Increased histone mRNA levels during inhibition of protein synthesis

Inhibition of protein synthesis by cycloheximide or puromycin specifically increases the amount of translatable histone mRNA in exponentially growing and in synchronous G₁ HeLa cells by 5-fold in 3 hours. In this case histone gene expression is uncoupled from DNA replication. We conclude that the level of histone mRNA is regulated by a labile protein and is only indirectly dependent on DNA synthesis.     

Effects of phenobarbital on protein synthesis and polysome levels in rat liver.

Administration of phenobarbital to rats increases the rate of synthesis of certain microsomal drug-metabolizing enzymes in a selective manner and promotes proliferation of smooth endoplasmic reticulum in the liver. Phenobarbital increased a number of factors by which protein synthesis could be enhanced in the liver. It produced a 30% increase in the amount of ribosomes and mRNA per cell. The proportion of ribosomes associated with polysomes was increased by 5-10% over normal liver. There was a 10-30% increase in the rate of ploypeptide elongation and a small increase or no change in polysome size, indicating that the rate of polypeptide initiation was increased proportionately. The product of these effects accounts for the 1.5-fold increase in the rate of total protein synthesis previously reported. The average polysome size, and the size of free polysomes in particular, was maintained when actinomycin D was administered to phenobarbital-pretreated rats, suggesting that the rate of mRNA degradation was decreased selectively. Phenobarbital did not, however, affect the distribution of ribosomes between the free and membrane-bound states or the activity of ribonucleases associated with isolated free and bound polysomes. Thus, we conclude that phenobarbital stimulates protein synthesis by expanding the mRNA pool, at least partially through effects on mRNA degradation, and by augmenting the rate of mRNA translation.     

Effects of exposure temperature on brown adipose tissue uncoupling protein mRNA levels

The effect of temperature on the amount of uncoupling protein mRNA in rat brown adipose tissue was examined after 1 and 14 days of exposure to cold. The relative amounts after 1 day, compared with rats kept at a thermoneutral temperature of 28 degrees C, were 3.2 at 19 degrees C, 3.3 at 11 degrees C, and 2.1 at 3 degrees C. This suggests that in warm-acclimated rats, a maximal response to a cold stimulus in brown adipose tissue is reached by 19 degrees C. In contrast to these results, the relative amounts of uncoupling protein mRNA after 14 days of cold exposure, compared with rats left at 28 degrees C, were 1.2 at 19 degrees C, 1.9 at 11 degrees C, and 2.1 at 3 degrees C. Since it is known that the amount of uncoupling protein in cold-acclimated rats increases continuously with decrease in temperature, the amount of protein reflects the mRNA levels during later times but not the initial time of exposure to cold.     

Sterol-mediated suppression of HMG-CoA reductase mRNA levels in cultured cells requires protein synthesis

The mRNA levels for HMG-CoA reductase were measured in Chinese hamster lung Dede cells or ovary CHO cells by Northern blot analysis. It was observed that 25-hydroxycholesterol decreased the level of reductase mRNA by 40 to 70% in a 6 hour treatment. Inclusion of cycloheximide in the culture prevented the decrease observed with 25-hydroxycholesterol alone. Pretreatment of cells with 25-hydroxycholesterol for 6 hours prior to cycloheximide addition reveals that the protein synthesis inhibitor can return the suppressed levels of reductase mRNA back to control levels. Thus, protein synthesis is required for 25-hydroxycholesterol dependent suppression of HMG-CoA reductase mRNA.     

Studies on protein content and yield levels in rice

Summary Crosses between low and high protein varieties revealed the dominance of low protein over high protein content. The number of desirable segregants with the double combination of high protein and yield were scored in each generation. The increasing frequency of desirable segregants from the F₂ to the F₃ generation in all the crosses increase the chances of selecting desirable recombinants for propagating improved rice varieties. Hybridisation followed by selection may help in developing varieties with high protein content and superior yield potential.     

