The effect of 20-hydroxyecdysone on synthesis of chromosomal and cytosol proteins in imaginal discs

Over 600 cytosol and 300 nonhistone chromosomal proteins of mass-isolated imaginal discs of Drosophila melanogaster have been resolved by two-dimensional electrophoresis. More than half of the nonhistone chromosomal proteins fall into families with effectively constant apparent molecular weight but varying isoelectric points. At least six chromosomal proteins differ distinctly in proportions between embryos and imaginal discs. The synthesis of six cytosol proteins is increased, and one decreased with incubation of the discs in vitro with 20-hydroxyecdysone. Two disc acidic chromosomal proteins are specifically synthesized in the presence of 20-hydroxyecdysone. Their isoelectric points and molecular weights are similar to those of the subunits of vertebrate steroid hormone receptor proteins. However, although ecdysteroid receptor activity is associated with purified chromatin, no ecdysteroid-dependent increase in receptor activity is detected during in vitro culture of discs.     

Properties of the genome in normal and SV-40 transformed WI-38 human diploid fibroblasts. III. Turnover of nonhisone chromosomal proteins and their phosphate groups.

The turnover of nonhistone chromosomal proteins and their phosphate groups was compared in normal and in SV-40 virus transformed WI-38 human diploid fibroblasts. Cells were pulse labelled with tryptophan-³H and ³²P for 30 minutes and the specific activities of tryptophan-³H and ³²P in the various molecular weight classes of nonhistone chromosomal proteins were determined during the first four hours following termination of labelling. While a rapid turnover of high molecular weight nonhistone polypeptides (142, 000 to 200, 000 Daltons) is evident after one hour in SV_40 transformed cells, the specific activities of these nonhistone chromosomal polypeptides are not significantly decreased in normal cells. In contrast, a rapid turnover of low molecular weight (30, 000 to 51, 000 Daltons) nonhistone chromosomal proteins occurs during the first hour in normal WI-38 cells with no corresponding decrease in the specific activities of these proteins in SV-40 transformed cells. There is no apparent net turnover of phosphate groups on nonhistone chromosomal proteins in either normal or SV-40 transformed cells four hours following pulse labelling. Rather, during the first four hours significant fluctuations are observed in the ³²P specific activities of defined molecular weight fractions. Taken together with previous reports of differences in the composition, synthesis and phosphorylation of nonhistone chromosomal proteins in normal and SV-40 transformed human diploid cells the present results further indicate the complex nature of the alterations in these proteins which accompany viral transformation.     

Effects of a 4-aminopyridine in calcium movements and changes of membrane potential in pinched-off nerve terminals from rat cerebral cortex

Abstract: The processes of acetylation, phosphorylation, and methylation of nuclear proteins in cerebral hemispheres of 10- and 30-day-old rats were investigated. The experiments were carried out in vitro by measuring the incorporation of labeled precursors into histones and nonhistone chromosomal proteins (NHP) extracted from nuclei and separated by polyacrylamide gel electrophoresis. The results obtained indicate that there are age-specific differences in the processes of phosphorylation and methylation of chromosomal proteins, whereas the acetylation process did not change significantly between 10 and 30 days of age. Electrophoretic analysis of histones indicated that the histone H₃ was labeled to a greater degree than the other fractions and showed major changes in the processes of phosphorylation and methylation during postnatal development. The electrophoretic analysis of NHP showed considerable changes between 10 and 30 days of age. Certain components of NHP became increasingly evident as the brain developed. The methylation of an as yet unidentified protein with a molecular weight of approximately 118,000 daltons occurred at both ages.     

