Importance of cytomatrix-bound polysomes to synthesis of lysine-containing proteins in triticale germs under ABA treatment

The influence of exogenous abscisic acid (ABA) on the content of free polysomes (FP), membrane-bound polysomes (MBP), cytoskeleton-bound polysomes (CBP) and cytomatrix-bound polysomes (CMBP) in triticale germs as well as in vitro protein synthesis by these four polysomal fractions were studied. During translation, proteins were biotinylated for chemiluminescence detection. We have found that ABA changed both the content of FP, MBP, CMP and CMBP in germ tissue, and their subsequent translation activity. At 100 μM ABA, the content of FP and MBP was over fourfold lower compared to the control, whereas the amounts of CBP and CMBP were about two- and threefold higher, respectively. Moreover, the estimation of the share of polysomes in each ribosomal fraction (sub-units, monosomes, polysomes) showed that, at 100 μM ABA, cytomatrix-bound polysomes, which constituted 90% of polysomes, were the predominant class in ABA-treated germs while membrane-bound polysomes, which made up 82% of polysomes, dominated in the control. A high level of CMBP in ABA-treated tissues may indicate that this class of polysomes participates in ABA-induced synthesis of proteins. In turn, the inhibition of MBP under ABA-treatment is probably due to the delayed protein synthesis which takes place on these polysomes. We identified two lysine-containing proteins synthesized on both of the above classes of polysomes, whose synthesis was altered due to ABA application. Synthesis of a 47 kDa protein on MBP was inhibited, while synthesis of a 79 kDa protein on CMBP is strongly enhanced by ABA influence. The importance of these findings is discussed.     

PROTEIN SYNTHESIS IN PANCREAS OF FASTED PIGEONS

The regulation of protein synthesis in the pigeon has been studied by comparing the capability of cell-free amino acid incorporating systems of membrane-bound and membrane-free polysomes prepared from fasted and fed birds. New methods were developed for isolating polysomes since techniques used for other tissues did not provide quantitative recovery of polysomal RNA. The sucrose gradient profile of polysomes from pigeon pancreas showed a predominance of trisome species. Although initiation factors are present on polysomes, it was found that polysomes in cell-free systems would not initiate protein synthesis without exogenous initiation factors. This suggested the presence of an inhibitor or regulator of protein synthesis. These studies show that fasting resulted in: (a) decreased amounts of polysomes; (b) disaggregation of polysomes to monosomes; (c) decreased capability of polysomes to synthesize nascent peptides and to initiate additional synthesis, apparently not related to concentration of initiation factors.     

Differential kinetics of changes in the state of phosphorylation of ribosomal protein S6 and in the rate of protein synthesis in MPC 11 cells during tonicity shifts

Mouse myeloma (MPC 11) cells respond rapidly to hypertonic conditions by shutting down protein synthesis at the level of polypeptide chain initiation. Translational activity recovers equally quickly upon a return to isotonicity. Disaggregation and reformation of polysomes occur in parallel to the changes in protein synthesis. Ribosomal protein S6 becomes dephosphorylated under hypertonic conditions and rephosphorylated when isotonic conditions are restored. The kinetics with which these changes occur are, however, too slow to account for the changes in protein synthesis. Treatment of the cells with a low concentration of cycloheximide allows reformation of polysomes under hypertonic conditions; conversely, puromycin prevents the restoration of polysomes which otherwise occurs on return to isotonicity. Neither inhibitor prevents the changes in S6 phosphorylation resulting from the tonicity shifts. We conclude that the overall extent of phosphorylation of S6 neither regulates nor is determined by the rate of protein synthesis and is not obligatorily related to the proportion of ribosomes in polysomes.     

Protein synthesis in homologous and heterologous cell-free systems from chick embryo connective tissues.

Two homologous systems for cell-free protein synthesis from chick embryo connective tissues are described. Both the skin polysomes and the wing-leg polysomes are active in collagen synthesis, but they have different requirements for optimum protein synthesis. Protein synthesis was not dependent on tissue-specific factors, since heterologous preparations of supernatant enzymes or initiation factors were able to stimulate maximum protein synthesis with each fraction of polysomes.     

