Equimolar proportions of somatic-type,core histones in chromatin of spermatogonia

[³H]Leucine incorporation into histones of seminiferous epithelial cells of hypophysectomized rats was used to calculate the molar proportions of the core histones of spermatogonia. The molar proportions H3:H2B:(H2A + protein A24):H4 are 1:1:1:1, viz. identical with those reported by others for somatic cells. Similar results were obtained when molar proportions of histones of seminiferous epithelial cells from immature rat testis (predominantly populated with spermatogonia) were determined by the dye-binding method. These data are relevant to mechanisms for the replacement of some of the core histones by variants during the primary spermatocyte stages.     

Exchange of histones H1, H2A, and H2B in vivo

We have asked whether histones synthesized in the absence of DNA synthesis can exchange into nucleosomal structures. DNA synthesis was inhibited by incubating hepatoma tissue culture cells in medium containing 5.0 mM hydroxyurea for 40 min. During the final 20 min, the cells were pulsed with [3H]lysine to radiolabel the histones (all five histones are substantially labeled under these conditions). By two electrophoretic techniques, we demonstrate that histones H1, H2A, and H2B synthesized in the presence of hydroxyurea do not merely associate with the surface of the chromatin but instead exchange with preexisting histones so that for the latter two histones there is incorporation into nucleosome structures. On the other hand, H3 and H4 synthesized during this same time period appear to be only weakly bound, if at all, to chromatin. These two histones have been isolated from postnuclear washes and purified. Some possible implications of in vivo exchange are discussed.     

Molar proportions and relative rates of synthesis of histones in rat testis

The molar proportions and relative rates of synthesis of histones in normal and hypophysectomized rat testis seminiferous epithelial cells were determined. After hypophysectomy the molar proportions of histones H1, H2B and (H2A + protein A24) in seminiferous epithelial cells of rat testis increased while their corresponding variants TH1-x, TH2B-x and X₂ decreased, but the molar proportions of major-class histones (i.e., sum of subfractions) remained relatively constant and similar to the proportions in somatic cells. The apparent molar proportions of the labeled histones, determined immediately after 2-h periods of [³H]leucine incorporation, were much higher relative to H4 than the proportions of total histones determined by dye binding. The values, however, approached the molar proportions of total histones when rats were killed 11 days after the [³H]leucine injection. Two-dimensional gel electrophoresis confirmed that the high initial molar proportions relative to H4 by [³H]leucine incorporation were not due to the possible contamination by highly-labeled non-histone proteins. The specific activity of histone H4 relative to the specific activity of DNA, determined immediately after 3-h periods of [³H]leucine and [¹⁴C]thymidine incorporations was similar to the value when rats were killed 13 days after the injections. It is proposed that histones of seminiferous epithelial cells are synthesized disproportionally relative to H4 and in excess of the quantities required for polynucleosome assembly. The excess histones are subsequently displaced or degraded slowly.     

Patterns of cell differentiation revealed by L-[3H]fucose incorporation in Dictyostelium

A study of the incorporation of l-[6-³H]fucose and d-[6-³H]glucosamine hydrochloride was conducted during the development of the cellular slime mold Dictyostelium discoideum 1-H. Autoradiographs revealed that pulse-labeled vegetative amoebae incorporated [³H]fucose intracytoplasmically within 15 min. The majority of the cells had randomly scattered silver grains but the remainder were distinguished by a dense localized labeling which suggested that oligo or polysaccharide synthesis was occurring. The localized pattern of labeling attributed to active synthesis declines at aggregation and early conus formation. As the pseudoplasmodium makes the developmental transition from the conus to the culmination stages the localized pattern of [³H]fucose labeling was restricted to the prespore cells while the prestalk cells were devoid of label. Prespore vacuoles were not present at the onset of this transition and consequently [³H]fucose incorporation occurred in the cells prior to their differentiation into prespore cells. In contrast to cells composing earlier stages, mature spores exhibited [³H]fucose-containing substances at the cell surface. At appropriate stages certain cells actively synthesize slime and stalk sheath which were labeled with either [³H]fucose or [³H]glucosamine.Prestalk isolates were obtained by transecting migrating slugs. [³H]Fucose was incorporated within 10 min among the basal cells of the isolate in the localized pattern typically found in prespore cells. The incorporation of [³H]fucose occurred prior to prespore differentiation as certain preparations were devoid of prespore vacuoles. Prespore isolates differentiate prestalk cells which have lost the capacity to incorporate [³H]fucose. This investigation suggests that cell contacts and interactions may affect the incorporation of [³H]fucose.     

