High Level Of Protein Synthesis Research Articles

Ultraviolet-inducible proteins in Chlamydomonas reinhardtii

DNA damage induces the synthesis of a number of proteins, many of which may also have a role in recombination, such as the RecA protein of Eschericha coli (McEntee, 1977). Irradiation with U.V. light has been found to induce this and other proteins in E. coli (Bagg et al., 1981; Witkin, 1976) and related proteins in yeast (Angulo et al., 1985; Rolfe, 1985). The present work forms part of an investigation into DNA recombination and repair mechanisms in plants and, in particular, attempts to identify proteins produced in response to, or associated with, DNA damage. Preliminary studies have been carried out using the alga Chlamydornonas reinhardtii because of the ease with which uniform irradiation of the organism could be obtained. This investigation will be extended to higher plants. C . reinhardtii cells were treated with U.V. light and subsequent protein synthesis was examined by labelling in vivo with [3sS]methionine followed by SDS/polyacrylamide-gel electrophoresis. The labelled proteins were then visualized using fluorography. In these experiments U.V. dosage was limited to achieve a high level of cellular survival (> 90% survival of wild-type cells), and therefore maintain a high level of protein synthesis. Such a protocol has previously been sucessfully employed with yeast to detect the induction of proteins after u.v.-induced DNA damage (Ruby et al., 1983; McClanahan & McEntee, 1984). In the present study, increases in the level of U.V. irradiation were not observed to cause any further alteration of the pattern of protein synthesis but did cause a large reduction in the incorporation of label. Protein synthesis was significantly reduced in cells immediately after treatment with even low levels of U.V. light so a short incubation period was employed between treatment and the addition of [.S]rnethionine to allow the level to return to near normal. This preincubation would also allow time for the synthesis of mRNAs of any proteins induced by the treatment. These experiments showed that the synthesis of two high molecular weight polypeptides is consistently specifically increased after low level irradiation (Fig. 1). The molecular masses of these polypeptides were estimated at approx. 71 kDa and 65 kDa. Inhibitors of protein synthesis on organellar or cytoplasmic ribosomes were used in conjunction with the U.V. irradiation and [S]methionine labelling to identify the site(s) of synthesis of these two polypeptides. The inhibitors used were anisomycin, which is known to inhibit cytoplasmic protein synthesis (Grollman, 1976); Neth et al., 1970), and lincomycin, which is known to inhibit organellar protein synthesis (Ellis & Hartley, 1971; Ellis, 1975). After treatment with U.V. light in the manner described above, cells of C . reinhardtii were labelled in vivo in the presence of neither, one or both of these compounds. Neither polypeptide was produced in cells labelled in the presence of anisomycin but both were present in lincomycin-treated cells (data not shown). Thus the results of these studies indicate that both the 7 1 kDa and 65 kDa polypeptides are synthesized on cytoplasmic rather than organellar ribosomes. A variety of DNA-damaging treatments are now being tested against cells of C. reinhardtii to determine the specificity of the conditions required to induce synthesis of these and any other polypeptides found to be associated with DNA damage. Attention is also being paid to proteins induced by heat shock, since a protein of 70 kDa has been reported to be induced in C. reinhardtii after such treatment 0 2 0 4 0

Localization and sequence of a vaccinia virus gene required for multiplication in human cells.

A vaccinia virus host range mutant (hr mutant) deleted of 18 kilobase pairs at the left end of the genome has been employed to precisely map a viral gene required for multiplication in human cells. DNA fragments from the wild-type virus were inserted into the thymidine kinase gene of mutant virus by means of in vivo homologous recombination, and the recombinants obtained were screened for their ability to multiply in human cells. A short sequence, 855 base pairs long, overlapping the HindIII M and K fragments was able to restore a normal host range on the mutant virus. A single long open reading frame that could encode a polypeptide of 32.5 kDa was found in the nucleotide sequence of the host range gene. The direction of transcription and the length of the open reading frame are in excellent agreement with previous mapping of mRNAs within this region of the genome. In vitro translation of infected cell early mRNA, selected by hybridization to the host range gene, yielded a prominent polypeptide product whose size (29 kDa) was close to that expected from the predicted amino acid sequence. The phenotype of the hr mutant suggests that the host range gene plays a role in maintaining a high level of protein synthesis in human cells. It may behave positively by complementing the lack of an analogous cellular activity or negatively by antagonizing a cell function that inhibits viral multiplication.

