Inhibition of host protein synthesis in vaccinia virus-infected cells in the presence of cordycepin (3''-deoxyadenosine).

Cordycepin inhibited efficiently viral mRNA and polyadenylic acid syntheses in vaccinia virus-infected cells, but allowed the shutoff of host protein synthesis to occur. Therefore, cordycepin was used to study this shutoff in the absence of gene expression. Ribosome transit time was increased in infected cells, revealing an inhibition at the level of elongation and/or release of polypeptide chains. However, the disappearance of heavy polysomes in vaccinia virus-infected cells showed that the inhibition of host protein synthesis resulted predominantly from a block at the stage of initiation. This conclusion was confirmed by the recovery of heavy polyribosomes when low levels of cycloheximide were added to slow down ribosome release from the mRNA. Similar amounts of cellular mRNA (present in the polyribosomes) were found in vaccinia virus-infected cells and in mock-infected cels (exposed to cordycepin), showing that the cellular mRNA was not inactivated in these conditions. It was concluded that a component of the vaccinia virion inhibits, in the absence of viral RNA and polyadenylic acid syntheses, host protein synthesis at the level of initiation and, to a lesser extent, at the level of elongation (and/or release) of polypeptide chains.     

Messenger RNA synthesis in the testis of immature rats: effect of gonadotropins and cyclic AMP.

Two hours after the intratesticular injection of FSH, hCG or cyclic AMP, the incorporation of labeled uridine into poly(A)-rich RNA was increased. Pretreatment with actinomycin D inhibited the incorporation of uridine into mRNA. After the seminiferous tubules and interstitial cells were separated by treatment with collagenase, FSH treatment increased mRNA synthesis only in the tubules whereas hCG stimulated mRNA synthesis only in the interstitial cells. Cyclic AMP increased the synthesis of mRNA in both interstitial cells and seminiferous tubules. These results suggest a differential action of the two gonadotropic hormones in the cells of the testis; both effects appear to be mediated by cyclic AMP.     

Heat-shock protein synthesis in Chlamydomonas reinhardi. Translational control at the level of initiation of a poly(A)-rich-RNA coded 22-KDa protein in a cell-free system

The effect of temperature on the in vitro translation of control and heat-shock poly(A)-rich RNA, obtained from Chlamydomonas reinhardi cells, incubated for 2 h at 25 degrees C respectively, was studied using the wheat-germ translation system. Incubation of the cells at 42 degrees C induces the synthesis of RNAs coding for several heat-shock proteins, including a 22-kDa major polypeptide as well as several proteins of 45-94 kDa, as demonstrated by run-off translation of polyribosomes isolated from intact cells. However, the high-molecular-mass heat-shock proteins are poorly translated in the wheat-germ system. The poly(A)-rich RNA coding for the 22-kDa heat-induced polypeptide has an apparent sedimentation coefficient higher than that expected from the molecular mass of its translation product, and was preferentially translated in vitro at temperatures above 31 degrees C as compared with pre-existing RNAs. Raising the temperature of translation, slightly inhibited (10%) the runoff translation of polyribosomes isolated from intact cells. However, when initiation was carried out in vitro for a short time at increasing temperatures and translation continued at 25 degrees C in the presence of aurintricarboxylic acid, the 22-kDa heat-shock polypeptides was preferentially translated. Aurintricarboxylic acid did not significantly inhibit incorporation of [35S]methionine when added to polyribosomes isolated from control or heat-shocked cells. From the above data we conclude that the translation of the 22-kDa heat-shock protein is controlled in vitro at the initiation level.     

Messenger RNA turnover during bone marrow erythroid cell differentiation

Incubation of bone marrow cells from anaemic rabbits in the presence of actinomycin D led to a decrease in total protein synthesis and an increase in the relative synthesis of globin. This increase in the proportion of globin was observed with in vivo labelling of cellular proteins and in vitro translation of isolated RNA, which indicates that the messenger RNA for globin is much more stable than the other bone marrow cell messages. This was further shown by pulse-labelling the RNA and characterization of the different species by separation on a cDNA-oligo(dT)-cellulose column. Within 12 h after pulse-labelling the relative levels of globin mRNA had risen 10-fold, while a rapid decrease in the level of the poly(A)-rich RNA fraction was observed. Investigations into the mechanisms of this differential stability indicate that the more metabolically active cells from the early stages of erythropoietic development are more susceptible to inhibitors of RNA synthesis such as actinomycin D and alpha-amanitin. A preliminary study using a lysosomal inhibitor, chloroquine, indicates that there appear to be at least two degradative mechanisms, involving a lysosomal and a non-lysosomal pathway, with selective specificity for different messages.     

