Purification of the Atlantic salmon hepatic 21 kDa protein and classification as a high mobility group chromatin protein.

The 21 kDa protein of liver from Atlantic salmon (Salmo salar) has been purified. Hepatic nuclei were extracted with 0.75 M HClO4. The extracted proteins were fractionated using reversed phase high performance liquid chromatography. The purity of the protein was analysed by isoelectric focusing in the first, and SDS-polyacrylamide gel electrophoresis in the 2nd dimension. Isoelectric focusing separated the protein into 5 spots. In gel trypsin digestion after isoelectric focusing followed by SDS-polyacrylamide gel electrophoresis resulted in identical migration of the tryptic peptides. The amino acid composition of the 21 kDa protein was similar to that of high mobility group (HMG) proteins C and D from rainbow trout (Oncorhynchus mykiss). The N-terminal sequence of the amino acids 1-19 revealed a conserved region characteristic for HMG 14/17 proteins of mammals and avians, and their equivalents in rainbow trout. Considering the electrophoretic mobility, amino acid composition and N-terminal amino acid sequence it is concluded that the 21 kDa protein of Atlantic salmon is a member of the HMG protein family resembling the HMG D protein of rainbow trout.     

Independent control during myogenesis of histone and high-mobility-group (HMGs) chromosomal protein heterogeneity

The ontogeny of high-mobility-group chromosomal protein and histone heterogeneity was followed in differentiating chicken skeletal muscle. The high-molecular-weight, high-mobility-group chromosomal proteins (HMGs 1, 2A, and 2B) showed a substantial change in their relative amounts during the period from 8 to 18 days. During this period HMG 2A decreases relative to HMG 1. There is also a decrease in the high-molecular-weight HMGs relative to the low-molecular-weight HMGs (HMGs 14 and 17). The microheterogeneity of the histones extracted from purified nuclei isolated from 8-, 11-, and 18-day embryonic myogenic tissue was analyzed by: (1) NaDodSO₄, (2) acid-urea, and (3) Triton acid-urea polyacrylamide gel electrophoresis. In contrast to the HMGs, the pattern of histone microheterogeneity for both H1 and H2A remained remarkably constant, despite the dramatic changes occurring in the rates of proliferation and the quality of gene expression in muscle cells during this developmental period. As would be expected from the decrease in cell proliferation occurring between days 8 and 18, increased amounts of phosphorylated H1 subspecies could be identified at the earlier stage. There was no change, however, in the proportion of the 4 major H1 parental variants. Thus, since the tissue-specific distribution of the H1s and HMGs are established at different times during myogenesis, it is likely that the two groups of chromatin proteins are involved in different events required for the establishment of the tissue-specific pattern of gene expression.     

Observations of the biology and parasites of postsmolt Atlantic salmon, Salmo salar, from the Norwegian sea

The main food items of postsmolt Atlantic salmon caught in the Norwegian sea during summer 1991 were Parathemisto spp. and herring ( Chipca harengus ). The most prevalent parasite species were Chloromyxion sp., Apatemon sp., H. adunction, A simplex and I. salmonis .     

Epitopes associated with mature spores not recognized on Kudoa thyrsites from recently infected Atlantic salmon smolts

Atlantic salmon Salmo salar skeletal muscle was examined for Kudoa thyrsites by polymerase chain reaction (PCR) and positive fish were further examined by in situ hybridization (ISH) and immunohistochemistry (IHC). The infection was detected in 42% of salmon by PCR following a 60 d exposure to infective seawater at a temperature of 10 degrees C (= 600 degree-days, degreeD). The parasite was detected by ISH in skeletal and cardiac muscle but not in gill, kidney, spleen, liver, stomach, intestine, pyloric caeca and skin. None of 4 monoclonal antibodies (2F4, 4H2, 1H2, 3E8) raised against mature K. thyrsites spores reacted with the stages identified by ISH following a 600 degreeD exposure, but they did react with ISH-identified stages following a 1600 degreeD exposure. In contrast, a polyclonal antibody reacted with K. thyrsites stages in salmon with both 600 and 1600 degreeD exposures, suggesting that the parasite observed in 600 degreeD infections represents an antigenically distinct developmental stage of K. thyrsites.     

Expression and synthesis of high mobility group chromosomal proteins in different rat skeletal cell lines during myogenesis.

