Nuclear Protein Synthesis Research Articles

Protein synthesis and the recovery of both survival and cytoplasmic “petite” mutation in ultraviolet-treated yeast cells. II. Mitochondrial protein synthesis

The contribution of mitochondrial proteins in the repair of UV-induced lethal and cytoplasmic genetic damages was studied in dark liquid held exponential and stationary phase yeast cells. This was performed by using the specific inhibitors, erythromycin (ER) and chloramphenicol (CAP). It was shown that mitochondrial proteins are involved in the recovery of survival of UV-treated exponential phase cells, but not in the recovery of stationary phase cells. Mitochondrial proteins are partly implicated in the mechanisms leading to the restoration of the ϱ + genotype in UV-irradiated dark liquid held exponential phase cells. Here again, in statonary phase cells, mitochondrial enzymes do not seem to participate in the negative liquid holding (NLH) processes for the ϱ − induction, as shown by inhibiting mitochondrial protein synthesis or both mitochondrial and nuclear protein synthesis. When cells are grown in glycerol, the response after dark liquid holding of UV-treated cells in the different growth stages are similar to that found for glucose-grown cells. In other words, the fate of cytoplasmic genetic damage, in particular, is not correlated with the repressedor depressed state of the mitochondria.

Protein synthesis and the recovery of both survival and cytoplasmic “petite” mutation in ultraviolet-treated yeast cells II. Mitochondrial protein synthesis

The contribution of mitochondrial proteins in the repair of UV-induced lethal and cytoplasmic genetic damages was studied in dark liquid held exponential and stationary phase yeast cells. This was performed by using the specific inhibitors, erythromycin (ER) and chloramphenicol (CAP). It was shown that mitochondrial proteins are involved in the recovery of survival of UV-treated exponential phase cells, but not in the recovery of stationary phase cells. Mitochondrial proteins are partly implicated in the mechanisms leading to the restoration of the ϱ+ genotype in UV-irradiated dark liquid held exponential phase cells. Here again, in statonary phase cells, mitochondrial enzymes do not seem to participate in the negative liquid holding (NLH) processes for the ϱ− induction, as shown by inhibiting mitochondrial protein synthesis or both mitochondrial and nuclear protein synthesis. When cells are grown in glycerol, the response after dark liquid holding of UV-treated cells in the different growth stages are similar to that found for glucose-grown cells. In other words, the fate of cytoplasmic genetic damage, in particular, is not correlated with the repressedor depressed state of the mitochondria.

Microsurgical Studies on the Formation of the Golgi Apparatus and Nuclear Envelope in Amebae

The means of formation of the Golgi apparatus is uncertain. In beginning these studies, it seemed that an important first step was to determine the degree of dependence of the Golgi apparatus on the nucleus. This was investigated in Amoeba proteus because these cells are readily enucleated microsurgically. Normal amebae contained multiple Golgi bodies, which declined in size and number in the absence of the nucleus, indicating that the Golgi apparatus in amebae depends upon the nucleus. The influence of the nucleus on the Golgi apparatus may be mediated through nuclear RNA synthesis and cytoplasmic protein synthesis, since the ultrastructural effects of enucleation were mimicked by treating amebae with actinomycin D to inhibit RNA synthesis and, in part, by treating cells with emetine, which inhibits protein synthesis in amebae.

Early Nuclear Events in the Induction of Lymphocyte Proliferation by Mitogens: EFFECTS OF CONCANAVALIN A ON THE PHOSPHORYLATION AND DISTRIBUTION OF NON-HISTONE CHROMATIN PROTEINS

Abstract The mitogen concanavalin A (Con A) initiates a series of alterations of chromatin proteins early after addition to cultures of purified equine lymphocytes. Within 15 min of addition, Con A induces cellular non-histone proteins to bind to the chromatin and stimulates the phosphorylation of specific nuclear acidic proteins. The rate of phosphorylation of phenol-soluble nuclear acidic proteins increases as much as 4-fold within 2 hours of Con A stimulation. Phosphorylation is maximal 8 hours after Con A addition. The amount of phenol-soluble non-histone protein associated with the DNA increases as much as 4-fold within 6 hours of Con A stimulation. Con A also stimulates the synthesis of nuclear acidic proteins. Cycloheximide inhibits nuclear acidic protein synthesis by more than 90% but does not appreciably diminish the Con A-induced increase in the amount of these proteins entering the nucleus. Prelabeling of lymphocytes with [14C]leucine followed by treatment with Con A in unlabeled medium results in increased labeling of nuclear acidic proteins, indicating that presynthesized proteins are induced to bind to the chromatin in the presence of Con A. Experiments performed with lymphocytes fractionated by nonaqueous procedures indicate that proteins associating with the chromatin in response to Con A are largely of cytoplasmic origin. These results suggest that both a flux of specific proteins from the cytoplasm to the nucleus and an increase in phosphorylation of nuclear acidic proteins precede the extensive gene activation promoted by mitogens.