Regulation of synthesis of larval serum proteins after transplantation of larval fat body into adult Drosophila melanogaster

The larval serum proteins, LSP1 and LSP2, of Drosophila melanogaster are synthesized by the fat body during the third instar. We examined the potential for LSP synthesis by fat body implants in adult flies. Fat body from third instar donors will continue to synthesize LSPs in both males and females. Implants from late second instar larvae will start synthesizing LSP1 and LSP2 in females but only LSP1 in males, suggesting that regulation of these proteins is not the same and that the physiological milieu in the two sexes differs. The newly synthesized LSPs are secreted into the hemolymph for approximately 48 hr when secretion stops but synthesis continues. This sequence follows the pattern for LSP secretion in situ. Fat body from mid second instar larvae is variable in its ability to synthesize LSPs. LSPs are not detected in implants from first instar larvae despite there being a high level of protein synthesis in the implant and considerable growth of the fat body cells. We conclude that there is a critical stage of differentiation during the latter half of the second instar when the fat body becomes independent of the larval milieu and can synthesize LSPs in the adult.

Mitochondrial DNA expression in Drosophila melanogaster: neosynthesized polypeptides in isolated mitochondria

The expression of mitochondrial genome of D. melanogaster in isolated mitochondria was followed by incorporation of 35S methionine in neosynthesized polypeptides. A high level of protein synthesis was obtained after optimization of all the incubation parameters. Two kinds of energy-generating systems were used: an endogenous system where an oxidizable substrate were added for ATP synthesis; an exogenous system with an energy-rich compound for ATP regeneration, the latter proved to be the most effective. The effect of the oxidative phosphorylation uncoupler (Clccp), and an ATPase inhibitor (oligomycine) allow us to postulate the role of the electrochemical potential in the expression of the mitochondrial genome. Electrophoresis and autoradiography of neosynthesized mitochondrial proteins exhibits 18 to 24 protein bands, ranging from 6.5 to 65 Kd; incubation of KC 0% drosophila cells with 35S methionine and cycloheximide gave similar results. Both our results and those published elsewhere suggest that the expression of mitochondrial genome in higher organisms could be more complex than simple translation of the 13 genes presents on these genomes.

Kinetic analysis of regulatory events in G1 leading to proliferation or quiescence of Swiss 3T3 cells.

Kinetic analysis of cellular response to serum deprivation or inhibition of protein synthesis was performed on Swiss 3T3 cells. Time-lapse cinematographic analysis of individual cells transiently exposed to serum-free medium (with or without the addition of purified growth factors) or cycloheximide enabled a detailed mapping of the magnitude and variability of cellular response in different parts of the cell cycle. In all cells, in all stages of the cell cycle, serum deprivation resulted in inhibition of protein synthesis, but only in postmitotic cells in the first 3-4 hr of G1 (here denoted the G1pm phase) did it produce cell-cycle arrest. During G1pm, the cells are highly dependent on the continuous presence of serum growth factors and a high level of protein synthesis in order to progress toward mitosis. A 1-hr exposure to serum-free medium or to cycloheximide was sufficient to force most G1pm cells into a state of quiescence (G0), from which the cells required 8 hr to return to G1pm. During G1pm the cells complete the growth factor-dependent processes leading to commitment for proliferation. Thereafter they enter the growth factor-independent pre-DNA-synthetic part of G1 (here denoted G1ps). The commitment process in G1pm could be successfully completed in the presence of platelet-derived growth factor as the only supplied growth factor. Epidermal growth factor and insulin were insufficient for the completion of the commitment processes in G1pm, although they were able to temporarily prevent the G1pm cells from entering G0 during serum starvation. Under conditions optimal for proliferation, the cells complete the commitment processes in G1pm within a remarkably constant time period. Almost all cells in the population left G1pm and entered G1ps between the third and fourth hour after mitosis. The duration of G1ps, on the other hand, showed a large intercellular variability consistent with a transition-probability event. In fact, G1ps accounts for most of the variability in G1 and cell cycle time.

