver HMG1 protein becomes able both to destabilize a double helix of DNA and to stimulate homologous DNA polymerases only when rat liver cells enter a phase of DNA synthesis, possibly after a specific modification.     

Evidence for the location of high mobility group protein T in the internucleosomal linker regions of trout testis chromatin.

Antibodies against the trout testis non"histone chromosomal protein, high mobility group protein T (HMG-T), have been elicited in goats. The antiserum was shown to be specific for HMG-T and did not cross-react with histone 1 or with the other two trout testis HMG proteins, H6 and ubiquitin. Purified anti-HMG-T IgG was used to determine the location of HMG-T within chromatin subunits separated on sucrose gradients. Binding of fluorescent labeled anti-HMG-T to these subunits clearly supports the notion that this protein is associated not with the nucleosome core but rather with the internucleosomal linker regions, and previously suggested (Levy W., B., Wong, N.C.W., and Dixon, G. H. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2810-2814).     

The effect of alpha-tocopherol on the lipid peroxidation of mitochondria and microsomes obtained from rat liver and testis.

The effect of intraperitoneal administration of alpha-tocopherol (100 mg/kg wt/24 h) on ascorbate (0.4 mM) induced lipid peroxidation of mitochondria and microsomes isolated from rat liver and testis was studied. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:6 n3 in liver and C20:4 n6 and C22:5 n6 in testis. The lipid peroxidation of liver mitochondria or microsomes produced a significant decrease of C20:4 n6 and C22:6 n3 in the control group, whereas changes in the fatty acid composition of the alpha-tocopherol treated group were not observed. The light emission was significantly higher in the control than in the alpha-tocopherol treated group. The lipid peroxidation of testis microsomes isolated from the alpha-tocopherol group produced a significant decrease of C20:4 n6 , C22:5 n6 and C22:6 n3, these changes were not observed in testis mitochondria. The light emission of both groups was similar. The treatment with alpha-tocopherol at the dose and times indicated showed a protector effect on the polyunsaturated fatty acids of liver mitochondria, microsomes and testis mitochondria, whereas those fatty acids situated in testis microsomes were not protected during non enzymatic ascorbate-Fe2+ lipid peroxidation. The protector effect observed by alpha-tocopherol treatment in the fatty acid composition of rat testis mitochondria but not in microsomes could be explained if we consider that the sum of C20:4 n6 + C22:5 n6 in testis microsomes is 2-fold than that present in mitochondria.     

High mobility group proteins in skeletal muscle of pre- and postsmolt salmon (Salmo salar)

HMG proteins were extracted from whole skeletal muscle of pre- and postsmolt salmon (Salmo salar). The proteins were identified by SDS and acid-urea polyacrylamide gel electrophoresis. Semi-quantitative analysis revealed an increase in the ratio of HMG H6 to F protein and of HMG T to HMG H6 protein in the postsmolt as compared with the presmolt stage of the salmon. The results demonstrate a change in relative amounts of skeletal muscle HMG proteins which was dependent on the developmental stage of the fish.     

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.     

Cloning and functional characterization of a novel mitochondrial N-ethylmaleimide-sensitive glycerol-3-phosphate acyltransferase (GPAT2)

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step in glycerolipid synthesis. Several mammalian GPAT activities have been recognized, including N-ethylmaleimide (NEM)-sensitive isoforms in microsomes and mitochondria and an NEM-resistant form in mitochondrial outer membrane (GPAT1). We have now cloned a second mitochondrial isoform, GPAT2 from mouse testis. The open-reading frame encodes a protein of 798 amino acids with a calculated mass of 88.8kDa and 27% amino acid identity to GPAT1. Testis mRNA expression was 50-fold higher than in liver or brown adipose tissue, but the specific activity of NEM-sensitive GPAT in testis mitochondria was similar to that in liver. When Cos-7 cells were transiently transfected with GPAT2, NEM-sensitive GPAT activity increased 30%. Confocal microscopy confirmed a mitochondrial location. Incubation of GPAT2-transfected Cos-7 cells with trace (3 microM; 0.25 microCi) [1-(14)C]oleate for 6h increased incorporation of [(14)C]oleate into TAG 84%. In contrast, incorporation into phospholipid species was lower than in control cells. Although a polyclonal antibody raised against full-length GPAT1 detected an approximately 89-kDa band in liver and testis from GPAT1 null mice and both 89- and 80-kDa bands in BAT from the knockout animals, the GPAT2 protein expressed in Cos-7 cells was only 80 kDa. In vitro translation showed a single product of 89 kDa. Unlike GPAT1, GPAT2 mRNA abundance in liver was not altered by fasting or refeeding. GPAT2 is likely to have a specialized function in testis.     

Non-random reconstitution of HMG1 and HMG2 in chromatin. Determination of the histone contacts

We have studied how non-histone proteins HMG1 and HMG2 interact with rat liver chromatin using reconstitution and chemical cross-linking procedures. Both proteins were found to associate to chromatin only to some extent and always with a marked preference for short oligonucleosomes, mainly mono- and dinucleosomes. However, a slight reconstitution with the long polynucleosomal fraction can be observed in H1-depleted chromatin. Reconstitution is non-random and a clear preference for regions highly sensitive to staphylococcal nuclease (EC 3.1.31.1) is observed. Chemical cross-linking has allowed us to identify H1, H2A and H2B as the histones contacted by HMG1 and HMG2 upon reconstitution. Also, we present evidence that HMG1 and HMG2 interact with the nucleosomal particle without replacing H1 or any other histone.