Formation Of Messenger RNA Research Articles

Inhibition by α-amanitin of messenger RNA formation in cultured fibroblasts: Potentiation by amphotericin B

α-Amanitin, a potent inhibitor of RNA polymerase II, is found inert against transformed fibroblasts in tissue culture. However, when α-amanitin is synergistically used with amphotericin B, RNA and protein synthesis are strongly blocked. Our data suggest that messenger RNA formation is preferentially inhibited since (1) the total inhibition by α-amanitin was greatly magnified when rRNA synthesis was first blocked with 0.03 μg/ml actinomycin D; (2) mRNA in polysomes was greatly reduced and the size of polysomes diminished after cells were exposed to 2 μg/ml α-amanitin plus 20 μg/ml amphotericin B for 5 h.

Mechanism of influenza recombination. I. Factors influencing recombination rates between temperature-sensitive mutants of strain WSN and the classification of mutants into complementation--recombination groups.

Mixed infection of chick embryo fibroblasts with ts mutants of WSN virus resulted in a number of reeombinant-yielding cells far in excess of what was predicted in terms of the adsorbed multiplicity of the plaque-forming virus employed. Some pairs of mutants had a strong tendency to form mixed aggregates which gave rise to recombinants by a single hit process. Reeombinant-yielding cells were also generated by a two-hit mechanism, in which case the yield of mixedly infected cells (recombinant yielders) exceeded the expectation (based on the number of plaque-forming particles adsorbed) by a factor of 30–60 times. These results reinforce the previously proposed conclusion that non-plaque-forming particles are the principal source of recombinants in mixed infection. The two-hit recombinant-yielding cells were obtained at exceedingly low virus inputs and were obtained in cells that were incubated solely at the restrictive temperature for the ts mutants employed. This undoubtedly means that the initial steps leading to recombinant production were the result of complementation, followed at maturation by an exchange of RNA pieces which in some cases gave wild-type particles. These results strongly suggest that non-plaque-forming virus has the capacity to enter cells and generate some syntheses such as the formation of messenger RNA and viral structural proteins. They also suggest that two non-plaque-forming particles, each bearing a mutation on a different RNA piece, may by means of complementation and recombination, give rise to wild-type virus. On the basis of complementation-recombination patterns a number of mutant strains have been classified into eight groups. Members of each group give high frequency complementation-recombination with each of the other groups. The physiological basis of the classification is being investigated.

Formation of Messenger RNA in Eukaryolic Cells

Formation of Messenger RNA in Eukaryolic Cells

Messenger RNA formation: Resistance to inhibition by 3′-deoxycytidine

Cordycepin (3′-deoxyadenosine) has been previously shown to inhibit nucleolar RNA synthesis and mRNA production. 3′-Deoxycytidine shares with cordycepin the ability to inhibit nucleolar RNA synthesis, but it does not affect the appearance of mRNA. Neither compound affects heterogeneous nuclear RNA (HnRNA). The results are interpreted to mean that cordycepin inhibits proper poly(A) metabolism.

Arrest of cell division and hemoglobin synthesis in differentiating yolk sac erythroid cells

Yolk sac derived erythroid cells of C57B1/Cas fetal mice which are X irradiated with 120 rads at the 10th day of fetal development perform three normal cell cycles and synthesize hemoglobin in a normal fashion until day 12. Between day 12 and 13 normal cells perform one final cell cycle and reduce to half the rate of hemoglobin synthesis per cell; conversely, irradiated erythroid cells do not divide and maintain the full rate of hemoglobin formation. At day 13 irradiated non-divided erythroid cells contain polyribosomes in the same amount and with the same size distribution as normally divided cells and yet synthesize hemoglobin at a two-fold faster rate. This difference of hemoglobin synthesizing capacity is not due to new formation of messenger RNA for hemoglobin, neither it is due to a faster rate of translation, but rather because at day 13 non-divided erythroid cells have maintained the full complement of messenger RNA-ribosomal complexes active for hemoglobin synthesis, while in normally divided erythroid cells the number of messenger RNA-ribosomal complexes active for hemoglobin synthesis is cleaved in the course of cell division. In the irradiated erythroid population the total amount of each globin chain produced in the course of differentiation is not decreased in spite of the arrest of cell division. These data suggest that the total amount of hemoglobin produced during the development of the progeny of a single erythroid cell is programmed in early stages of erythroid differentiation.