Gibberellic acid and abscisic acid modulate protein synthesis and mRNA levels in barley aleurone layers

Using in vivo pulse labeling, changes in the pattern of protein synthesis were detected in isolated barley aleurone layers treated with fibberellic acid (GA₃). GA₃ greatly altered the relative rates of synthesis of many polypeptides, increasing some, notably -amylase, and decreasing others. -Amylase synthesis increased until it was the major product (over 60%) of protein synthesis after 24h. The pulse-labeled pattern of secreted polypeptides was also changed by GA₃. There was the expected increase in -amylase together with a number of other polypeptides but there was reduced secretion of several polypeptides also.Cell-free translation of RNA isolated from control and hormone-treated tissues was used to measure changes in mRNA levels. GA₃ caused many changes, particularly in the level of mRNA for -amylase. In vitro synthesized -amylase, identified by immunoaffinity chromatography, had an Mr of 46 000. This polypeptide was partially processed to a polypeptide with Mr 44 000 by the addition of dog pancreas membranes to the in vivo translation mixture. The level of mRNA for -amylase began to increase 2–4 h after GA₃ was added and reached a maximum level of about 20% of total mRNA after 16 h. Thus after 16 h, the synthesis of -amylase as a proportion of total protein synthesis, continued to increase while the level of its mRNA as a proportion of total mRNA remained constant. These results indicate that protein synthesis was modified more extensively than we can account for by changes in mRNA.Abscisic acid (ABA) reversed all of the effects of GA₃ on protein synthesis and mRNA levels. It also promoted synthesis of a small number of new polypeptides and increased the level of some mRNAs. GA₃ reversed the accumulation of ABA-promoted mRNAs. Although, ABA strongly suppressed the increase in the level of translatable mRNA for -amylase, there was an even stronger inhibition of enzyme synthesis and accumulation.We conclude that both GA₃ and ABA regulate protein synthesis both positively and negatively in aleurone cells largely by regulating levels of mRNA and in the case of -amylase, possibly also by changing the efficiency of translation of its mRNA.     

Requirement of protein synthesis for the coupling of histone mRNA levels and DNA replication

H1 and core histone mRNA levels have been examined in the presence of protein synthesis inhibitors with different mechanisms of action. Total HeLa cell RNAs were analyzed by Northern Blot hybridization using cloned human histone genes as probes. Inhibition of DNA replication resulted in a rapid decline in histone mRNA levels. However, in the presence of cycloheximide or puromycin, H1 and core mRNAs did not decrease in parallel with DNA synthesis, but were stabilized and accumulated. Inhibition of DNA synthesis with hydroxyurea after the inhibition of protein synthesis did not lead to a decline in histone mRNA levels. These results suggest that synthesis of a protein(s)--perhaps a histone protein(s)--is required for the coordination of DNA synthesis and histone mRNA levels.     

Effects of protein kinase C activation on prostaglandin production and cyclooxygenase mRNA levels in ovine astroglia

We examined effects of protein kinase C (PKC) activation by phorbol dibutyrate (PDB) on prostaglandin production in astroglia. Astroglia were cultured from sheep fetal cortex and grown in Eagle's basal media supplemented with 10% fetal calf serum (BME-C). Prostaglandin F_2a (PGF_2α) levels in media were determined at 2–24 hours after exposure to PDB. PDB increased production of PGF_2α at 10⁻⁸M and 10⁻⁶M. In addition, PDB increased the ratio of membrane to cytosolic PKC. Coapplication of H7 [1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine] (10⁻⁴M) with PDB (10⁻⁶M) inhibited PDB-induced PGF_2a production. To investigate the role of protein synthesis in increased prostaglandin production by PDB, astroglia were coincubated with actinomycin D (1 mg/ml) or cycloheximide (10 mg/ml). At 4 hrs, both actinomycin D and cycloheximide inhibited increases in PGF_2a in response to PDB application. In addition, COX-2 mRNA levels and COX activity levels were examined. PDB increased COX-2 mRNA levels by 2 hours, and COX activity tripled after 12 hr exposure to PDB. In addition, the increase in COX activity was blocked by cycloheximide. In summary, PKC activation promotes enhanced prostaglandin production via an increase in COX synthesis.     

Effects of blue and red light on unrolling of rice leaves

Unrolling of the second leaf of 8-day-old rice (Oryza sativa L.) seedlings was promoted by weak blue light (B), but not by red light (R). The effect of B was counteracted by irradiation with R just before or after the B. The counteracting effect of R was reversed by subsequent irradiation with far-red light but not by B, even if B was applied for 10 h. The B was effective when the region 0.5–2 cm from the tip of the leaf was irradiated. These results indicate that in rice photoreceptors for blue light located in the region 0.5–2 cm from the tip of the leaf play a key role in leaf unrolling and that a B-absorbing pigment and phytochrome participate in leaf unrolling in a closely related manner.Abbreviations B blue light - R red light - FR far-red light - W white light - D dark This work was presented at the Annual Meeting of the Japanese Society of Plant Physiologists on April 4, 1978, in Hiroshima     

Effects of castration on the epididymis of a non-mammalian vertebrate: evolution of morphology, protein synthesis, and of specific mRNA levels