Effects of cholecystokinin octapeptide and hydrocortisone on the exocrine pancreas of fed and fasted 24-hour-old rats: An electron microscopic study

We evaluated by electron microscopy the effects of two hormones, cholecystokinin octapeptide (CCK-8) and hydrocortisone, on the development of the pancreatic acinar cell in newborn rats. The newborn rat pancreas contains abundant zymogen granules of variable size but is unresponsive to secretagogues. In contrast the 24-hr-old pancreas is depleted of granules, indicating emptying of the acinocytes in response to feeding. Zymogen granule content is moderate in the 24-hr-old rat that is fasted from birth. The pancreas from the 24-hr-old rat that is fasted and treated with either hydrocortisone or CCK-8 has an intermediate number of granules. In the animals fasted and treated with hormones, the rough endoplasmic reticulum is arranged into parallel lamellae suggesting a higher level of protein synthesis which may contribute to the formation of abundant zymogen stores. We conclude that zymogen stores are lost earlier postnatally than previously understood, and that zymogen seen in the 24-hr-old animals may represent newly synthesized materials.

MORPHOLOGICAL AND PHYSIOLOGICAL VARIATIONS IN CLONES OF ASTERIONELLA FORMOSA HASSALL

SummaryThe morphology of Asterionella formosa isolated from two contrasting lakes has been studied. During four months in culture the valve length of five isolates decreased. Greater variation in valve length was observed in the strain from Llyn Coron, resulting in the separation of two morphologically distinct clones from the original isolate. Two cultures of the diatom from Llyn Padarn exhibited a more regular decrease in cell size. All strains had similar chlorophyll‐a levels in relation to cell volume.Most rapid growth, but smallest yields, in batch cultures, were characteristic of the two large‐celled clones from Llyn Coron, whereas the small‐celled clone from this lake exhibited the slowest growth rate, but maximum yield. Thus morphological change which is accompanied by physiological differences may occur in a culture derived from a single parent colony. The growth characteristics of the two strains from Llyn Padarn were more similar than those of the three isolates from Llyn Coron.The rapidly growing large‐celled strain from Llyn Coron had a high photosynthetic rate. So had one isolate from Llyn Padarn, but a large proportion of fixed carbon was lost extra‐cellularly by this strain, which may account for its slower growth rate. Photosynthetic rates of the small‐celled isolate from Llyn Coron and the second isolate from Llyn Padarn were low. A range of photosynthetic efficiencies were recorded and these were related positively to the maximum photosynthetic rate. The rapidly photosynthesizing Padarn strain, which had the lower chlorophyll‐a content per cell, was the most efficient. The pattern of distribution of photosynthate for this strain from Padarn differed from the other isolates. In addition to the large proportion lost extracellularly, it had a higher level of protein synthesis. The presence of extracellular products reduced the photosynthetic rate, and differences were noted in the response to light intensity between A. formosa from the two lakes.Morphological and physiological variations were confirmed in A. formosa. The situation is complex, since morphologically similar strains isolated on different occasions from Llyn Padarn showed contrasting physiological behaviour and, during culture, morphological types were distinguished in the isolate from Llyn Coron, which also differed physiologically.

Intensity of RNA and protein synthesis in fibroblasts during wound healing in mice

RNA and protein synthesis in fibroblasts was studied by a double-labeling method on the 3rd and 7th days of wound healing in mice by electronmicroscopic autoradiography. The ratio of protein synthesis in the cytoplasm and RNA synthesis in the nucleus was shown to differ significantly in individual cells, with the result that fibroblasts with relative predominance of one of the two types of synthesis were found at both stages of wound healing. On average fibroblasts showed a higher level of protein synthesis on the 3rd day of healing. This high mean level was maintained by an increase in the proportion of fibroblasts in which protein synthesis predominated over RNA synthesis.

Nucleolar reorganization in liver cells of the aging rat.

The nucleoli of rat liver cells duplicate in great detail the life-long series of reorganizational changes encountered in kidney and intestinal epithelial cells. The ultrastructural components of the large, loosely organized polymorphous nucleoli, which are dominant in the rapidly multiplying stem cells of embryos, are readily accessible for chemical activities. Smaller, more compact amphinucleoli are dominant in more mature cells, which were characterized by Smetana ('70) as "idling" cells, showing slowly continuing ribosome formation and RNP synthesis. In older cells bipartite nucleoli become dominant and are reorganized in increasing numbers from the younger amphinucleoli. These, however, are not replaced in equal numbers from the shrinking pool of polymorphs of young cells which have greatly reduced mitotic potential. Paralleling the shifts in dominant nucleolar types, the high level of protein synthesis declines in older cells not only in the quantity of proteins synthesized but also in kinds of enzymes produced. These fail to meet the structural and functional requirements of aging cells leading ultimately to the onset of age-related degenerative changes. Again it is noted that separation of the karyosomal DNA from the plasmosomal RNA-protein complex of the nucleolus may lead to possible breakdown of the DNA-dependent RNA-protein transcription system ultimately bringing protein synthesis to a very low level in the senescent animal.

