EFFECT OF AUXIN AND ANTIAUXIN ON THE GROWTH AND RNA SYNTHESIS OF ETIOLATED PEA INTERNODE

Using etiolated Alaska pea internode segments the followingresults were obtained. An auxin, 2,4-dichlorophenoxyacetic acid, 1 mg/liter, inducedthe elongation growth of the segment and stimulated the biosynthesisof RNA, particularly of messengar RNA, in 3 hr incubation. Thesimilar stimulation of RNA synthesis and of elongation due toindole-3-acetic acid was found in 1 hr incubation. The stimulativeeffect of 2,4-dichlorophenoxyacetic acid on the elongation andon the synthesis of messenger RNA was inhibited by the additionof an antiauxin, transcinnamic acid. Significance of the auxin-inducedsynthesis of messenger RNA in producing protein responsiblefor the stress relaxation of cell wall is discussed. (Received June 13, 1967; )     

Ribonucleic acid and protein synthesis in Rhizophlyctis rosea zoospores

LéJohn, Herbert B. (Purdue University, Lafayette, Ind.), and James S. Lovett. Ribonucleic acid and protein synthesis in Rhizophlyctis rosea zoospores. J. Bacteriol. 91:709-717. 1966.-The uniflagellate zoospores of Rhizophlyctis rosea display active motility and a high endogenous respiratory metabolism, but neither growth nor net ribonucleic acid (RNA) or protein synthesis can be measured by ordinary procedures. Nevertheless, synthesis can be detected with isotopic precursors. Uracil-C(14) is incorporated slowly into both the soluble and ribosomal RNA. Analysis of zoospore extracts (on diethylaminoethyl cellulose columns or sucrose gradients) after various periods of labeling suggested that most of the uracil incorporation represents slow synthesis of ribosomal precursor RNA and, ultimately, ribosomes. Actinomycin D caused an 80% inhibition of uracil incorporation. The most rapidly labeled RNA was susceptible to extensive degradation in cells treated with actinomycin, but the percentage of stable RNA increased with the time of incorporation before addition of the antibiotic. Neither the effects of actinomycin nor the results of chase experiments have established unequivocally the existence of turnover or the presence of a short-lived "messenger" fraction in motile spores. Both leucine and methionine were slowly incorporated into a spectrum of cellular proteins. The methyl group of C(14)-methylmethionine also served as a methyl donor for the methylation of soluble RNA but not of ribosomal RNA. The observations that some of the newly synthesized RNA and protein occur in the intact 82S ribosomes and that actinomycin inhibits the low level of protein synthesis provide some indirect evidence for a very low rate of "messenger" synthesis and turnover in zoospores.     

THE RELATIONSHIP BETWEEN RNA SYNTHESIS AND HEMOGLOBIN SYNTHESIS IN AMPHIBIAN ERYTHROPOIESIS : Cytochemical Evidence

A cytochemical study of the relationship between RNA synthesis and hemoglobin synthesis has been performed on splenectomized newts, Triturus viridescens. Employing radioautography, labeled cytidine was incorporated into the RNA of the early developmental stages but was not incorporated in the later stages. Labeled leucine was incorporated into the cellular protein of all stages except mature erythrocytes but was incorporated at a higher level in the later stages. Microphotometric measurements of azure B binding to cytoplasmic RNA revealed a sharp initial increase between the stem cell and proerythroblast followed by a rapid decrease between the basophilic and polychromatophilic stages. The loss of cytoplasmic RNA became more gradual in the late stages and, in the mature erythrocyte, little or no cytoplasmic RNA could be detected. Measurements of cytoplasmic total protein, using fast green staining at pH 2.0, and of heme showed that both curves increased similarly with development, indicating net hemoglobin synthesis. The results are compatible with the hypothesis that, as the stem cell differentiates along erythrocytic lines, a stable "messenger" RNA specifying the production of a given type or types of hemoglobin is formed. This complex probably becomes associated with ribosomal RNA and is retained throughout the process of RBC differentiation.     