The lizard epididymis is an androgen-dependent organ undergoing large variations in its structure and in protein synthesis ability during its annual cycle. It produces a major androgen-dependent protein, the L protein. This work reports the effects of castration performed at 3 prominent points of the sexual cycle: stage 1 (epithelium reorganization) stage 3 (onset of secretory activity) stage 6 (full secretory activity). Evolution of various parameters (organ weight, histology, amount of soluble proteins, rate of soluble protein synthesis and of specific protein synthesis: L protein, and mRNA levels) was considered over a period ranging from 7 days to 15, 30 and 60 days. Deprivation of the testis was followed by an organ involution which was more or less accentuated or more or less rapid according to the stage of the operation but some peculiarities need to be emphasized. At first, the evolution of the organ was not stopped but it proceeded: at stage 1, there was cell division and a correlated increase in total protein synthesis (without L protein synthesis), at stage 3 total protein synthesis and L protein mRNA levels increased (synthesis of L protein proceeded up to 30 to 60 days), at stage 6 the involution was accelerated. These effects concerning stage 1 and particularly stage 3 appeared as a kind of a paradoxical induction. Secondly, the epithelium underwent phases of destruction and regeneration which were obviously not controlled by the testis.     

高温对水稻叶片蛋白质表达的影响

为明确高温对耐热性不同水稻品种叶片蛋白质表达的影响,以耐热性不同的2个籼稻品种双桂1号(不耐热)和黄华占(耐热)为材料,分别于苗期、减数分裂期及抽穗(始穗后0—10d)和灌浆早期(始穗后11—20d)进行高温处理,之后取材并采用双向凝胶电泳技术研究高温对不同水稻品种叶片蛋白质表达的影响。结果表明,高温胁迫导致叶片中蛋白质的变化呈4种状况:新蛋白质的产生,一些蛋白质表达量上调,一些蛋白质的表达被抑制,一些蛋白质表达量下调。蛋白质表达变化在两品种以及4个处理时期的表现不同,总体表现为在热敏感品种中表达谱发生变化的蛋白质总数高于耐热品种。质谱分析表明,差异蛋白质主要涉及光合作用和信号转导,该类蛋白质在热敏感品种中表现为不表达或表达量下降,而在耐热品种则表现为有新诱导的蛋白质的产生或表达量上调,表明参与光合作用和信号转导的蛋白质在水稻耐热机制中发挥了重要作用。     

Effects of acid stress on polyamine levels,ion efflux,protective enzymes and macromolecular synthesis in cereal leaves

Wheat or oat leaf segments were floated on acid solutions and the changes in several parameters measured. The putrescine content of wheat leaves was 12.8-fold greater at pH 3.5 than at pH 6 after 72 h. The increase in putrescine was accompanied by an increase in the amount of K⁺ efflux from the leaf tissue to the external solution. The activities of superoxide dismutase and catalase of wheat leaves at pH 3.5 decreased to 51% and 40%, respectively, of their levels at pH 6 by 24 h. [³H]Uridine and [³H]leucine incorporation into macromolecules in oat leaves (with the lower epidermis removed) decreased to 58% and 28% of the control in response to acid stress in 8 h, at the same time as a 5-fold increase in putrescine. When DL--difluoro-methyarginine was added to the pH 3.5 buffered solution, the effect of acid was slightly less, with the incorporation into macromolecules being 64% and 35% of the pH 6 control. The results indicated that the putrescine accumulation under acid stress was concomitant with ion efflux, and a decrease in both macromolecular synthesis and the activities of superoxide dismutase and catalase in cereal leaves.Abbreviations Put putrescine - Spd spermidine - Spm spermine - SOD superoxide dismutase - CAT catalase - ADC arginine decarboxylase - DFMA DL--difluoromethylarginine This paper is dedicated to Dr. A.W. Galston in honor for his great achievements and in recognition of his continuing contributions to the study of plant science.     

适于蛋白双向电泳的水稻叶片样品提取方法初探

在水稻基因组测序完成后,利用蛋白质组学技术揭示水稻基因功能的研究,已成为水稻分子生物学研究的热点之一。水稻叶片作为DNA研究的便利材料被经常使用,但对蛋白质研究来说,占叶片全蛋白50%～60%的核酮糖二磷酸羧化酶（RuBP羧化酶）对低丰度蛋白常常造成掩盖。以水稻叶片为材料,用不同浓度的聚乙二醇（PEG）去除叶片中RuBP羧化酶。通过SDS-PAGE垂直电泳比较发现,浓度为17%的PEG对去除RuBP羧化酶效果最好,所获得的蛋白质样品可以得到质量较高的双向电泳图谱。