Synthesis Of New RNA Research Articles

Polyribosomal protein synthesis in fertilized sea urchin eggs: The effect of actinomycin treatment

The protein synthesis initiated at fertilization in sea urchin eggs occurs, at least in part, on polyribosomes. Eggs treated with actinomycin D show a normal response to fertilization. The actinomycin-treated embryos suffer an 80–90% loss in the synthesis of new RNA with potential template function. In these cells, protein synthesis attains rates as high as those in controls. The actinomycin-resistant protein synthesis also occurs on polyribosomes, and on the basis of ribonuclease sensitivity, such units do not differ in any important way from those found in the normal early embryo. The unfertilized egg seems not to contain polyribosomes which are extracted by the methods used for embryos. These facts support the hypothesis that certain kinds of messenger RNA are present in the unfertilized egg, and that free ribosomes are unable to attach in a way that allows translation to take place.

Ribosomes and polyribosomes in Brassica pekinensis

Leaves of Brassica pekinensis plants contain 83-S ribosomes which occur free in the cytoplasm rather than in chloroplasts. A 68-S class of ribosomes present in 20–35% the amount of the 83-S, occurs largely if not entirely in chloroplasts. Both classes may occur as polyribosomal aggregates which can be temporarily preserved in leaf extracts using polyvinyl sulphate as a nuclease inhibitor. The major environmental influence on polyribosome levels in leaves is a diurnal cycle in which light is the predominant factor. Polyribosomes decrease in amount during the night and reach their lowest level before dawn. 2 or 3 h after sunrise 80–90% of the 83-S ribosomes may be in polyribosomes. If the natural dark period is extended for 2–3 h no polyribosomes can be detected. When such plants are exposed to sunlight a rise in polyribosomes and a drop in 83-S ribosomes can be detected within 4 min. This light-dependent increase in polyribosomes appears to be associated, at least in part, with the synthesis of new RNA.

Fate of the ribosomal RNA produced by a “relaxed” mutant of Escherichia coli

The fate of the RNA produced during methionine starvation by a strain of Escherichia coli with “relaxed” control of RNA synthesis has been investigated. This RNA consists of amino acid transfer RNA (4 s), messenger RNA and ribosomal RNA (16 s and 23 s); the ribosomal RNA forms part of a particle (18 s to 25 s). The RNA of the “relaxed” particles is not stable: it disappears slowly (half-life approximately 20 minutes) when RNA synthesis is inhibited by 2,4-dinitrophenol. When methionine is restored to cells that have accumulated the “relaxed” particles, the RNA of these particles is rapidly incorporated intact into newly formed ribosomes. This incorporation also occurs when cells that have accumulated the “relaxed” particles in a glucose medium free of methionine are transferred to a succinate medium containing methionine: the “shift-down” prevents synthesis of new RNA, but permits the synthesis of the protein required for the conversion of the particles to complete ribosomes.

Prevention by 2,4-Dichlorophenoxyacetic Acid of an Increase in the Rate of Phosphate Utilization, caused by the Prolonged Treatment of Segments of Pea Stems in Maleate Buffer

WHILE examining the influence of 2,4-dichlorophenoxyacetic acid (2,4-D) on the absorption of ions by epicotyl segments of etiolated peas, it was established that if freshly cut segments are first placed in aerated, distilled water or 0.01 M maleate buffer (pH 5.2) for 12 h, their ability to absorb and esterify inorganic phosphate in a given time (1 h) is increased 12-fold. This marked increase in the rate of phosphate absorption necessitated further study before the effects of 2,4-D on the uptake of ions could be further elucidated. On the basis of this work, which has been reported elsewhere1, it is concluded that one of the basic changes occurring in the segments during the period in buffer or water is an increase in the rate of synthesis of protein, which in turn results in an increased rate of absorption of phosphate. An increased turnover of protein when pea segments were placed in water for 24 h was reported by Christiansen and Thimann2 in 1950. Recently, Click and Hackett3 have suggested that the increased respiratory rate and other metabolic changes occurring when potato slices are similarly treated depend on the synthesis of new RNA and protein.