Glucocorticoid coordinate regulation of type I procollagen gene expression and procollagen DNA-binding proteins in chick skin fibroblasts

Nuclei were isolated from control and dexamethasone-treated (2 h) embryonic chick skin fibroblasts and transcribed in vitro. Nuclei isolated from dexamethasone-treated fibroblasts transcribed less pro alpha 1(I) and pro alpha 2(I) mRNAs but not beta-actin mRNA. Fibroblasts receiving dexamethasone and [5,6-3H]uridine also demonstrated decreased synthesis of nuclear type I procollagen mRNAs but not beta-actin mRNA. In fibroblasts treated with cycloheximide the newly synthesized nuclear type I procollagen mRNA species were markedly decreased. An enhanced inhibitory effect was observed when fibroblasts were treated with cycloheximide plus dexamethasone. Since the studies above demonstrate that active protein synthesis is required to maintain the constitutive expression of the type I procollagen genes, we determined if glucocorticoids regulate DNA-binding proteins with sequence specificity for the alpha 2(I) procollagen gene. Nuclear protein blots were probed with the 32P-end-labeled pBR322 vector DNA and 32P-end-labeled alpha 2(I) procollagen promoter containing DNA. Nonhistone proteins remained bound to labeled DNA at stringency washes of 0.05 and 0.1 M NaCl. As the ionic strength was increased to 0.2 and 0.3 M NaCl, the nonhistone-protein DNA binding was preferentially lost. Only the low molecular weight proteins remained bound to labeled DNA at the highest ionic strength, indicating nonspecific binding of these nuclear proteins. Dexamethasone treatment resulted in an increase of binding of nonhistone proteins to vector- and promoter-labeled DNAs over that observed in control fibroblasts at stringency washes of 0.05 and 0.1 M NaCl and to a lesser extent at 0.2 M NaCl. The binding specificities of nonhistone proteins for the alpha 2(I) procollagen promoter containing DNA were calculated. Three nonhistone DNA-binding proteins of Mr 90,000, 50,000, and 30,000 had altered specificities following dexamethasone treatment.     

NONHISTONE NUCLEAR PROTEINS OF RAT BRAIN

Abstract— The rat brain was dissected into cerebral cortex, cerebellum and the remaining regions. From the nuclei, isolated from these three brain sections, were extracted two fractions of nuclear sap proteins (proteins soluble in 014 M NaCl and proteins soluble in 01 M Tris-HCl buffer pH 7-6) and two fractions of nonhistone chromosomal proteins (one soluble in 0-35 M NaCl and one which is not soluble at this salt concentration). Each of these four fractions of the nonhistone nuclear proteins was further separated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the studied fractions of nuclear proteins are qualitatively identical regardless of the brain section from which the analysed protein fraction was isolated. In addition, there arc no qualitative differences in the electrophoretic patterns of nonhistone chromosomal proteins which are and which are not soluble in 0-35 M NaCl. In contrast to the qualitative similarity of the electrophoretic patterns of proteins from different sections of the brain, the amount of the nonhistone nuclear proteins is characteristic for each studied brain section. The ratio of the total nonhistone nuclear proteins to DNA is highest in the brain cortex and lowest in the cerebellum. The most expressed difference between the nuclei is in the ratio of the nonhistone chromosomal proteins soluble in 0-35 M NaCl to DNA. This ratio is 0-52 in the cortex. 0-38 in the mixture of noncortical and noncerebel-lar regions and only 0-18 in the cerebellum. The amount of the three fractions of nonhistone nuclear proteins in the nuclei of individual brain sections is proportional to the activity of the genome in these nuclei. The only exception are the nonhistone chromosomal proteins which are not soluble in 0-35 M NaCl. These proteins and the histones are present in the same amounts in nuclei isolated from all three studied sections of the brain. The results support a proposal that the nonhistone nuclear proteins are involved in the expression of the genetic activity of the cell, without the majority of the proteins in any of the four fractions being the specific regulatory molecules.     

