Ribosomal subunits and ribosomal proteins of Tetrahymena thermophila. Effect of the presence of iodoacetamide during ribosome extraction on the properties of the subunits

Proteolytic degradation of ribosomal proteins occurs during the preparation of subunits of the cytoplasmic ribosomes of the protozoa Tetrahymena thermophila and the isolated subunits are inactive. Addition of 5 mM iodoacetamide to cell suspensions before extraction inhibits proteolytic activity and permits isolation of active subunits. The protein complements of these subunits have been characterized in two different two-dimensional electrophoretic systems, and their molecular weights have been determined.     

Regulation of protein synthesis in Tetrahymena: isolation and characterization of polysomes by gel filtration and precipitation at pH 5.3.

The fraction of ribosomes loaded on polysomes is about 95% in logarithmically growing Tetrahymena thermophila, and about 4% in starved cells. Cytoplasmic extracts from cells in these two physiological states were used to develop column chromatographic methods for the purification of polysomes. Bio-Gel A 1.5 m was found to separate total cytoplasmic ribosomes from many soluble proteins, including RNAse, with no detectable change in the polysome size distribution. Polysomes can be separated from monosomes and non-polysomal mRNA by chromatography on Bio-Gel A 15 m without size selection. These methods can easily be adapted to large scale preparations of polysomes, even from cells where a small fraction of the ribosomes is on polysomes. A method is described for reversible precipitation of polysomes and monosomes from dilute solutions at pH 5.3 which greatly facilitates polysome isolation. Hybridization of 3H-labeled polyU to RNA isolated from column fractions has been used to demonstrate that purification of EDTA released polysomal mRNA can be performed using the column chromatography procedures described here. These methods have been employed to demonstrate that most of the cytoplasmic mRNA in log-phase Tetrahymena is loaded onto polysomes while most of the mRNA is starved cells exists in a non-polysomal form.     

The influence of fixation on the relative amount of cytoplasmic ribosomes in mouse epidermal basal keratinocytes

The nature and significance of so-called dark keratinocytes in the epidermis during chemical carcinogenesis is still a matter of concern and debate. Based on ultrastructural observations it has been suggested that dark cells most often are shrunken cells. Reports on skin carcinogenesis, however, claim that dark cells are a sign of ongoing tumor promotion and represent those stem cells in the epidermis from which the tumors originate. It is therefore important to find out whether these cells are simply injured and shrunken cells, or vital cells of great importance for carcinogenesis. Dark cells are assumed to be rich in ribosomes. There is evidence, however, that the observed number of dark cells is highly dependent on tissue fixation. In the present ultrastructural study, morphometric methods were used to compare the effects of two different fixation procedures on the amount of cytoplasmic ribosomes in dark cells from both untreated and carcinogen-treated hairless mouse epidermis. The results show that the ultrastructural features of both dark and clear cells vary considerably with different fixation procedures. In acetone-treated controls typical dark cells are only observed when the fixative has a lower osmotic activity than the plasma. With iso-osmolal fixation typical dark cells are not observed. After an abortive two-stage carcinogenesis treatment, in which a single application of 9,10-dimethyl-l,2-benzanthracene (DMBA) in acetone was followed by a single application of 12-O-tetradecanoyl-13-acetate (TPA) in acetone, signs of cell injury could be found after both fixation procedures. With DMBA/TPA and hypo-osmolal fixation the number of dark cells seemed to increase, whereas only signs of cell injury with occurrence of some heavily altered “clear cells” dominated the picture with iso-osmolal fixation. Morphometry showed that both the numerical and the volumetric densities of cytoplasmic ribosomes in basal keratinocytes varied most significantly with the fixation procedure used. The cytoplasmic volumes did not vary in a way that could explain these differences. One might therefore assume that the number of ribosomes depends on the fixative. Large swelling artifacts occurred when a fixative with low osmotic activity was used, leading to compression of neighboring cells. Hence, an increased ribosomal density reported previously in dark cells is probably related to such cell volume artifacts and does not reflect an actually increased quantity of ribosomes. With both fixation procedures, a single application of DMBA followed by one of TPA appeared to produce an increased number of ribosomes in basal keratinocytes. When hypo-osmolal fixation was used, however, treatment with DMBA/TPA did not influence the cytoplasmic volume or the numerical density of ribosomes, in dark cells. This might indicate that so-called dark keratinocytes following DMBA/TPA treatment are functionally inactive cells that appear more vulnerable than active cells to compression during hypo-osmolal fixation.     