Studies on the form and synthesis of messenger ribonucleic acid in the rat ventral prostate gland, including its tissue-specific stimulation by androgens

1. When prostate polyribosomes are labelled with radioactive precursors in vivo and subsequently dissociated with sodium dodecyl sulphate, a heterogeneous 6-15S RNA species may be identified that possesses all of the distinctive properties of mRNA. 2. Apart from the selective incorporation of 5'-fluoro-orotic acid into this 6-15S RNA component, it is bound by nitrocellulose filters under experimental conditions where only poly(A)-rich species of RNA are specifically retained. Most importantly, however, only the 6-15S RNA fraction is capable of promoting the incorporation of amino acids into peptide linkage in an mRNA-depleted cell-free system derived from ascites-tumour cells. 3. With the development of a simpler method for labelling the total RNA fraction of the prostate gland in vitro, the poly(A)-enriched RNA fraction may be readily isolated by adsorption and elution from oligo(dT)-cellulose. The synthesis of the poly(A)-enriched 6-15S RNA fraction is stringently controlled by androgens in a highly tissue- and steroid-specific manner. 4. From an analysis of the proteins synthesized in the ascites cell-free system in the presence of the poly(A)-rich RNA fraction, it appears that protein synthesis in the prostate gland is stimulated in a rather general way, even during the earliest phases of the androgenic response. This conclusion may require modification when more specific means of analysis are available than those used in the present investigation. 5. The implications of these findings to the mechanism of action of androgens are discussed.     

Metabolic Properties of Early and Late Vaccinia Virus Messenger Ribonucleic Acid

In vaccinia-infected cells, 60% of the viral messenger ribonucleic acid (mRNA) was associated with polyribosomes, and the remainder sedimented in a broad peak in the 30 to 74S region. The quantity of mRNA in polyribosomes was sharply reduced late in the infectious cycle [9 hr postinfection (PI)] to less than 30% of the 2-hr value. However, protein synthesis proceeded at a nearly constant rate from 2 to 13 hr PI. This ability of small quantities of late mRNA to support as much protein synthesis as do the much larger quantities of early mRNA was not due to an increase in stability, since late mRNA decays with a half-life of 13 min, whereas early mRNA has a half-life of 120 min. A similar decrease in viral mRNA synthesis without an accompanying decrease in viral protein synthesis was observed when deoxyribonucleic acid synthesis is inhibited. In contrast to the rapid decay of the late mRNA which was present in polyribosomes, the mRNA which sedimented in the 30 to 74S region remained unchanged even after a 2-hr period of exposure to actinomycin. The rate at which infected cells lose the capacity to synthesize specific viral proteins after exposure to actinomycin D was consistent with the half-life values of early and late mRNA that were observed.     

Flagellar synthesis in Salmonella typhimurium: requirement for ribonucleic acid synthesis

The micro-complement-fixation assay has been demonstrated to be a sensitive assay for flagella which occur in nanogram amounts. By use of this assay, it was found that flagellar synthesis occurs during starvation of Salmonella typhimurium for tryptophan, an amino acid not present in flagellar protein. Under these conditions net ribonucleic acid (RNA) synthesis was reduced to approximately 10% of the control rate. Less than 1 mug of actinomycin D per ml further reduced RNA synthesis to less than 1% of the control rate in a culture sensitized by prior treatment for 5 min at 37 C with 5 x 10(-4)m ethylenediaminetetraacetate in 0.33 m tris(hydroxymethyl)aminomethane-chloride (pH 8.0). In the presence of actinomycin D, no synthesis of flagellar protein could be detected. Analysis of fractions of RNA separated by zone centrifugation indicated that actinomycin D reduces the production of template RNA as well as of ribosomal RNA. This suggests that in S. typhimurium the production of flagellar protein requires the concomitant synthesis of RNA. There is no evidence that a stable messenger RNA specific for flagellar synthesis is present.     

Structural difference between the 5'' termini of viral and cellular mRNA in poliovirus-infected cells: possible basis for the inhibition of host protein synthesis.