Chromosomal protein synthesis during erythropoiesis in the mouse spleen

Induced erythropoiesis in the mouse spleen was employed to study chromosomal protein synthesis during erythroid cell development. Splenic erythropoiesis occurring after phenylhydrazine induced hemolysis can be divided into an early phase during which nuclear RNA polymerase activity and RNA production are maximal and a late phase in which hemoglobin synthesis and DNA accumulation are maximal. Chromatin was isolated from splenic tissue during both the early and late phases of erythropoiesis as well as from non-anemic animals. The total protein content of chromatin from the early erythroid phase was greater than that of chromatin from the late erythroid phase or from non-anemic controls. The increase was due to a coordinate increase in the concentration of both histone and nonhistone proteins. During late erythropoiesis, the concentration of each returned to pre-anemic levels. Total histone synthesis increased 2.6-fold during early erythropoiesis as compared with the pre-anemic state and remained elevated in late erythropoiesis. The increase in histone synthesis was due to an increase in the synthesis of all five major histone proteins. Nonhistone protein synthesis was more active than that of histones in the pre-anemic spleen and rose only slightly during early erythropoiesis, returning to preanemic levels during late erythropoiesis. Fractionation of nonhistone proteins on SDS-urea polyacrylamide gels revealed complex patterns with significant differences between the pattern of erythroid spleen non-histone proteins and that of the pre-anemic spleen. Analysis of the incorporation of ³H-valine into the non-histone proteins indicated that during early erythropoiesis there was a generalized increase in nonhistone protein synthesis. During the late erythroid phase, the decline in non-histone protein synthesis was most marked for the higher molecular weight proteins.     

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.     

In vivo and in vitro phosphoarylation of nuclear proteins in rat liver.

The incorporation of 32P into nuclear nonhistone proteins was compared in rat liver in vivo, in liver slices incubated in vitro, and in isolated nuclei incubated with gamma-[32P]ATP. The highest specific activities of nuclear phosphorproteins were obtained by incubating isolated nuclei. However, the Radioactivity profiles of polyacrylamide gel electrophoretograms of these proteins differed from those obtained in vivo or in liver slice experiments. A group of low molecular weight nonhistone proteins exhibited a very high incporation of labelled phosphate. These proteins could be obtained from the interface when the phosphoproteins were isolated by the buffered phenol extraction procedure. Phosphorylated proteins were also obtained from three cytoplasmic fractions (mitochondria, microsomes, and cytosol). The specific activities of these proteins were much lower than of the nuclear phosphoproteins.     

Partial purification and properties of a chromatin-associated phosphoprotein kinase from rat liver nuclei.

A phosphoprotein kinase (EC 2.7.1.37) KIVb, from rat liver nuclei, was purified 75-fold by phosphocellulose chromatography and gel filtration on Sephadex G-200. The enzyme, which has an apparent molecular weight of 55 000, phosphorylates casein and chromatin-bound nonhistone proteins more readily than histones or ribosomal proteins. It exhibits an absolute requirement for divalent cation with optimum activity at 15--20 mM Mg2+. Maximal kinase activity is achieved at 100 mM NaCl. The pH vs. activity curve is biphasic with optima at pH 6.5 and pH 8.0. The Km value for casein is 280 mug/ml and the Km for ATP is 6-10(-6) M. Kinase KIVb phosphorylates numerous nonhistone nuclear proteins as shown by electrophoretic analysis. The addition of kinase KIVb to reaction mixtures containing nonhistone proteins results in the phosphorylation of a spectrum of polypeptides similar to those that are phosphorylated by endogenous nuclear kinases. Nonhistone proteins bound to chromatin appear to be better substrates for KIVb than nonhistones dissociated from chromatin. A comparison of nuclear phosphoproteins phosphorylated either in the intact animal or in vitro (by the addition of kinase KIVb) indicates some differences and some similarities in the patterns of phosphorylation.     

Hepatoma-associated nuclear matrix nonhistone antigens

Polyclonal antibodies generated against a group of high molecular weight nonhistone proteins from Morris hepatoma 7777 were used in immunological studies of hepatoma-associated nonhistone proteins in rat and hamster. We revealed the presence of cross-reactive antigens in rat Morris hepatomas 7777 and 8994, and in hamster Kirkman-Robbins hepatoma, but not in normal rat or hamster livers. These specific nonhistone proteins were found to be preferentially localized in the nuclear matrix of rat Morris hepatoma 7777 as well as hamster Kirkman-Robbins hepatoma.     