Soluble factor requirements for the Tetrahymena peptide elongation system and the ribosomal ATPase as a counterpart of yeast elongation factor 3 (EF-3)

Peptide elongation factor 3 (EF-3), which is widely present in yeasts and fungi (Eumycota), does not occur in another lower eukaryote, the unicellular protozoan Tetrahymena pyriformis, as was shown by the following findings: (a) there is no activity to satisfy the EF-3 requirement of yeast ribosomes in the post-ribosomal supernatant fraction from Tetrahymena, and (b) the Tetrahymena ribosomes displayed their full capacity for polyphenylalanine synthesis with purified EF-1 alpha and EF-2 alone from either Tetrahymena or yeast, and their activity on the Tetrahymena ribosomes was not further enhanced by the addition of yeast EF-3, in contrast to the case of the yeast ribosomes. However, as a substitute for the ribosome-activated nucleotidase activity of EF-3, Tetrahymena ribosomes were shown to harbor strong, firmly bound ATPase and GTPase activities, which probably involve the same active site. The ribosome-bound ATPase activity was inhibited by a polyclonal antibody raised against yeast EF-3 with the same inactivation profile as that of polyphenylalanine synthesis on Tetrahymena ribosomes, indicating that the ribosomal ATPase plays an essential role in the elongation process on Tetrahymena ribosomes as previously revealed in the yeast system. It was also shown that the ribosomal nucleotidase plays a pivotal role in the elongation cycle in other eukaryotes.     

The influence of fixation on the relative amount of cytoplasmic ribosomes in mouse epidermal basal keratinocytes. A morphometric study of so-called "dark cells" and their putative role in epidermal carcinogenesis

The nature and significance of so-called dark keratinocytes in the epidermis during chemical carcinogenesis is still a matter of concern and debate. Based on ultrastructural observations it has been suggested that dark cells most often are shrunken cells. Reports on skin carcinogenesis, however, claim that dark cells are a sign of ongoing tumor promotion and represent those stem cells in the epidermis from which the tumors originate. It is therefore important to find out whether these cells are simply injured and shrunken cells, or vital cells of great importance for carcinogenesis. Dark cells are assumed to be rich in ribosomes. There is evidence, however, that the observed number of dark cells is highly dependent on tissue fixation. In the present ultrastructural study, morphometric methods were used to compare the effects of two different fixation procedures on the amount of cytoplasmic ribosomes in dark cells from both untreated and carcinogen-treated hairless mouse epidermis. The results show that the ultrastructural features of both dark and clear cells vary considerably with different fixation procedures. In acetone-treated controls typical dark cells are only observed when the fixative has a lower osmotic activity than the plasma. With iso-osmolal fixation typical dark cells are not observed. After an abortive two-stage carcinogenesis treatment, in which a single application of 9,10-dimethyl-1,2-benzanthracene (DMBA) in acetone was followed by a single application of 12-O-tetradecanoyl-13-acetate (TPA) in acetone, signs of cell injury could be found after both fixation procedures. With DMBA/TPA and hypo-osmolal fixation the number of dark cells seemed to increase, whereas only signs of cell injury with occurrence of some heavily altered "clear cells" dominated the picture with iso-osmolal fixation. Morphometry showed that both the numerical and the volumetric densities of cytoplasmic ribosomes in basal keratinocytes varied most significantly with the fixation procedure used. The cytoplasmic volumes did not vary in a way that could explain these differences. One might therefore assume that the number of ribosomes depends on the fixative. Large swelling artifacts occurred when a fixative with low osmotic activity was used, leading to compression of neighboring cells. Hence, an increased ribosomal density reported previously in dark cells is probably related to such cell volume artifacts and does not reflect an actually increased quantity of ribosomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ribosomal proteins in growing and starved Tetrahymena pyriformis. Starvation-induced phosphorylation of ribosomal proteins.