Regulation of glucose 6-phosphate dehydrogenase formation in mammary organ culture

Abstract 1. 1. We have continued our study of the stimulation of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in mammary tissue from pregnant C 3 H mice in organ culture. Our results are based on the finding that the complex culture Medium 199 can be replaced by a medium containing only insuling, glucose, amino acids and inorganic salts. 2. 2. With 1 mg/ml glucose at least 5 μg/ml insulin is needed for maxiumum increases in activity. Insulin may be replaced by 1 mM cysteine. Glucose may be replaced by mannose or fructose but not glycerol or pyruvate. With 7.5 mg/ml glucose insulin is not required for increases in enzyme activity. 3. 3. The process which leads to the increases in enzyme activity is initiated by the addition of glucose to the culture medium, and becomes insensitive to actinomycin D after 4 h, although the enzyme activities only begin to rise after 12 h. Although glucose must be present during the initial 4-h period it may afterwards be replaced by glycerol or pyruvate as a source of energy. Moreover, the addition of amino acids may be delayed until 4 h. 4. 4. The increases in enzyme activity are unaffected by inhibition of DNA synthesis and mitosis. 5. 5. We suggest that the increases in enzyme activity result from a stimulated uptake of glucose by the mammary tissue, and that they are caused by a metabolic product of glucose stimulating the formation of messenger RNA for the two enzymes over the first 4 h of culture.

Hybridizable ribonucleic acid of rat brain.

1. Cerebral RNA of adult and newborn rats was labelled in vivo by intracervical injection of [5-(3)H]uridine or [(32)P]phosphate. Hepatic RNA of similar animals was labelled by intraperitoneal administration of [6-(14)C]orotic acid. Nuclear and cytoplasmic fractions were isolated and purified by procedures involving extraction with phenol and repeated precipitation with ethanol. 2. The fraction of pulse-labelled RNA from cerebral nuclei that hybridized to homologous DNA exhibited a wide range of turnover values and was heterogeneous in sucrose density gradients. 3. Base composition of the hybridizable RNA was similar to that of the total pulse-labelled material; both were DNA-like. 4. Pulse-labelled cerebral nuclear RNA hybridized to a greater extent than cytoplasmic RNA for at least a week after administration of labelled precursor. This finding suggested that cerebral nuclei contained a hybridizable component that was not transferred to cytoplasm. 5. The rates of decay of the hybridizable fractions of cerebral nuclei and cytoplasm were faster in the newborn animal than in the adult. Presumably a larger proportion of labile messenger RNA molecules was present in the immature brain. 6. Cerebral nuclear and cytoplasmic RNA fractions from newborn or adult rats, labelled either in vivo for periods varying from 4min. to 7 days or in vitro by exposure to [(3)H]-dimethyl sulphate, uniformly hybridized more effectively than the corresponding hepatic preparation. These data suggested that a larger proportion of RNA synthesis was oriented towards messenger RNA formation in brain than in liver.

Kinetic aspects of ribosomal and messenger RNA formation in young rat brain

RNA was isolated from purified nuclei and polysomes obtained from brains of 2-week-old rats which were injected with 32P 1 at various intervals of time prior to sacrifice. The RNA was separated into components of different S values by centrifugation through a sucrose-density gradient and the distribution of A 260m μ and radioactivity along the gradient was determined. The results indicate that the RNA synthesized in the nucleus immediately after injection consisted of messenger RNA (mRNA) and ribosomal precursor RNA. The mRNA was immediately transferred to the cytoplasm while the ribosomal precursors which consisted of both heavy and comparatively smaller molecules were fabricated into the normal 18-S and 28-S ribosomal RNA (rRNA). There was a lag period of 40–80 min before the heavier RNA in the polysome became labeled. The entry of polysomal RNA into the cytoplasm followed the time sequence, mRNA, 18-S rRNA and finally 28-S, rRNA. An RNA fraction heavier than 28 S was found in the brain nuclei, but not in the polysomes. The kinetic characteristics of its labeling and its absence from the polyribosomes suggest that it may constitute a rRNA precursor rather than polycistronic mRNA. Sedimentation analysis of mRNA isolated from polysomes by mild EDTA treatment showed that it consisted of a number of heterogeneous RNA's with sedimentation values between 4S and 18 S. No RNA heavier than 28 S was detected. Synthesis of both rRNA and mRNA were inhibited by the administration of antinomycin D.