Nuclear protein synthesis in hamster kidney cells: the effect of pseudorabies virus infection.

Incorporation of [35S] methionine into the nuclei of BHK21-C13 cells after infection with pseudorabies virus indicated that acidextractable protein synthesis diminished rapidly while salt- and urea-extractable protein synthesis remained constant for up to 3 hours after infection. The proteins synthesized and extracted under the above conditions in both exponentially-growing and serum-depleted cells were analyzed by polyacrylamide gel electrophoresis. The major qualitative changes in nuclear protein synthesis resulting from pseudorabies infection are discussed in relation to possible function.

Acidic nuclear protein synthesis in Rous Sarcoma virus infected chick embryo fibroblasts

Acidic nuclear protein synthesis in Rous Sarcoma virus infected chick embryo fibroblasts

Long-term stimulatory effects of adenosine 3',5'-cyclic monophosphate on nuclear and cytoplasmic protein synthesis of rat hepatocytes in primary culture.

Long-term stimulatory effects of adenosine 3',5'-cyclic monophosphate on nuclear and cytoplasmic protein synthesis of rat hepatocytes in primary culture.

Nuclear metabolism. II protein synthesis by isolated calf thymus nuclei

Isolated nuclei from calf thymus gland actively incorporate [ 14C]-amino acids into the nuclear proteins. The energy for protein synthesis in the isolated nuclei is derived from both glycolysis and oxidative phosphorylation. Our results suggest that 2,4-dinitrophenol inhibition of protein synthesis not only occurs because it is an uncoupler of nuclear oxidative phosphorylation, but it may also inhibit the “transport” of necessary constituents in the incubation mixture to the intranuclear sites of synthesis. Amino acid incorporation by the isolated nuclei into protein responded differently to some of the inhibitors of nuclear phosphorylation phorylation such as malonate and maleate. Cytidine and guanosine, which were reported to inhibit nuclear phosphorylation (9), can bring about a substantial stimulatory effect on nuclear protein synthesis especially when ribose-5-phosphate is added to the incubation mixture. Fructose-1,6-diphosphate, on the other hand, stimulated nuclear phosphorylation (9), but caused 50% inhibition of nuclear protein synthesis. The differential effect on nuclear phosphorylation and protein synthesis indicates that the nucleus has different sites or compartments for biochemical activities which are dependent upon or connected with each other and that certain cytoplasmic metabodites can exert a profound effect on the rate and magnitude of various nuclear pathways.

Synthesis of immunoglobulin and nuclear protein in synchronized mouse myeloma cells.

AbstractThe synthesis of immunoglobulin and of nuclear proteins has been studied in synchronized mouse myeloma cells of the C1 line. Synchronization has been obtained by a double thymidine block. C1 cells synthesize immunoglobulin at a relatively constant rate throughout the cell cycle except for mitosis, when a decrease in the rate of synthesis of total protein and of immunoglobulin is observed. Cell synchrony around mitosis is not sufficiently good to determine whether immunoglobulin is synthesized at all. Nuclear protein and in particular histones appear to be synthesized synchronously with DNA during the S phase of the cell cycle.

Unit gravity sedimentation analysis of basic nuclear protein synthesis during the HeLa cell cycle.

Exponentially growing HeLa cells have been separated according to their cell cycle age by sedimenting at unit gravity for 3 hr on a phosphate-buffered sucrose density gradient. Measurements of cell size, cell number, DNA content, and tritiated thymidine incorporation in consecutive portions of the gradient showed that cells in upper fractions were in G1, cells in middle fractions were in S, and cells in lower fractions were in G2. Basic amino acids were rapidly incorporated into nuclear protein during late G1 and S; some incorporation also took place during G2.

Time sequence of nuclear pore formation in phytohemagglutinin-stimulated lymphocytes and in HeLa cells during the cell cycle.