Studies on the preparation and properties of ribonucleoprotein particles from rat liver nuclei

Ribonucleoprotein particles of 38 S were extracted from rat liver nuclei with isotonic salt buffer under concomitant sonication. The fate of the endogeneous nuclear RNAases assayed with poly(A), high molecular weight yeast RNA and rapidly labeled hnRNA was followed during the preparation of 38-S nuclear ribonucleoprotein (nRNP) particles. Essentially all the RNAase activity could be removed from the particle preparation. The effect of synthetic RNAase inhibitors on the nRNP particles was studied. Upon extraction of nuclei with 0.14 M NaCl, approximately 38% of the total nuclear radioactivity was found in the 38-S nRNP particles. By two successive extractions of the remaining chromatin with either isotonic or 0.22 and 0.3 M NaCl, an additional 25 and 9% of rapidly labeled hnRNA of 38 S particle were dissociated from chromatin, respectively. The chromatin components, DNA, nonhistone proteins, histones and RNA were determined after successive salt extractions. Particularly alterations in the nonhistone proteins and RNA were found. The protein patterns upon SDS-acrylamide gel electrophoresis of the salt-extracted chromatin preparations were compared with those of the 38-S nRNP particles. Particularly proteins in the molecular weight range of 32 000-43 000 were dissociated from chromatin after treatment with 0.22 or 0.3 M NaCl.     

Changes in template activity and structure of nuclei from WI-38 cells in the prereplicative phase.

Quiescent confluent monolayers of WI-38 fibroblasts were stimulated to proliferate by either adding 10% fetal calf serum or by trypsinization and replating at lower density. The length of the prereplicative phase was 12 hr after serum stimulation and 18 hr after trypsinization and replating at lower density. Nuclei were isolated from WI-38 cells at different time intervals after either type of stimulation and their template activity, circular dichroism spectra, and ability to bind ethidium bromide were investigated. All these parameters were similarly increased after either type of stimulation. However, these changes, like the onset of DNA synthesis, were delayed 6 hr in cells trypsinized and replated at lower density. While there were no detectable changes in nuclear protein content after serum stimulation, at least 40% of nuclear protein, mostly nonhistone chromosomal proteins, were lost after trypsinization. The amount of nuclear proteins returned to prestimulation levels only 6-8 hr after replating. These data seem to suggest that nonhistone chromosomal proteins lost by trypsinization are essential for the entrance of WI-38 cells into the "prereplicative phase".     

Adenovirus gene function required for induction of nuclear acidic protein synthesis: binding of these proteins to adenovirus DNA.

Two different viral DNA-defective temperature-sensitive mutants of adenovirus 12 (H 12) were defective in their ability to induce the synthesis of various molecular weight classes of nuclear acidic proteins, both virion and nonvirion components, after lytic infection of human embryo kidney (HEK) cells at the restrictive temperature. This finding indicates that the induction of nuclear acidic protein synthesis is an adenovirus gene function(s). Treatment of infected cells with actinomycin D at an early stage of virus maturation suppressed the synthesis of an acidic virion protein (hexon), but allowed the synthesis of other classes of nuclear nonvirion acidic proteins during the subsequent late maturation period, suggesting that different mechanisms control virion and nonvirion polypeptide synthesis. The interaction of the nuclear acidic proteins isolated from H 12-infected cells with native-labeled H 12 DNA was studied using the membrane filter technique. Measurements of the ability of different DNA preparations to inhibit the H 12 DNA-acidic protein complex formation suggest that the nuclear acidic proteins bound to native H 12 or HEK cell DNA with much higher affinity than to native calf thymus DNA. Moreover, native H 12 DNA was able to bind the acidic proteins more efficiently than did denatured H 12 DNA. The acidic proteins isolated from the cytoplasm of H 12-infected cells bound approximately 100-fold less to native H 12 DNA than did the nuclear proteins. Furthermore, the H 12 DNA binding affinity of the nuclear acidic proteins from uninfected cells, or from H 12-infected and 1-beta-D-arabinofuranosylcytosine-treated cells, was somewhat lower than that of the nuclear proteins from infected (untreated) cells.     