The complements of ribosomal proteins in growing and starved cells of Tetrahymena pyriformis strain GL were examined by two-dimensional gel electrophoresis. In growing cells, the 40-S ribosomal subunit contained 30 proteins, 4 of which migrated toward the anode at pH 8.6, while the 60-S ribosomal subunit contained 46 proteins, 9 of which migrated toward the anode at pH 8.6. When exponentially growing cells were transferred into a non-nutrient medium pronounced phosphorylation of a single 40-S ribosomal subunit protein, S6, was induced. The phosphorylation was very specific; more than 99.5% of the [32P]phosphate incorporated into ribosomal proteins was associated with S6. Phosphate was incorporated into S6 as O-phosphoserine and O-phosphothreonine. Two-dimensional gel electrophoresis indicated that the complement of proteins associated with the ribosomes isolated from starved cells differed from that of growing cells. Careful examination, however, suggested that except for the phosphorylation of certain ribosomal proteins in starved cells, the observed differences did not reflect starvation-induced changes in vivo, but most probably different levels of artifactual modifications (limited proteolysis) during the preparation of the ribosomes.     

Isolation of prolactin from lyophilized porcine pituitary glands]

The optimal conditions for lyophilization of porcine pituitary glands and isolation of pure prolactin from lyophilized preparation have been investigated. The isolation method consisted in the extraction of crude pituitary preparation with acidified acetone followed by precipitation of crude prolactin preparation (acid acetone powder) by increasing the concentration of acetone in the extract to 92%. Further purification of prolactin was achieved by fractional precipitation at varying pH values and gel filtration on Sephadex G-75 column in a pH 7.5 phosphate buffer. This final procedure resulted in obtaining the monomeric form of prolactin. The identity of the isolated hormone was confirmed by spectrophotometric and radioimmunological methods as well as by polyacrylamide gel electrophoresis.     

The ribosomal proteins of Drosophila melanogaster. I. Characterization in polyacrylamide gel of proteins from larval, adult, and ammonium chloride-treated ribosomes

Summary Ribosomes were isolated from larvae and adult flies, and the purity of the preparation was checked by electron microscopy. The ribosomal proteins were extracted with cold dilute hydrochloric acid, and precipitated with cold acetone. The proteins were characterized by polyacrylamide gel electrophoresis. At pH 3.0 at least 25 bands of different color intensities were resolved, forming a complex pattern.On the basis of electrophoretic mobilities, it was shown that some ribosomal proteins are species-specific, and that larval ribosomes have three protein components more than ribosomes from adult flies.Incubation of the ribosomes with 0.75 M NH₄Cl at a low Mg⁺⁺ concentration lead to detachment of 64% of the ribosomal protein. This detachment of protein molecules was considerably reduced by a five-fold increase of Mg⁺⁺ ions.     

Polypeptide release factor eRF1 from Tetrahymena thermophila: cDNA cloning, purification and complex formation with yeast eRF3.

The first cDNA for the translational release factor eRF1 of ciliates was cloned from Tetrahymena thermophila. The coding frame contained one UAG and nine UAA codons that are reassigned for glutamine in Tetrahymena. The deduced protein sequence is 57% identical to human eRF1. The recombinant Tetrahymena eRF1 purified from a yeast expression system was able to bind to yeast eRF3 as do other yeast or mammalian eRF1s as a prerequisite step for protein termination. The recombinant Tetrahymena eRF1, nevertheless, failed to catalyze polypeptide termination in vitro with rat or Artemia ribosomes, at least in part, due to less efficient binding to the heterologous ribosomes. Stop codon specificity and phylogenetic significance of Tetrahymena eRF1 are discussed from the conservative protein feature.     

