Initiation of Protein Synthesis in HeLa Cells

Initiation of protein synthesis in HeLa cells has been synchronized by exposure of the cells to fluoride. Double-labeling of such cells for short pulses with [(35)S]methionine and a tritiated amino acid, followed by Edman degradation of the puromycin-released nascent peptides, has shown that the percent of N-terminal methionine incorporated compared to total incorporation is significantly higher than the value obtained with any of the other amino acids tested. The results suggest that the bulk of the nascent proteins synthesized in vivo by HeLa cells are initiated with methionine.     

Atypical Pattern of Utilization of Amino Acids for Mitochondrial Protein Synthesis in HeLa Cells

The capacity of HeLa cell mitochondria, either isolated or in intact cells, to incorporate different labeled amino acids into proteins was investigated. Eight amino acids (alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, and lysine), which include most of the charged polar ones, showed a very low amount, if any at all, of chloramphenicol-sensitive incorporation, relative to that expected for an "average" HeLa-cell protein. By contrast, the most hydrophobic amino acids (leucine, isoleucine, valine, phenylalanine, and methionine) were the most actively incorporated by HeLa mitochondria. The available evidence suggests that pool effects cannot account for this general pattern of utilization of amino acids; furthermore, this pattern is in good agreement with the known hydrophobic properties of proteins synthesized in mitochondria.     

The Regulation of Protein Synthesis in Mammalian Cells VI. Soluble and Polyribosome Associated Components Controlling In Vitro Polypeptide Initiation in HeLa Cells

The in vitro initiation of polypeptides on endogenous polyribosomes has been studied in extracts from HeLa cells. Regulation of the rate of initiation of polypeptides can be examined. In these experiments an assay using [(35)S]fMet-tRNA(f) (Met) has been developed, and the system further characterized.The system has been separated into a fraction containing polyribosomes with subunits and a fraction containing soluble components. The regulation of initiation has at least two distinct components.There is one factor in the soluble fraction which develops a stimulated response after protein synthesis has been inhibited in intact cells. This stimulation does not require new RNA synthesis during the period of cell "stress."A second component is associated with ribosomes. This factor is necessary for the initiation of polypeptides on endogenous polyribosomes. It disappears gradually when cells are exposed to actinomycin. The disappearance is first manifested by an inability of polyribosomes to respond to stimulated supernatants. This unstable component, which decays in the presence of actinomycin, has no apparent counterpart in systems that measure initiation on exogenous mRNA.     

Stimulation of Polypeptide Initiation In Vitro After Protein Synthesis Inhibition In Vivo in HeLa Cells

Crude cytoplasmic extracts prepared from HeLa cells actively incorporate amino acids but show little initiation of new peptides (as seen by labeling of N-terminal amino acids). In contrast, extracts prepared from cells subjected to prior inhibition of protein synthesis show a significant amount of polypeptide initiation indicated by formation of peptides with radioactive N-terminal methionine. The same result was obtained whether prior inhibition occurred with cycloheximide or by starvation for an essential amino acid. Cellular response to suppression of protein synthesis appears to be mediated through production of RNA, since it is inhibited by actinomycin but appears in the presence of cycloheximide. The crude extracts continue initiating new polypeptides for at least 10 min in vitro. It is postulated that enhancement of in vitro initiation described here is related to the apparent stimulation of initiation of translation seen in vivo.     

Prebiotic Synthesis of Hydrophobic and Protein Amino Acids

The formation of amino acids by the action of electric discharges on a mixture of methane, nitrogen, and water with traces of ammonia was studied in detail. The presence of glycine, alanine, alpha-amino-n-butyric acid, alpha-aminoisobutyric acid, valine, norvaline, isovaline, leucine, isoleucine, alloisoleucine, norleucine, proline, aspartic acid, glutamic acid, serine, threonine, allothreonine, alpha-hydroxy-gamma-aminobutyric acid, and alpha,gamma-diaminobutyric acid was confirmed by ion-exchange chromatography and gas chromatography-mass spectrometry. All of the primary alpha-amino acids found in the Murchison Meteorite have been synthesized by this electric discharge experiment.     