The time sequence of nuclear pore frequency changes was determined for phytohemagglutinin (PHA)-stimulated human lymphocytes and for HeLa S-3 cells during the cell cycle. The number of nuclear pores/nucleus was calculated from the experimentally determined values of nuclear pores/micro(2) and the nuclear surface. In the lymphocyte system the number of pores/nucleus approximately doubles during the 48 hr after PHA stimulation. The increase in pore frequency is biphasic and the first increase seems to be related to an increase in the rate of protein synthesis. The second increase in pores/nucleus appears to be correlated with the onset of DNA synthesis. In the HeLa cell system, we could also observe a biphasic change in pore formation. Nuclear pores are formed at the highest rate during the first hour after mitosis. A second increase in the rate of pore formation corresponds in time with an increase in the rate of nuclear acidic protein synthesis shortly before S phase. The total number of nuclear pores in HeLa cells doubles from approximately 2000 in G(1) to approximately 4000 at the end of the cell cycle. The doubling of the nuclear volume and the number of nuclear pores might be correlated to the doubling of DNA content. Another correspondence with the nuclear pore number in S phase is found in the number of simultaneously replicating replication sites. This number may be fortuitous but leads to the rather speculative possibility that the nuclear pore might be the site of initiation and/or replication of DNA as well as the site of nucleocytoplasmic exchange. That is, the nuclear pore complex may have multiple functions.

Speculative review: A teleological argument for the function of nuclear protein synthesis

1. 1. There is considerable circumstantial evidence that nuclei synthesize proteins. 2. 2. Cellular hypertrophy and hyperplasia are both associated with increased incorporation by isolated nuclei of amino-acids into nuclear proteins. 3. 3. This ability, widely distributed in nature, may be important in the evolution of eukaryotes and for the maintenance of cellular differentiation. 4. 4. If this is so, it must also participate in the genesis of cancer and its persistence. 5. 5. It can be argued that until the role of nuclear protein labelling is comprehended, any conception of gene function in higher organisms will be necessarily incomplete.

Early increase in nuclear acidic protein synthesis after SV40 infection.

WHEN the replication of density-inhibited cultures of fibroblasts1,2 is stimulated by a change of medium3–5, the rate of nuclear acidic protein synthesis10–13 is immediately increased. We wanted to determine whether the induction of cellular DNA synthesis by an oncogenic virus6–9 is also preceded by nuclear acidic protein synthesis. We report here the effect of SV40 virus infection of a permissive and a non-permissive cell line on their synthesis of total cellular proteins and nuclear acidic proteins in the first hours after infection.

Evidence for IgG-immunoglobulin synthesis by isolated rat spleen nuclei

Rat spleen nuclei, isolated with isotonic sucrose solution containing 3.0 mM MgCl 2, were found to incorporate radioactive amino acid into protein which was precipitable with rabbit antiserum against rat IgG-immunoglobulin. This finding was closely examined from various points of view. The antiserum-precipitable radioactivity was strongly inhibited by deoxyribonuclease, respiratory poisons, and puromycin. Ribonuclease did not significantly block this radioactivity Antiserum-precipitable radioactivity which was inhibited by deoxyribonuclease was completely restored by the addition of polyanion. These facts were all characteristic of nuclear protein synthesis in agreement with the observation on bulk acid-insoluble radioactivity and the results observed in other systems of nuclear protein synthesis. The method of identification by immunological technique was extensively examined excluding possible nonspecific inclusion of intrusive radioactive materials and excluding also possible spurious coprecipitation of irrelevant radioactive polypeptides. The ratio of IgG-immunoglobulin to whole labeled nuclear proteins was approx. 3%, judging from radioactivity counted in the supernatant after ultracentrifugation of incubated and sonicated nuclei.

Depressed RNA synthesis in the brains and livers of thyroidectomized, normal and hormone injected rats

Depression of rat brain and liver RNA synthesis was seen at 22 and 90 days subsequent to neonatal radiothyroidectomy. Cerebral RNA synthesis (per unit DNA) was depressed by 16% ( P < 0.001) while hepatic RNA synthesis was reduced by 43% ( P < 0.01). Nuclear protein synthesis was not detectably altered. RNA and protein synthesis determinations were conducted, using isolated nuclei in standardized in vitro assay systems which circumvented complications (often seen in vivo approaches) such as pool size changes, cell membrane transport alterations and mediation of blood-brain barrier related effects. Chronic daily administration in vivo of l-thyroxine (10 μg/100 g body weight) restored the depressed cerebral synthesis of RNA to normal levels while daily administration of bovine growth hormone (100 μg per rat) did not.

