Initiation of mammalian protein synthesis. II. The assembly of the initiation complex with purified initiation factors

The assembly of initiation complexes is studied in a protein synthesis initiation assay containing ribosomal subunits, globin [¹²⁵I]mRNA, [³H]Met-tRNA_f, seven purified initiation factors, ATP and GTP. By omitting single components from the initiation assay, specific roles of the initiation factors, ATP and GTP are demonstrated. The initiation factor eIF-2 is required for the binding of Met-tRNA_f to the 40 S ribosomal subunit. The initial Met-tRNA_f binding to the small ribosomal subunit is a stringent prerequisite for the subsequent mRNA binding. The initiation factors eIF-3, eIF-4A, eIF-4B and eIF-4C together with ATP promote the binding of mRNA to the 40 S initiation complex. The association of the 40 S initiation complex with the 60 S ribosome subunit to form an 80 S initiation complex is mediated by the initiation factor eIF-5 and requires the hydrolysis of GTP. The factor eIF-1 gives a twofold overall stimulation of initiation complex formation. A model of the sequential steps in the assembly of the 80 S initiation complex in mammalian protein synthesis is presented.     

Stimulation of differentiation of several murine embryonal carcinoma cell lines by retinoic acid

Retinoic acid stimulates several murine embryonal carcinoma (EC) cell lines, even those previously considered to be incapable of differentiating, to give rise to cell types distinguishable from the parental phenotype in morphology, production of plasminogen activator and surface protein properties. Retinoic acid promotes these changes over a range of low concentrations (10⁻⁹–10⁻⁵ M) which are generally non-toxic to the cells. The effects are clearly demonstrated when EC cells are aggregated prior to exposure to retinoic acid. It is concluded that the observed phenotypic alterations induced by retinoic acid reflect differentiation of the EC cells since non-EC cell characteristics are maintained by cloned cells several generations after retinoic acid is removed from the cultures. Our studies suggest that although retinoic acid stimulates the conversion of EC cells to differentiated derivatives, it does not influence the direction of differentiation. Furthermore, the effectiveness of retinoic acid in stimulating differentiation of EC cells from lines such as Nulli-SCC1 raises the question of whether true ‘nullipotent’ EC lines really exist.     

Commitment of hydra interstitial cells to nerve cell differentiation occurs by late S-phase

Nerve cells in hydra differentiate from the interstitial cell, a multipotent stem cell. Decapitation elicits a sharp increase in the fraction of the interstitial cells committed to nerve cell differentiation in the tissue which forms the new head. To investigate when during the cell cycle nerve cell commitment can be stimulated, hydra were pulse-labeled with [³H]thymidine at times from 18 hr before to 15 hr following decapitation; the resulting cohorts of labeled interstitial cells were in the various phases of the cell cycle at the time of decapitation. Increased commitment to nerve cell differentiation within a single cell cycle (≈24 hr) was observed in those cohorts which were at least 6 hr before the end of S-phase (12 hr) at the time of decapitation. The lag time required for decapitation to produce an effective stimulus for nerve cell differentiation was measured by transplanting the stem cells from the regenerating tissue to a neutral environment. Following decapitation, 3 to 6 hr were required for increased nerve cell commitment to be stable to such transplantation. These results suggest that interstitial cells must be stimulated by late S-phase to become committed to undergo nerve cell differentiation following the subsequent mitosis. However, when head regeneration was reversed by grafting a new head onto the regenerating surface, nerve cell differentiation by such committed stem cells was greatly reduced. This indicates that an appropriate tissue environment is required for committed interstitial cells to complete the nerve cell differentiation pathway.     

The mechanism of cell elongation during lens fiber cell differentiation

Lens fiber formation is characterized by extensive cell elongation. Earlier studies have shown that lens cell elongation in vitro can occur in the absence of microtubules and is associated with a proportional increase in cell volume. We have previously suggested that lens fiber cell elongation is directly caused by an increase in cell volume. In this report, lenses from 3- and 6-day-old chicken embryos were three-dimensionally reconstructed from serial sections to provide a measure of cell volume and length during various stages of primary and secondary lens fiber formation. In both cases, cell volume was highly correlated with cell length during lens cell elongation. In addition, during primary lens fiber formation, large intercellular spaces between lens vesicle cells disappeared as these cells began to elongate to form lens fibers. Loss of intercellular spaces would be expected if increasing cell volume were responsible for cell elongation. Finally, results of experiments in which the lens capsule was cut with a fine tungsten needle suggested that the capsule was elastic and normally under tension. These findings were used to formulate a model which accounts for the major events in lens morphogenesis based on (1) the regulation of cell volume, (2) the junctions present between lens cells, and (3) the constraint provided by the elasticity of the lens capsule.     