Effect of osmotic stress on the formation of a population of polysomes and their stability in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) seeds

Plants growing under natural conditions are constantly exposed to various stress factors, which can restrain their productivity and limit yields. This paper deals with the effect of long- and short-term osmotic stress followed by recovery on the formation of polysomes and their stability during germination of pea (Pisum sativum L.) seeds. By isolating polysomes, it is possible to obtain an index which evidences the ability of tissues to synthesize proteins. Changes in the distribution of polysomes often precede measurable changes in amounts of proteins. Under osmotic stress, the dominant population of polysomes was the population of free polysomes (FP). The share of membrane-bound polysomes (MBP) and cytoskeleton-bound polysomes (CBP) and cytoskeleton-membrane-bound polysomes (CMBP) in the total fraction of ribosomes increased under intensive (−1.0 and −1.5 MPa) osmotic stress. These results can suggest that the bound forms of polysomes play an important role in the synthesis of stress proteins. In addition, the stability of polysomes isolated from pea early seedlings growing under unstressed control and osmotic stress conditions was tested. It turned out that polysomes formed under osmotic stress conditions (especially the CMBP) were more resistant to the activity of exogenous ribonucleases than the polysomes in the control samples. Under stress conditions it is highly likely that ribosomes become more densely packed on mRNA thus making it more resistant to ribonuclease. This is just one of the many mechanisms regulating stability of mRNA.     

Effect of Water Deficit on the Growth of Squash Fruit

The growth of squash fruit was followed in the field under normal and drought conditions. The following phenomena were observed: a) under drought conditions the fruit growth is normal in the night while it becomes more and more reduced with progress of time in the daylight period; b) the decrease of growth capacity in the daylight period does not correspond to a dehydration of the fruit tissues and it is accompanied by a conversion of polysomes to monosomes; c) the water deficit also causes a decrease of RNA and protein content in the fruit tissues, but this situation does not affect the rate of growth in the night. The data suggest that under conditions the fruit growth is controlled by water availability through protein synthesis; under ample water supply, water availability or some other factor, rather than RNA and protein level, is limiting the fruit growth.     

Respiration and protein synthesis in nongrowing cultured pear fruit cells in response to ethylene and modified atmospheres: a model system for fruits postharvest

The respiration of pear fruit (Pyrus communis L. Passe Crassane) cells was monitored after subculture into an auxin-free, mannitol-enriched medium in which the cells remained viable but did not grow. Respiration rates were affected by the presence or absence of sucrose in the medium even though the cells retained reserves of sucrose and starch. Provided the medium contained respirable carbohydrate, exposure to ethylene (1-10 microliters per liter) increased the respiration rate with some acceleration of cell death. In the range from 10 to 2% oxygen by volume, the respiration rate of the cells decreased with oxygen concentration resulting in some prolongation of cell life. Thus, in their responses to ethylene and modified atmospheres, the cells reflected the behavior of harvested fruits. Having defined conditions under which respiration rate could be varied without apparent influence on the quiescent state of the cells, we sought a connection between maintenance respiration and protein turnover. Relative rates of protein synthesis were assessed by measuring ribosome distribution between monosomes and polysomes. In general, the higher the respiration rate the higher the proportion of polysomes supporting the thesis that protein turnover is a variable component of maintenance metabolism. Protein turnover in cells incubated in the presence or absence of sucrose was measured as retained α-amino-³H following a pulse of ³H₂O. Turnover was shown to be a quantitatively important component of the maintenance budget and to be more rapid in cells in media supplemented with sucrose through the chase period. The experiments illustrate that cultured cells may be used to explore aspects of the maintenance metabolism of resting or senescent cells that are not amenable to study in bulky fruit tissues.     

Possible involvement of the cytoskeleton in the regulation of barley caryopsis dormancy and germination

This study was conducted on barley cv. Ars. caryopses collected at full ripeness and divided into two batches. From one batch (dormant caryopses) polysomes were isolated from embryos immediately after harvesting and after two days of germination. From the other batch (non-dormant caryopses) the same was done after eight months storage in a dry state. A low ionic strength cytoskeleton-stabilizing buffer was used for the isolation of polysomes. Four different fractions of polysomes were examined: free polysomes (FP), membrane-bound polysomes (MBP), cytoskeleton-bound polysomes (CBP) and cytoskeleton-membrane-bound polysomes (CMBP). In germs grown from non-dormant caryopses, the first two fractions (FP + MBP) made up about 78 % of the total ribosomal material, whereas in embryos of dormant, imbibed caryopses, two last fractions (CBP + CMBP) made up about 71 %. The percentage of polysomes after 48 hours of imbibition of dormant caryopses in the FP, MBP and CBP was only about 13 % (i.e., 87 % monosomes), whereas a greater proportion (19.4 %) was found in the CMBP. The highest incorporation of ³H-uridine and ¹⁴C-amino acids (after 48 hours of germination and 0.5, 3 and 6 hrs incubation with precursors) took place in trhc CMBP both in dormant and non-dormant caryopses The major amount of the two polysome fractions associated with the cytoskeleton (CBP and CMBP) and the higher activity of CMBP in protein synthesis in embryos of dormant, imbibed triticale caryopses may indicate a significant role for polysomes associated with the cytoskeleton in the control of protein synthesis in dormant and germinating caryopses.     