Resistance against cycloheximide in cell lines from Chinese hamster and human cells is conferred by the large subunit of cytoplasmic ribosomes

Summary Cell lines from Chinese hamster ovary [CHO-K1-D3] and human fibroblast cells [46, XX, 18p-] were mutagenized with N-nitrosomethylurea followed by a selection for cycloheximide resistance. Two mutants resistant against the durg were selected from either wildtype. 80S ribosomes and their ribosomal subunits were isolated from all mutant and wildtype cells. 80S ribosomes reassociated from the isolated subunits were as active as isolated 80S couples in the poly (U) dependent poly (Phe) synthesis. Hybrid 80S ribosomes constructed from subunits of the various cell lines of the same species were fully active, whereas the interspecies 80S hybrids were not active at all in poly (Phe) synthesis.Hybrid 80S ribosomes from subunits of mutant and the ocrresponding wildtype cells were tested in the poly (U) assay in the presence and absence of cycloheximide. The results strikingly indicate that in all four mutant cell lines the resistance against cycloheximide is conferred by the large subunit of cytoplasmic ribosomes.Abbreviations CHM Cycloheximide - CHO Chinese hamster ovarien - FBS foetal bovine serum - Eagle MEM Eagle minimal essential medium - EMS Ethyl-metansulfonate - NMU N-nitrosomethylurea     

Lipoprotein lipase activity of rat cardiac muscle. The intracellular distribution of the enzyme between fractions prepared from cardiac muscle and cells isolated from the hearts of fed and starved animals.

1. Subcellular fractions, characterized by using morphological, compositional and enzymic markers, were prepared from rat heart tissue and cells isolated from the hearts of fed and 24 h-starved rats. 2. The lipoprotein lipase activity of fractions from whole tissue and isolated cells was determined in either fresh fractions or in acetone/diethyl ether powders of the fractions. 3. Lipoprotein lipase activity was present in all the fractions from tissue and cells, but was found to be of highest relative specific activity in the microsomal () fractions. 4. In fractions prepared from the isolated cells of hearts from starved rats the proportion of the total lipoprotein lipase present and its relative specific activity in the microsomal fraction were greater than in the equivalent fractions from fed animals. 5. The enhancement of lipoprotein lipase activity as a result of the acetone/diethyl ether powder preparation of fractions was most extensive in the microsomal fractions. 6. Investigation of the microsomal fraction showed that the lipoprotein lipase activity present was in two pools, one of which was within endoplasmic-reticulum vesicles. 7. The observations were consistent with the possibility that the cardiac-muscle cell could be the origin of the lipoprotein lipase activity functional in triacylglycerol uptake by the heart.     

Studies of Messenger RNA during the Cell Cycle in Synchronized Mass Cultures of Tetrahymena pyriformis GL by Hybridization

SYNOPSIS RNA isolated from free ribosomes, from cell structures and from soluble cell phase after indole lysis of synchronized Tetrahymena cells showed different abilities to hybridize with DNA. The supraoptimal temperature (34 C) caused a decrease in the ability to hybridize in all 3 RNA fractions. The same effect was noted at the time of cell division. Synthesized messenger RNA as a proportion of the total quantity of RNA was roughly constant during the whole cell cycle. However, in contrast to synchronized mammalian cells the messenger RNA synthesis did not proceed at a constant rate thruout the cell cycle.     