Cell cycle-dependent expression of surface antigens in murine T-lymphoma cells : II. Murine leukemia virus gp70 increases in S phase

Studies were undertaken to identify cell surface markers specific for different phases of the cell cycle. Antisera were prepared in rabbits against membrane protein preparations from synchronized BW 5147 cells, an AKR mouse T-lymphoma cell line, in the G1, S, G2 or M phases of the cell cycle. These antisera were used to precipitate radioiodinated surface proteins from synchronized cells in the different phases. The immunoprecipitates were quantitatively analyzed by sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Cells in S phase had significantly higher concentrations of proteins weighing 70 × 10³ and 165 × 10³ D than cells in G1 or G2 phase. The other major labeled surface components did not vary. These results were confirmed by quantitative absorption of the antisera with synchronized cells. Comparative analysis of the antisera showed that the 165 × 10³ D peak contained at least two antigens, one recognized by both a-G1 and a-S and the other by a-G1 only. Though cells in S phase had large quantities of the 70 × 10³ D protein, intact and SDS-solubilized membrane preparations from S phase could not elicit in rabbits any antibody against that protein. These antisera did, however, have good antibody titers to the other major protein peaks and the antisera developed against cells in G1, G2 or M had good anti-70 × 10³ activity. The results suggest a qualitative molecular change in the 70 × 10³ protein during S phase.     

Temperature sensitivity of muscle and neuron differentiation in embryonic cell cultures from the Drosophila mutant, shibire.

The sex-linked temperature-sensitive mutation, shibire^ts1, which causes, at the restrictive temperature, adult paralysis and pleiotropic morphological defects in embryonic, larval, and pupal development, has been shown to exhibit temperature-sensitive inhibition of differentiation in embryonic cultures in vitro. When shi cultures were incubated at 30°C for 24 hr, both muscle and neuron differentiation were inhibited more than 90% compared to control shi cultures incubated at 20°C. Heat shift experiments showed that the temperature-sensitive periods for neuron and muscle differentiation occurred at 11 to 18 and 14 to 16 hr, respectively, where zero time was the initiation of gastrulation in donor embryos. Short heat pulses (4 and 8 hr) which extended into the temperature-sensitive period resulted in moderate inhibition of differentiation; greater inhibition occurred as the duration of the pulses increased. In contrast, heating wild-type Oregon-R cultures at 30°C for 24 hr did not inhibit muscle cell differentiation and inhibited neuron differentiation relatively little. The temperature-sensitive period in shibire for muscle differentiation occurred well after myoblast division, during the period of myocyte elongation, aggregation, and fusion, whereas that for neuron differentiation took place during a period of enzyme synthesis (acetylcholinesterase and choline acetyltransferase) and axon elongation. Thus, the shi temperature-sensitive gene product affects at least two different cell types, in vitro, at different times during differentiation.     

Mitotic cell populations obtained from a micro-carrier culturing system

Colcemid treatment of cell culture beads confluent with cells released a mitotic population of cells which was as efficient as that obtained by conventional shake-off procedures. The bead system provides an easy and efficient means for harvesting large quantities of mitotic cells, costs about one-tenth as much as conventional culturing techniques, and is at least 100 times more rapid.     

Inhibition of cell aggregation by specific carbohydrates.