Insulin effects on protein synthesis and secretion in primary cultures of amphibian hepatocytes

The objective of the present study was to determine the effects of insulin on amphibian hepatocytes in primary culture. Hepatocytes were isolated from adult bullfrogs by collagenase perfusion and maintained as monolayers in serum-free medium. Cells cultured in the continuous presence of insulin exhibited a relatively constant rate of protein secretion over the first four to five days, whereas controls showed an almost three-fold decrease over the same time period. The decline in secreted proteins was equally represented in most exported proteins, except that serum albumin secretion showed twice as much of a decrease relative to the other proteins. The maintenance of protein secretion by insulin was the result of its effect on protein synthesis. The rate of protein synthesis was measured by the incorporation of (3H)-leucine into protein using culture medium containing 0.5 mM leucine, a condition where the specific radioactivity of leucyl-tRNA was shown to be equal to that of (3H)-leucine in the medium. Cultures maintained with insulin for 60 hours synthesized protein at two to three times the rate found in non-insulin treated controls whose rate of protein synthesis was first detectably decreased after nine hours of culture in the insulin-free medium. Sedimentation profiles of polyribosomes from hepatocytes maintained for 60 hours without insulin showed proportionately fewer ribosomes in large polysomes and more in monosomes and free ribosomal subunits than ribosomes from cells cultured with insulin. This result suggests that the decrease in protein synthesis found in the absence of insulin is due to a defect in initiation. Insulin does not exert its effect by regulating cellular levels of ATP; no change in ATP content was found in cells maintained with or without insulin. The results show that insulin maintains high levels of protein synthesis and secretion in amphibian hepatocytes. The hepatocytes in monlayer culture provide a system to study the molecular mechanisms involved in the translational control of protein synthesis by insulin.

The Influence of Some Metabolic Inhibitors On the Response of Susceptible/Resistant Cultivars of Tomato To Meloidogyne Incognita

The effect of cycloheximide and puromycin (protein synthesis inhibitors) and actinomycin D (RNA synthesis inhibitor) was studied on host parasite interaction between Meloidogyne incognita and susceptible and resistant cultivars of tomato, Lycopersicon esculentum. The concentrations of cycloheximide (1 ppm) and puromycin (40 ppm) which caused great inhibition of root growth, did not affect the galling response or nematode establishment and development in the infected, susceptible cultivar. Thus, a high level of protein synthesis may not be required for initiation of susceptible response to M. incognita infection. However, relatively high concentrations of cycloheximide (2.5 ppm) and puromycin (55 ppm) did inhibit galling and larval development, suggesting that, a certain minimum level of protein synthesis is necessary for establishment of the nematode within the susceptible host. The concentrations of Actinomycin D that were inhibitory to root growth were also inhibitory to galling. In the resistant cultivar, cycloheximide treated infected roots did not exhibit any browning (hypersensitive reaction, HR) of cells surrounding the nematodes within the roots. Our results suggest that lack of HR in these roots many have led to exit of the nematodes from the roots. Hence, inhibition of HR does not confer susceptibility to the resistant plants. Some other factor seems to be necessary for the plants to develop susceptibility to M. incognita. Alternatively, some mechanism of resistance other than HR may be operative within the resistant plants. Puromycin does not show any inhibitory effect on the visible browning of the root tips in the resistant plants.

Effect of actinomycin D on the structure of the chromatoid body in the rat spermatids.

The effect of actinomycin D on the chromadoid body of rat spermatids has been studied by light and electron microscopy, high resolution autoradiography and biochemical methods. Actinomycin D caused structural changes in the chromatoid body of young round nucleated spermatids. The normal irregularily lobulated chromatoid body acquired a ring-like configuration 12 h after an intratesticular injection of 2 microgram of the drug. The labelling of the chromatoid body with 3H-uridine which can normally be seen after 12 h was also abolished by actinomycin D. These observations lead to the suggestion that the chromatoid body contains a store of long-lived mRNA molecules that are activated later during spermiogenesis when transcription in the spermatid nucleus has ceased but a high level of protein synthesis still persists.