Potassium Requirement for Synthesis of Macromolecules in Bacillus subtilis Infected with Bacteriophage 2C

A mutant of Bacillus subtilis 168 (strain 168 KW), defective in its ability to concentrate K(+) from low levels in the growth medium, was used to study the role of K(+) in the development of phage 2C. Both the final burst size and the duration of the rise period depended on the K(+) concentration in the medium. During normal infection (in the presence of K(+)), host deoxyribonucleic acid (DNA) synthesis stopped. The synthesis of host messenger ribonucleic acid (RNA) continued throughout infection, albeit at a steadily decreasing rate. The synthesis of ribosomal RNA and its subsequent incorporation into mature ribosomes also proceeded. In contrast to these findings, host DNA and messenger RNA synthesis were not inhibited in cells infected in the absence of K(+). Only "early" phage messenger RNA was synthesized under these conditions of infection. Phage DNA synthesis was dependent on K(+) irrespective of the requirement for this cation in protein synthesis.     

The effect of cycloheximide upon polyribosome stability in two yeast mutants defective respectively in the initiation of polypeptide chains and in messenger RNA synthesis

Summary The effect of cycloheximide upon protein synthesis, RNA metabolism, and polyribosome stability was investigated in the parent and in two temperature-sensitive mutant yeast strains defective respectively in the initiation of polypeptide chains and in messenger RNA synthesis. Cycloheximide at high concentrations (100 g/ml) severely inhibits but does not completely stop protein synthesis (Fig. 1); the incorporation of ¹⁴C-amino acids into polyribosome-associated nascent polypeptide chains continues at a slow but measurable rate (Figs. 2 and 3). Polyribosome structures are stable in the parent strain at 36° whether or not cycloheximide is present (Fig. 5). However, in Mutant ts^- 136, a mutant defective in messenger as well as in stable RNA production, polyribosomes decay at the restrictive temperature (36° C) at the same rate whether or not cycloheximide is present (Fig. 5). Thus the maintenance of polyribosome structures is dependent upon the continued synthesis of messenger RNA even under conditions of extremely slow polypeptide chain elongation. In mutant ts^- 187, a mutant defective in the initiation of polypeptide chains, all of the polyribosomes decay to monoribosomes within 2 minutes after a shift to the restrictive temperature; cycloheximide completely prevents this decay demonstrating that this mutant is capable of continued messenger RNA synthesis at 36° C. Consistent with these observations is the fact that a newly synthesized heterogeneously sedimenting RNA fraction continues to enter polyribosomes in the presence of cycloheximide whereas the entrance of newly synthesized ribosomal RNA is severely inhibited (Figs. 7, 8, 9). The decay or lack of decay of polyribosomes at the restrictive temperature is, therefore, a rapid and discriminating test for the analysis of mutants defective in macromolecule synthesis. Mutants which exhibit a decay of polyribosomes in the presence of cycloheximide are likely to be defective directly or indirectly in the synthesis of messenger RNA whereas mutants in which decay is prevented or slowed by cycloheximide are likely to be defective in some factor required for the association of ribosomes and messenger RNA.     

THE REGULATION OF RNA SYNTHESIS AND PROCESSING IN THE NUCLEOLUS DURING INHIBITION OF PROTEIN SYNTHESIS

The effect of protein synthesis inhibition by cycloheximide on nucleolar RNA synthesis and processing has been studied in HeLa cells. Synthesis of 45S RNA precursor falls rapidly after administration of the drug. However, the nucleolar content of 45S RNA remains relatively constant for at least 1 hr because the time required for cleavage of the precursor molecule into its products is lengthened after treatment with cycloheximide. The efficiency of transformation of 45S RNA to 32S RNA remains constant with approximately one molecule of the 32S RNA produced for each cleavage of a molecule of 45S RNA. However, shortly after the cessation of protein synthesis the formation of 18S RNA becomes abortive. The amount of 32S RNA present in the nucleolus remains relatively constant. After long periods of protein synthesis inhibition the 28S RNA continues to be synthesized and exported to the cytoplasm but at a greatly reduced rate. When the protein synthesis inhibitor is removed, a prompt, although partial, recovery in the synthesis rate of 45S RNA occurs. The various aspects of RNA synthesis regulation and processing are discussed.     