One approach to investigating the potential role of surface carbohydrates in mediating intercellular adhesion is to study cell reaggregation in the presence of defined concentrations of specific saccharides. Fifteen different exogenously added saccharides were tested for their effect on the reaggregation of 24 h sea urchin embryo cells (Strongylocentrotus purpuratus) dissociated by removal of divalent cations. Aliquots (0.2 ml) of cell suspension were rotated at 68 rpm, 17 °C, pH 8.0, with varying concentrations (0.5 × 1^?1?0.5 × 10^?5 M) of the sugars. Relative percents of cell aggregation were determined using an electronic particle counter assay. In all experiments cell viability using trypan blue was over 95.8%. Among the sugars tested, in 15 separate experiments, d-galactose and N-acetyl-d-galactosamine consistently inhibited aggregation to the greatest extent at early time points. d-Galactose, at all concentrations tested, at 10, 20, 30, 40, and 60 min rotation, showed mean decreases of aggregation over control values in the absence of sugar of 59.3, 53.6, 43.2, 35.0 and 36.4%, respectively. N-Acetyl-d-galactosamine also caused mean decreases in aggregation of 73.5, 54.5, 40.8, 42.2 and 45.6%, respectively. Each difference over the control is significant to the p value of less than 0.01. In three experiments, β-galactosidase substantially inhibited reaggregation of these cells. These results suggest that galactopyranosyl-like groups may be implicated in mediating adhesion of 24 h sea urchin embryo cells to each other.     

Tubulin synthesis and heat shock-induced cell synchrony in Tetrahymena

This paper offers the suggestion that heat shock inhibition of tubulin synthesis accounts for the molecular mechanism by which periodic heat shocks induce cell synchrony in Tetrahymena. Each heat shock (34 °C) represses tubulin synthesis and blocks the division cycle at the point when the oral structure, rich in microtubules, would normally begin to assemble. Recovery (at 28 °C) from each heat shock is characterized by parallel derepression of tubulin synthesis and of oral development. Changes in protein synthesis patterns are complex when the temperature is shifted up and down between 28 and 34 °C and further experimental support is required in support of the hypothesis here forwarded.     

Carbohydrate composition of central nervous system synapses: Analysis of isolated synaptic junctional complexes and postsynaptic densities

The composition of specialized structures present at synapses within the central nervous system was elucidated by biochemical analysis of fractions enriched in synaptic junctional complexes and postsynaptic densities. The results indicate that the synaptic junctional complex is primarily protein together with some glycoproteins. The synaptic junctional complex proteins are similar in amino acid composition to synaptic membrane proteins; they are not especially rich in basic residues, as previously suggested. The major carbohydrates present in the synaptic junctional complex and postsynaptic density glycoproteins are mannose, galactose, and glucosamine, with lesser amounts of fucose, $\text{N-}\text{acetylneuraminic}$ acid, and galactosamine. Comparison with the synaptic membrane fraction indicates that galactose is more concentrated in the synaptic junctional complex and mannose in the postsynaptic density. Glucose is dramatically enriched in both these fractions. Sucrose binding during isolation may partially account for the glucose enrichment.     

Biosynthesis of transcobalamin II by adult rat liver parenchymal cells in culture.

The biosynthesis of transcobalamin II was investigated in primary cultures of adult rat liver parenchymal cells maintained in serum-free media. The data indicate that these hepatocytes secrete a vitamin B₁₂-binding substance into the culture medium which is identical to rat serum transcobalamin II as judged by the following criteria: (i) gel filtration on columns of Sephadex G-200; (ii) ion-exchange chromatography on columns of diethyl aminoethyl cellulose and carboxymethyl cellulose; (iii) polyacrylamide-gel electrophoresis at pH 9.5; and (iv) the ability to facilitate cellular vitamin B₁₂ uptake by HeLa cells and mouse L-929 fibroblasts in culture. The secretion of transcobalamin II by the liver parenchymal cells was blocked by cycloheximide, puromycin, and p-fluorophenylalanine. The inhibition by cycloheximide, but not that of the other inhibitors, was partially reversed upon removal of the drug. The liver parenchymal cells incorporated radioactive amino acids into transcobalamin II which was absorbed from the growth medium using affinity chromatography on Sepharose containing covalently linked B₁₂. Collectively, these data indicate that rat liver parenchymal cells, in culture, are capable of the biosynthesis de novo of transcobalamin II and the subsequent secretion of this protein into the culture media.     

Lowry determination of protein in the presence of Triton X-100.

The Lowry procedure has been modified for use in the presence of Triton X-100 (TX-100) by the addition of 10% sodium dodecyl sulfate. The method is applicable to samples containing 40–120 μg protein.     