Dynamic histone acetylation in alfalfa cells. Butyrate interference with acetate labeling

Dynamic histone acetylation of alfalfa (Medicago sativa) was studied in suspension cultures by short-term labeling with radioactive acetate. The relative labeling rates for the acetylated histones were in order of decreasing incorporation; H3.2 greater than H3.1 greater than H4 greater than H2B.1 greater than H2A.3. Histone H3 showed at least seven sites of acetylation, histone H2B.1 had six sites and histone H4 had five sites. Low numbers of acetylation sites were observed for histone H2B.2 and all histone H2A variants. The mass ratio, steady state acetylation and dynamic acetylation between major variant H3.1 and minor variant H3.2 were approx. 2:1, 1:2 and 2:5, respectively. Treatment of alfalfa cells with 50 mM n-butyrate did not lead to histone hyperacetylation, but instead interfered with histone acetylation labeling by acetate. The extent of apparent inhibition increased with time and concentration of butyrate. It is likely that the conversion of butyrate to acetylCoA results in dilution of the specific radioactivity of [3H]acetate in the acetylCoA pool thereby inhibiting the labeling reaction. This interpretation is supported by 14C-labeling of alfalfa acetylated histones by [1-14C]butyrate.     

Effect of of distamycin A on histone H1 methylation, extraction and formation of UV-inducible crosslinks with DNA in the interphase rat liver nucleus

Incubation in vitro of rat liver nuclei in the presence of S-adenosyl[methyl-(3)H]methionine ([(3)H] SAM) leads to incorporation of the radioactive label not only into core-histones H3 and H4, but also into linker histone H1. Addition of distamycine A to the incubation medium stimulates label incorporation into histone H1 ~ in 6 times and into histone H3 ~ in 2 times. The presence of distamycine facilitates histone H1 extraction by polyglutamic acid (poly(Glu)) and decreases of UV-induced DNA-histone cross-links formation. These effects give evidence of weakening of H1-chromatin interaction by distamycin to be results of histone H1 position change relative to nucleosome and(or) disturbance of histones H1-H3 interactions so as these histones are exposed to additional methylation.     

Alterations in nicotinamide and adenine nucleotide systems during mixed-function oxidation of p-nitroanisole in perfused livers from normal and phenobarbital-treated rats

When rat liver nuclei were incubated with [adenine-3H]NAD, besides histone 1, histone 2A and especially histone 2B accepted 3H radioactivity. 3H radioactivity was also found on the non-histone proteins and on the small amounts of histones 1 and 3 released into the supernatant during incubation. [14C]Adenine uptake in vivo by liver and thymus nuclei showed radioactivity in histones 1 and 3. After digestion with Pronase and leucine aminopeptidase 14C- or 32P-labelled histone 3 released a serine phosphate-containing nucleotide, which on acid hydrolysis yielded ADP-ribose and serine phosphate. Serine phosphate was also found in the material from the nucleotide peaks from histones 2A and 2B. ADP-ribosylated histones 1 and 3 were more easily released from nuclei than their unmodified forms and showed higher [32P]Pi and [3H]lysine uptakes in vivo [Ord & Stocken (1975) FEBS Meet. Proc. 34, 113-125].     

Metabolism of Functional Groups Modifying the CNS Myelin-Associated Glycoprotein

We have examined the metabolic turnover of the peptide backbone of the CNS myelin-associated glycoprotein (MAG) and of the fucose and sulfate groups modifying this protein. Rats (20 or 90 days old) were injected intracranially with mixtures of [3H]fucose and [14C]glycine, [3H]glycine and [35S]sulfuric acid, or [3H]fucose and [35S]sulfuric acid. At times ranging from 30 min to 4 weeks later, myelin was isolated, and radioactivity in MAG was determined following electrophoretic separation. Following the peak of incorporation, glycine-derived radioactivity in the MAG peptide backbone declined several-fold during the first week and was then metabolically stable (half-life much greater than 1 month). Declines with time in [3H]fucose- and [35S]sulfate-derived radioactivity in MAG were similar to that of [3H]glycine, an observation indicating that the fucose and sulfate groups modifying MAG are metabolized together with the peptide backbone as a single metabolic entity. These results were confirmed by experiments involving selective immunoprecipitation of MAG. The rates of incorporation of labeled glycine, fucose, and sulfate into MAG all decreased approximately 12-fold between 20 days of age and adulthood, a finding providing further evidence for concerted turnover of the entire molecule. Because of this concerted turnover, we suggest that functional groups modifying MAG serve some permanent structural role in protein configuration.     