Activation of amino acids during sea urchin development

Previous work from this laboratory has demonstrated that unfertilized sea urchin eggs have very little, if any, ability to incorporate in vivo labelled amino acids into their proteins. Incorporation begins immediately following fertilization but its rate becomes especially high between blastula and midgastrula (1, 10, 12). Experiments carried out in vitro (4, 5) indicate that the ribosomes isolated from unfertilized eggs are unable to incorporate amino acids into proteins but acquire this ability within 30 minutes after fertilization. However, the ribosomes from unfertilized eggs can be induced in vitro to synthesize polyphenylalanine by the addition of synthetic polyuridylic acid (13, 16, 17). These results seem, therefore, to warrant the suggestion that the lack of synthetic ability on the part of the ribosomes of the unfertilized egg may depend on the nonavailability of messenger RNA. That a new synthesis of RNA begins shortly after fertilization is indicated by the recent work of Gross and Cousineau (2). Another important step, and indeed the first one, in the process of protein synthesis to which, in the case of the sea urchin embryo, almost no attention has thus far been paid is the activation of amino acids.

THE ROLE OF RNA SYNTHESIS IN EARLY ESTROGEN ACTION.

Previous studies from this laboratory have demonstrated that in the rat uterus the early response to estradiol is characterized by a rapid acceleration of synthetic reactions leading to the accumulation of phospholipid, ribonucleic acid, and protein.'-6 Levels of puromycin which blocked protein synthesis prevented these responses, suggesting that the primary action of this hornmone was to accelerate synthesis of rate-limiting enzymes for these anabolic pathways.7 Since it has been demonstrated in other biological systems that the synthesis of specific proteins depends on the antecedent synthesis of new RNA species,8- it was of interest to inquire whether or not the early estrogenic response required the synthesis of new RNA. The recent studies of a number of investigators have documented the ability of actinomycin D to block the DNA-dependent synthesis of RNA in cells and in isolated enzyme systemns.l-4 This paper will describe the use of this agent to prevent RNA synthesis in the uterus of the intact rat. It will be shown that under conditions in which the synthesis of new RNA was blocked by actinomycin D, the early acceleration of phospholipid and protein synthesis, as well as a major portion of the imbibition of water resulting from in vivo action of estradiol, failed to occur. The requirement for the synthesis of new RNA in the early estrogenic response is discussed. Methods.-Female Holtzman rats of the same age and weighing approximately 180 gm were ovariectomized through the dorsal approach and maintained on a diet of Purina dog chow for at least 3 weeks prior to experimentation. Only rats which were ovariectomized on the same day were used in any one experiment. The rats were injected intraperitoneally with actinomycin D (375 pg in 0.5 ml of 0.154 Ml NaCI) or with saline alone as indicated in the figures. At zero hr 10 pg of estradiol-17, in 1.0 ml of buffered saline containing 1% ethanol' or the control solution of buffered saline and ethanol was injected via the tail vein. To assess the in vivo synthesis of RNA, protein, and phospholipid, a combination of 25 uc of uridine-H3 (specific activity = 1.0 mc/0.036 mg) and 6 Jc of glycine-2C4 (specific activity = I mc/12.4 mg) dissolved in 0.5 ml of saline was injected intraperitoneally at 2 and 3 hr. In certain experiments only glycine-2CT was administered. The rats were killed 4 hr after the injection of hormone by decapitation, and the uteri were removed, stripped of accessory fat, and weighed on a Roller-Smith torsion balance. The uteri were then homogenized in cold distilled water, and the resulting dispersion was treated with 4%0 perchloric acid (PCA) to remove the acid-soluble fraction. The sedimented tissue residue was washed twice with 4% PCA and then was extracted successively with 90% ethanol, absolute ethanol, and twice with anhydrous ethyl ether. The combined ethanol-ether extracts, making up the crude lipid fraction, were counted in a liquid scintillation counter. The tissue residue remaining after ethanol-ether extraction was suspended in 0.01 M Tris (trishydroxymethylaminomethane) buffer at pH 7.4 and incubated at 37?C for 30 min with 100 pg of RNAse. The reaction mixture was treated with 4% PCA to obtain the RNAse-released material; this fraction was counted for H3 content in a liquid scintillation counter. The results were expressed as counts per min (CPM) per uterus. The PCA-insoluble residue was washed successively with 90% ethanol, absolute ethanol, and twice with anhydrous ethyl ether. The residual protein was spread on aluminum planchets with the aid of a small amount of formic acid, and after drying it was counted in a gas flow counter 256

Altered base ratios in RNA synthesized during enterovirus infection of human cells.