Control of Single Ribosome Formation by an Initiation Factor for Protein Synthesis

30 and 50S ribosomal subunits, released from polysomes upon polypeptide chain termination, possess a high affinity for each other and readily form single ribosomes. Highly purified initiation factor F3(B), acting stoichiometrically, prevents the formation of single ribosomes without promoting their dissociation. These findings are interpreted in terms of a ribosome cycle in which a limiting amount of factor F3(B) controls the number of ribosomes active in protein synthesis, in response to metabolic changes in the cell, by regulating the flow of ribosomal subunits into polysomes or into a sidetrack pool of synthetically inactive single ribosomes. The reported apparent ribosome dissociation activity of F3(B) is explained.     

Regulation of the Nucleolar DNA-Dependent RNA Polymerase by Amino Acids in Ehrlich Ascites Tumor Cells

Experiments were performed to ascertain the degree to which the amount of amino acids might be one of the regulatory factors that control the activity of the nucleolar RNA polymerase. Assays of the enzymatic activity were done with isolated nuclei from cells incubated with low and high concentrations of amino acids. Soon after the cells were exposed to a medium enriched in amino acids, a rapid increase of nucleolar RNA polymerase activity occurred. A similar result was obtained in cells incubated with lower concentrations of amino acids. However, the rate of ribosomal RNA synthesized was regularly much higher in cells incubated in a medium enriched with amino acids than in a medium low in amino acids. Apparently, the amino acids only controlled ribosomal RNA synthesis. Thus, neither maturation, processing, and transport of nuclear precursors into cytoplasmic ribosomal RNA, nor the synthesis of rapidly labeled RNA was affected.     

Protein Synthesis in Rat Lymphocytes: Radioautographic Studies of Availability and Utilization of Labeled Amino Acids in Vivo

Injections of H³-methionine and H³-leucine were combined with radiochemical and radioautographic technics to study the availability time ofH³-methionine and the protein synthetic ability of rat lymphocytes in vivo.

Although 98.5 per cent of H³-methionine was removed from the serum5 minutes after injection, sufficient quantities persisted and/or re-entered theserum from tissues to cause increasing grain counts in radioautographs oflarge lymphocytes for 1 hour after isotope administration. A small amount ofadditional labeling occurred during the 2nd hour, but it is calculated thatlabeling is 97-98 per cent complete by 1 hour.

All of the large and medium lymphocytes were labeled in the thymus, lymphnode, and thoracic duct lymph at short intervals after injection of 4 µc./Gm.body weight of H³-methionine. Evidence is presented that protein synthesisoccurs in the nucleus as well as in the cytoplasm and that newly formed protein is equally distributed between daughter cells following mitosis. Previousimmunochemical studies are combined with information on generation timeand disappearance rates of radioactivity to suggest that large and mediumlymphocytes are constantly producing and releasing proteins. Large andmedium cells in lymph and lymph node are more active in this than aresimilar cells in the thymus. Evidence of reutilization of labeled metabolitesin the lymph node and especially in the thymus is discussed.

Although not all small lymphocytes were labeled by 4 µc./Gm. body weightof H³-methionine, it was shown that larger doses of isotope would label 100per cent of them. Small lymphocytes in thoracic duct lymph evidenced significant turnover of labeled protein during the 1st day after isotope administration.