Chromatin core particle obtained by selective cleavage of histones H3 and H4 by clostripain

A new chromatin core particle characterized by a half-proteolyzed octamer is obtained by controlled digestion of the native core particle by clostripain. The proteolyzed histones correspond to four polypeptide fragments which are tentatively assigned to H2A[4-129], H2B[1-125], H3[27-135] and H4[18-102] on the basis of electrophoretic evidence and the known specificity of clostripain for arginyl residues. Despite the loss of the N-terminal regions of histones H3 and H4, the partially proteolyzed core particle retains the structural conformation of the native one as shown by circular dichroism. As expected, this half-proteolyzed core particle presents an intermediate accessibility to polycations, such as spermidine, in comparison with that observed with the native core particle and a fully proteolyzed core particle. The latter includes the polypeptide fragments H2A[12-129], H2B[21-125], H3[27-135] and H4[20-102].     

Radiolabelling of Mycobacterium avium oligosaccharide determinant and use in macrophage studies

Internal radiolabelling procedures were used to radiolabel the oligosaccharide determinant of the glycopeptidolipids (GPL) from serovars 4 and 20 of the Mycobacterium avium complex. Mycobacteria were cultured in the presence of [6-3H]fucose, [2-3H]mannose or [methyl-3H]methionine, after which radiolabelled native lipid was extracted and distribution of radioactivity in native and deacetylated lipid was determined by thin-layer chromatographic methods. Incorporation of radiolabel was confirmed by examining acid hydrolysates of purified GPL for 3H-labelled sugars on cellulose thin-layer plates. Least incorporation of radiolabel into GPL was observed with [6-3H]fucose, whereas better incorporation was obtained with [2-3H]mannose and [methyl-3H]methionine. Use of [methyl-3H]methionine resulted in the radiolabelling of the methylated sugars in both the oligosaccharide determinant and the 3,4-di-O-methylrhamnose located at the terminus of the peptide core. Use of [2-3H]mannose resulted in the incorporation of radioactivity into the oligosaccharide determinant as 2-O-methylfucose, found in the GPL of both serovars 4 and 20. GPL radiolabelled with [2-3H]mannose were subsequently examined in macrophage cultures and found to be relatively inert to degradation by those phagocytic cells. These results substantiate earlier findings with the GPL of serovar 20 and indicate that these mycobacterial components may play a role in pathogenesis.     

Influence of distamycin A on the methylation,extraction, and formation of UV-inducible DNA-protein cross links of histone H1 in the interphase nuclei of rat liver cells

The incubation in vitro of rat liver nuclei in the presence of S-adenosyl[methyl-³H]methionine ([³H] SAM) leads to the incorporation of a radioactive label not only into core histones H3 and H4, but also into linker histone H1. The addition of distamycin A to the incubation medium stimulates label incorporation into histone H1 by approximately six times and into histone H3 by around two times. The presence of distamycin facilitates histone H1 extraction by polyglutamic acid (poly(Glu)) and decreases UV-induced DNA—histone cross-link formation. These effects give evidence that the weakening H1—chromatin interaction by distamycin may be the result of a histone H1 position change relative toward the nucleosome and (or) a disturbance of the histone H1–H3 interactions, as these histones are exposed to additional methylation.     

Covalent binding of benzo[a]pyrene metabolites to DNA, RNA and chromatin proteins in the AKR mouse embryo cell-line

A specific fraction from the nuclei of the AKR mouse embryo cell-line (fraction I) displayed a much greater localization of radioactivity compared to fraction II and III when the chemical carcinogen, [3H]benzo[a]pyrene (B[a]P) was incubated with the cells for 24 h. The radioactivity in fraction I consisted of both covalently and non-covalently bound metabolites. Isolation of the DNA, RNA and protein of fraction I revealed that 94% of the covalently bound radioactivity was to protein, 5% to RNA and 1% to DNA. Analysis of the fraction I proteins by SDS gel electrophoresis revealed that there was more radioactivity covalently bound to the larger proteins than to smaller proteins. Isoelectric focusing (IEF) of the purified proteins displayed two peaks of radioactivity, one at a pH of 5 and the other at 11. The former proteins bound more radioactivity per mass of protein than the latter proteins. Analysis of fraction I histones on acid urea polyacrylamide gels showed that the radioactivity coincided with histones H3 and H2B and low levels of radioactivity associated with histones H1, H2A and H4. Two significant peaks of radioactivity closely migrated near but did not co-migrate with histone H1. The distribution of the bound radioactivity is probably a reflection of the availability of the proteins to the reactive carcinogen metabolites. The possible binding of B[a]P metabolites to phosphorylated histones and to the high mobility of group (HMG) proteins 1 and 2 is discussed.     

