Protein synthesis during a nutritional shift-up in Escherichia coli

The rate of synthesis of ribosomal protein increased immediately following a nutritional shift-up in $\text{Escherichia coli}$; while the rate of synthesis of elongation factors did not increase until 5–10 minutes had elapsed. The relative rates of synthesis of EFG and EFTs were constant at all times following shift-up. This constancy was not maintained between elongation factors and ribosomal protein early during shift-up. These data suggest that ribosomal and elongation factor proteins are not co-ordinately synthesized and argue against the postulate that their genes are part of one polycistron.     

Synthesis of individual ribosomal proteins in Escherichia coli B/r

The differential synthesis rates of individual ribosomal proteins (r-proteins) were measured in Escherichia coli B/r during the transition period following a nutritional shift-up from succinate minimal to glucose/ammo acids medium. These rates were observed to respond sequentially to the shift-up; the differential synthesis rate of protein L28 begins to increase within 0.1 of a minute following the shift-up, while the protein L29 synthesis rate begins to increase only after a lag of 2.5 minutes. The onset of induction of the remaining r-proteins occurs within this 2.5-minute interval. Furthermore, there was a twofold variation in the acceleration of the differential synthesis rates of individual r-proteins. Within the initial two to ten-minute period following the shift-up the differential synthesis rates of most r-proteins reached values ranging from 2.2 to 3.0-fold higher than the pre-shift rates, before declining to the post-shift steady-state values. It is suggested that the increases in the differential synthesis rates of r-proteins result in part from increases in the translational efficiency of messenger RNA in the post-shift growth medium and in part from increases in the amount of r-protein mRNA that is present.     

Inefficiency of the stringent response in the fungus Mucor

Removal of a required amino acid from the growth medium or addition of cycloheximide caused an immediate stoppage of growth and protein synthesis in the fungus $\text{Mucor}\text{racemosus}$. However, RNA synthesis persisted for several hours at rates that only gradually decreased under the same circumstances. An analysis of the major classes of RNA synthesized during the first hour of treatment showed that cycloheximide preferentially inhibited rRNA synthesis, whereas amino acid starvation slowed synthesis of all RNA species uniformly. Neither treatment affected the percentage of mRNA synthesized. The partial and delayed effects of amino acid starvation and cycloheximide treatment on RNA synthesis reported here suggest the absence of or the gross inefficiency of a classical stringent response in $\text{M.}\text{racemosus}$.     

Denovo synthesis of prolactin by human decidua

Relatively large amounts of immunoreactive prolactin were measured in homogenates of human decidual tissue obtained immediately after delivery of normal term pregnancies. In order to study the release and possible synthesis of prolactin by this tissue, explants of decidua were incubated for 24 hours at 37°C in oxygenated Gey's buffer containing 20% fetal calf serum. When cycloheximide was added to the medium in concentrations sufficient to prevent $\text{in}$$\text{vitro}$ protein synthesis, 85–90% of the prolactin present in the tissue was released into the medium during the first 3 hours of incubation. No additional prolactin accumulated in either the medium or the tissue during the remainder of the incubation period. In the absence of cycloheximide, the prolactin concentration in the medium increased progressively during incubation, so that after 24 hours the total amount of hormone present in the tissue and medium was significantly greater than that in the tissue and medium prior to incubation (37.6 ± 9.6 ng/ml at 0 time vs 82.2 ± 7.7 ng/ml at 24 hours). When ³H-1-leucine (100 u Ci) was supplied during incubation, radioactive proteins were detected in the medium at 24 hr, 14–20% of which were specifically precipitated by antiserum to human pituitary prolactin. When aliquots of this medium were chromatographed on Sephadex G-100, 80–95% of the ³H-proteins precipitated by antiserum to pituitary prolactin eluted in the same position as did purified, iodinated pituitary prolactin. These data indicate that a species of prolactin which is identical to pituitary prolactin by the criteria of immunoprecipitation and gel chromatography is synthesized by human decidual tissue $\text{in}$$\text{vitro}$.     

DNA-dependent in vitro synthesis of Escherichia coli ribosomal protein L10 and the formation of an L10L12 complex

The $\text{in vitro}$ synthesis of ribosomal protein L10 has been demonstrated using λrif^d18 DNA as template. The L10 synthesized $\text{in vitro}$ forms a complex with ribosomal protein L12 and the L10 in this complex can be immunoprecipitated with L12 antiserum.     

Differential translation of turnip yellow mosaic virus mRNAs in vitro

Total TYMV RNA was incubated in a reticulocyte lysate, and the initiation peptides of the main proteins synthesized $\text{in vitro}$ (195 K, 150 K and 20 K daltons) analyzed after tryptic digestion. The 195 K and the 150 K dalton proteins present analogous patterns, different from the one obtained with the 20 K dalton protein (coat protein), suggesting that only one initiation site exists on the genomic RNA for the synthesis of the two high molecular proteins. The results of competition experiments between genomic and coat protein mRNA indicate that the ribosomes have a much greater affinity for the coat protein mRNA. This may represent a regulatory mechanism for the preferential amplification of coat protein synthesis in the infected cells.     