Submitted on August 21, 1963 Accepted on November 9, 1963     

Initiation of Picornavirus Protein Synthesis in Ascites Cell Extracts

The current model of picornavirus protein formation implies that initiation of protein synthesis occurs at a single site on the viral RNA, and that the large polypeptide formed is later cleaved. A direct test of this model was made in vitro by studying the incorporation of [(35)S]methionine from rabbit liver Met-tRNA(M) (Met) and fMet-tRNA(F) (Met) into encephalomyocarditis virus RNA-coded proteins in extracts of Ehrlich ascites cells. The incorporation of N-formylmethionine was complete within 5 min, while utilization of Met-tRNA(M) (Met) continued for 20 min. Tryptic digests of [(35)S]methionine-labeled products from Met-tRNA(M) (Met) analyzed by anion-exchange chromatography yielded more than 30 peptides, as compared to about 15 [(35)S]methionine-labeled peptides from purified encephalomyocarditis virus. In contrast, products labeled with fMet-tRNA(F) (Met) yielded one major (26)S-labeled tryptic peptide. The N-terminal location of methionine in this peptide was verified by Edman degradation. One predominant N-terminal tryptic peptide was also obtained with fMet-tRNA(F) (Met) when mouse Elberfeld and mengo-virus RNAs were used as messengers. On the basis of N-terminal compared with internal labeling of the products, no evidence for in vitro post-translational cleavage was found. The results are consistent with a single initiation site for synthesis of picornavirus proteins.     

Nucleic Acids and Protein Metabolism in Acute Leukemia Cells

DNA, RNA and protein metabolism was investigated by means of a highresolution autoradiographic technic in normal and acute leukemia blast cellsby studying the incorporation of tritiated thymidine, uridine, leucine andphenylalanine. A strikingly lower percentage of cells labelled with thymidinewas demonstrated in acute leukemia and was interpreted as evidence of adecreased proliferative capacity. A very significantly lower uptake of uridine,leucine and phenylalanine was detected in acute leukemia cells.

In normal and leukemic cells, amino acid incorporation occurred both inthe nucleus and in the cytoplasm; uridine was incorporated exclusively inthe nucleus during the first hour of incubation and the cytoplasm becamelabelled only in a later period.

The constant ratio between uridine and amino acid incorporation detectedin normal myeloblasts was always altered in acute leukemia cells.

The lower RNA and protein metabolism and its dissociation in acute leukemia cells was discussed as related to the well-known maturation defectof these cells.

Submitted on May 20, 1960 Accepted on August 8, 1960     

Aurintricarboxylic Acid: Inhibitor of Initiation of Protein Synthesis

Aurintricarboxylic acid prevents the attachment of bacteriophage messenger RNA to ribosomes. As a consequence, initiation of protein synthesis in cell-free extracts prepared from Escherichia coli or rabbit reticulocytes is inhibited at concentrations of dye that do not prevent chain extension. Its properties can be distinguished from other agents that inhibit protein synthesis, including sodium fluoride, cycloheximide, and pactamycin.     

Increased Uptake of Amino Acids and 2-Deoxy-D-Glucose by Virus-Transformed Cells in Culture

Transformed and nontransformed cells in tissue culture differ in their rate of uptake of certain nutrients, as determined by a polyester-coverslip technique. A 2.5- to 3.5-fold increased rate of uptake of alpha-aminoisobutyric acid, cycloleucine, and 2-deoxy-D-glucose was observed with polyoma virus-transformed baby hamster kidney (BHK) 21 cells and simian virus 40 (SV40)-transformed BALB/3T3 (mouse fibroblast) cells, compared to their nontransformed counterparts. Kinetic analysis suggested that the increased uptake by cells transformed with virus was associated with a 3-fold greater V(max), with no detectable changes in apparent K(m). Limited studies also revealed increased initial rates of uptake by murine sarcoma virus-transformed rat liver cells, as compared to the parental line. Exposure of cells to concanavalin A and wheat-germ agglutinin led to significant reductions in amino-acid uptake by both transformed and nontransformed cells; however, transformed cells showed a greater decrease in uptake after exposure to wheat-germ agglutinin. Increased initial rates of uptake of certain amino acids and sugars may be a feature common to transformed cells, compared to their parental control.     