Ackermann et al.' reported increased synthesis of RNA and protein in resting (nondividing) HeLa cells after infection with poliovirus. The RNA produced showed normal base ratios. Salzman et al.,2 however, found no significant increase in synthesis of RNA after poliovirus infection of actively multiplying HeLa cells, and we have confirmed this. In a preliminary report,' it has been shown that in actively multiplying HeLa cells poliovirus infection led to altered base ratios in newly synthesized (P32-labeled) RNA without causing a significant shift in total cell RNA base ratios or rate of RNA synthesis. The base ratios in newly synthesized RNA shifted in the direction of the base composition of RNA reported for purified poliovirus by Schaffer et al.4 This suggests that RNA virus can cause infected cells to produce mainly viral type RNA, just as the DNA of bacterial viruses induces production of viral DNA. Whether this RNA is mainly virus RNA or nonviral RNA whose synthesis is directed by viral RNA (as phage DNA was shown to direct synthesis of new RNA by Volkin and Astrachan') or simply host celldirected RNA of different composition is not certain. The present report provides evidence that the altered RNA is virus RNA and/or virus-directed RNA. It is also shown that poliovirus RNA metabolism may involve a high specific activity fraction of cell RNA (r-RNA or residual RNA)6-9 which is not extractable with aqueous phenol in addition to the phenol-extractable RNA (p-RNA), which is most studied. No shift in base composition in HeLa cell DNA could be detected during poliovirus infection, which is further evidence that DNA does not participate in RNA virus infection.?0' 1

A CYTOCHEMICAL INVESTIGATION OF OÖGENESIS AND DEVELOPMENT TO THE SWIMMING LARVAL STAGE IN THE CHITON, CHITON TUBERCULATUM L

1. Oöcyte growth and development and the subsequent development of the egg to the swimming larva stage were investigated in the chiton, Chiton tuberculatum, using topological cytochemical methods for nucleic acids, proteins and mucopolysaccharides. 2. The growing oöcytes exhibited two phases of synthetic activity: the first phase was chiefly concerned with RNA production and the second phase with protein yolk synthesis. 3. A probable alteration in the cytoplasmic RNA-protein complex prior to the initiation of yolk synthesis was detected by an alteration of the affinity of RNA-associated proteins for binding acid dyes in mixtures. 4. Oöcyte accessory cells were probably responsible in some way for chorion formation. After the chorion was formed, ornate hollow processes grew out of the chorion, each beneath an accessory cell. When development of the processes was completed, the accessory cells had disappeared. 5. There was no evidence of new synthesis of RNA in any of the cells of the developing embryo except the cells of the prototroch and apical tuft. This applies to the complete developmental history of the oöcyte from the cessation of RNA synthesis prior to protein yolk formation in the oöcyte to the swimming trochophore larva. 6. Since primary differentiation occurs in mosaic embryos in the absence of synthesis of new RNA, the possibility that primary differentiation is controlled by the RNA produced in the oöcyte was discussed. 7. Although there was no differential segregation of protein yolk into the blastomeres—both RNA-basophilia and protein yolk being evenly distributed in all blastomeres—the posterior blastomeres received a larger proportion of yolk material than the micromeres. This was particularly evident in the yolk-laden cells of the trochophore gut.

フアージ増殖の生化学的研究

An active turnover of RNA-phosphorus assimilated after phage infection has been revealed. It was also found that chloramphenicol, when added 10 minutes after T2 infection, blocked the breakdown of RNA formed after infection, but not the synthesis of new RNA, indicating a correlation between RNA breakdown and protein synthesis. From these results it is concluded that the RNA, which is formed after infection, is distinct from the RNA of the host cell in metabolic activity.

フアージ増殖の生化学的研究

It was found that chloramphenicol, when added before T2 infection, blocked the formation of protein and DNA, but not the RNA synthesis after infection. The RNA synthesis after infection seems to be slightly stimulated by the addition of chloramphenicol. This RNA, which is formed without a priori formation of protein after T2 infection, is also characteristic of T2 infection in its mononucleotide composition. These results show that T2 infection induces a synthesis of new RNA, or at least a specific change in RNA metabolism at first without the prior synthesis of new protein.