Conformation of free and ribosome bound rRNAs from E.coli

The effect of protein moiety on the conformation of 16S and 23S RNA of the $\text{E.}\text{coli}$ ribosome has been studied by circular dichroic spectroscopy. Both rRNAs possess a comparable net content of ordered secondary structure which remains unchanged after association with ribosomal proteins into “core” particles or into complete 30S and 50S subunits, respectively. However, differences found in the stability and the cooperativity of melting of free and protein-associated rRNAs imply protein-caused variations in the distribution of the intramolecular hairpin stems and loops and/or changes in long range tertiary interactions which appear to be different for both rRNAs. While 23S RNA is maximally stabilized on the large subunit by the full set of proteins, 16S RNA on the complete small subunit shows lower stability but higher cooperativity in melting.     

Effect of cycloheximide on the induction of tryptophan oxygenase mRNA by hydrocortisone invivo

Hydrocortisone increases rat liver tryptophan oxygenase mRNA activity as measured by a translational assay. Pretreatment of rats with cycloheximide thirty minutes before hydrocortisone administration largely prevents the hormonal induction of tryptophan oxygenase mRNA. Tryptophan oxygenase mRNA activity begins to increase after a lag of at least 30 to 60 minutes after hydrocortisone injection. These results suggest that the synthesis of intermediary protein(s) is required for the induction of tryptophan oxygenase mRNA by glucocorticoids.     

Defective 30S ribosomal particles in a polyamine auxotroph of Escherichia coli

Polypeptide synthesis directed by poly(U) or MS 2 phage RNA is several fold more active in cell-free systems prepared from polyamine supplemented bacteria than in extracts of polyamine depleted cells. This effect depends on the presence of defective 30S ribosomal subunits in the starved bacteria. It is concluded that polyamines play a role in the normal biosynthesis, maturation and/or assembly of the small ribosomal subparticles.     

An invitro assay system for chitin synthesis in insect tissue

The system described utilizes the abdominal integument of the grasshopper $\text{Melanoplus}$$\text{sanguinipes}$ as the tissue and radioactive glucose, glucosamine or UDP-N-acetyl glucosamine as substrate. A micromethod is described that makes it possible to use as little as 2 μl total volume of incubation. After an initial lag period, incorporation rates become linear for at least 2 hrs. Cell disruption leads to immediate and complete loss of activity.     

Regulation of nitrate reductase level in pea: Invivo stability by ammonium

Ammonium, the end-product of nitrate-reduction, causes a marked increase in nitrate-dependent formation of nitrate reductase activity in pea shoot apices. The ammonium effect is mediated via a decrease in the rate of nitrate reductase decay. The increased stability of the enzyme in the presence of ammonium is indirect and depends upon protein synthesis. A regulatory role for ammonium-induced protein(s) is suggested.     

OKY-1581: A selective inhibitor of thromboxane synthesis invivo and invitro

OKY-1581 is an effective inhibitor of thromboxane synthesis $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. The generation of thromboxane B₂ (TxB₂), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$ caused a reduction in TxB₂ generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB₂ was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB₂ in atherosclerosis.     

Extra components in kinetoplast DNA preparations from Crithidiafasciculata

Kinetoplast DNA from the order Kinetoplastidae (trypanosomatids) exists as large associations (molecular weight 4 × 10¹⁰), made up of about 104 small, probably circular, molecules, commonly known as ‘minicircles’. These minicircles were originally thought to be identical in base composition, suggesting that the coding capacity of kinetoplast DNA is very restricted. However, linear molecules have also been observed in preparations of kinetoplast DNA, which, if they contain unique sequences, could represent additional genetic information. This linear DNA has been assumed to be derived from the kinetoplast, but the possibility of it being nuclear contamination has not been definitely ruled out. Work presented in this paper demonstrates that nuclear DNA contamination may indeed be present in kinetoplast DNA prepared by a commonly used method.     

Biological activity of invitro synthesised protein: Binding of Semliki Forest virus capsid protein to the large ribosomal subunit

Cell-free translation of the Semliki Forest virus-specific 26S RNA yielded primarily capsid protein. After treatment of the protein synthesising reaction with 25 mM EDTA, the capsid protein cosedimented with the large ribosomal subunit in sucrose gradients, and banded with the subunit at a density of 1.54 gm/cm³ in CsCl. Exposure to 0.5 M KCl released the protein from the subunit. Similar binding of the virus capsid protein to the large ribosomal subunit has been observed in infected HeLa cells, although its function is not clear. The nonstructural proteins, which are the major products translated from the virion 42S RNA, did not associate with sedimenting structures.     

Regulation of argF mRNA synthesis,performed in vitro

The specific synthesis of $\text{arg}$F mRNA directed by the $\text{arg}$F gene carried on the specialized transducing bacteriophage $\text{λh80C}{}_1\text{857darg}$F, performed $\text{in vitro}$, is described with the use of an S180 extract from a strain carrying $\text{arg}$R^?. Synthesis of $\text{arg}$F mRNA is biphasic at approximately 7 minutes. The regulation of $\text{arg}$F mRNA synthesis by the specific arginine holorepressor present in an S180 extract prepared from a strain carrying the $\text{arg}$R⁺ allele is described.