The synthesis, turnover, and expression of all the major high mobility group (HMG) chromosomal proteins was studied in different rat skeletal myogenic cell lines. Whereas pulse-chase experiments revealed a similar half-life (greater than 2 cell generations) for all the HMG proteins in both L8 myoblasts and myotubes, [3H]lysine incorporation data indicated a 2- to 4-fold greater incorporation of the label in the HMG proteins in proliferating myoblasts relative to the nondividing myotubes. Analysis of the HMG-1, -14, and -17 mRNAs during myogenesis showed a significant down-regulation in L6 and L8 myotubes compared to the myoblasts. However, the timing of the shift and the extent of down-regulation was cell type-dependent, being more pronounced in L6 myotubes at fusion compared to 4 days postfusion in L8 myotubes. By contrast, L8-derived fusion-defective fu-1 cells over the same period of growth showed no change in HMG-14/17 mRNA levels. HMG-I(Y) protein isoforms, noted for the first time in rat myoblasts, like their counterparts, seemed to be stable and showed a precipitous reduction in their mRNAs during myogenesis. The results suggest a cell type-specific correlation between HMG expression and cell proliferation; they also argue for their role in maintenance of the cell's state of differentiation.     

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.     

Evidence for a quantitative tissue-specific distribution of high mobility group chromosomal proteins

The quantitative tissue specificity of the high mobility group (HMG) chromosomal proteins was investigated. Perchloric acid (PCA) extracts of four different chicken tissues and erythrocytes contained three proteins which comigrated on NaDodSO4-polyacrylamide gels with the HMG's 1,2, and E from erythrocyte nuclei. These three HMG's from embryonic skeletal muscle and erythrocytes also comigrated on two-dimensional gels, employing an acid-urea system in the first dimension and an NaDodSO4 system in the second. Interpretation of the two-dimensional gels suggested that the two low molecular weight proteins of this triplet arose from the HMG 2 band of the acid-urea gels. These have been designated HMG 2A and HMG 2B. Three proteins of similar molecular weights were also found in the PCA extracts of calf thymus. They were arranged in a similar but not identical pattern on two-dimensional gels. Thus, these three HMG's appear to be neither tissue nor species specific. In addition, the 2.0% PCA extracts of all chicken tissues examined contain a 38 000-dalton (38K) nuclear protein which coisolates with the HMG's. These four proteins are found in different relative amounts in each of the four chicken tissues and erythrocytes. They are found in the same relative amounts, however, in embryonic skeletal muscles from different chicken strains with widely different highly repetitive sequence content, suggesting that none of these individual proteins is selectively localized to constitutive heterochromatin. The quantitative tissue specificity of the HMG's and the 38K protein, however, suggests that they may participate in regulating cell-specific gene expression.     

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 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.     

Alterations in amounts and covalent modifications of low-molecular-weight chromosomal proteins in Chinese hamster ovary cells during polyamine depletion

The effect of polyamine depletion on phosphorylation and ADP-ribosylation of low-M_r chromosomal proteins was studied in intact, mutant Chinese hamster ovary cells (CHO-P22) devoid of ornithine decarboxylase activity. When starved of polyamines for 6 days, severe polyamine deficiency develops and the cells gradually stop growing. The rate of DNA synthesis was retarded to 16% of the control value and to 29% in density-inhibited cells. The synthesis of high-mobility-group (HMG) proteins was decreased by 65% in polyamine-depleted cells and by 40% in density-inhibited cells. The synthesis of core histones was decreased by 40% both in polyamine-depleted and density-inhibited cells. In polyamine-depleted cells the molar ratio of the higher-M_r HMG proteins (HMG 1 + 2) to the lower-M_r HMG proteins (HMG 14 + P) was about one-half of that found in cells grown in the presence of putrescine or in density-inhibited cells. In contrast to HMG proteins, no major differences were found in the content of core histones in these cell populations. In the perchloric acid-soluble fraction of nuclear proteins, ³²P was incorporated mainly into histone H1, HMG P and a protein migrating more slowly than HMG 1 (protein P1). Specific changes in the ³²P-labeling and migration of a number of protein bands, including histone H1, was observed in polyamine-depleted cells as compared to cells grown in the presence of putrescine or to density-inhibited cells. ADP-ribosylation experiments using [³H]adenosine showed a different pattern of label distribution; the higher-M_r HMG proteins from polyamine-depleted cells contained about one-half the amount of label found in the proteins from control cells. The lower-M_r HMG proteins and histone H1 were the preferentially labeled proteins in polyamine-depleted cells. Labeling of core histones with [³²P]orthophosphate or [³H]adenosine did not differ markedly in the two cell populations. The results obtained using intact polyamine auxotrophic cells indicated that polyamine depletion is connected with more severe alterations in amounts and covalent modifications (phosphorylation and ADP-ribosylation) of HMG chromosomal proteins and histone H1 than core histones.     