Host protein synthesis in poliovirus-infected HeLa cells is interrupted, but the host mRNA appears to remain completely intact and unmodified. The average size and poly (A) content of host mRNA was previously known to be unchanged (Koschel, 1974; Leibowitz and Penman, 1971), and this was confirmed. In addition, the 5' terminal methylated "cap" structures remained intact, and no further base modifications at the level of 1 base in 1,000 could be detected. Poliovirus RNA from viruses was previously shown not to have "caps" (Wimmer, 1972), and in this work poliovirus RNA from polyribosomes was found to have pUp at its 5' end. Since, initiation of protein synthesis is probably the basis for the inhibition of cellular protein synthesis in infected cells, the difference in the 5' ends of the host cell and viral RNA could be the basis of selective translation of viral RNA during infection.     

Studies on the intracellular segregation of polyribosome-associated messenger ribonucleic acid species in the lactating guinea-pig mammary gland.

1. Free and membrane-bound polyribosomes were isolated and the associated mRNA species characterized by cell-free protein synthesis, RNA-complexity analysis and polyribosome run-off in vitro. 2. Of the recovered polyribosomal RNA 85% was associated with membrane-bound polyribosomes and contained 87--93% of the total milk-protein mRNA species as assessed by cell-free protein synthesis or RNA-complexity analysis. 3. RNA-complexity analysis showed that the abundant (milk-protein mRNA assumed) species constituted 55% of the post-nuclear poly(A)-containing RNA population, the remainder consisting of a moderately abundant population (18%) and a low abundance population (27%). Calculations suggest that each population contained up to 2, 48 and 5000 different species respectively. 4. RNA-complexity analysis of the free polyribosomal poly(A)-containing RNA demonstrated that all the species in the post-nuclear fraction were present, though in different proportions, the abundant, moderately abundant and low-abundance groups representing 38, 30 and 32% of this population. 5. RNA-complexity analysis of the membrane-bound polyribosomal poly(A)-containing RNA revealed a more limited population, 72% consisting of the abundant (milk-protein mRNA) species, and 28% a population of up to 900 RNA species. 6. Polyribosome run-off confirmed that milk-protein mRNA was associated with the membrane-bound and free polyribosomes, but represented only a small fraction of the total protein synthesized by the latter. 7. Comparative analysis of milk proteins synthesized in mRNA-directed cell-free systems, or by run-off of free and of membrane-bound polyribosomes, is consistent with the interpretation that in vivo the initiation of protein synthesis occurs on free polyribosomes, followed by the attachment of a limited population to the endoplasmic reticulum. After attachment, but before completion of peptide synthesis, the detachable N-terminal peptide sequence of one of these(pre-alpha-lactalbumin) is removed. 8. The results are discussed in terms of the mechanisms involved in the intracellular segregation of mRNA species in the lactating guinea-pig mammary gland.     

Differences in sequence content of nuclear and cytoplasmic polyribosomal RNA from adenovirus-infected cells.

RNA molecules from nuclear and cytoplasmic polyribosomes of adenovirus-infected HeLa cells were compared by hybridization to analyse the sequence content. Nuclear polyribosomes were released by exposure of intact detergent-washed nuclei to poly(U) and purified. Cytoplasmic polyribosomes were also purified from the same cells. To show that nuclear polyribosomes contain ribosomes linked by mRNA, polyribosomes were labelled with methionine and uridine in the presence of actinomycin D in adenovirus-infected cells. Purified nuclear polyribosomes were treated with EDTA under conditions which dissociate polyribosomes into ribosomes and subunits with a simultaneous release of mRNA, and sedimented. The treatment dissociated these polyribosomes, releasing the mRNA from them. Radiolabelled total RNA from each polyribosome population was fractionated in sucrose gradients into several pools or hybridized to intact adenovirus DNA to select virus-specific RNA. Sucrose-gradient-fractionated pool-3 RNA (about 28S) and virus-specific RNA were then hybridized to fragments of adenovirus DNA cleaved by restriction endonucleases SmaI, HindIII and EcoRI by the Southern-blot technique and by filter hybridization. The results showed that nuclear RNA contained sequences, from about 0 to 18 map units, which were essentially absent from cytoplasmic RNA. Furthermore, the amount of virus-specific RNA for a particular sequence was also different in the two populations.