Identification of ethanol-inducible P-450 isozyme 3a as the acetone and acetol monooxygenase of rabbit microsomes

Treatment of rabbits with 1% (v/v) acetone for 1 week resulted in the appearance in blood serum of 88 +/- 14 14 nmol/ml 1-hydroxyacetone (acetol) and 70 +/- 9 nmol/ml 1,2-propanediol. Untreated rabbits had no detectable 1,2-propanediol or acetol. Hepatic microsomes from control, ethanol-, and acetone-treated rabbits catalyzed the hydroxylation of acetone at rates of 0.32 +/- 0.01, 2.01 +/- 0.43, and 3.64 +/- 0.23 nmol/min/mg of protein, respectively. The same microsomal preparations catalyzed the hydroxylation of acetol at rates of 0.33 +/- 0.04, 0.94 +/- 0.20, and 1.08 +/- 0.12 nmol/min/ mg of microsomal protein, respectively. Isozyme 3a purified from acetone- or ethanol-treated rabbits was identical as judged by comparison of the high performance liquid chromatographic profiles of tryptic digests of the two proteins. Antibody to isozyme 3a inhibited greater than 90% of the acetone monooxygenase activity from untreated, acetone-, or ethanol-treated rabbits. In contrast, the antibody only inhibited 30% of the acetol monooxygenase activity of microsomes from untreated rabbits. The inhibition was increased to about 70% after acetone or ethanol treatment. Although the activities were inhibited to different extents, a comparison of the rates attributable to isozyme 3a from antibody inhibition experiments indicated that both activities were induced to a similar extent by ethanol. Similarly, acetone also increased both activities to the same extent but was more effective than ethanol. In a reconstituted system, isozyme 3a was the only isozyme of six forms from rabbit liver to exhibit acetone monooxygenase activity. Isozyme 3a was the most active enzyme in the hydroxylation of acetol, but isozymes 2, 3b, and 4 also were able to catalyze the reaction. Antibody to isozyme 3a also inhibited greater than 90% of the acetone hydroxylase activity and 70% of the acetol hydroxylase activity of microsomes from acetone-treated rats. Two proteins were immunochemically stained on Western blots of microsomes from untreated and acetone-treated rats, one of which was increased by acetone treatment. These results suggest that isozyme 3a in rabbit and an immunochemically homologous enzyme in rat are responsible for acetone and acetol hydroxylation, the initial steps in the proposed gluconeogenic pathways for acetone.     

Effect of heat shock on ribosome structure: appearance of a new ribosome-associated protein.

After a nonlethal but heat shock protein-inducing hyperthermic treatment, ribosomes isolated from Tetrahymena thermophila contained an additional 22-kilodalton protein (p22). When maximally ribosome associated, this protein was found to be on the small subunit in a 1:1 stoichiometric ratio with other ribosomal proteins. Using an antiserum directed against the purified 22-kilodalton protein, we found that non-heat-shocked and heat-shocked cells contain identical amounts of this protein, the only difference being that in the stressed cells p22 is entirely ribosome bound, whereas in the unstressed cells p22 has little or no detectable ribosome association. Because the two-dimensional electrophoretic properties of p22 showed no alterations after heat shock, this change in state of ribosome-p22 interaction does not appear to be caused by a chemical modification of p22. When not strongly ribosome associated, p22 is not found free in the cytoplasm. During that time in heat shock when p22 is first becoming ribosome associated, it is found preferentially on polysomal ribosomes. Subsequently, all ribosomes, whether polysome bound or not, obtain a bound p22. The functional significance of this association is discussed.     

Nonidentity of ribosomal structural proteins in growing and starved Tetrahymena

We have examined the ribosomal structural proteins isolated from vegetatively growing Tetrahymena pyriformis and from cells that had been starved of all nutrients for 24 h. Reproducible, nonartifactual differences in protein complement, primarily associated with the large ribosomal subunit, were found. The kinetics of change in ribosomal protein complement were followed both in refed and in newly starved cells. Furthermore, attempts at correlating a certain protein "phenotype" with a particular functional state of the ribosome were made. It was concluded that the alterations seen could not be correlated with a specific stage in the normal ribosome cycle. We did show, however, that the change in protein complement could occur as a result of altering preexisting ribosomes. In addition, we showed that the change correlates with a decrease in growth rate rather than being caused by the starvation conditions themselves. Speculations as to the functional significance of the protein changes are presented.     