Regulation of Ferritin Synthesis in Rat Liver

Abstract Ferritin synthesis in rat liver is greatly increased by prior treatment of intact animals with iron salts. Liver slices from treated rats incorporate increased amounts of 14C-leucine into ferritin in vitro, with net synthesis of the protein. Slices of spleen, testis, intestine, and kidney also show increased incorporation following iron. The response of the liver to iron has been studied to determine whether increased transcription of genes and the formation of messenger RNA are involved, or whether control is at a later stage in the biosynthetic pathway. Three lines of evidence support the existence of transcriptional control. (a) Actinomycin D inhibits the response to iron. (b) Iron treatment stimulates incorporation of 14C-orotic acid into a rapidly labeled fraction of liver nuclear RNA. (c) Direct addition of iron to liver slices in vitro has failed to stimulate ferritin synthesis, although 59Fe penetrates liver cells.

Effects of actinomycin D on development in pupae of Tenebrio molitor.

Ilan, Ilan and Quastel have reported1 that modified adults emerge when 0.16 μg of actinomycin D is injected into the pupae of Tenebrio molitor at any time during the pupal period. The duration of pupation is said to be unaffected. The adults which emerge are reported to have an adult head and thorax, but the abdomen has pupal gin traps, urogomphi and pupal cuticle on the posterior segments. Injected pupae do not develop wings. The interest in the effects of actinomycin D on development stems from the way in which the drug is known to interfere with DNA dependent RNA synthesis. The pupal development of insects must involve the transcription of the genetic information coded in DNA into the specific proteins of the various tissues by the formation of messenger RNA (mRNA). Ilan et al. interpret their observations by supposing that the mRNA for the development of the head and thorax is present from the first day of pupation and that abdominal development is sensitive to actinomycin D until the last day of the pupal period.

Nuclear changes in the course of development of the sea urchin studied by means of Hale staining

The nuclear changes occurring in the course of the early development of Paracentrotus lividus have been studied by means of the ferric acetate staining method of Hale. If the sections of Carnoy fixed eggs or embryos are subjected to the Hale procedure, very few interphase nuclei become stained. Treatment of the sections with a buffer or a buffered trypsin solution prior to the Hale procedure may induce a staining of the interphase nuclei, which varies according to pH of the buffer, stage of development and position in the embryo, in a way, shown in Text-Figs 1–3. During the early development, the interphase nuclei remain unstained even after pretreatment. At an early blastula stage (6 hr after fertilization), the interphase nuclei become Hale positive, following a pretreatment with 0.0025 per cent trypsin (Figs 1c, d). Only at the stage of free swimming blastulae (14 hr after fertilization), pretreatment with acetate buffer gives a certain staining of the interphase nuclei, a staining that then increases in intensity and becomes less pH dependent during the following stages of development (Figs 2 e-j). The prophase chromosomes are not directly stained by the Hale reagent, but staining may be induced in any stage of development by pretreatment with buffer at pH above 3, or with trypsin within the pH range of activity of this enzyme. At pH values above 3, the meta- and anaphase chromosomes become stained without any pretreatment of the sections. At early blastula stage, the Hale staining of the interphase nuclei is not influenced by exposure to DNase or RNase. If, however, the sections are pretreated with trypsin and thereafter exposed to a mixture of DNase and RNase, the staining capacity of the interphase nuclei is abolished. In stages from the late blastula stage onwards, DNase decreases or abolishes the Hale reaction without any previous pretreatment. RNase may affect, but does not alone abolish the staining. The inference from the data is that Fe 3+ in the Hale reagent competes with nuclear proteins for reactive sites in the nucleic acids. These sites may correspond mainly to the phosphate groups. In the interphase nuclei, the proteins block the access of Fe 3+ to the phosphate groups with a strength varying according to the circumstances defined above. In prophase nuclei, the effect of the proteins is weaker and the Hale staining is therefore easily provoked upon pretreatment. In the meta- and anaphase chromosomes, Fe 3+ eliminates without pretreatment the action of proteins. The Hale staining of interphase nuclei is different in different embryonic regions and it often seems to reflect different intensities in synthetic activity. The propensity of interphase nuclei for Hale staining seems to parallel their nucleolar activity and consequently their production of ribosomal RNA. It may also parallel certain changes in pattern of messenger RNA formation during development.