Characterization of a macrophage chemotactic lymphokine produced by purified protein derivative stimulation in vitro and in vivo

Using an immunoadsorbent column conjugated with anti-macrophage chemotactic factor-c (anti-MCF-c), MCF-c which has been separated and highly purified from a delayed-type hypersensitivity reaction (DHR) site, shares common antigenicity with the major macrophage chemotactic lymphokine released from purified protein derivative (PPD)-stimulated lymphocytes and also macrophage chemotactic lymphokine from phytohemagglutinin (PHA)-stimulated lymphocytes. Using a combination of the immunoadsorbent column and Sephadexgel chromatography these two lymphokines are purified to homogeneity from PPD- or PHA-stimulated guinea pig lymphocyte culture supernatants. These observations, taken in conjunction with the similarity in biological activities, physicochemical properties, and antigenicities, suggest that these two mediators are very similar, or possibly identical. MCF-c with chemotactic activity for macrophages seemed to exist as complexes with serum protein at the skin site of PPD-induced DHR in guinea pigs. The active substance, separated from the complexes under acid conditions, is indistinguishable from the major macrophage chemotactic lymphokine released by PPD stimulation with respect to antigenicity, heat stability, and non-diffusibility. They both have a molecular weight of about 12, 500. The chemotactic lymphokine formed comparable complexes with serum protein under neutral conditions; however, this complex dissociated in acid without loss of activity.     

Membrane differentiation and de Nova synthesis of the (Na+ + k+)-activated adenosine triphosphatase during development of Artemia salina nauplii.

The developing brine shrimp, Artemia salina, nauplius is explored as a new model for the study of the biogenesis of the cation transport enzyme, (Na⁺ + K⁺)-activated adenosine triphosphatase [(Na, K)-ATPase]. (Na, K)-ATPase activity develops from undetectable levels in preemergent cysts (embryos prior to 12 hr of development) to very high levels in the nauplius after 40 hr of incubation in sea water [Conte, F. P., Droukas, P. C., and Ewing, R. D. 1977). J. Exp. Zool.202, 339], then declines between 44 and 72 hr. Similar ontogenic patterns of enzyme activity development are observed for Mg-ATPase, 5′-nucleotidase, glucose-6-phosphatase, NADH oxidase (rotenone insensitive), and cytochrome oxidase. However, these enzymes show measurable activity in the early cyst stage, and the points at which the activity increases and then reaches a maximum are usually different from those of the (Na, K)-ATPase. These enzyme ontogeny studies demonstrate that membrane differentiation is extensive during the period in naupliar development when (Na, K)-ATPase activity appears, and that the appearance of specific enzymes is asynchronous during embryogenesis. Pulse-chase experiments with NaH¹⁴CO₃ show an increase in the specific radioactivity of the partially purified (Na, K)-ATPase which is maximum when the label is administered at 12–18 hr after the initiation of development. At this time the specific radioactivity increases with purity of the enzyme, whereas in earlier pulse periods the specific radioactivity is higher in the more crude enzyme fractions, suggesting that preferential synthesis of the (Na, K)-ATPase occurs between 12 and 18 hr. Radioactivity is found in the subunits of the partially purified (Na, K)-ATPase isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and is maximum for the 12- to 18-hr pulse experiment. These pulse-chase experiments demonstrate that the large increase in (Na, K)-ATPase activity is due to de novo synthesis and establish that the brine shrimp is a workable new model for the study of the biogenesis of the (Na, K)-ATPase.     

Density labeling of proteins with 13C-labeled amino acids: no accumulation of an inactive alpha-amylase precursor in barley aleurone cells.