Nuclear RNA and protein synthesis during early sea urchin development

RNA and protein synthesis in isolated nuclei from developing embryos of Lytechinus variegatus was investigated between the two-cell stage and the hatched blastula. Both in vivo and in vitro incorporation studies were performed, and comparison between the two types of experiment shows that the observed labelling of nuclear RNAs cannot be attributed to a transfer of RNA from cytoplasm to nucleus during mitosis, or to cytoplasmia contaminants. Both RNA and protein synthesis increase gradually from the two-cell stage. Messenger RNA can be detected from the first mitosis and rRNA appears soon thereafter. Protein synthesis increase simultaneously in an exponential way. The questions of the onset of RNA and protein synthesis and of the participation of the newly-made messengers are discussed.

Dissociation of nuclear DNA replication from concomitant protein synthesis in synchronous cultures of chlorella

The interrelationship between nuclear DNA replication and cytoplasmic protein synthesis in Chlorella was studied by use of hydroxyurea and cycloheximide as specific inhibitors. 15 μM cycloheximide completely blocked the synthesis of protein and depressed DNA and RNA formation by about 90 %. Protein synthesis resumed immediately after removal of the inhibitor, while DNA synthesis was fully restored after a lag time of about 2 h. 10 mM hydroxyurea completely inhibited DNA synthesis. A decrease in protein and RNA synthesis gradually ensued after about 3 h of exposure. DNA synthesis was restored immediately after removal of the inhibitor, but the final DNA levels were markedly lower than in control cultures. During the recovery from the hydroxyurea treatment about 50 % increase of the DNA content was observed in the presence of cycloheximide. The results suggest that simultaneous synthesis of specific proteins required for DNA replication occurs during the S phase. These proteins continue to be synthesized when nuclear DNA replication is stopped by specific inhibitors, and can then be used to support subsequent DNA replication in the absence of protein synthesis.

Development of herpes simplex virus in the isolated nuclei of HEp-2 cells. I. Viability of isolated HSV-infected nuclei.

A study was made to determine whether herpes simplex virus (HSV)-infected HEp-2 cell nuclei, when isolated post infection (p.i.) and incubated in the absence of cytoplasm, were capable of maintaining synthesis of protein and deoxyribonucleic acid (DNA). Demonstration of synthesis in nuclei isolated post infection was measured by the incorporation of 14C-glutamate into protein or 3H-thymidine into DNA. Both uninfected and HSV-infected nuclei synthesized nuclear protein and DNA for 2 h post isolation (p.e.). Similar rates of in vitro synthesis of nuclear protein were found in uninfected nuclei and nuclei isolated 2 h or 4 h post infection. In vitro synthesis of DNA occurs at about one-fourth the rate in virus-infected nuclei as compared to uninfected nuclei. These data serve as a determination of nucleus viability preceding further studies involving the maturation of HSV in isolated nuclei when incubated free from cytoplasmic control.

Inhibition of nuclear protein synthesis in BHK-21 cells infected with arboviruses.

Host-cell protein synthesis in BHK-21 cells infected with Chikungunya virus (ChV) was markedly inhibited. Strongest inhibition was observed in nuclear and microsomal fractions of the infected cells. Protein synthesis of nuclei isolated from ChV-infected BHK-21 cells has also been suppressed profoundly, whereas infection by dengue virus (DV) did not result in a clear inhibition of nuclear protein synthesis. In addition, infection by these arboviruses brought about a rapid and marked inhibition of DNA-dependent RNA polymerase activity in BHK-21 cells. However, the inhibitory effect of DV was apparently less marked than that of ChV. From the results of preliminary studies, it was suggested that an inhibitory factor produced during the early stage of ChV infection is responsible for the inhibition of nuclear protein synthesis and RNA polymerase activity in host cells. This inhibitor is found mostly in the microsomal fraction of infected cells.

Labelling of proteins by isolated rat liver nuclei.

Rat liver nuclei, isolated in hypertonic sucrose solution and washed with Triton X-100, incorporate radioactive amino acids into hot trichloroacetic acid insoluble materials.Optical and biochemical evidence of nuclear purity is presented. The temperature-dependent incorporation continued for 20–30 min, and was proportional to the concentrations of both nuclear protein between 0.5–1.5 mg/ml, and radioactive amino acid. The radioactive product was degraded by pronase, and a number of inhibitors reduced incorporation, but only if present at [Formula: see text]. Proteins extracted from labelled nuclei and microsomes and examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis at pH 7.2 exhibited different patterns of radioactivity. This provides further support for the concept of protein synthesis intrinsic to rat liver nuclei.A teleological argument for the function of nuclear protein synthesis is discussed.