The effect of actinomycin D on the insulin-mediated enhancement of incorporation of 14C-labeled precursors into protein and RNA by fetal RAT thyroid glands in organ culture

Insulin enhanced the incorporation of 14C of uniformly labeled leucine into proteins and of [8- 14C]adenine into RNA of fetal rat thyroid glands in organ culture. Actinomycin D suppressed the formation of protein by about 65 % and of RNA by about 90 %. The influence of insulin on 14C incorporation into protein persisted even in the presence of actinomycin D. This indicates that insulin-mediated enhancement of protein formation by the embryonic rat thyroid gland may not be dependent entirely on the increased formation of messenger RNA.

Effect of interferon on early interferon production.

Chick embryo cells given prior treatment with interferon make new interferon earlier and in larger quantities upon stimulation with Chikungunya virus than cells not so treated. By the criterion of loss of sensitivity to actinomycin, the time needed for formation of messenger RNA for interferon was decreased in the primed cells. Thus interferon affects virus action within 1 hour after infection.

Studies on the mode of action of vitamin K

The regulation of cell metabolism involves controls imposed at three general levels: (1) modification of the activity of existing enzymes, (2) modification of the permeability and function of cell and organelle membranes, and (3) the control of the synthesis of enzymes and other proteins of biological significance mediated by genetic mechanisms. Attempts to explain the action of the fat-soluble vitamins in terms of the first two mechanisms cited above have not been successful. In view of the fact, however, that the fat-soluble vitamins as a class do influence the concentrations of enzymes and other proteins in various organisms, it appeared possible that these changes reflected an action at the genetic level. It has been shown that actinomycin D inhibits vitamin K-induced prothrombin formation in chicks deficient in vitamin K. Doses of actinomycin which inhibited the normal response to vitamin K inhibited incorporation of adenine-8- 14C into hepatic RNA. These results were consistent with the hypothesis that vitamin K acts by stimulating messenger RNA formation for the synthesis of clotting proteins. Puromycin was also shown to inhibit the response to vitamin K in proportion to its inhibitory effect on liver protein synthesis. Since the coumarin drugs antagonize vitamin K, it seemed possible that the site of antagonism was a molecule which regulates this expression of genetic information. To test this hypothesis, rats were fed a diet containing 0.05 per cent dicumarol for 48 hr, at which time they showed prolonged clotting and one-stage prothrombin times. This clotting defect could be corrected in 6 hr by the administration of 1 mg of vitamin K 1 to each rat by mouth. The administration of actinomycinD in doses from 30–240 μg/100 g body weight 4 hr prior to the vitamin K 1 either modified or prevented the action of the vitamin. If actinomycin was given after the vitamin, or given to normal rats, no anticoagulant effect was noted. The dose-response curve of vitamin K-deficient chicks to vitamin K 3 in the presence and absence of prior dicumarol was suggestive of an allosteric interaction. These data are consistent with the view that the site of antagonism between vitamin K and the coumarin drugs is a molecule which controls DNA-dependent RNA replication.

Synthesis of Deoxyribonucleic Acid in the Liver of Hypophysectomized Rats After Partial Hepatectomy.

DNA synthesis in regenerating liver following partial hepatectomy of normal and hypophysectomized rats was measured following administration of thymidine-6-T(n). Such DNA synthesis in the hypophysectomized animals exhibits a delayed start and a prolonged period before its peak is reached, as compared with intact animals. However, there is a prolongation of maximal incorporation of DNA precursor, and a slower decline of DNA synthesis in hypophysectomized rats. The promotion of enzyme activity involved in DNA synthesis may be linked to the pituitary effects upon messenger RNA formation.