Gibberellic acid enhances α-amylase (EC 3.2.1.1) production in isolated barley aleurone layers after a lag period of 4 to 8 h, and most of the enzyme is produced after 12 h of hormone treatment. Amino acids necessary for protein synthesis in barley aleurone layers are derived from the degradation of storage proteins in this tissue. Since bromate is an inhibitor of barley protease, in the presence of bromate the production of α-amylase in aleurone layers becomes dependent on exogenous amino acids. We have incubated aleurone layers with bromate plus ¹³C-labeled amino acids and [³H]leucine from 0 to 24, 0 to 12, and 12 to 24 h after the application of gibberellic acid. The chemical quantity of [³H]leucine was negligible in comparison to that of ¹³C-labeled amino acids. Therefore, any density shift of proteins observed must be due to the incorporation of ¹³C-labeled amino acids. The density shift of α-amylase and that of newly synthesized proteins (radioactivity profile) were determined by isopycnic centrifugation in CsCl density gradients. The density shift of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 12 to 24 h after the addition of hormone was much larger than that of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 0 to 12 h of hormone treatment. By comparing the density shift of α-amylase with that of newly synthesized proteins, it is apparent that essentially all the amylase molecules are de novo synthesized. We can conclude that there is little or no accumulation of an inactive α-amylase precursor in barley aleurone cells between the time of the application of gibberellic acid and the time of the rapid increase in α-amylase activity.     

Specificity of codon recognition by Escherichia coli tRNALeu isoaccepting species determined by protein synthesis in vitro directed by phage RNA.

Codon-anticodon recognition and transfer RNA utilization for the leucine tRNA isoaccepting species of Escherichia coli have been studied by protein synthesis in vitro directed by sequenced bacteriophage MS2 RNA. We have added radioactive Leu-tRNA^Leu isoaccepting species as tracers, rather than use a tRNA-dependent system, since in the presence of an excess of non-radioactive leucine, there is no transfer of radioactive leucine from one isoaccepting species to another. MS2-specific peptides containing leucine residues encoded by known codons were isolated and identified, and the relative abilities of the Leu-tRNA^Leu isoaccepting species to transfer leucine into these peptides compared. Sequenced tRNA₁^Leu and sequenced tRNA₃^Leu are of roughly equal efficiency in their ability to recognize CUC and CUA codons, while tRNA₃^Leu is highly preferred for the CUU codon; tRNA₄^Leu and tRNA₅^Leu both recognize UUA and UUG codons, with tRNA₄^Leu slightly preferred for the UUA codon. We conclude that: (1) wobble is greater than permitted by the wobble hypothesis; (2) there is still some discrimination in the third code letter, and that the CUX₄ (CUC, CUA, CUU, CUG) portion of the leucine family of six codons is not read by a simple “two out of three” mechanism; (3) a Watson-Crick pair (C · G) between codon and anticodon does not appear to be preferred over an unorthodox pair (C · C) in the wobble position; (4) a standard wobble pair (U · G) between codon and anticodon is preferred over an unorthodox pair (U · C); and (5) the extensive wobble observed in the CUX₄ leucine codon series is not paralleled in the UUX₄ leucine (UUG, UUA) and phenylalanine (UUU, UUC) codon series, where mistranslation would be the consequence of such wobble.     

Enzymology of long-chain base synthesis by liver: characterization of serine palmitoyltransferase in rat liver microsomes

Serine palmitoyltransferase [palmitoyl-CoA:L-serine C-palmitoyltransferase (decarboxylating) EC 2.3.1.50] catalyzes the initial and committed step in the biosynthesis of the long-chain bases of sphingolipids. A simple assay, based upon the incorporation of [3H]serine into the chloroform-soluble product 3-ketosphinganine, has been developed and demonstrated to be valid for analyzing this enzyme in rat liver microsomes. More than 75% of the serine palmitoyltransferase of rat liver was associated with the microsomal subfraction. The dependencies of activity on the incubation time, pH, temperature, other assay components (e.g., dithiothreitol, EDTA, and pyridoxal 5'-phosphate), and the concentrations of microsomal protein, L-serine, and palmitoyl-CoA were investigated. The requirement of pyridoxal 5'-phosphate for activity was established by formation of the apoenzyme by dialysis against cysteine, and recovery of full activity upon reconstitution with the coenzyme. Activities with fatty acyl-CoA's of varying alkyl chain length were distributed nearly symmetrically around a maximum at 16 carbons (palmitoyl-CoA) for the fully saturated substrates. Less activity was obtained with the CoA thioesters of cis-unsaturated fatty acids, but trans-9-hexadecenoyl-CoA yielded essentially the same activity as palmitoyl-CoA. Hence, this enzyme is capable of initiating the synthesis of the major long-chain bases, as well as compounds that may constitute the unidentified bases reported in analyses of mammalian sphingolipids.     