AUTORADIOGRAPHIC STUDIES OF SYNTHESIS AND INTRACELLULAR MIGRATION OF GLYCOPROTEINS IN THE RAT ANTERIOR PITUITARY GLAND

The incorporation of [³H]fucose in the somatotrophic and gonadotrophic cells of the rat adenohypophysis has been studied by electron microscope autoradiography to determine the site of synthesis of glycoproteins and to follow the migration of newly synthesized glycoproteins. The pituitaries were fixed 5 min, 20 min, 1 h, and 4 h after the in vivo injection of [³H]fucose and autoradiographs analyzed quantitatively. At 5 min after [³H]fucose administration, 80–90% of the silver grains were localized over the Golgi apparatus in both somatotrophs and gonadotrophs. By 20 min, the Golgi apparatus was still labeled and some radioactivity appeared over granules. At 1 h and 4 h, silver grains were found predominantly over secretory granules. The kinetic analysis showed that in both protein-secreting cells (somatotrophs) and glycoprotein-secreting cells (gonadotrophs), the glycoproteins have their synthesis completed in the Golgi apparatus and migrate subsequently to the secretory granules. It is concluded from these in vivo studies that glycoproteins which are not hormones are utilized for the formation of the matrix and/or of the membrane of the secretory granules. The incorporation of [³H]fucose in gonadectomy cells (hyperstimulated gonadotrophs) was also studied in vitro after pulse labeling of pituitary fragments in medium containing [³H]fucose. The incorporation of [³H]fucose was localized in both the rough endoplasmic reticulum (ER) and the Golgi apparatus. Later, the radioactivity over granules increased while that over the Golgi apparatus decreased. The concentration of silver grains over the dilated cisternae of the rough ER was not found to be modified at the longest time intervals studied.     

Sialic acid, galactose and fucose on the surface of Ehrlich ascites tumor cells grown in glucose-free medium in the presence of uridine

1) The content and accessibility of terminal sialic acid and galactose residues as well as the incorporation of [3H]fucose into glycoconjugates were determined in 48-h cultures of Ehrlich ascites tumor cells in a glucose-free medium supplemented with uridine, a compound which can fulfil the necessary functions of glucose. 2) Sialic-acid residues accessible to sialidase cleavage were reduced from 695 +/- 80 nmol/10(9) cells (controls) to 284 +/- 22 nmol/10(9) cells (43% of controls). In situ labeling using periodate oxidation followed by sodium borotritiide reduction revealed a tritium incorporation of 47 +/- 11% that of controls (= 4.1 x 10(5) cpm/mg protein). 3) Labeling of galactose residues of 80-90% of that of controls was achieved after treatment of the cells with galactose oxidase/sodium borotritiide. A nearly six-fold enhancement of tritium incorporation into galactose of control cells was observed after sialidase/galactose oxidase treatment and sodium borotritiide reduction (1.5----8.8 x 10(5) cpm/mg protein); only a 3.6-fold increase (1.2 x 10(5)----4.3 x 10(5) cpm/mg protein) was found with glucose-free cultured cells. It is concluded that the galactose content of the cell surface is reduced to about 50% of controls. 4) The incorporation of tritium into acid-insoluble precipitate after 24 h incubation with [3H]fucose and the activity of the acid-soluble fraction were enhanced by about 85% as compared to controls. The pattern of inhibition by tunicamycin of [3H]fucose uptake and incorporation was the same in glucose-containing standard medium and in glucose-free uridine medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of [3H]galactose and [3H]fucose uptake with the morphological and histochemical changes observed in mucous secretion in chemically induced rat colonic carcinoma

Summary The alterations in carbohydrate metabolism which occur in the distal colon of rats during carcinogenesis induced by dimethylhydrazine were investigated using [³H]galactose and [³H]fucose as glycoprotein precursors.A statistically significant decrease in [³H]galactose uptake was observed in dysplastic epithelia. These findings are consistent with the alterations in mucin composition with predominance of sialomucins shown in these areas by histochemical methods. Furthermore, changes in the gradient of [³H]galactose incorporation along the crypt epithelium were also found in the histological and histochemically non-involved colonic mucosa of dimethylhydrazine-treated rats, as compared with controls.No significant variations were seen in [³H]fucose incorporation.These results correlate well with our previous histochemical observations and are further evidence of the profound alterations in glycoprotein synthesis affecting the whole colonic mucosa during carcinogenesis.     