The regulatory function of the fat-soluble vitamins.

The regulation of cell metabolism involves controls imposed at three general levels: (a) modification of the activity of existing enzymes, (b) modification of the permeability and function of cell and organelle membranes, and (c) the control of the synthesis of enzymes and other proteins of biological significance mediated by genetic mechanisms. Attempts to explain the action of the fat-soluble vitamins in terms of the first two mechanisms cited above have not been successful. In view of the fact, however, that the fat-soluble vitamins, as a class, do influence the concentrations of enzymes and other proteins in various organisms, it appeared possible that these changes reflected an action at the genetic level.It has been shown that actinomycin D inhibits vitamin K induced prothrombin formation in chicks deficient in vitamin K. Doses of actinomycin which inhibited the normal response to vitamin K inhibited incorporation of adenine-8-C14into hepatic RNA. These results were consistent with the hypothesis that vitamin K acts by stimulating messenger RNA formation for the synthesis of clotting proteins. Since the coumarin drugs antagonize vitamin K, it seemed possible that the site of antagonism was a molecule which regulates this expression of genetic information. To test this hypothesis, rats were fed a diet containing 0.05% dicumarol for 48 hours, at which time they showed prolonged clotting and one-stage prothrombin times. This clotting defect could be corrected in 6 hours by the administration of 1 mg of vitamin K1to each rat by mouth. The administration of actinomycin D in closes from 30 to 240 μg per 100 g body weight 4 hours before vitamin K1either modified or prevented the action of the vitamin. If actinomycin was given after the vitamin, or given to normal rats, no anticoagulant effect was noted. These data are consistent with the view that the site of antagonism between vitamin K and the coumarin drugs is a molecule which controls DNA-dependent RNA replication.

Characteristics of virus-cell complexes

T HE interaction of a virus particle with a cell can be subdivided into a series of steps. At first the virus particle establishes contact with the cell (attachment), then the nucleic acid of the particle becomes released from its coats and reaches the cellular site where its replication is going to take place (penetration). The nucleic acid then replicates (replication), and the protein units which constitute the viral envelopes are synthesized. The nucleic acid and these coat units become assembled together into virus particles (maturation). Finally, the progeny virus particles are freed from the cell in which they were produced (release). Not all these steps must necessarily take place in all cases. For instance, naked nucleic acid can penetrate the cell, thus eliminating attachment and part of the penetration process; or release may be absent. In some cases the viral nucleic acid reaches a cell from the parent cells; in other cases the multiplication of the nucleic acid and the sy-nthesis of coat material may not take place. Every virus, however, must be able to cause the complete series of steps, since this is the basis for the definition of the virus: i.e., a particle which can infect a cell and can multiply in a cell, giving a product which is identical with the infecting particle itself. The fact that in specific cases the interaction of a virus with a cell may not involve the complete series of steps delineated shows that the type of interaction between the virus and the cell may be different. The consequences of the presence of the virus for the cell vary greatly, according to the type of interaction: in some cases, the cell is destroyed, in some it may be essentially unchanged, in others it may be changed into a cancer cell. Thus, also, the consequences of the infection of an organism depend on the type of virus-cell interactions which become established between the virus and the cells of the body. Knowledge of virus-cell interactions is, therefore, fundamental to understanding the biology and the pathology of virus infection. The step which most markedly distinguishes different types of virus-cell interaction is the replication of the viral nucleic acid. In order to appreciate this, the following points are worth considering. The viral nucleic acid must be considered to be endowed with two fundamental properties. One is that of a source of information for protein synthesis. In the case of DNA viruses, the genes of the virus are reproduced in the form of messenger RNA which, upon association with ribosomes, then causes the synthesis of polypeptide chains of specific sequences. In the case of RNA viruses, the viral RNA itself functions as messenger. Thus, in both cases viral proteins are produced in the cells, and the structure of these proteins is determined by the sequence of nucleotides in the viral genes. These proteins in turn have specific functions in the infected cells. Some of them constitute the envelope of the progeny virus particles. Others alter the cellular functions in a wa) which is suitable for the reproduction of the virus. Thus, a virus-infected cell becomes different from a similar but uninfected cell, because of the presence of these viral proteins. The other fundamental property of the viral nucleic acid is that of reproduction. The results of this reproduction are molecules of nucleic acid endowed with the characteristics of the infecting molecules. Reproduction, however, is not a simple act; it follows a complex path and has complex requirements. The complexity of the process is indicated by the fact that the nucleic acid must change its conformation in order to replicate. For instance, double-stranded molecules must separate into single strands, a process which can take place at the moment reproduction takes place. Singlestranded molecules must build their doublestranded counterparts. The detailed mechanisms involved in all these cases are still obscure for the most part. The complexity of the requirements derives from the multiplicity and