Changes in the synthesis and intracellular localization of nuclear proteins during embryogenesis in the sea urchin Strongylocentrotus purpuratus

A rapid, gentle technique is described for the isolation of nuclei from sea urchin embryos. Using this technique, we have analyzed the synthesis and accumulation of nonhistone nuclear proteins during sea urchin development by two-dimensional gel electrophoresis. Most nuclear proteins fall into one of three patterns of synthesis, which are distinguished by maximal rates of accumulation at early (prior to hatching blastula), middle (hatching blastula/gastrula), or late (prism/pluteus) stages of development. Over 60% of observed nuclear proteins undergo apparent qualitative changes in synthesis and accumulation between the 64-cell and pluteus stages. Most of these changes represent appearances of new proteins. A large number of qualitative changes occur very early in development; the period of greatest change is between the 64-cell and 200-cell stages. Over half of the proteins which first appear in the nucleus subsequent to the 64-cell stage are synthesized at stages prior to the time of their initial appearance in nuclei, but are excluded from nuclei for some time.     

Effects on in vitro brain protein synthesis of a translational inhibitor isolated from rabbit brain following intravenous administration of LSD

Inhibition of brain protein synthesis following intravenous administration of d-lysergic acid diethylamide (LSD) is accompanied by generation of a translational inhibitor protein in the postribosomal supernatant of cerebral hemispheres. Addition of an enriched preparation of this factor to a brain cell-free translation system resulted in a selective reduction in the level of phosphorylation of proteins of molecular weight 55K, 41K, and 25K. A similar set of changes was also observed in a brain cell-free system prepared 1 hr subsequent to drug injection. The brain inhibitor reduced the translational capacity of a messenger RNA-dependent reticulocyte lysate programmed with brain polysomes isolated from saline-injected animals however little effect was apparent when polysomes were prepared from LSD-treated animals. The translational inhibitor did not affect the spectrum of translation products from either set of polysomes.Abbreviations used HRI heme-regulated inhibitor - K 1000 Molecular weight - LSD d-lysergic acid diethylamide - PMS postmitochondrial supernatant - TCA trichloroacetic acid     

The rapid intranuclear accumulation of preexisting proteins in response to high plasmodial density in Physarum polycephalum

When actively growing microplasmodia of the lower eukaryote Physarum polycephalum are gently pelleted and allowed to stand at high plasmodial densities for 45 min, three specific nuclear acidic proteins undergo dramatic quantitative changes. Two major proteins of molecular weight 46 000 and 94 000 increase 110 and 320%, respectively. The increase in these two proteins is not markedly attenuated during periods when 88% total protein synthesis is blocked by cycloheximide, and the specific radioactivities of these proteins from prelabeled and continuously labeled control and pelleted plasmodia are essentially identical. A third protein of molecular weight 34 000 decreases by 51 % during the 45 min period and when cycloheximide is present, a 36% decrease in this protein still occurs. The rapid changes which occur in these three proteins in response to high plasmodial density also develop, together with many other changes, during plasmodial differentiation, but only after about 6 h of starvation. It is concluded that the rapid increase in the 46 000 and 94 000 mol. wt proteins results from protein transfer phenomena rather than de novo synthesis and that these proteins perhaps function in the early reorganization of cell metabolism rather than in structural differentiation. In further comparative studies it has been observed that mature spherules of P. polycephalum contain a major acidic protein not present in growing or differentiating plasmodia and also that the complement of residual acidic proteins differs in starvation-induced vs cold-induced spherules.