Utilization of stored messenger RNA during early aging of Jerusalem artichoke tuber slices

Using dissociation in 0.8 M KCl, it was established that in freshly excised Jerusalem artichoke (Helianthus tuberosus L.) tuber slices less than 8% of the ribosomes were in polysomes. The first hour of aging in water was the period of most rapid polysome accumulation; over 32% of the ribosomes carried nascent polypeptide chains at the end of this time. Thereafter polysome accumulation continued to increase, but more gradually. While synthesis of high-molecular-weight RNA (presumed mRNA) was inhibited more than 95% by -amanitin during the first hour of aging, the inhibitor had no effect on polysome formation. As determined by [³H]polyuridylic acid hybridization, unaged cells contained polyadenylated RNA with a size range of 6–30S. The amount of polyadenylated RNA did not change during the first hour of aging. In control cells in water the in-vivo rate of protein synthesis increased exponentially during the first 4 h of aging without a comparable increase in polysomes. In -amanitintreated tissues a similar increase in protein synthesis was not observed despite the presence of near control levels of polysomes. It is suggested that early polysome formation depends on stored mRNA. Inhibition of mRNA synthesis by -amanitin prevents the normal development of an enhanced rate of protein synthesis which is not directly related to numbers of ribosomes in polysomes.Abbreviations Poly(A) polyadenylic acid - Poly(A)+RNA polyadenylated RNA - Poly(U) polyuridylic acid - TCA trichloroacetic acid     

Intramolecular homologous recombination in Bacillus subtilis 168

Summary Ribosomal protein synthesis is regulated by controlling the fraction of mRNA associated with polysomes. It is known that this value changes in different developmental stages during Xenopus embryogenesis or, more generally, with changing cell growth conditions. We present here an analysis of the proportion of mRNA loaded on polysomes, carried out with probes for five different ribosomal proteins on several batches of Xenopus embryos obtained from different individuals. The results obtained indicate the existence of probe-dependent and individual differences, which reflect genetic variations in the cis- and trans-acting regulatory elements responsible for translational regulation. The fraction of ribosomal protein mRNA loaded onto polysomes can be used as an index of an individual's capacity for ribosome production.     

Cytokinin effect on protein synthesis in vivo in higher plants

The influence of naphthalene-1-acetic acid and kinetin on protein synthesis in vivo was investigated by measuring the incorporation of radioactive amino acids into polypeptides of synthesizing polysomes. The second subculture of sterile pith tissue of Nicotiana tabacum L. cv. Wisconsin 38 grown on a medium containing only a minimum of growth substances was further starved in a small volume of medium lacking auxin and cytokinin for 12 h. An incubation period of 4 h with [¹⁴C]amino acids followed. The last 30 min of those incubations were carried out in the presence of actinomycin D and the last 20 min were performed under different conditions: a) without any growth substances, b) with naphthalene-l-acetic acid, c) with kinetin. By measuring the differences of the specific radioactivities of the polysomes the following results were revealed: 1) Kinetin increases the protein synthesis by an average of 35%-2) Auxin has no effect on protein synthesis.Abbreviations Act. D actinomycin D - NAA naphthalene-1-acetic acid Part of a Diplomarbeit, University of Bonn 1976     

Parallel changes in protein synthesis and messenger RNA content in growing and resting epithelial cells of Xenopus laevis

The regulation of cell growth can be achieved at many levels but ultimately the regulatory factors must alter protein synthesis since growing cells always exhibit an increased rate of protein synthesis compared to resting cells. Some studies using growing and nongrowing mammalian cells have shown that the rate of protein synthesis compared to resting cells. Some studies using growing and nongrowing mammalian cells have shown that the rate of protein synthesis is directly dependent on mRNA content. Other studies have shown that growing and resting cells have similar amounts of mRNA and that protein synthesis is regulated by the proportion of mRNA in polysomes. We have analyzed mRNA content in growing and resting epithelial cells of Xenopus laevis. Quantitation of poly(A)⁺ mRNA by uniform labeling with ³H-uridine and by ³H-poly(U)hybridization demonstrated a direct relationship between mRNA content and the relative rate of protein synthesis in growing and resting cells. Likewise, after serum stimulation of resting cells the increase in mRNA content closely paralleled the increase in protein synthesis. Our results suggest that control of protein synthesis in growing and nongrowing cells is exerted before the translational level.     

Helical polysomes induced by aflatoxin B1 in vivo : A new hypothesis for helix formation by chemicals and carcinogens

We have studied the induction of helical polysomes by aflatoxin B₁ in liver and kidney cells from rat and mouse. We succeeded in giving to reticulocyte polysomes a shape resembling helices after in vitro treatment with O-methylthreonine which is used as an inhibitor of polypeptide chain termination. From this and knowing the site of action of aflatoxin B₁ on rat liver polysomes, we hypothesize that the induction of helical polysomes in tissues from adult animals treated by chemicals or carcinogens is due to the inhibition of release of ribosomes from the messenger RNA (mRNA). Theoretical studies of protein synthesis inhibition are in agreement with this new hypothesis.     