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.     

Astaxanthin binding protein in Atlantic salmon

The rubicund pigmentation in salmon and trout flesh is unique and is due to the deposition of dietary carotenoids, astaxanthin and canthaxanthin in the muscle. The present study was undertaken to determine which protein was responsible for pigment binding. Salmon muscle proteins were solubilized by sequential extractions with non-denaturing, low ionic strength aqueous solutions and segregated as such into six different fractions. Approximately 91% of the salmon myofibrillar proteins were solubilized under non-denaturing conditions using a protocol modified from a method described by Krishnamurthy et al. [Krishnamurthy, G., Chang, H.S., Hultin, H.O., Feng, Y., Srinivasan, S., Kelleher. S.D., 1996. Solubility of chicken breast muscle proteins in solutions of low ionic strength. J. Agric. Food Chem. 44: 408-415.] for the dissolution of avian muscle. To our knowledge, this is the first time this solubilization approach has been applied to the study of molecular interactions in myofibrillar proteins. Astaxanthin binding in each fraction was determined using an in vitro binding assay. In addition, SDS-PAGE and quantitative densitometry were used to separate and determine the relative amounts of each of the proteins in the six fractions. The results showed that alpha-actinin was the only myofibrillar protein correlating significantly (P<0.05) with astaxanthin binding. Alpha-actinin was positively identified using electrophoretic techniques and confirmed by tandem mass spectroscopy. Purified salmon alpha-actinin bound synthetic astaxanthin in a molar ratio of 1.11:1.00. The study was repeated using halibut alpha-actinin, which was found to have a molar binding ratio of astaxanthin to alpha-actinin of 0.893:1. These results suggest that the difference in pigmentation between white fish and Atlantic salmon is not due to binding capacity in the muscle, but rather differences in the metabolism or transport of pigment.     

Effects of 3,5,3'-triiodothyroacetic acid (TRIAC) on protein metabolism of genetically obese or non-obese Zucker rats

Genetically obese female rats (fa/fa) and their lean littermates (Fa/-) were given oral administration of 3,5,3-triiodothyroacetic acid (TRIAC) (20 micrograms/ 100 g of body weight/ day) during 4 weeks. Metabolism of proteins was evaluated in several organs and in skeletal muscle after intraperitoneal injection of 14C and 3H-leucine 6 days and 16 hrs respectively before the sacrifice of animals. We have determined radioactivity of 14C and 3H and the 3H/14C ratio. No significant differences were found in lean and obese rats except in skeletal muscle. The relative protein turnover in skeletal muscle is significantly higher in the obese rats than in the lean rats. Treatment by TRIAC decreases the body weight gain in obese rats compared with controls but it has no statistically significant effect on the relative protein turnover in either obese or lean rats.     

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.     

Characterization of DHP binding protein in crayfish striated muscle

The dihydropyridine calcium channel blocker, [3H]PN 200-110, binds specifically also to crayfish muscle membranes, though with a binding capacity smaller than that measured with rabbit or human skeletal muscle membranes. [3H]PN 200-110 binding proteins from the crayfish T-tubules were solubilized and purified on WGA Sepharose or extracted from gel. The purified protein has a molecular mass of approximately 190 kDa under nonreducing conditions and was able to transport calcium after reconstitution. Polyclonal antibodies against crayfish T-tubules enriched with purified DHP-binding protein were shown to bind to DHP-binding protein from both the crayfish and the rabbit skeletal muscle, although not with the same intensity. Electron microscopy showed the presence of ovoid particles. Our results suggest that a voltage-dependent calcium channel may be present in crayfish skeletal muscle, which is homological with the L-type calcium channel in rabbit skeletal muscle.