Changes in an intranucleolar body in hamster facial neurons following axotomy

Facial motor neurons of adult golden hamsters were studied at frequent intervals up to 25 days after severance of the right facial nerves at their exit from the stylomastoid foramen. All brains were perfusion-fixed and prepared for light microscopy; sections were stained with either thionine, Feulgen reagent, or mercuric bromphenol blue. The intranucleolar body was observed as a rounded, intensely basophilic, Feulgen-negative structure located centrally in the nucleolus of normal adult facial neurons. After axotomy, chromatolysis resulted within 1 day and peaked at about 4 days, with gradual restoration of stainable Nissl substance thereafter. The intranucleolar body in severed neurons was apparently unaffected at 1 and 2 days, but by 4 days was represented by three or four densely staining particles forming a ringlike configuration, which persisted even after complete return of Nissl substance. Only after a postoperative period of 25 days did the intranucleolar body again appear as a single, densely staining structure comparable to that in normal cells. The disappearance of the intranucleolar body during chromatolysis coincided with what is known to be a period of heightened nucleolar synthetic activity. Delay in returning to its definitive appearance, despite a rather prompt reconstitution of Nissl substance, may reflect a persistently high level of protein synthesis in neurons which are prevented from reestablishing their original connections. Evidence indicates that the existence of the body does not depend upon the establishment of terminal connections per se, even in the developing neuron.

Incorporation of 14C-Photosynthate into Protein during Leaf Development in Young Populus Plants

Gas exchange and protein metabolism were studied in expanding, mature, and near-senescent leaves of young clonal Populus x euramericana cv. Wisconsin-5 plants. Dark respiration, CO(2) evolution in the light, and CO(2) compensation concentrations were highest in unexpanded leaves but declined markedly as leaves matured and aged. Net photosynthesis was highest in nearly mature leaves. Fresh weight continued to increase after leaf expansion was complete, whereas soluble protein levels declined. Changes in the distribution of photosynthetically incorporated (14)C indicated that a high level of protein synthesis and rapid formation of structural components occurred only in expanding leaves. Protein turnover was slight in expanding leaves but was substantial after leaves were mature. Expanding leaves synthesized predominantly fraction I protein (ribulose diphosphate carboxylase). However, formation of this protein from photosynthate was slight once leaves matured.

Polyribosome disaggregation and cell-free protein synthesis in preparations from cerebral cortex of hyperphenylalaninemic rats.

Abstract— Seven‐day‐old rats were injected intraperitoneally with l‐phenylalanine (1 g/kg) and the time course of brain polyribosome disaggregation and changes in brain levels of phenylalanine, tryptophan and tyrosine were determined. Disaggregation of brain polyribosomes preceded the increase in levels of phenylalanine in brain, and followed the same time course as depletion of tryptophan from brain.The effects of several metabolites of phenylalanine (which are formed in phenylketonuria) on protein synthesis in vitro was determined for brain and liver systems. None of the compounds tested was inhibitory at concentrations below 10 mM and in all cases hepatic protein synthesis was more sensitive to inhibition than was the corresponding system from brain.Ribosomal dimers, formed in brain after injection of phenylalanine, were incapable of supporting high levels of protein synthesis in vitro, a finding that suggested that the inhibition of protein synthesis in vitro in cell‐free systems of brain tissue after injection of phenylalanine into young rats was mediated by disaggregation of brain polyribosomes associated with tryptophan deficiency in brain.

Liver RNA polymerase activity in genetically obese and lean rats fed cholesterol

Hepatic nuclear RNA polymerase activity (C 14-ATP incorporated into RNA) as well as microsomal protein synthesis (C 14-leucine incorporated into protein) was studied in vitro in groups of genetically obese female rats of the Zucker strain before and after the introduction of a diet containing cholesterol. A similar study was carried out in normal lean rats of the same strain. The obese homozygotes were found to demonstrate a higher level of protein synthesis as well as nuclear RNA polymerase activity than did the lean homozygotes. Following the feeding of the hypercholesteremic diet for either 7 or 28 days, protein synthesis and RNA polymerase activity were shown to be lower in both groups of females. However, these changes in protein synthesis induced by this diet were relatively greater among the obese rats, whereas the concomitant changes in polymerase activity were of the same order of magnitude between genotypes. These findings have been discussed in terms of the suggested “allosteric” nature of the RNA polymerase. Furthermore, the possibility of more than one specific RNA polymerase existing in the rat liver has been speculated to explain certain of the above differences.