玉米早期根系构型及其生理特性对土壤水分的响应

为了探明玉米早期根系结构及其对土壤水分的生理响应,揭示玉米幼苗的抗旱机理,以蠡玉18为材料,采用盆栽试验,设置轻度胁迫(LS)、中度胁迫(MS)、重度胁迫(SS)和正常供水(CK)4个水分处理,系统研究从播种开始持续水分处理对夏玉米苗期根系形态结构及活力、保护酶系统及生理调节物质的影响。结果表明:随着水分胁迫程度的加剧,玉米根长、根表面积、根体积和根干重等各形态指标较CK下降幅度逐渐增大,不同水分胁迫使夏玉米苗期根系结构存在差异。轻度和中度胁迫显著增加了细根(0.05—0.25 mm)根长和根表面积比例,重度水分胁迫显著降低粗根(0.50 mm)根长与根表面积比例。玉米苗期根冠比、根系活力和丙二醛(MDA)含量随水分胁迫程度的增强而上升,随着胁迫时间的延长,根冠比逐渐降低。根系可溶性蛋白含量随土壤水分含量的下降而下降,MS、SS处理较CK显著降低(P0.05)。夏玉米根系中SOD对水分胁迫较CAT、POD更敏感,轻度水分胁迫下主要依赖CAT、中度水分胁迫下主要依赖POD、重度水分胁迫下主要依赖SOD来降低氧化伤害;且重度胁迫下,随着胁迫时间的延长保护酶活性下降。苗期玉米通过增加根冠比、增强根系活力和不同保护酶活性及降低可溶性蛋白等渗透调节物质来协同减少水分胁迫的危害。     

Initiation of endogenous messenger RNA translation on hen oviduct polysomes.

The eIF-2A fraction of reticulocyte ribosomal salt wash is capable of maximally stimulating the translation of endogenous messenger RNA by hen oviduct polysomes. The factor increases the initiation of protein synthesis 2--3-fold when measured by the factor-dependent synthesis of NH2-terminal peptides. The addition to these polysomes of elongation factor, EF-1, also increases protein synthesis but at a distinctly different rate and Mg2+ concentration optimum than the eIF-2A fraction. Moreover, there is no stimulation of NH2-terminal peptide synthesis with EF-1 alone. In contrast, all the known initiation factors are required for the translation of exogenous globulin mRNA on oviduct polysomes. Reticulocyte polysomes isolated by an identical procedure to that used for oviduct polysomes or by standard methods also require all the initiation factors for the translation of either endogenous mRNA or exogenous ovalbumin mRNA. Addition of 7-methylguanosine 5'-monophosphate does not inhibit the factor-dependent stimulation of oviduct polysomes except at high concentrations (1.0 mM) indicating that the sites with which 7-methylguanosine 5'-monophosphate normally competes are already occupied. These findings suggest that the messenger RNA remains bound to the oviduct polysomes or initiation factors. Hence the addition of exogenous factors which are involved with mRNA recognition and binding to the ribosome are not required. It has been previously shown that eIF-2A is capable of binding in vitro the initiatior tRNA to an existing Ado-Urd-Gua-40 S complex and initiating protein synthesis when such a complex is present. These present studies indicate that such an initiation complex may exist within the oviduct cell on membrane-associated polysomes. Under these circumstances eIF-2A mediates binding of the initiator tRNA and initiates protein synthesis.     

Brain ribosomes in intracranial hypertension

Abstract— Increased intracranial pressure was produced by perfusion of cerebrospinal fluid (CSF) at various pressures into the lateral ventricles of adult Sprague-Dawley rats with bilateral chronic intraventricular cannulas. When CSF perfusion was carried out at pressures of 150, 300 or 600 mm of water, brain polysomal profiles were similar to controls. Rats perfused under a pressure of 1500 mm water for 30 min were comatose, had slow electroencephalograms and showed a fall in brain polysomes from 66 to 24 per cent of the total ribosomes (P < 0.01) while ribosomal monomers and dimers increased. These monomers and dimers were completely and reversibly dissociated into subunits in 500 niM KC1 buffers, unless prefixed in formaldehyde. [³H]leucine incorporation into brain ribosomes in vivo was decreased by severe intracranial hypertension. In cell-free systems in vitro, pathological ribosomes were less active in protein synthesis than controls (P < 0.01) but were at least as readily stimulated by poly U. After intracranial pressure was returned to normal, there was a progressive reassociation of ribosomes into polysomes, even in the presence of Actinomycin D. These findings suggest that during severe intracranial hypertension cerebral protein synthesis is inhibited, perhaps through reversible inactivation of the translation of messenger RNA.