Effects of the Amnesic Agent 2-Deoxygalactose on Incorporation of Fucose into Chick Brain Glycoproteins

The interaction of the amnesic agent 2-deoxygalactose with fucose incorporation into glycoproteins in day-old chick forebrain has been studied with the aim of identifying glycoproteins whose synthesis is modified during memory formation. 2-Deoxygalactose inhibited total exogenous [14C]fucose incorporation into the forebrain glycoproteins by 26%. Sodium dodecyl sulphate-polyacrylamide gradient gel analysis revealed that intracerebrally injected 2-[3H]deoxygalactose labelled the same eight major glycoprotein bands as were identified using [14C]fucose labelling. Subsequent investigations focussed on these selected components. Subcellular fractionation showed that between 4 and 24 h after administration of the deoxy-sugar, the incorporated radioactivity was found predominantly at the synaptic sites, some glycoproteins being more abundant in synaptic plasma membranes and others in postsynaptic densities. This distribution pattern varied according to the time after injection. The effect of passive avoidance training, using a methylanthranilate-coated bead, on [14C]fucose incorporation into forebrain was to decrease fucose uptake into components of molecular mass 150-180 kilodaltons but to increase significantly labelling of glycoproteins of molecular mass 33 and 28 kilodaltons. The possible implications of these training-induced changes are discussed.     

Demonstration that some of the nonhistone proteins, inducible to translocate into the nucleus, are glycosylated

Con A, NaF, and eserine (lysosomotropic agents) induced marked translocation of acidic [3H] nonhistone proteins (NHP) from the cytoplasm to the nucleus in lymphocytes prelabeled with [3H]-2-mannose. The nuclear [3H] NHP contents were 38-120% higher in cells treated with these agents than in control cells. Tunicamycin, a strong inhibitor of N-glycosylation via the dolichol pathway, caused a concentration-dependent inhibition of [3H]-2-mannose incorporation into the nuclear [3H] NHP. Considerable amounts of nuclear [3H] NHP from lymphocytes labeled with either [3H]-2-mannose or [3H] leucine, bound specifically to Con A-Sepharose and could be eluted by alpha-methyl mannoside. Con A and NaF caused also nuclear translocation of acidic [3H] NHP in cells labeled with [3H] glucosamine, [3H] galactose, or [3H] fucose. Fractionation of the nuclear proteins by isoelectric focusing in a pH gradient of 2.5-6.5 showed that multiple species of acidic NHP were labeled with each of the four 3H-sugars. These results indicate that a fraction of the acidic nuclear NHP are N-glycosylated proteins and that gene activation and mitogenesis are associated with the translocation of these glycoproteins to the nucleus. Considering the known intracellular traffic of nascent glycoproteins our results suggest that at least some of the acidic NHP are synthesized and glycosylated in the endoplasmic reticulum and the Golgi (secretory pathway). It is likely that these proteins, after completion of synthesis and glycosylation, emerge from the trans-stack of the Golgi packaged in vesicles and accumulate in the cytoplasm. Induction of nuclear translocation of such NHP by various agents may be mediated by a vesicular transport mechanism.     

Sites of in vivo histone methylation in developing trout testis.

Specific lysyl residues of trout testis histones H3 and H4 are methylated partially during rainbow trout spermatogenesis. Histones H1, H2A, H2B, and protamine are not methylated. The single site (lysine 20) in histone H4 and the two major sites (lysines 9 and 27) in histone H3 are homologous to those determined for other organisms, but an additional minor site (lysine 4) occurs in histone H3. As described for calf thymus, both histones H3 and H4 contain epsilon-N-mono- and dimethyllysine, while histone H3 contains in addition, epsilon-N-trimethyllysine. The trout-specific histone H6, which accounts for 0.5 to 1.0% of total histone, contains a sequence for residues 3 to 5,-Arg-Lys-Ser-, which is the same as one methylated in histones H3, at lysines 9 and 27. However, histone H6 yields only trace amounts of [3H]methyl incorporation and no detectable methyllysines on amino acid analysis.     

Turnover of chromatin proteins in rat liver during induction of RNA synthesis by electrostimulation of the hypothalamus

It has been shown that the induction of D-RNA synthesis in rat liver nuclei by electrostimulation of hypothalamus is accompanied by a decrease in chromatin protein synthesis and an increase in phosphorylation and acetylation of chromatin proteins. The decrease of the histone synthesis is mainly due to the decrease of [14C]lysine and [14C]alanine incorporation into histones H1 and H4. The relationship between H1, H2b-H3, H2a and H4 histone fractions remains unchanged. Electrostimulation of hypothalamus increases acetylation of H2a and H4 histone fractions and phosphorylation of all histones with the exception of histone H1.