Protein and ribonucleic acid syntheses in dispersed rat-liver cells

Using dispersed rat-liver cells, protein and RNA syntheses were studied. Dispersed cells did not show any obligatory requirements for protein synthesis. Among the various media tested, 10 % polyvinylpyrrolidone and Locke's solution were the most suitable for protein synthesis, while 0.1 M sucrose was the best for lipid synthesis. The rates of protein and RNA syntheses in dispersed cells proceeded linearly during the first 2 h of incubation, whereas those of a homogenate decreased with time reaching a steady level after 30 min. The incorporation of amino acid into protein was inhibited by 2,4-dinitrophenol and anaerobiosis. Puromycin also strongly inhibited amino acid incorporation into protein but not RNA synthesis. Actinomycin strongly inhibited the incorporation of uridine into RNA, but, when not preincubated with the cells, it was less inhibitory for protein synthesis. However, when it was preincubated with the cells, the degree of inhibition of amino acid incorporation into protein by this antibiotic increased and reached a maximal value when the incubation period was 1 h. These results support the idea that protein synthesis in dispersed cells is controlled by the formation of messenger RNA.

The intracellular uncoating of poxvirus DNA: II. The molecular basis of the uncoating process

The uncoating of poxvirus DNA is a two-step process. The first stage begins immediately after penetration of virus particles and is effected by enzymes present in the uninfected cell; the products of this stage are virus cores. Viral DNA within cores is not accessible to DNase. The second stage of uncoating results in the breakdown of the cores to release naked poxvirus DNA. This stage is inhibited by fluorophenylalanine and puromycin, inhibitors of protein synthesis, as well as by actinomycin D and irradiation with u.v. light, inhibitors of messenger RNA synthesis. It is concluded that in order for virus cores to be broken down a protein has to be synthesized after infection. This protein is synthesized only in response to infection with virus particles containing un-denatured protein. Evidence is presented suggesting that the synthesis of this protein is under the control of the host-cell genome. A scheme is presented which is capable of explaining all known facts concerning poxvirus reactivation, initiation and uncoating. It postulates that a viral protein released during the first stage of uncoating causes derepression of a portion of host cell DNA, permitting the synthesis, through the mediation of messenger RNA formation, of a protein which is instrumental in degrading virus cores and thus releasing viral DNA in the free state.

Zellwachstum und RNS-Synthese im Ei-N�hrzellverband von Ophryotrocha puerilis

During its growth phase, the oocyte is associated with a single highly polyploid nurse cell. Although the nurse cell contains high amounts of cytoplasmic ribonucleic acid, no RNA seems to be transferred to the oocyte. Autoradiographs prepared after pulse labelling with 3H-uridine indicate that the nuclei of both cell types are actively engaged in RNA synthesis during the whole period of oogenesis. Chromosomal RNA synthesis is most intense in oogonia before onset of the growth period. It still proceeds, although at a lower rate, after termination of oocyte growth when all RNA is lost from the nucleoli. Nucleolar RNA synthesis, on the other hand, is strictly correlated with the growth phase proper. Grain counts on chromatin and nucleoli of both egg cells and nurse cells at all stages of oogenesis indicate that nucleolar and chromosomal RNA synthesis are independent of each other to a large extent. It is thought that the type of RNA synthesized within the nucleolus is essentially ribosomal while the chromosomes are primarily engaged in the formation of messenger RNA.