Translational Control of Protein Synthesis in Staphylococcus aureus.

The calculated in vivo polypeptide chain growth rate for Staphylococcus auteus MF-31 grown in nutritionally rich medium assuming all the ribosomes were functional was found to be approximately 16 amino acids/s/ribosome, but decreased to 10.2 amino acids/s/ribosome for cells grown in poor medium. An in vitro analysis revealed that 70S ribosomes isolated from rich medium cells were more active than similar 70S ribosomes derived from cells grown in poor medium. The 30S subunit was found responsible for the increased activity of the rich monosomes, whereas the 50S subunit appeared to be capable of either high or low activity.     

One step purification procedure of elastase from pancreatic powder by affinity chromatography

Pancreatic elastase was isolated from acetone powder of porcine pancreas by a one step purification procedure on a trialanyl CH-Sepharose 4B affinity column. This column exhibited affinity not only for active elastase but also for trypsin and chymotrypsin which were present in the same pancreatic powder. However, as the extent of affinity toward elastase is considerably higher, the proper conditions were determined with which the adsorbed elastase was isolated in a highly purified form. The yield of elastolytic activity ranged from 60–85% and the purified elastase was shown to be one component by polyacrylamide disc electrophoresis.     

A Mutation in the Large Subunit Ribosomal RNA Gene of Tetrahymena Confers Anisomycin Resistance and Cold Sensitivity

Anisomycin, an antibiotic that specifically inhibits the peptidyl transfer function of eukaryotic ribosomes, has been used to select resistant mutants in Tetrahymena thermophila. A mutation conferring anisomycin resistance (an-r) has been localized to a 1.2-kb fragment of the large subunit ribosomal RNA (rRNA) gene by transformation via microinjection. A single base pair change was detected within this region. Nine independently isolated an-r mutants had the same base pair change. T. thermophila strains that are homozygous for this mutation are cold sensitive, unable to mate and grossly abnormal in cell morphology.     

RIBOSOMAL SUBUNITS FROM NEONATAL MOUSE BRAIN HIGHLY ACTIVE IN POLYPHENYLALANINE SYNTHESIS

Abstract— A highly active subcellular protein synthesising system is described, in which uncomplexed ribosomes isolated from 5 to 7 day old mouse brain can be reprogrammed with polyuridylic acid. Either purified free polyribosomes or microsomes were used as the starting material for the preparation of uncomplexed ribosomes by treatment with 0.5 m -KCl and puromycin. After reduction of the salt concentration 80S ribosomes were isolated by washing through sucrose. When, subsequently, zonal centrifugation in equivolumetric sucrose gradients containing 0.5 m -KCI was performed, purified ribosomal subunits were obtained. Cross-contamination of subunits was less than 5%. Re-associated ribosomes and recombined isolated ribosomal subunits both showed high activities in vitro. Incorporation levels of 50–60 phenylalanine residues per ribosome could be reached, at a rate of 0.5–2.0 residues/min/ribosome, depending on the activity of the high speed supernatant enzymes added. It was shown by paper chromatography of the cell-free product that only oligophenylalanine formation takes place. It was estimated that 6&70% of the ribosomes present in vitro were actively participating in the protein synthesis process.     

Tetrahymena cells secrete a low-molecular weight factor which inhibits RNA synthesis in vivo and in vitro.

Medium which has been dialyzed against a dense population of Tetrahymena cells contains a factor which reduces the rate of RNA synthesis in other Tetrahymena cells inoculated on that medium. Furthermore, this extracellular factor inhibits the ribosomal RNA synthesis in vitro when added to an RNA synthesizing r-chromatin preparation.