Characterization of RNA synthesized during germination of Blastocladia ramosa zoospores

The synthesis of RNA was studied during the synchronous germination of Blastocladia ramosa zoospores. Comparison of RNA synthesis during germination of B. ramosa and Blastocladiella emersonii zoospores revealed that B. ramosa has a longer lag time before RNA synthesis is initiated and, in addition, the rate of RNA synthesis is ten-fold lower in B. ramosa. Zoospores of B. ramosa were shown to contain pre-formed messenger RNA but this messenger RNA directs only a portion of the protein synthesis which occurs during early germination. The conclusion that the remainder of the protein synthetic activity of the germinating spores is due to new message synthesis was supported by demonstrating that the timing of the initation of protein synthesis on new messages correlates with the time RNA synthesis is initiated. New message synthesis was also demonstrated by the incorporation of label into RNA which contains a poly (A) fragment. Synthesis of all classes of RNA including ribosomal, messenger, and transfer RNA was shown to be initiated at the same time. The implications of this observation are discussed.     

Protein synthesis by long-lived messenger ribonucleic acid in bacteria

1. Experiments were performed to investigate two hypotheses about the function of long-lived messenger RNA in bacteria. After RNA synthesis had been stopped by the addition of actinomycin, continuing protein synthesis was used as a measure of persistent messenger RNA. 2. The hypothesis that messenger RNA responsible for the synthesis of membrane protein is exceptionally long-lived was tested in experiments with protoplasts of Bacillus megaterium. However, this messenger RNA proved to be of approximately average stability. 3. The hypothesis that long-lived messenger RNA is responsible for the synthesis of constitutive proteins was tested by comparing the synthesis of penicillinase in an inducible and a constitutive strain of Bacillus licheniformis. After the addition of actinomycin, penicillinase synthesis continued for far longer in the constitutive than in the inducible strain. This difference is attributed to a difference in stability of the penicillinase-messenger RNA in the two strains, which does not extend to all messenger RNA indiscriminately. 4. A model is tentatively proposed to account for the altered stability of messenger RNA in the constitutive mutant.     

REVERSIBLE DEGRADATION OF POLYRIBOSOMES IN CHANG CELLS CULTURED IN A GLUTAMINE-DEFICIENT MEDIUM

The effects of temporary glutamine deficiency on the protein and nucleic acid metabolism of Chang's liver cells in suspension cultures have been studied. It was observed that cells maintained in a glutamine-free medium showed a reduced incorporation of labeled precursors into protein and RNA. At the same time, the activity of the ribosomes and the proportion of polyribosomal aggregates in cell extracts diminished. These effects were reversed when the glutamine content of the medium was restored. The restoration of a normal rate of amino acid incorporation by intact cells as well as by cell-free systems was time dependent, and took place within a few hours after glutamine addition without preceding increase in the prevailing low rate of RNA synthesis. The addition of actinomycin D at concentrations that strongly inhibited the RNA metabolism of the cells did not prevent the increase in protein synthesis or the reappearance of polyribosomal aggregates. These facts suggest that the restoration of protein synthesis in the cells after glutamine starvation was not dependent on a production of new messenger RNA. The experimental data are consistent with the hypothesis that previously synthesized messenger RNA, preserved in the cells in a stable form, was brought into action in response to the reestablishment of an adequate cellular environment.     