5-Bromodeoxyuridine inhibition of differentiation. Kinetics of inhibition and reversal in myoblasts

This paper describes experiments on the kinetics of inhibition of muscle differentiation in vitro in the presence of 5-bromodeoxyuridine (BrdUrd) and the recovery phenomena that occur when such inhibited cells are permitted growth in normal medium. The studies consist of a quantitation of cell fusion in the presence of the analog and during recovery in its absence coupled with simultaneous studies on changes in buoyant density of cellular DNA. We find that if myoblasts are exposed to BrdUrd during the last doubling before cell fusion would normally occur, most cells do not differentiate, but as many as 18% of the cells can fuse in spite of the incorporation of BrdUrd into their nuclei. These nuclei contain approximately the amount of BrdUrd expected for a full round of DNA synthesis. Studies on the rate of recovery of inhibition of cell fusion following one generation in BrdUrd reveal that after one doubling of inhibited cells in the presence of normal medium. fusion reaches about 50% of the control value; after two doublings it reaches 75% of control value; and after 2.5 doublings of reversal, recovery is essentially complete. We find that both the degree of inhibition after approximately one round of BrdUrd incorporation and the rate of cell differentiation after two generations of reversal are consistent with a model which assumes that BrdUrd “sensitivity” resides on single pair of chromosomes and that inhibition occurs in a dominant fashion if approximately 30% or more of the thymidine is replaced by BrdUrd in the readout strand of either chromosome.     

Glucocorticoid requirement for tyrosine aminotransferase induction by adenosine 3':5'-cyclic phosphate analogs in somatic cell hybrids.

The ability of adenosine 3′:5′-cyclic phosphate (cyclic AMP) analogs to induce l-tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5; TAT) in a rat hepatoma (H35)-rat liver cell (BRL) hybrid (BF5) and a subclone which has lost 29 chromosomes (BF5-1-1) has been analyzed. Cyclic AMP analogs alone were unable to increase TAT activity in either hybrid cell line or in the “normal” liver cells despite three- to fivefold induction of this enzyme in the hepatoma parental cells. In contrast, dexamethasone by itself reproducibly increased TAT activity both in BF5-1-1 cells and in the parental H35 hepatoma cells. Pretreatment of the hybrid cells with dexamethasone revealed a synergistic increase in TAT activity when a cyclic AMP analog was added. From studies of the thermal stability and immunological inhibition of TAT activity, it is concluded that the low basal activity in BRL, BF5, and BF5-1-1 cells represents tyrosine transamination catalyzed by a different aminotransferase, whereas all the induced activity does represent bona fide TAT. The results suggest that functional TAT mRNA may not be present in significant quantities in the hybrid cells in the absence of adrenal steroids and that this could account for the inability of cyclic AMP analogs to exert their presumably translational effect on TAT synthesis.     

Antigen modulation of the secondary immune response. VI. Contribution of cells recruited from precursor pool to expression of memory.

The adoptive transfer system has been used extensively to study the ability of antigen triggered memory cells to become antibody forming cells and/or to proliferate and expand the memory cell population. Selective antigen triggering of the memory cells for low and high affinity antibody formation has also been studied in this way. One of the main counter-arguments to the interpretation of these data is that the presence of antigen in the adoptive host may lead to recruitment of new memory cells from either a host or donor precursor population. In this paper we examined the contribution of both host and donor precursor cells to the total antibody response in adoptive secondary recipients. The following donor-host combinations were used in which the recipients were given 1 mg fluid antigen intravenously: (A) normal (non-immune) donors to normal irradiated recipients; (B) normal donors to carrier primed irradiated recipients; (C) carrier primed donors to normal irradiated recipients; (D) normal donors to carrier primed recipients with challenge and subsequent transfer to additional carrier primed recipients; (E) carrier primed donor to normal recipients to carrier primed recipients; (F) repeat of B and C above with multiple antigen administration; (G) purified immune (DNP-BGG) donor T cells mixed with normal B cells transferred to normal irradiated recipients. In most cases recruitment was seen but this represented less than 4% of the responses seen with immune cells. Thus we conclude that this level of recruitment does not compromise the use of the adoptive transfer system for studying selective antigen triggering of memory cells.