Met-tRNAfMet Binding to 40S Ribosomal Subunits: A Site for the Regulation of Initiation of Protein Synthesis by Hemin

On incubation of reticulocyte lysates at 30 degrees in the absence of added hemin, protein synthesis declines sharply within 4-6 min, due to the action of a translational inhibitor. Partially purified preparations of this inhibitor, in concentrations that inhibit protein synthesis in the lysate, cause reduced binding of Met-tRNA(f) (Met) to derived 40S ribosomal subunits in a ribosomal-salt-wash-dependent assay system. Neither the association of salt wash proteins with the subunits nor the level of Met-tRNA(f) (Met) bound in preformed 40S complexes is reduced by the inhibitor. No Met-tRNA(f) (Met) deacylase activity could be detected in the inhibitor preparation. Protein synthesis in reticulocyte lysates lacking added hemin or containing exogenous inhibitor is maintained by addition of small amounts of an initiation preparation factor, "F-MP," which may be involved in the binding of Met-tRNA(f) (Met) to 40S subunits. This binding constitutes a site for control of protein synthesis by hemin in reticulocytes.     

Double-Stranded Poliovirus RNA Inhibits Initiation of Protein Synthesis by Reticulocyte Lysates

Infection of HeLa cells with poliovirus results in an inhibition of host-cell protein synthesis. Cytoplasmic extracts from infected cells contian an activity that inhibits initiation of protein synthesis in vitro by rabbit reticulocyte lysates. This inhibitory activity has been purified and characterized, and is shown to reside in poliovirus double-stranded RNA. The intact double-stranded molecule is not essential for inhibitory activity.     

Role of Protein Synthesis on Ethanol Regulation of Adenylyl Cyclase Activity in Wild-Type S49 Murine Lymphoma Cells

Adenylyl cyclase activity was determined in membranes from wild-type S49 murine lymphoma cells that had been exposed to ethanol for 4 hr. Mn-, NaF-, and forskolin-stimulated adenylyl cyclase activities of cells—pretreated with cycloheximide, puromycin, or serum deprivation—were significantly decreased by treatment with 50 mM of ethanol. As demonstrated for Mn-stimulated activity, the decrease was dose-dependent on ethanol and was temporal; a normal activity recovered after 16–24 hr treatment, even in the presence of cycloheximide and ethanol. Studies with a cell-free membrane system of S49 cells revealed a similar activity decrease after treatment of the membranes with ethanol. In contrast, cells treated with 50 mM of ethanol in a regular culture condition showed no decrease in adenylyl cyclase activity over 24 hr. These results indicate that ethanol regulation of adenylyl cyclase activity in S49 cells depends on reduced or impaired protein synthesis.     

Thymidylate Synthesis in a Folate Deprived Cell Line

Summary . Abnormalities of the de novo and salvage pathways of thymidylate synthesis have been investigated in a folate deprived lymphoblastoid cell line. Impaired DNA synthesis was observed, with an increased percentage of cells in S and G₂ phase, whereas the mitotic index was decreased. Thymidylate synthesis along the salvage pathway was markedly increased, with a higher activity of thymidine kinase and higher uptake of ³H-thymidine (³H-TdR). The same abnormalities were observed when cells were treated with 5-fluorodeoxyuridine or methotrexate. The de novo pathway was slightly modified with a nearly normal incorporation of ³H-6 deoxyuridine (³H-UdR) and a moderate decrease of thymidylate synthetase activity; in contrast, the uptake of ³H-dU was markedly inhibited in drug-treated cells. Preincubation with cold deoxyuridine (10^?4 M) did not suppress the uptake of ³H-TdR as efficiently as in control cells; with increasing concentration of dU to 10^?2 M, this suppressive effect became almost complete. This high concentration of cold dU exerted a competitive inhibition on thymidine kinase. The deoxythymidine triphosphate (dTTP) pool was increased in deficient cells and it was only slightly increased by addition of cold dU (10^?4 M) in the culture medium whereas a substantial expansion of this pool was observed in control cells treated under the same conditions. These data do not necessarily exclude a defect of thymidylate synthesis along the de novo pathway in the folate deficient cells. The normal incorporation of ³H-dU could be explained by a decreased isotope dilution due to a reduced deoxyuridine monophosphate (dUMP) pool. This pool could be decreased by feedback inhibition by dTTP on some enzymatic activities, mainly deoxycytidylate deaminase. The enlarged dTTP pool which probably derives mainly from the salvage pathway could be poorly functional for DNA replication according to the model of compartmentation of DNA precursors.