Streptomycin Action: Greater Inhibition of Escherichia coli Ribosome Function with Exogenous than with Endogenous Messenger Ribonucleic Acid

Inhibition of protein synthesis by streptomycin was tested in extracts from a strain of Escherichia coli sensitive to streptomycin. Three kinds of messenger ribonucleic acid (RNA) were employed: endogenous cellular RNA, extracted cellular RNA, and phage R17 RNA. Protein synthesis directed by extracted cellular RNA was inhibited three- to fourfold more than protein synthesis directed by endogenous RNA. With R17 RNA as messenger, nearly total inhibition of protein synthesis at initiation was again observed. The greater inhibition of function of extracted RNA, which must initiate new polypeptide chains in vitro, is in accord with the observation that in whole cells streptomycin blocks ribosomes at an early stage in protein synthesis. When streptomycin was added at successively later times during protein synthesis, the subsequent inhibition was progressively less. This was observed with either extracted cellular RNA or phage R17 RNA. A model is presented that can explain the less drastic inhibition by streptomycin of messenger RNA that is already functioning on ribosomes.     

PERSISTENCE OF MESSENGER RNA THROUGH MITOSIS IN HELA CELLS

The decrease in protein synthesis which occurs in mammalian cells during cell division is associated with significant disaggregation of polyribosomes. For determining whether messenger RNA survives this disaggregation, the reformation of polyribosomes was investigated in synchronized HeLa cells as they progressed from metaphase into interphase in the presence of 2 µg/ml Actinomycin D. The persistence of messenger during cell division was evidenced by: (1) a progressive increase in the rate of protein synthesis in both treated and untreated cells for 45 min after metaphase; (2) reformation of polyribosomes, as determined by both sucrose gradients and electron microscopy, within 30 min after the addition of Actinomycin D to metaphase cells; (3) the persistence of approximately 50% of the rapidly labeled nonribosomal RNA which had associated with polyribosomes just before metaphase; (4) the resumption of synthesis, following cell division, of 6 selected peptides in Actinomycin-treated cells.     

Accumulation of 70S Monoribosomes in Escherichia coli After Energy Source Shift-Down

When Escherichia coli is shifted from glucose-minimal to succinate-minimal medium, a transient inhibition of protein synthesis and a time-dependent redistribution of ribosomes from polysomes to 70S monosomes occurs. These processes are reversed by a shift-up with glucose. In a lysate made from a mixture of log-phase and down-shifted cells, the 70S monosomes are derived solely from the down-shifted cells and are therefore not produced by polysome breakage during preparation. This conclusion is supported by the absence of nascent proteins from the 70S peak. The monosomes are not dissociated by NaCl or by a crude ribosome dissociation factor, so they behave as "complexed" rather than "free" particles. When down-shifted cells are incubated with rifampin to block ribonucleic acid (RNA) synthesis, the 70S monosomes disappear with a half-life of 15 min. When glucose is also added this half-life decreases to 3 min. The 70S particles are stable in the presence of rifampin when chloramphenicol is added to block protein synthesis. We interpret these data to mean that the existence of the 70S monosomes depends on the continued synthesis of messenger RNA and their conversion to free ribosomes (which dissociate under our conditions) is a result of their participation in protein synthesis. Finally, a significant fraction of the RNA labeled during a brief pulse of (3)H-uracil is found associated with the 70S peak. These results are consistent with the hypothesis that the 70S monosomes are initiation complexes of single ribosomes and messenger RNA, which do not initiate polypeptide synthesis during a shift-down.     

Assembling an intermediate filament network by dynamic cotranslation

We have been able to observe the dynamic interactions between a specific messenger RNA (mRNA) and its protein product in vivo by studying the synthesis and assembly of peripherin intermediate filaments (IFs). The results show that peripherin mRNA-containing particles (messenger ribonucleoproteins [mRNPs]) move mainly along microtubules (MT). These mRNPs are translationally silent, initiating translation when they cease moving. Many peripherin mRNPs contain multiple mRNAs, possibly amplifying the total amount of protein synthesized within these "translation factories." This mRNA clustering is dependent on MT, regulatory sequences within the RNA and the nascent protein. Peripherin is cotranslationally assembled into insoluble, nonfilamentous particles that are precursors to the long IF that form extensive cytoskeletal networks. The results show that the motility and targeting of peripherin mRNPs, their translational control, and the assembly of an IF cytoskeletal system are linked together in a process we have termed dynamic cotranslation.